Propargyl-linked antifolates are dual inhibitors of Candida albicans and Candida glabrata

Article abstract of DOI:10.1021/jm401916j

Species of Candida, primarily C. albicans and with increasing prevalence, C. glabrata, are responsible for the majority of fungal bloodstream infections that cause morbidity, especially among immune compromised patients. While the development of new antifungal agents that target the essential enzyme, dihydrofolate reductase (DHFR), in both Candida species would be ideal, previous attempts have resulted in antifolates that exhibit inconsistencies between enzyme inhibition and antifungal properties. In this article, we describe the evaluation of pairs of propargyl-linked antifolates that possess similar physicochemical properties but different shapes. All of these compounds are effective at inhibiting the fungal enzymes and the growth of C. glabrata; however, the inhibition of the growth of C. albicans is shape-dependent with extended para-linked compounds proving more effective than compact, meta-linked compounds. Using crystal structures of DHFR from C. albicans and C. glabrata bound to lead compounds, 13 new para-linked compounds designed to inhibit both species were synthesized. Eight of these compounds potently inhibit the growth of both fungal species with three compounds displaying dual MIC values less than 1 μg/mL. Analysis of the active compounds shows that shape and distribution of polar functionality is critical in achieving dual antifungal activity.

Full text of DOI:10.1021/jm401916j

Article
pubs.acs.org/jmc
Propargyl-Linked Antifolates are Dual Inhibitors of Candida albicans
and Candida glabrata
Narendran G-Dayanandan,†,§ Janet L. Paulsen,†,§ Kishore Viswanathan,† Santosh Keshipeddy,†
Michael N. Lombardo,† Wangda Zhou,† Kristen M. Lamb,† Adrienne E. Sochia,‡ Jeremy B. Alverson,‡
Nigel D. Priestley,‡ Dennis L. Wright,*,† and Amy C. Anderson*,†
†Department of Pharmaceutical Sciences, University of Connecticut, 69 N. Eagleville Road, Storrs, Connecticut 06269, United States
‡Department of Chemistry, University of Montana, Missoula, Montana 59812, United States
S
* Supporting Information
ABSTRACT: Species of Candida, primarily C. albicans and with increasing prevalence, C. glabrata, are responsible for the
majority of fungal bloodstream infections that cause morbidity, especially among immune compromised patients. While the
development of new antifungal agents that target the essential enzyme, dihydrofolate reductase (DHFR), in both Candida species
would be ideal, previous attempts have resulted in antifolates that exhibit inconsistencies between enzyme inhibition and
antifungal properties. In this article, we describe the evaluation of pairs of propargyl-linked antifolates that possess similar
physicochemical properties but different shapes. All of these compounds are effective at inhibiting the fungal enzymes and the
growth of C. glabrata; however, the inhibition of the growth of C. albicans is shape-dependent with extended para-linked
compounds proving more effective than compact, meta-linked compounds. Using crystal structures of DHFR from C. albicans
and C. glabrata bound to lead compounds, 13 new para-linked compounds designed to inhibit both species were synthesized.
Eight of these compounds potently inhibit the growth of both fungal species with three compounds displaying dual MIC values
less than 1 μg/mL. Analysis of the active compounds shows that shape and distribution of polar functionality is critical in
achieving dual antifungal activity.
of therapy to which the strain is sensitive. While C. albicans
remains relatively sensitive to azoles, flucytosine, and
echinocandins, C. glabrata exhibits decreased sensitivity for
fluconazole, with evidence of cross-resistance to other azoles
such as voriconazole;8,9 11% of fluconazole-resistant strains are
now also resistant to echinocandins.10 The increased incidence
of C. glabrata as a causative agent of candidiasis along with the
increasing drug resistance in this strain makes new antifungals
that target C. glabrata a clear priority. However, an ideal agent
would target both C. albicans and C. glabrata as C. albicans
infections continue to be a major health risk and the two are
difficult to distinguish in a clinical setting.
INTRODUCTION
■
Although bloodstream infections (BSI) are frequently attrib-
uted to bacterial pathogens, fungal infections caused by
Candida species actually represent the fourth leading cause of
BSI in the United States and present a specific risk for immune
compromised patients.1−3 The incidence of candidiasis has
increased dramatically over the previous two decades,2,4,5
resulting in significant morbidity, mortality (40−49%6), and
increased healthcare costs.2 Among the Candida spp., C.
albicans is the primary cause of BSI (45.6%), followed by C.
glabrata (26.0%).7 However, C. glabrata represents an
increasing threat as studies show that while C. glabrata
accounted for 18% of BSI candidemia between 1992 and
2001, that fraction rose to 26% in the time period 2001−2007.
The administration of effective empirical therapy for fungal
BSI significantly reduces mortality (27% vs 46%).6 Unfortu-
nately, however, there is often a significant delay in the correct
diagnosis of candidiasis,2,6 identification of the species, and start
Targeting the essential enzyme dihydrofolate reductase
(DHFR) has proven to be an effective strategy for both
prokaryotic (e.g., trimethoprim) and protozoal (e.g., pyrimeth-
Received: December 13, 2013
Published: February 25, 2014
© 2014 American Chemical Society
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Figure 1. Shape of the propargyl-linked antifolates affects the antifungal activity. Enzyme inhibition is shown per species as an abbreviation (e.g.,
CgDHFR IC50) with 50% inhibition concentrations (IC50 values) reported in nM; MIC values are reported in μg/mL. The positional isomers for
rings B and C are shown in the center of the figure.
amine) pathogens but is not widely used clinically in the
treatment of invasive fungal infections. DHFR plays a critical
role in the turnover of folate cofactors; effective inhibition of
DHFR produces a blockade in thymidine synthesis leading to
“thymineless” death. As humans are also dependent on active
DHFR, it is important that there is selective inhibition of the
pathogenic enzyme. Fortunately, there are several important
active site differences between human and Candida species that
can be exploited for selectivity.
to a meta-linked biphenyl14,15 or biaryl16 system (example
compounds 1, 2, and 4 in Figure 1) that show potent and
selective inhibition of DHFR from C. albicans and C. glabrata.
However, while potent inhibition of the growth of C. glabrata
was observed with these antifolates, enzyme inhibition did not
translate to antifungal activity against C. albicans, in a manner
similar to that in previously reported studies.
As results in the literature show that target potency did not
exclusively drive antifungal activity, we re-examined previously
abandoned leads in the propargyl-linked antifolate series to
search for potentially active chemotypes against C. albicans. In
doing so, we identified three para-linked compounds
(compounds 3, 5, and 6) that inhibit both Candida species.
Building on this promising discovery, herein we report the
synthesis and evaluation of 13 additional para-linked inhibitors
and show that eight of these compounds inhibit the growth of
both Candida species, with three showing very potent
antifungal activity (MIC values of <1 μg/mL). Analysis of
crystal structures of DHFR from both species bound to para-
linked antifolates correlates with structure−activity relation-
ships to reveal that hydrophobic functionality at the C-ring
improves the potency of enzyme inhibition. These develop-
ment studies represent a significant advance toward achieving a
propargyl-linked antifolate as a single agent that potently
targets both major species of Candida. Furthermore, prelimi-
nary studies reported here suggest that in addition to inhibitor
potency at the enzyme level, there is a second critical
relationship between the shape of the inhibitor, dictated here
by the positional isomers of the ring systems, and antifungal
activity. These compounds may also be useful to permit
comparative studies between the two Candida species.
It is widely recognized that the development of antimetabo-
lites targeting C. albicans can be complicated by pronounced
inconsistencies between target inhibition and antifungal
activity.11−13 Attempts to study whether the cell wall or
membrane permeability affects the uptake of six unrelated
antibiotics targeting intracellular proteins failed to derive a
direct relationship.13 These same inconsistencies have also
complicated the development of antifungal antifolates. For
example, Glaxo researchers hypothesized that molecular weight
was inversely related to antifungal activity and pursued the
synthesis and evaluation of over 150 low molecular weight
analogues. Although the Glaxo effort produced potent, albeit
nonselective inhibitors with good antifungal activity, lead
optimization of the antifolates against C. albicans was hindered
by a lack of correlation between enzyme inhibition and
antifungal activity. The researchers concluded that there was no
relationship between activity and inhibitor size or lipophilicity
but that differences in transport phenomenon could still play an
important role in antifungal activity.11 More recently, a German
company12 reported a group of potent C. albicans DHFR
inhibitors based on a benzyl(oxy)pyrimidine scaffold. However,
these compounds did not exhibit in vitro antifungal activity.
After showing that the compounds were not generally
susceptible to efflux, the authors of this study also speculated
that the compounds were unable to enter C. albicans. While
these studies were conducted with C. albicans, it is unclear
whether the same phenomenon would be observed with C.
glabrata.
RESULTS
■
The meta-heterobiaryl propargyl-linked antifolates (such as
compound 1 in Figure 1) are potent inhibitors of DHFR from
both C. glabrata and C. albicans, with many compounds having
50% inhibition concentrations (IC50) under 100 nM16 and a
large number of interactions with active site residues
(Supporting Information, Figure S1). However, despite the
Previously, we reported a new class of antifolates possessing a
2,4-diaminopyrimidine ring linked through a propargyl bridge
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Table 1. Biological Evaluation of Propargyl-Linked Antifolates
a
b
c
d
Selectivity is calculated as IC50 for the fungal enzyme/IC50 for the human enzyme. Compound number/MW/clogP. ND: not determined. NA:
not active at 100 μg/mL.
fact that these compounds are also potent inhibitors of the
growth of C. glabrata, these meta-linked compounds were
unable to potently inhibit C. albicans. For example, compound
1 inhibits C. glabrata and C. albicans DHFR with IC50 values of
89 and 60 nM yet inhibits C. glabrata and C. albicans with MIC
values of 1.3 μg/mL and 25 μg/mL, respectively. In an attempt
to determine whether permeability may explain the differential
antifungal activity of the propargyl-linked antifolates, we
measured MIC values for compound 1 in the presence of
0.01% Triton X-100. Triton X-100 is known to increase
membrane permeability without denaturation.17 The experi-
ments show that in the presence of Triton X-100, the MIC
values for compound 1 significantly decreased (25 to 6.25 μg/
mL). These results suggest that permeability may influence
antifungal activity.
As our prior work had shown that compounds with different
physicochemical properties or shapes displayed differential
antifungal activity against C. glabrata (for example, compare
compounds 1−6 in Figure 1),16 we re-examined the C. albicans
activity of several earlier scaffold types. This investigation
showed that compounds containing a para-biphenyl moiety as
the hydrophobic domain (e.g., compound 3) had promising
(MIC 1.6 μg/mL) activity against C. albicans while maintaining
activity against C. glabrata (MIC 0.39 μg/mL) (Figure 1).
These results suggested the intriguing possibility that alteration
of the molecular shape greatly influences the C. albicans activity
without diminishing activity against C. glabrata. This improve-
ment in the C. albicans activity was then shown to extend to
two other compounds in the para-biphenyl series (e.g., 5 and
6). Also encouraging, the compounds remained selective for the
fungal cells over human cells. For example, compounds 3 and 5
inhibit the growth of MCF-10 cells at 74 and 80 μM,
respectively (Table 1). These results prompted the exploration
of this para-linked shape with a goal of identifying compounds
that maintain enzyme inhibition and have superior antifungal
activity against both Candida species.
Crystal Structures of Candida DHFR Bound to para-
Linked Antifolates. In order to elucidate the structural basis
of the affinity of the para-linked compounds for C. glabrata and
C. albicans DHFR and to design more potent analogues in this
series, we determined the ternary structures of the two enzymes
bound to NADPH and compound 3 as well as the complex of
C. albicans DHFR bound to NADPH and 6. The structures
were determined by molecular replacement using diffraction
amplitudes extending to 1.76 Å (CaDHFR/NADPH/3 and
CaDHFR/NADPH/6) or 2.0 Å (CgDHFR/NADPH/3)
(Supporting Information, Table S1). All structures contain
two molecules in the asymmetric unit. Despite the fact that the
crystallization conditions included a racemic mixture of the
ligand, the R-enantiomer is the only one observed in the
electron density. Interestingly, one of the two inhibitor
molecules of CgDHFR/NADPH/3 adopts a different con-
formation from the other inhibitor in the same asymmetric unit.
One conformation points the 3′-methoxy down into the pocket
enclosed by Phe 36, Leu 69, and Met 33 (Figure 2a), and the
other points the methoxy toward Ser 61 to form a water-
mediated hydrogen bond (Figure 2b). Similarly, one of the two
molecules of CaDHFR/NADPH/3 in the asymmetric unit
exhibits the “down” conformation of the methoxy toward Phe
36 and Leu 69 at 100% occupancy (Figure 2c); the other
inhibitor molecule has two conformations of the methoxy
group with split 75%/25% occupancy. The “up” conformation
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Figure 2. Crystal structures of (a) C. glabrata DHFR bound to NADPH and 3 (PDB ID: 4HOG) showing one conformation of the inhibitor and
(b) a second conformation of the inhibitor; (c) C. albicans DHFR bound to 3 (PDB ID: 4HOF, magenta) and 6 (PDB ID: 4HOE, teal). Compound
3 in PDB ID 4HOF also shows two conformations of the inhibitor in chain A that are similar to those observed in the structure with C. glabrata
DHFR.
a
Scheme 1
a
(a) Aryl-boronic acid, Pd(PPh3)2Cl2, Cs2CO3, dioxane, 80°C; (b) Ph3PCHOMe, THF; (c) Hg(OAc)2, Kl, THF/H2O; (d) dimethyl(1-diazo-2-
oxopropyl)phosphonate, K2CO3, MeOH; (e) CISO2NCO, CH2Cl2; (f) 6-ethyl,5-iodo-2,4-diaminopyrimidine, Pd(PPh3)2Cl2, Cul, Et3N, DMF.
(75% occupancy) forms a water-mediated hydrogen bond
between the methoxy group and Ser 61; the “down”
conformation (25% occupancy) interacts with Phe 36 and
Leu 69. Overall, the inhibitors form the conserved set of
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hydrogen bonds and hydrophobic interactions between the
pyrimidine ring and Glu 32, Ile 9, Phe 36, Met/Ile 33, and Ile
121. The propargyl linker forms van der Waals interactions with
Ile 121 and Leu 25 as well as NADPH. The biphenyl moiety
forms important hydrophobic contacts with Ile 62, Pro 63, and
Phe 66. The para position of the distal C-ring appeared to offer
an ideal location for the introduction of functionality that could
alter the physicochemical properties of the molecule without
being deleterious to enzyme inhibition.
compounds 28 and 37 shows that the polar 2′-methoxy is well-
tolerated in this region but is not required for potency. Three
new derivatives (46−48) were prepared from available pyridyl
or pyrimidyl building blocks (38 and 39) using an analogous
series of transformations as previously described (Scheme 2).
a
Scheme 2
Chemistry. The dual inhibition of C. glabrata and C.
albicans encouraged us to design and synthesize 10 new
biphenyl inhibitors in the para-linked series of compounds with
varying substitutions at the 4′ position of the distal phenyl ring
designed to probe the dependence of antifungal activity on
physicochemical properties or to increase polarity. The
synthesis of the compounds follows from previously developed
routes and in brief involves the use of a central 4-bromo-
acetophenone moiety such as compounds 7 and 8 (Scheme 1).
Suzuki cross-coupling with various aryl boronic acids gives a
diverse group of biaryl derivatives (9−17) with a key acetyl
group that can be taken on to the propargylated intermediates
(18−27) through a three-step process. Final cross-coupling
with 6-ethyl-5-iodo-2,4-diaminopyrimidine yields the panel of
inhibitors (28−37).
a
(a) Aryl-boronic acid, Pd(PPh3)2Cl2, Na2CO3, CH3CN, H2O, 80°C;
(b) Ph3PCHOMe, THF; (c) Hg(OAc)2, Kl, THF/H2O; (d)
dimethyl(1-diazo-2-oxopropyl)phosphonate, K2CO3, MeOH; (e) 6-
ethyl,5-iodo-2,4-diaminopyrimidine, Pd(PPh3)2Cl2, Cul, Et3N, DMF.
Biological Evaluation. Evaluation of a series of nine
biphenyls with variable substitution on the C-ring (compounds
28 and 30−37) clearly indicates that diverse substitution at this
position is well-tolerated as all compounds maintained good
enzyme inhibitory activity against both species (IC50 values are
6−31 nM for CgDHFR and 18−64 nM for CaDHFR).
However, only those compounds substituted with hydrophobic
functionality at the 4-position of the distal C-ring (28, 31, 32,
36, and 37) possess significant antifungal activity against C.
albicans with MIC values ranging from 1.8−7.5 μg/mL. These
results suggest that not only the shape (para-linked C-ring) but
also the para-substitution on the C-ring affects C. albicans
activity. As we had previously observed, the activity of
compound 29 against C. glabrata improved slightly (1.6 to
0.78 μg/mL); however, this was accompanied by a significant
diminution in activity for C. albicans (6.3 to 25 μg/mL).
There appear to be two clusters of activities. In one cluster,
compounds 35, 29, 30, and 33 with polar substituents NMe2,
endo-N, OH, and CO2NH2 exhibit a significant decrease in
activity. This decrease is particularly large for C. albicans but is
also apparent for C. glabrata, with the noted exception of
compound 29. Additionally, the compounds with polar
substitutions showed decreased selectivity at the enzyme
level, most likely because of interactions with the human
residue, Asn 64 (Phe in both fungal species). In a second
cluster, compounds 28, 37, 31, 32, and 36 with hydrophobic or
electron-withdrawing substituents H, CH3, CN, and F maintain
or show improvement in activity with noted variation between
the two species.
Excitingly, compounds 46−48 display a striking improve-
ment in antifungal activity against both species (MIC = 0.2−
0.78 μg/mL). As expected with the more permeable
compounds and in contrast with compound 1, the antifungal
activity of compound 47 was not significantly changed in the
presence of 0.01% Triton X-100. Additionally, compounds 46
and 47 are highly selective for the fungal enzymes (13−30-fold;
sequence alignment in Supporting Information, Figure S2). In
contrast to the distal pyridines, incorporation of pyridine in the
B-ring (compounds 46 and 47) did not provide a significant
increase in solubility (20 and 15 μg/mL, respectively).
However, installation of the much more polar pyrimidine
group (48) increased solubility to a very good level (60 μg/
mL). While compound 48 exhibited a decrease in selectivity for
the fungal enzymes, it maintains an excellent level of selectivity
at the cellular level with an IC50 against mammalian cells of 216
μM. On the basis of docking models of CaDHFR and
CgDHFR bound to compound 48 (Supporting Information,
Figure S3) superimposed with human DHFR, it is apparent
that additional hydrophobic substituents to the C-ring may
enhance selectivity by increasing interactions with Phe 66 in the
fungal enzymes and decreasing interactions with Asn 64 in the
human enzyme.
DISCUSSION
■
As reported here, the shape and distribution of polar
functionality in the compounds significantly impacts the C.
glabrata and C. albicans antifungal activity independent of the
enzyme inhibitory potency. One hypothesis for these changes
in activity could relate to differences in permeability as
ineffective compounds fail to reach the intracellular target.
While membrane permeability is generally thought to be related
to the hydrophobicity of the compounds, the isomeric pairs
shown in Figure 1 (e.g., compounds 2/3 and 4/5) possess the
same clogP values, suggesting the involvement of more subtle
relationships between structure and permeability. Alternative
explanations for the differential antifungal activity of com-
While the SAR clearly indicated that hydrophobic function-
ality was preferred for activity against both species, these
compounds are only moderately soluble. For example,
compound 3 is soluble in water in the presence of 0.02%
hydroxypropyl methylcellulose (HPMC) at 25 μg/mL.
Knowing that the shape of the molecule and the position of
polar functionality is a more important determinant of activity
than overall molecular properties, we investigated the option of
adding solubility-enhancing basic nitrogen to the proximal
aromatic B-ring. Interestingly, the comparison of the activity of
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triethylamine was purchased from Aldrich and degassed by purging
with argon. All reagents were used directly from commercial sources
unless otherwise stated. Boronic acids for Suzuki coupling were
purchased from Frontier Scientific, Inc. 4′ Bromoacetophenone and 5-
acetyl-2-bromopyridine were purchased from Sigma Aldrich. The
starting bromo ketones 7,28 40,29 the Ohira−Bestmann reagent,30 and
2,4-diamino-6-ethyl-5-iodopyrimidine31 were synthesized according to
literature procedures. Purity analyses were performed with reversed
phase high performance liquid chromatography (RP-HPLC) per-
formed on a Shimadzu Prominence 20 instrument fitted with a Luna 5
μm C18(2) 100 Ao column (5 μM, 4.6 mm × 250 mm, Phenomenex)
and detected using a UV diode array at 254 nm. Two separate
determinations, method A with isocratic 50% (v/v) MeCN in 50%
mM potassium phosphate monobasic at pH 7.0 and method B with
isocratic 80% (v/v) MeOH in 20% mM potassium phosphate
monobasic at pH 7.0, were employed to determine compound purity.
Compounds were diluted in HPLC grade methanol and filtered prior
to analysis. Sample concentrations were 1 mg/mL, and injection
volumes were 1 μL−3 μL. The purity of the final compounds were
found to be ≥95% except for that of compound 35, the purity of which
could not be determined exactly because of instability.
General Procedure for Suzuki Coupling. 1-(3-Methoxy-
biphenyl-4-yl)-ethanone (9). Ketone 7 (0.52 g, 2.20 mmol),
phenylboronic acid (0.55 g, 4.50 mmol), Cs2CO3 (2.23 g, 6.84
mmol), Pd(PPh3)2Cl2 (0.16 g, 0.22 mmol, 10% Pd), and anhydrous
dioxane (8 mL) were added to 50 mL of screw cap pressure vessel.
The mixture was stirred, degassed by purging with argon for 15 min,
sealed, and placed in an 80 °C oil bath for 5 h. The dark colored
mixture was cooled, diluted with ether, and filtered through a pad of
Celite and rinsed with ether. The filtrate was concentrated and the
residue purified by flash column chromatography (SiO2, 20 g, 5%
EtOAc/hexanes) to afford the biphenyl ketone 9 as a white solid (0.41
g, 79%): TLC Rf = 0.5 (25% EtOAc/hexanes); mp 72.8−74.5 °C; 1H
NMR (500 MHz, CDCl3) δ 7.83 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.5
Hz, 2H), 7.45 (dd, J = 7.3 Hz, 7.3 Hz, 1H), 7.45 (dd, J = 7.3 Hz, 7.3
Hz, 1H), 7.38 (m, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 3.96 (s,
3H), 2.64 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 199.3, 159.5,
147.0, 140.3, 131.1, 129.0, 128.3, 127.3, 126.9, 119.5, 110.5, 55.7, 32.0;
IR (neat cm−1) 3059, 2994, 2953, 2920, 1665, 1467, 1220, 1014, 761,
694; HRMS (DART, M+ + H) m/z 227.1088 (calculated for
C15H15O2, 227.1072).
pounds 1 and 2 could include isomer-specific sequestration by
extracellular components of the cell, differences in intracellular
proteins that selectively degrade some of the antifolates, or
differences in efflux pump activity between the two species.
Preliminary experiments to increase membrane permeability
show that previously ineffective compounds (such as 1 or 2) do
inhibit the growth of C. albicans when treated with 0.01%
Triton X-100. These results suggest that the extracellular effect
on the membrane is critical and decreases the likelihood of any
significant role of intracellular proteins or efflux pumps. The
Triton X-100 may inactivate extracellular proteins that interfere
with antifolate penetration, although this would need to be in
an isomer-specific manner.
An alternative hypothesis is that ineffective compounds
become sequestered in the unique cell wall of C. albicans. The
cell wall of C. albicans possesses more than 20 cell wall proteins
covalently attached to the skeletal layer18 and are tightly packed
together, thus providing the organism with a protective protein
coat and also limiting permeability.19−21 Cell wall proteins also
tend to form phosphodiester linkages via carbohydrate side
chains, giving the surface a net negative charge.22,23 C. glabrata
is also known to express cell wall proteins, but much less is
known about the composition of these proteins in the cell
wall.24 One working hypothesis is that in situations where a
concentration of polar functionality is symmetrically distrib-
uted, the compound may have strong, nonselective binding to
the cell wall and hence poor permeability. In contrast,
compounds such as 28, 46, 47, and 48 are amphipathic in
their distribution of polar functionality, which may limit their
sequestration and increase their permeability. Interestingly,
similar trends are apparent in the Glaxo work.11 In that work,
potent compounds were also amphipathic with hydrophobic
domains attached to the diaminopyrimidine ring; potency was
decreased when these domains were di- or trimethoxybenzyl
groups. The differences in activity between C. albicans and C.
glabrata may relate to differences in the composition of their
cell walls.
1-(2-Methoxy-4-pyridin-4-yl-phenyl)-ethanone (10). According to
the general Suzuki coupling procedure, ketone 7 (0.50 g, 2.10 mmol),
pyridine-4-boronic acid (0.54 g, 4.36 mmol), Cs2CO3 (2.15 g, 6.55
mmol), Pd(PPh3)2Cl2 (0.15 g, 0.22 mmol, 10% Pd), and anhydrous
dioxane (6 mL) were heated at 80 °C for 12 h (overnight). Following
the general workup and flash chromatography (SiO2, 20 g, 50%
EtOAc/hexanes), biaryl ketone 10 was obtained as a pale white solid
(0.45 g, 89%): TLC Rf = 0.2 (50% EtOAc/hexanes); mp 88.8−92; 1H
NMR (500 MHz, CDCl3) δ 8.72 (d, J = 5.3 Hz, 2H), 7.86 (d, J = 7.9
Hz, 1H), 7.55−7.52 (m, 2H), 7.27 (dd, J = 8.0, 1.3 Hz, 1H), 7.20 (s,
1H), 4.02 (s, 3H), 2.67 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
199.1, 159.3, 150.2, 132.1, 132.0, 131.2, 128.5, 121.7, 119.3, 110.2,
55.7, 31.8; IR (neat cm−1) 3375, 3037, 2922, 1660, 1398, 1227, 1025,
813, 537; HRMS (DART, M+ + H) m/z 374.2003 (calculated for
C22H24N5O, 374.1981).
Herein we describe a significant advance in the development
of propargyl-linked antifolates targeting fungal pathogens. This
work has identified a new generation of analogues that are
highly potent inhibitors of the DHFR enzymes as well as the
growth of both C. albicans and C. glabrata. We have shown that
the shape and exposed polar functionality of the compounds
strongly affect the antifungal activity. These compounds may be
used for further development of potent antifungal antifolates.
EXPERIMENTAL PROCEDURES
■
The synthesis and characterization of compounds 1-6 were previously
reported in refs 25−27.
The 1H and 13C NMR spectra were recorded on Bruker instruments
at 500 MHz. Chemical shifts are reported in ppm and are referenced
1-[3-Methoxy-4′-(tetrahydro-pyran-2-yloxy)-biphenyl-4-yl]-etha-
none (11). According to the general Suzuki coupling procedure,
ketone 7 (0.49 g, 2.17 mmol), 4-(tetrahydro-2H-pyran-2-yloxy)-
phenylboronic acid (0.96 g, 4.34 mmol), Cs2CO3 (2.12 g, 6.51 mmol),
Pd(PPh3)2Cl2 (0.15 g, 0.22 mmol, 10% Pd), and anhydrous dioxane (6
mL) was heated at 80 °C for 14 h (overnight). Following the general
workup and flash chromatography (SiO2, 20 g, 10% EtOAc/hexanes),
biaryl ketone 11 was obtained as a pale white solid (0.430 g, 61%):
to the residual CHCl3 solvent; 7.24 and 77.23 ppm for H and 13C,
1
residual solvent MeOH; 4.78, 3.31, and 49.15 ppm, respectively.
Melting points were recorded on a Mel-Temp 3.0 apparatus and are
uncorrected. The high-resolution mass spectrometry was provided by
the Notre Dame Mass Spectrometry Laboratory and University of
Connecticut Mass Spectrometry Laboratory using an AccuTOF mass
spectrometer and/or using a DART source. IR data were obtained
using an Alpha diamond ATR probe. TLC analyses were performed on
Sorbent Technologies silica gel HL TLC plates. All glassware was
oven-dried and allowed to cool under an argon atmosphere.
Anhydrous dichloromethane, ether, and tetrahydrofuran were used
directly from Baker Cycle-Tainers. Anhydrous dimethylformamide was
purchased from Acros and degassed by purging with argon. Anhydrous
1
TLC Rf = 0.5 (25% EtOAc/hexanes); mp 86.7−89.7 °C; H NMR
(500 MHz, CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 7.9 Hz,
2H), 7.30−6.97 (m, 4H), 5.46 (s, 1H), 3.95 (s, 3H), 3.92−3.87 (m,
1H), 3.62−3.59 (m, 1H) 2.62 (s, 3H), 2.05−1.97 (m, 1H), 1.87−1.86
(m, 2H), 1.72−1.65 (m, 2H), 1.60−1.58 (m, 1H); 13C NMR (125
MHz, CDCl3) δ 199.2, 159.5, 157.5, 146.7, 133.5, 131.2, 128.4, 126.3,
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119.1, 116.9, 110.0, 96.4, 62.1, 55.6, 32.0, 30.4, 25.3, 18.8; IR (neat
cm−1) 2940, 2872, 2847, 1656, 1598, 1358, 1280, 1177, 1019, 813,
518; HRMS (DART, M+ + H) m/z 327.1612 (calculated for
C20H23O4, 327.1596).
(2.13 g, 6.54 mmol), Pd(PPh3)2Cl2 (0.15 g, 0.22 mmol, 10% Pd), and
anhydrous dioxane (8 mL) were added to a 50 mL screw-cap pressure
vessel. The mixture was stirred, degassed by purging with argon for 15
min, sealed, and placed in an 80 °C oil bath for 12 h. Following the
general workup and flash chromatography (SiO2, 20g, 50% EtOAc/
hexanes), ketone 16 was obtained as a white solid (0.49 g, 92%); TLC
1-(3-Methoxy-4′-methyl-biphenyl-4-yl)-ethanone (12). According
to the general Suzuki coupling procedure, ketone 7 (0.465 g, 2.03
mmol), 4-tolylboronic acid (0.551 g, 4.06 mmol), Cs2CO3 (1.98 g,
6.09 mmol), Pd(PPh3)2Cl2 (0.142 g, 0.20 mmol, 10% Pd), and
anhydrous dioxane (6 mL) were heated at 80 °C for 5 h. Following the
general workup and flash chromatography (SiO2, 20 g, 5% EtOAc/
hexanes), biaryl ketone 12 was obtained as a white solid (0.375 g,
1
Rf = 0.52 (15% EtOAc/hexane); mp 89.5−90.0 °C; H NMR (500
MHz, chloroform-d) δ 7.83 (d, J = 8.0 Hz, 1H), 7.59−7.56 (m, 2H),
7.17−7.11 (m, 4H), 3.99 (s, 3H), 2.65 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 199.2, 163.0 (d, J = 246.2 Hz, 1C), 159.6, 145.9, 136.4,
131.3, 129.0 (d, J = 7.5 Hz, 2C) 126.9, 119.4, 116.1 (d, J = 21.2 Hz.
2C), 110.4, 55.7, 32.1; IR (neat cm−1) 3064, 3009, 1666, 1604, 1558,
841; HRMS (DART, M++H) m/z 245.1002 (calculated for
C15H14FO2, 245.0978).
1
77%): TLC Rf = 0.57 (25% EtOAc/hexanes); mp 84.2−86.1 °C; H
NMR (500 MHz, CDCl3) δ 7.87 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.6
Hz, 2H), 7.31 (d, J = 7.6 Hz, 2H), 7.24 (d, J = 8.0 Hz, 1H), 7.18 (s,
1H), 4.01 (s, 3H), 2.69 (s, 3H), 2.45 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 198.9, 159.4, 146.8, 138.1, 137.1, 130.9, 129.6, 126.9, 126.4,
119.1, 110.0, 55.4, 31.8, 21.1; IR (neat cm−1) 3028, 2949, 2921, 1657,
1551, 1223, 1013, 808, 510; HRMS (DART, M+ + H) m/z 241.1251
(calculated for C16H17O2, 241.1229).
1-Biphenyl-4-yl-ethanone (17). According to the general Suzuki
coupling procedure, 4′-bromoacetophenone 8 (0.600 g, 3.02 mmol),
phenylboronic acid (0.736 g, 6.04 mmol), Cs2CO3 (2.95 g, 9.06
mmol), Pd(PPh3)2Cl2 (0.211 g, 0.30 mmol, 10% Pd), and anhydrous
dioxane (6 mL) were heated at 80 °C for 5 h. Following the general
workup and flash chromatography (SiO2, 20 g, 5% EtOAc/hexanes),
biaryl ketone 17 was obtained as a white solid (0.415 g, 70%): TLC Rf
4′-Acetyl-3′-methoxy-biphenyl-4-carbonitrile (13). According to
the general Suzuki coupling procedure, ketone 7 (0.500 g, 2.18 mmol),
4-cyanophenylboronic acid (0.640 g, 4.36 mmol), Cs2CO3 (2.14 g,
6.55 mmol), Pd(PPh3)2Cl2 (0.154 g, 0.22 mmol, 10% Pd), and
anhydrous dioxane (6 mL) were heated at 80 °C for 14 h (overnight).
Following the general workup and flash chromatography (SiO2, 20 g,
10% EtOAc/hexanes), biaryl ketone 13 was obtained as a white solid
(0.470 g, 86%): TLC Rf = 0.4 (25% EtOAc/hexanes); mp 125−127.2
1
= 0.6 (25% EtOAc/hexanes); mp 118−120 °C; H NMR (500 MHz,
CDCl3) δ 8.01 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 7.5 Hz, 2H), 7.61 (d, J
= 7.6 Hz, 2H), 7.46 (dd, J = 7.3 Hz, 7.3 Hz, 1H), 7.46 (dd, J = 7.3 Hz,
7.3 Hz 1H), 7.40−7.37 (m, 1H), 2.62 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 197.8, 145.8, 139.9, 136.0, 129.1, 129.1, 128.4, 127.4, 127.3,
26.8; IR (neat cm−1) 3072, 2996, 2916, 2047, 1676, 1599, 1402, 958,
763; HRMS (DART, M+ + H) m/z 197.0984 (calculated for C14H13O,
197.0966).
1
°C; H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 8.0 Hz, 1H), 7.76−
7.73 (m, 2H), 7.71−7.68 (m, 2H), 7.20 (dd, J = 8.0, 1.6 Hz, 1H), 7.13
(d, J = 1.5 Hz, 1H), 4.00 (s, 3H), 2.64 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 199.2, 159.5, 144.8, 144.7, 132.9, 131.5, 128.2, 128.1, 119.8,
118.8, 112.1, 110.7, 55.9, 32.0; IR (neat cm−1) 3073, 3046, 2923, 2225,
1652, 1601, 1468, 1225, 1023, 812,543; HRMS (DART, M+ + H) m/z
252.1032 (calculated for C16H14NO2, 252.1024).
General Procedure for the Suzuki Coupling of Pyridine
Compounds. 1-(6-Phenyl-pyridin-3-yl)-ethanone (40). 5-Acetyl-2-
bromopyridine 38 (0.656 g, 3.28 mmol), phenylboronic acid (0.800 g,
6.56 mmol), Na2CO3 (0.243 g, 2.29 mmol), Pd(PPh3)2Cl2 (0.115 g,
0.16 mmol, 5% Pd), acetonitrile (26 mL), and H2O (26 mL) were
added to a 50 mL screw-cap pressure vessel. The mixture was stirred,
degassed by purging with argon for 15 min, sealed, and placed in an 80
°C oil bath for 14 h (overnight). The dark colored mixture was cooled
and extracted with ether. The organic layer was filtered through a pad
of Celite, rinsed with ether, washed with brine, dried over MgSO4, and
filtered. The filtrate was concentrated, and the residue purified by flash
column chromatography (SiO2, 20 g, 5% EtOAc/hexanes) to afford
the coupled ketone 40 as a white solid (0.33g, 52%): TLC Rf = 0.4
4′-Acetyl-3′-methoxy-biphenyl-4-carboxylic Acid Methyl Ester
(14). According to the general Suzuki coupling procedure, ketone 7
(0.506 g, 2.21 mmol), 4-(methoxycarbonyl)phenylboronic acid (0.795
g, 4.42 mmol), Cs2CO3 (2.16 g, 6.63 mmol), Pd(PPh3)2Cl2 (0.155 g,
0.22 mmol, 10% Pd), and anhydrous dioxane (6 mL) were heated at
80 °C for 14 h (overnight). Following the general workup and flash
chromatography (SiO2, 20 g, 15% EtOAc/hexanes), biaryl ketone 14
was obtained as a white solid (0.490 g, 78%): TLC Rf = 0.4 (25%
1
1
EtOAc/hexanes); mp 140.4−142.6 °C; H NMR (500 MHz, CDCl3)
(25% EtOAc/hexanes); mp 124.9−126.2 °C; H NMR (500 MHz,
δ 8.09 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0
Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 3.97 (s, 3H), 3.91 (s,
3H), 2.62 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 199.3, 166.9,
159.5, 145.6, 144.7, 131.3, 130.3, 129.9, 127.5, 127.4, 119.8, 110.7,
55.8, 52.4, 32.1; IR (neat cm−1) 2997, 2955, 2929, 2845, 2162, 1714,
1657, 1604, 1554, 1280, 1247, 831, 768. HRMS (DART, M+ + H) m/z
285.1111 (calculated for C17H17O4, 285.1127).
CDCl3) δ 9.22 (d, J = 2.0 Hz, 1H), 8.28 (dd, J = 8.3, 2.2 Hz, 1H),
8.10−7.99 (m, 2H), 7.83 (d, J = 8.3 Hz, 1H), 7.55−7.40 (m, 3H), 2.65
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 196.6, 161.1, 150.2, 138.2,
136.7, 130.9, 130.4, 129.2, 127.6, 120.4, 26.9; IR (neat cm−1) 3062,
2999, 2960, 2322, 1673, 1557, 1382, 1016, 736; HRMS (DART, M+ +
H) m/z 198.0906 (calculated for C13H12NO, 198.0919).
1-(6-p-Tolyl-pyridin-3-yl)-ethanone (41). According to the general
Suzuki coupling of pyridine compounds 5-acetyl-2-bromopyridine 38
(0.394 g, 1.97 mmol), 4-tolylboronic acid (0.535 g, 3.94 mmol),
Na2CO3 (0.146 g, 1.38 mmol), Pd(PPh3)2Cl2 (0.042 g, 0.06 mmol, 3%
Pd), acetonitrile (7.8 mL), and water (7.8 mL) were heated at 80 °C
for 14 h (overnight). Following the general workup and flash
chromatography (SiO2, 20 g, 5% EtOAc/hexanes), coupled ketone was
obtained as a white solid 41 (0.381 g, 92%); TLC Rf = 0.4 (25%
EtOAc/hexanes); mp 106−107.5 °C; 1H NMR (500 MHz, CDCl3) δ
9.19 (d, J = 2.2 Hz, 1H), 8.24 (dd, J = 8.3, 2.3 Hz, 1H), 7.95 (d, J = 8.2
Hz, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 2.63 (s,
3H), 2.40 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 196.6, 161.0,
150.2, 140.5, 136.4, 135.5, 130.5, 129.8, 127.4, 26.8, 21.5; IR (neat
cm−1) 3024, 2911, 2853, 2042, 1671, 1589, 1555, 1371, 1279, 1141,
819, 762; HRMS (DART, M+ + H) m/z 212.1093 (calculated for
C14H14NO, 212.1075).
1-(4′-Dimethylamino-3-methoxy-biphenyl-4-yl)-ethanone (15).
According to the general Suzuki coupling procedure, ketone 7 (0.50
g, 2.18 mmol), N,N-dimethylamine phenylboronic acid (0.43 g, 2.62
mmol), Cs2CO3 (2.13 g, 6.54 mmol), Pd(PPh3)2Cl2 (0.15 g, 0.22
mmol, 10% Pd), and anhydrous dioxane (8 mL) were added to a 50
mL screw-cap pressure vessel. The mixture was stirred, degassed by
purging with argon for 15 min, and placed in an 80 °C oil bath for 12
h. Following the general workup and flash chromatography (SiO2, 20
g, 50% EtOAc/hexanes), ketone 15 was obtained as a yellow solid
(0.42 g, 72%); TLC Rf = 0.41 (20% EtOAc/hexanes); mp 107.7−
1
108.0 °C; H NMR (500 MHz, chloroform-d) δ 7.80 (d, J = 8.1 Hz,
1H), 7.52 (d, J = 8.7 Hz, 2H), 7.17 (dd, J = 8.1, 1.4 Hz, 1H), 7.10 (s,
1H), 6.78 (d, J = 8.8 Hz, 2H), 3.96 (s, 3H), 3.00 (s, 6H), 2.62 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 199.2, 159.8, 150.8, 147.2, 131.3,
128.1, 125.6, 118.6, 112.7, 109.3, 55.7, 40.6, 32.1; IR (neat cm−1)
2894, 2816, 1657, 1588, 1564, 807; HRMS (DART, M+ + H) m/z
270.1488 (calculated for C17H20NO2, 270.1494).
1-(4′-Fluoro-3-methoxy-biphenyl-4-yl)-ethanone (16). According
to the general Suzuki coupling procedure, ketone 7 (0.50 g, 2.18
mmol), 4-fluorophenylboronic acid (0.37 g, 2.62 mmol), Cs2CO3
General Procedure for the Suzuki Coupling of Pyrimidine
Compounds. 1-(2-Phenyl-pyrimidin-5-yl)-ethanone (42). Ketone
39 (0.72 g, 4.6 mmol), phenylboronic acid (0.841 g, 6.9 mmol),
Pd(OAc)2 (0.041 g, 0.184 mmol, 4% Pd), PPh3 (0.241 g, 0.92 mmol),
sat. Na2CO3 (23 mL), and anhydrous dioxane (30 mL) were added to
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a 100 mL screw-cap pressure vessel. The mixture was stirred, degassed
by purging with argon for 15 min, sealed, and placed in a 110 °C oil
bath for 14 h. The dark colored mixture was cooled and extracted with
ether. The organic layer was filtered through a pad of Celite, rinsed
with ether, washed with brine, dried over MgSO4, and filtered. The
filtrate was concentrated and the residue purified by flash column
chromatography (SiO2, 20 g, 5% EtOAc/hexanes) to afford the
coupled ketone 42 as a white solid (0.66 g, 74%): TLC Rf = 0.4 (25%
K2CO3 (0.337 g, 2.70 mmol) were stirred at 0 °C. Following the
general workup and flash chromatography (SiO2, 15g, 25% EtOAc/
hexanes), alkyne 19 was obtained as a pale yellow solid (0.100 g, 23%
yield over 3 steps). TLC Rf = 0.1 (25% EtOAc/hexanes); mp 111.6−
1
113.6 °C; H NMR (500 MHz, CDCl3) δ 8.64 (d, J = 7.6 Hz, 2H),
7.67 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 6.0 Hz, 2H), 7.07 (d, J = 1.3 Hz,
1H), 4.21 (qd, J = 7.0, 2.5 Hz, 1H), 3.91 (s, 3H), 2.27−2.21 (m, 1H),
1.47 (d, J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 156.5, 150.1,
148.5, 138.0, 132.2, 128.4, 121.7, 119.5, 109.0, 87.1, 70.0, 55.6, 25.3,
22.6; IR (neat cm−1) 3156, 3042, 2930, 2099, 1596, 1454, 1227, 1032,
810; HRMS (DART, M+ + H) m/z 238.1259 (calculated for
C16H16NO, 238.1232).
1
EtOAc/hexanes); mp 154−156 °C; H NMR (500 MHz, CDCl3) δ
9.23 (s, 2H), 8.49 (dd, J = 8.1, 1.6 Hz, 2H), 7.72−7.36 (m, 3H), 2.61
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 195.2, 167.2, 157.5, 136.7,
132.1, 130.0, 128.9, 127.4, 26.8; IR (neat cm-1) 3077, 3036, 2049,
1681, 1537, 1429, 1376, 1271, 954, 744, 688; HRMS (DART, M+ +
H) m/z 199.0868 (calculated for C12H11N2O, 199.0871).
3′-Methoxy-4′-(1-methyl-prop-2-ynyl)-biphenyl-4-ol (20). Ac-
cording to the general procedure for homologation, methoxymethyl
triphenylphosphonium chloride (4.71 g, 13.73 mmol) in dry THF (37
mL), NaOtBu (1.58 g, 16.5 mmol), and ketone 11 (1.79 g, 5.49 mmol)
in THF (7 mL) were stirred at 0 °C. Following the general workup,
the mixture of enol ethers (1.77 g, 5.00 mmol) in THF/H2O (9:1, 17
mL) was hydrolyzed using Hg(OAc)2 (4.8 g, 14.97 mmol) at room
temperature. After the general extraction procedure, aldehyde (1.564
g, 4.65 mmol) in MeOH (20 mL), the Ohira−Bestmann reagent (1.60
g, 8.4 mmol) dissolved (11 mL) in MeOH, and powdered K2CO3
(1.337 g, 9.7 mmol) were stirred at 0 °C. Following the general
workup and flash chromatography (SiO2, 30 g, 15% EtOAc/hexanes),
O-THP alkyne was obtained as a pale white solid (0.84 g).
Deprotection of O-THP alkyne was carried out by dissolving the
protected alkyne (0.84 g, 2.5 mmol) in MeOH (150 mL) and cooled
to 0 °C. p- Toluenesulfonic acid (0.951 g, 2.5 mmol) was added and
the reaction allowed to warm to room temperature. The reaction was
followed by TLC, diluted with water, neutralized with sat NaHCO3,
and extracted with ether. The organic extracts were washed with brine,
dried over MgSO4, and filtered. The filtrate was concentrated and
purified by flash column chromatography (SiO2, 30g, 15% EtOAc/
hexanes) to give the terminal acetylene 20 as a white solid (0.61 g,
43% yield over 4 steps); TLC Rf = 0.3 (25% EtOAc/hexanes); mp
90.2−91 °C; 1H NMR (500 MHz, CDCl3) δ 7.57 (d, J = 7.9 Hz, 1H),
7.45 (d, J = 8.6 Hz, 2H), 7.11 (dd, J = 7.9, 1.7 Hz, 1H), 6.98 (d, J = 1.6
Hz, 1H), 6.88 (d, J = 8.6 Hz, 2H), 4.18 (qd, J = 7.0, 2.4 Hz, 1H), 3.88
(s, 3H), 2.20 (d, J = 2.5 Hz, 1H), 1.45 (d, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 156.5, 155.3, 141.0, 134.3, 129.7, 128.6, 128.2,
119.4, 115.8, 109.3, 87.8, 69.5, 55.7, 25.3, 22.9; IR (neat cm−1) 3334.
3300, 2938, 1606, 1494, 1218, 807, 633; HRMS (DART, M+ + H) m/
z 253.1224 (calculated for C17H17O2, 253.1229).
General Procedure for Wittig Homologation/Enol Ether
Hydrolysis/Ohira−Bestmann Homologation. 3-Methoxy-4-(1-
methyl-prop-2-ynyl)-biphenyl (18). A 50 mL round bottomed flask
with a stir bar was flame-dried under argon and allowed to cool to
room temperature. Methoxymethyl triphenylphosphonium chloride
(1.23 g 3.6 mmol) was added to the flask, followed by dry THF (9.7
mL) and cooled to 0 °C. To the cold suspension was added NaOtBu
(0.434 g, 4.5 mmol) in one portion and stirred for 0.5 h at 0 °C.
Ketone 9 (0.409 g, 1.8 mmol) in THF (3 mL) was added dropwise to
the red-orange suspension and allowed to stir for 20 min. The reaction
was followed by TLC and quenched with water and diluted with ether.
The organic phase was separated and the aqueous phase extracted with
ether. The combined extracts were washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated and passed through
a column of silica gel to afford a mixture of enol ethers (0.409 g) that
were immediately hydrolyzed in the subsequent step.
To a solution of 0.409 g (1.6 mmol) of enol ether in THF/H2O
(9:1, 6 mL), which was degassed by purging with argon, Hg(OAc)2
(1.54 g, 4.8 mmol) was added. The reaction mixture was stirred for 2 h
under argon at RT and then 5 mL of saturated solution of KI was
added, stirred for 15 min. The reaction mixture was then extracted
with ether, and the combined extracts were washed with a saturated
solution of NaHCO3, sat. Na2S2O3, and brine, dried over MgSO4, and
filtered. The filtrate was concentrated to afford the aldehyde (0.107 g)
as a colorless oil, which was taken to the next step without further
purification.
Aldehyde (0.1 g 0.41 mmol) in MeOH (3 mL) under argon was
cooled to 0 °C, and the Ohira−Bestmann reagent (0.120 g, 0.62
mmol) dissolved in MeOH (1 mL) was added. Powdered K2CO3
(0.12 g, 0.87 mmol) was added to the homogeneous pale greenish
solution and stirred for 2 h. The reaction was followed by TLC and
diluted with water, and the aqueous phase was extracted with ether.
The combined organic extracts were washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated and purified by
flash column chromatography (SiO2, 10 g, 2% EtOAc/hexanes) to
afford acetylene 18 as a colorless oil (0.034g, 10% yield over 3 steps):
3-Methoxy-4′-methyl-4-(1-methyl-prop-2-ynyl)-biphenyl (21).
According to the general procedure for homologation, methoxymethyl
triphenylphosphonium chloride (1.07 g, 3.12 mmol) in dry THF (9
mL), NaOtBu (0.37g, 3.9 mmol), and ketone 12 (0.376 g, 1.56 mmol)
in THF (3 mL) were stirred at 0 °C. Following the general workup,
the mixture of enol ethers (0.375 g, 1.39 mmol) in THF/H2O (9:1, 5
mL) was hydrolyzed using Hg(OAc)2 (1.30g, 4.1 mmol) at room
temperature. After the general extraction procedure, aldehyde (0.141 g
0.60 mmol) in MeOH (3 mL), the Ohira−Bestmann reagent (0.160 g,
0.83 mmol) dissolved in MeOH (1 mL), and powdered K2CO3 (0.161
g, 1.16 mmol) were stirred at 0 °C. Following the general workup and
flash chromatography (SiO2, 6 g, 2% EtOAc/hexanes), alkyne 21 was
obtained as a colorless oil (0.017 g, 5% yield over 3 steps); TLC Rf =
0.2 (2% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.60 (d, J =
7.8 Hz, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 7.17
(dd, J = 7.8, 1.6 Hz, 1H), 7.03 (d, J = 1.5 Hz, 1H), 4.20 (qd, J = 7.0,
2.4 Hz, 1H), 3.89 (s, 3H), 2.39 (s, 3H), 2.21 (d, J = 2.5 Hz, 1H), 1.47
(d, J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 156.5, 141.4,
138.6, 137.3, 129.9, 129.7, 128.1, 127.2, 119.6, 109.5, 87.8, 69.5, 55.7,
25.3, 22.9, 21.3; IR (neat cm−1) 3305, 3028, 2970, 2932, 2357, 1607,
1
TLC Rf = 0.3 (2% EtOAc/hexanes); H NMR (500 MHz, CDCl3) δ
7.64 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 7.2 Hz, 2H), 7.44 (dd, J = 7.6 Hz,
7.6 Hz, 1H), 7.44 (dd, J = 7.6 Hz, 7.6 Hz, 1H), 7.35 (m, 1H), 7.20 (dd,
J = 7.8, 1.4 Hz, 1H), 7.06 (s, 1H), 4.23 (qd, J = 7.0, 2.4 Hz, 1H), 3.91
(s, 3H), 2.24 (d, J = 2.5 Hz, 1H), 1.49 (d, J = 7.1 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 156.5, 141.5, 141.5, 130.3, 128.9, 128.2, 127.5,
127.4, 119.8, 109.7, 87.8, 69.6, 55.7, 25.4, 22.9; IR (neat cm−1) 3290,
3029, 2972, 2931, 2107, 1608, 1566, 1484, 1402, 1220, 1036, 761, 698,
642; HRMS (DART, M+ + H) m/z 237.1264 (calculated for C17H17O,
237.1279).
4-[3-Methoxy-4-(1-methyl-prop-2-ynyl)-phenyl]-pyridine (19).
According to the general procedure for homologation, methoxymethyl
triphenylphosphonium chloride (1.208 g, 3.52 mmol) in dry THF (4
mL), NaOtBu (0.432 g, 4.4 mmol), and ketone 10 (0.400 g, 1.76
mmol) in THF (3 mL) were stirred at 0 °C. Following the general
workup, the mixture of enol ethers 0.350 g (1.37 mmol) in THF/H2O
(9:1, 6 mL) was hydrolyzed using Hg(OAc)2 (1.30 g, 4.1 mmol) at
room temperature. After the general extraction procedure, aldehyde
(0.330 g, 1.36 mmol) in MeOH (3 mL), the Ohira−Bestmann reagent
(0.422 g, 2.0 mmol) dissolved in MeOH (1 mL), and powdered
+
1582, 1225, 1031, 804; HRMS (DART, M+ + H ) m/z 251.1422
(calculated for C18H19O, 251.1436).
3′-Methoxy-4′-(1-methyl-prop-2-ynyl)-biphenyl-4-carbonitrile
(22). According to general procedure for homologation, methox-
ymethyl triphenylphosphonium chloride (1.09 g, 3.18 mmol) in dry
THF (4 mL), NaOtBu (0.382 g, 3.9 mmol), and ketone 13 (0.400 g,
1.59 mmol) in THF (3 mL) were stirred at 0 °C. Following the
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general workup, the mixture of enol ethers (0.330 g, 1.18 mmol) in
THF/H2O (9:1, 5 mL) was hydrolyzed using Hg(OAc)2 (1.13g, 3.5
mmol) at room temperature. After the general extraction procedure,
aldehyde (0.285 g, 1.1 mmol) in MeOH (3 mL), the Ohira−Bestmann
reagent (0.442 g, 2.15 mmol) dissolved in MeOH (1 mL), and
powdered K2CO3 (0.370 g, 2.68 mmol) were stirred at 0 °C.
Following the general workup and flash chromatography (SiO2, 10 g,
5% EtOAc/hexanes), alkyne 22 was obtained as a white solid (0.200 g,
47% yield over 3 steps); TLC Rf = 0.2 (5% EtOAc/hexanes); mp
103.7−104 °C; 1H NMR (500 MHz, CDCl3) δ 7.72 (m, 2H), 7.67 (m,
3H), 7.19 (d, J = 7.9 Hz, 1H), 7.03 (s, 1H), 4.23 (q, J = 7.0, 1H), 3.92
(s, 3H), 2.25 (d, J = 2.4 Hz, 1H), 1.48 (d, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 156.7, 145.8, 139.3, 132.7, 131.9. 128.6, 127.9,
119.9, 119.9, 119.1, 111.1, 109.5, 87.3, 69.9, 55.8, 25.4, 22.8; IR (neat
cm−1) 3285, 3069, 2935, 2221, 1604, 1488, 1393, 1225, 1024, 807,
657; HRMS (DART, M+ + H) m/z 262.1253 (calculated for
C18H16NO, 262.1232).
Carbamic acid 3′-methoxy-4′-(1-methyl-prop-2-ynyl)-biphenyl-
4-yl ester (23). To a 25 mL flame-dried flask with a stir bar cooled to
room temperature was added alkyne 20 (0.115 g, 0.46 mmol)
dissolved in anhydrous CH2Cl2. The reactant was cooled to 0 °C, and
chlorosulfonyl isocyanate (0.08 mL, 0.92 mmol) was added dropwise.
After 15 min, the reaction mixture was brought to room temperature
and followed by TLC. The reaction was quenched with water and
extracted with ether. The organic extracts were washed with brine,
dried over MgSO4, and filtered. The filtrate was concentrated and
purified by flash column chromatography (SiO2, 7 g, 25% EtOAc/
hexanes) to give the terminal acetylene 23 as a white solid (0.092 g,
68% yield): TLC Rf = 0.1 (25% EtOAc/hexanes); mp 113.6−115.3
°C; 1H NMR (500 MHz, CDCl3) δ 7.61 (d, J = 7.8 Hz, 1H), 7.56 (d, J
= 8.5 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 7.15 (dd, J = 7.8, 1.4 Hz, 1H),
7.01 (d, J = 1.7 Hz, 1H), 4.21 (qd, J = 7.0, 2.3 Hz, 1H), 3.88 (s, 3H),
2.23 (d, J = 2.4 Hz, 1H), 1.47 (d, J = 7.0 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 189.2, 156.4, 155.6, 150.3, 140.6, 138.9, 133.1, 130.3,
128.3, 128.2, 122.1, 119.7, 109.6, 87.7, 69.6, 60.6, 55.7, 25.3, 22.8, 21.2,
14.4; IR (neat cm−1) 3423, 3308, 3268, 3199, 2969, 2341, 2105, 1698,
1606, 1494, 1378, 1213, 586; HRMS (DART, M+ + H) m/z 296.1300
(calculated for C18H18NO3, 296.1287).
Ohira−Bestmann reagent (0.36 g, 1.86 mmol) dissolved in MeOH (2
mL), and powdered K2CO3 (0.26 g, 1.86 mmol) were stirred at 0 °C.
Following the general workup and flash chromatography (SiO2, 10g,
15% EtOAc/hexanes), alkyne 25 was obtained as a white solid (0.015
g, 6% yield over 3 steps); TLC Rf = 0.52 (10% EtOAc/hexanes); mp
1
60.8- 61.1 °C; H NMR (500 MHz, chloroform-d) δ 7.56 (d, J = 7.9
Hz, 1H), 7.48 (d, J = 8.9 Hz, 2H), 7.14 (dd, J = 7.9, 1.7 Hz, 1H), 7.01
(d, J = 1.6 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 4.18 (qd, J = 7.1, 2.6 Hz,
2H), 3.88 (s, 3H), 2.98 (s, 6H), 2.20 (d, J = 2.5 Hz, 1H), 1.46 (d, J =
7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 156.4, 150.2, 141.5,
129.5, 128.9, 128.0, 127.9, 118.9, 112.9, 108.9, 88.0, 69.4, 55.6, 40.8,
25.3, 22.9; IR (neat cm−1) 3292, 2972, 2932, 1605, 1574, 1397, 804;
HRMS (DART, M+ + H) m/z 280.1703 (calculated for C19H22NO,
280.1701).
4′-Fluoro-3-methoxy-4-(1-methyl-prop-2-ynyl)-biphenyl (26). Ac-
cording to the general procedure for homologation, methoxymethyl
triphenylphosphonium chloride (1.70 g, 4.97 mmol) in dry THF (10
mL), NaOtBu (0.57 g, 5.97 mmol), and ketone (0.49 g, 1.99 mmol) in
THF (3 mL) were stirred at 0 °C. Following the general workup, the
mixture of enol ethers (0.51 g, 1.87 mmol) in THF/H2O (9:1, 10 mL)
was hydrolyzed using Hg(OAc)2 (1.78 g, 5.61 mmol) at room
temperature. After the general extraction procedure, aldehyde (0.48 g,
1.87 mmol) in dry MeOH (6 mL), the Ohira−Bestmann reagent (0.72
g, 3.74 mmol) dissolved in MeOH (2 mL), and powdered K2CO3
(0.78 g, 5.61 mmol) were stirred at 0 °C. Following the general
workup and flash chromatography (SiO2, 30g, 15% EtOAc/hexanes),
alkyne 26 was obtained as a white solid (0.23 g, 45% yield over 3
1
steps); TLC Rf = 0.41 (10% EtOAc/hexane); mp 57.8−58.0 °C; H
NMR (500 MHz, chloroform-d) δ 7.62 (d, J = 7.8 Hz, 1H), 7.56−7.49
(m, 2H), 7.15−7.08 (m, 3H), 6.99 (d, J = 1.7 Hz, 1H), 4.21 (qd, J =
7.0, 2.4 Hz, 1H), 3.90 (s, 3H), 2.23 (d, J = 2.5 Hz, 1H), 1.48 (d, J = 7.1
Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 162.6 (d, J = 245.0 Hz, 1C);
156.5, 140.5, 137.5, 130.3, 128.8 (d, J = 7.5 Hz, 2C), 128.3, 119.7,
115.7 (d, J = 21.2 Hz, 2C), 109.5, 87.7, 69.6, 55.7, 25.3, 22.8; IR (neat
cm−1) 3306, 3297, 2975, 2957, 2931, 1607, 1492; HRMS (DART, M+
+ H) m/z 255.1199 (calculated for C17H16FO, 255.1185).
4-(1-Methyl-prop-2-ynyl)-biphenyl (27). According to the general
procedure for homologation, methoxymethyl triphenylphosphonium
chloride (1.16g, 3.4 mmol) in dry THF (10 mL), NaOtBu (0.409 g,
4.2 mmol), and ketone 17 (0.330 g, 1.70 mmol) in THF (3 mL) were
stirred at 0 °C. Following the general workup, the mixture of enol
ethers (0.388 g, 1.73 mmol) in THF/H2O (9:1, 6 mL) was hydrolyzed
using Hg(OAc)2 (1.650 g, 5.2 mmol) at room temperature. After the
general extraction procedure, aldehyde (0.150 g, 0.71 mmol) in
MeOH (3 mL), the Ohira−Bestmann reagent (0.205 g, 1.07 mmol)
dissolved in MeOH (2 mL), and powdered K2CO3 (0.207 g, 1.49
mmol) were stirred at 0 °C. Following the general workup and flash
chromatography (SiO2, 5 g, 2% EtOAc/hexanes), alkyne 27 was
obtained as a colorless oil (0.034 g, 24% yield over 3 steps); TLC Rf =
3′-Methoxy-4′-(1-methyl-prop-2-ynyl)-biphenyl-4-carboxylic
Acid Methyl Ester (24). According to the general procedure for
homologation, methoxymethyl triphenylphosphonium chloride (1.05
g, 3.06 mmol) in dry THF (9 mL), NaOtBu (0.367 g, 3.9 mmol), and
ketone 14 (0.434 g, 1.59 mmol) in THF (3 mL) were stirred at 0 °C.
Following the general workup, the mixture of enol ethers (0.214 g,
0.69 mmol) in THF/H2O (9:1, 5 mL) was hydrolyzed using
Hg(OAc)2 (0.656 g, 2.1 mmol) at room temperature. After the
general extraction procedure, aldehyde (0.194 g, 0.65 mmol) in
MeOH (3 mL), the Ohira−Bestmann reagent (0.224 g, 1.17 mmol)
dissolved in MeOH (2 mL), and powdered K2CO3 (0.188 g, 1.36
mmol) were stirred at 0 °C. Following the general workup and flash
chromatography (SiO2, 7 g, 2% EtOAc/hexanes), alkyne 24 was
obtained as a white solid (0.111 g, 25% yield over 3 steps); TLC Rf =
1
0.3 (2% EtOAc/hexanes); H NMR (500 MHz, CDCl3) δ 7.70−7.53
(m, 4H), 7.52−7.41 (m, 4H), 7.36−7.33 (m, 1H), 3.83 (qd, J = 7.2,
2.4 Hz, 1H), 2.30 (d, J = 2.5 Hz, 1H), 1.57 (d, J = 7.2 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 141.9, 141.1, 140.0, 128.9, 127.6, 127.4,
127.4, 127.3, 87.3, 70.5, 31.5, 24.4; IR (neat cm−1) 3288, 3032, 2974,
2869, 2111, 1595, 1293, 835, 762, 627, 531; HRMS (DART, M+ + H)
m/z 207.1161 (calculated for C16H15, 207.1174).
1
0.3 (5% EtOAc/hexanes); mp 106−108.5 °C; H NMR (500 MHz,
CDCl3) δ 8.09 (d, J = 8.1 Hz, 2H), 7.72−7.57 (m, 3H), 7.20 (d, J = 7.7
Hz, 1H), 7.06 (s, 1H), 4.21 (q, J = 5.0 Hz, 1H), 3.92 (s, 3H), 3.90 (s,
3H), 2.23 (d, J = 2.0 Hz, 1H), 1.47 (d, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 167.1, 156.5, 145.8, 140.1, 131.3, 130.2, 129.1,
128.4, 127.2, 119.9, 109.5, 87.5, 69.7, 55.7, 52.3, 25.4, 22.8; IR (neat
cm−1)3255, 2970, 2950, 2929, 2108, 1698, 1605, 1430, 1393, 1104,
1281, 769, 676; HRMS (DART, M+ + H) m/z 295.1329 (calculated
for C19H19O3, 295.1334).
5-(1-Methyl-prop-2-ynyl)-2-phenyl-pyridine (43). According to
general procedure for homologation, methoxymethyl triphenylphos-
phonium chloride (1.01 g, 2.94 mmol) in dry THF (9 mL), NaOtBu
(0.353 g, 3.7 mmol), and ketone 40 (0.290 g, 1.47 mmol) in THF (3
mL) were stirred at 0 °C. Following the general workup, the mixture
of enol ethers (0.298 g, 1.33 mmol) in THF/H2O (9:1, 5 mL) was
hydrolyzed using Hg(OAc)2 (1.272 g, 3.9 mmol) at room temperature.
After the general extraction procedure, aldehyde (0.175 g, 0.83 mmol)
in MeOH (4 mL), the Ohira−Bestmann reagent (0.238 g, 1.24 mmol)
dissolved in MeOH (2 mL), and powdered K2CO3 (0.240 g, 1.74
mmol) were stirred at 0 °C. Following the general workup and flash
chromatography (SiO2, 5g, 2% EtOAc/hexanes), alkyne 43 was
obtained as a white solid (0.102 g, 34% yield over 3 steps): TLC Rf =
0.3 (5% EtOAc/hexanes); mp 90.3−92 °C; 1H NMR (500 MHz,
3′-Methoxy-4′-(1-methyl-prop-2-ynyl)-biphenyl-4-yl)-dimethyl-
amine (25). According to the general procedure for homologation,
methoxymethyl triphenylphosphonium chloride (0.67 g, 1.95 mmol)
in dry THF (10 mL), NaOtBu (0.22 g, 2.34 mmol), and ketone (0.21
g, 0.78 mmol) in THF (3 mL) were stirred at 0 °C. Following the
general workup, the mixture of enol ethers (0.18 g, 0.62 mmol) in
THF/H2O (9:1, 10 mL) was hydrolyzed using Hg(OAc)2 (0.30 g,
0.93 mmol) at room temperature. After the general extraction
procedure, aldehyde (0.17 g, 0.62 mmol) in dry MeOH (6 mL), the
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CDCl3) δ 8.67 (d, J = 2.3 Hz, 1H), 7.97−7.95 (m, 2H), 7.79 (dd, J =
8.2, 2.3 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.49−7.42 (m, 2H), 7.42−
7.36 (m, 1H), 3.83 (qd, J = 7.2, 2.5 Hz, 1H), 2.30 (d, J = 2.5 Hz, 1H),
1.55 (d, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 156.3, 148.6,
139.3, 136.6, 135.3, 129.0, 128.9, 127.0, 120.5, 85.9, 71.1, 29.2, 24.2; IR
(neat cm−1) 3292, 2976, 2930, 2870, 2325, 2107, 1594, 1473, 1293,
1018, 841, 740, 693, 644; HRMS (DART, M+ + H) m/z 208.1144
(calculated for C15H14N, 208.1126).
1H), 7.07 (s, 1H), 5.18 (s, 2H), 4.86 (s, 2H), 4.43 (q, J = 6.9 Hz, 1H),
3.92 (s, 3H), 2.70 (q, J = 7.6 Hz, 2H), 1.54 (d, J = 7.0 Hz, 3H), 1.24 (t,
J = 7.6 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 173.5, 164.5, 160.8,
156.6, 141.5, 130.8, 128.9, 128.1, 127.6, 127.3, 119.8, 109.8, 102.3,
91.1, 74.6, 55.7, 29.9, 26.9, 23.1, 12.8; IR (neat cm−1) 3459, 3372,
3304, 3160, 2970, 2930, 2870, 1967, 1547, 1435, 1219, 761, 696.
HRMS (ESI, M+ + H) m/z 373.2012 (calculated for C23H25N4O,
373.2023). HPLC (a) tR = 22.9 min, 99.3%; (b) tR = 20.9 min, 98.9%.
6-Ethyl-5-[3-(2-methoxy-4-pyridin-4-yl-phenyl)-but-1-ynyl]-pyri-
midine-2,4-diamine (29). According to the general Sonogahisra
coupling procedure, ethyl-iodopyrimidine (0.070 g, 0.26 mmol) CuI
(0.0075 g, 0.039 mmol, 15 mol %), Pd(PPh3)2Cl2 (0.019 g, 0.026
mmol, 10 mol %), and alkyne 19 (0.094 g, 0.40 mmol) were reacted in
DMF/Et3N (1 mL each) at 70 °C for 12 h. After the mixture was
cooled, the dark reddish brown solution was concentrated, and the
product was purified by flash chromatography (SiO2, 10 g, 3% MeOH/
CH2Cl2) to afford coupled pyrimidine 29 as a pale white powder
(0.085 g, 86%) followed by reverse phase flash chromatography (NH2
capped SiO2, 3g, 100% CH2Cl2, 1% MeOH/CH2Cl2) for biological
evaluation: TLC Rf = 0.05 (5% MeOH/CH2Cl2); (500 MHz, CDCl3)
δ 8.70−8.56 (m, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.49 (dd, J = 4.5, 1.5
Hz, 2H), 7.24(dd, J = 7.8, 1.5 Hz, 1H), 7.10 (d, J = 1.4 Hz, 1H) 5.27
(s, 2H), 5.01 (s, 2H), 4.45 (q, J = 6.9 Hz, 1H), 3.94 (s, 3H), 2.70 (q, J
= 7.6 Hz, 2H), 1.55 (d, J = 7.02 Hz, 3H), 1.24 (t, J = 7.6 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 173.4, 164.5, 160.8, 156.8, 150.4, 148.4,
138.3, 132.9, 128.5, 121.8, 119.7, 109.3, 101.9, 90.8, 74.8, 55.7, 29.8,
26.9, 22.9, 12.7; IR (neat cm−1) 3446, 3312, 3137, 2928, 2220, 1631,
1571, 1440, 1223, 857; HRMS (DART, M+ + H) m/z 374.2003
(calculated for C22H24N5O, 374.1981). HPLC (a) tR = 5.4 min, 99.1%;
(b) tR = 7.9 min, 99.2%.
5-(1-Methyl-prop-2-ynyl)-2-p-tolyl-pyridine (44). According to the
general procedure for homologation, methoxymethyl triphenylphos-
phonium chloride (4.47 g, 3.61 mmol) in dry THF (10 mL), NaOtBu
(0.434 g, 4.5 mmol), and ketone 41 (0.381 g, 1.81 mmol) in THF (5
mL) were stirred at 0 °C. Following the general workup, the mixture
of enol ethers (0.418 g, 1.75 mmol) in THF/H2O (9:1, 6 mL) was
hydrolyzed using Hg(OAc)2 (1.670 g, 5.26 mmol) at room
temperature. After the general extraction procedure, aldehyde (0.197
g, 0.87 mmol) in MeOH (4 mL), the Ohira−Bestmann reagent (0.252
g, 1.31 mmol) dissolved in MeOH (2 mL), and powdered K2CO3
(0.254 g, 1.84 mmol) were stirred at 0 °C. Following the general
workup and flash chromatography (SiO2, 7 g, 2% EtOAc/hexanes),
alkyne 44 was obtained as a pale yellow solid (0.140 g, 33% yield over
1
3 steps): TLC Rf = 0.3 (5% EtOAc/hexanes); mp 84.1−84.2 °C; H
NMR (500 MHz, CDCl3) δ 8.65 (d, J = 2.3 Hz, 1H), 7.86 (d, J = 8.2
Hz, 2H), 7.77 (dd, J = 8.2, 2.3 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.26
(d, J = 7.9 Hz, 2H), 3.82 (qd, J = 7.1, 2.5 Hz, 1H), 2.39 (s, 3H), 2.29
(d, J = 2.5 Hz, 1H), 1.54 (d, J = 7.2 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 156.3, 148.6, 139.1, 136.6, 136.3, 135.3, 129.7, 126.9, 120.2,
86.1, 71.0, 29.3, 24.2, 21.5; IR (neat cm−1) 3214, 2973, 2928, 2867,
2109, 1679, 1474, 1386, 1293, 1087, 1014, 818, 764, 697, 534; HRMS
(DART, M+ + H) m/z 222.1303 (calculated for C16H16N, 222.1283).
5-(1-Methyl-prop-2-ynyl)-2-phenyl-pyrimidine (45). According to
the general procedure for homologation, methoxymethyl triphenyl-
phosphonium chloride (2.3 g, 6.62 mmol) in dry THF (18 mL),
NaOtBu (0.797 g, 8.3 mmol), and ketone 42 (0.655 g, 3.31 mmol) in
THF (6 mL) were stirred at 0 °C. Following the general workup, the
mixture of enol ethers (0.398 g, 1.76 mmol) in THF/H2O (9:1, 6 mL)
was hydrolyzed using Hg(OAc)2 (1.680 g, 5.28 mmol) at room
temperature. After the general extraction procedure, aldehyde (0.300
g, 1.41 mmol) in MeOH (4 mL), the Ohira−Bestmann reagent (0.407
g, 2.12 mmol) dissolved in MeOH (2 mL), and powdered K2CO3
(0.410 g, 2.96 mmol) were stirred at 0 °C. Following the general
workup and flash chromatography (SiO2, 5 g, 5% EtOAc/hexanes),
alkyne 45 was obtained as a white solid (0.066 g, 10% yield over 3
4′-[3-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-1-methyl-prop-2-
ynyl]-3′-methoxy-biphenyl-4-ol (30). According to the general
Sonogahisra coupling procedure, ethyl-iodopyrimidine (0.036 g, 0.14
mmol), CuI (0.0075 g, 0.039 mmol, 21 mol %), Pd(PPh3)2Cl2 (0.009
g, 0.014 mmol, 10 mol %), and alkyne 20 (0.037 g, 0.15 mmol) were
reacted in DMF/Et3N (0.5 mL each) at 60 °C for 14 h. After the
mixture was cooled, the dark reddish brown solution was concentrated,
and the product was purified by flash chromatography (SiO2, 5 g, 3%
MeOH/CH2Cl2) to afford coupled pyrimidine 30 as a pale white
powder (0.043 g, 79%) followed by reverse phase flash chromatog-
raphy (NH2 capped SiO2, 3g, 100% CH2Cl2, 1% MeOH/CH2Cl2) for
biological evaluation: TLC Rf = 0.06 (5% MeOH/CH2Cl2); mp
1
188.1−189.3 °C; H NMR (500 MHz, CDCl3) δ 7.52 (d, J = 7.8 Hz,
1
1H), 7.42 (d, J = 8.6 Hz, 2H), 7.11 (dd, J = 7.9, 1.7 Hz, 1H), 7.01 (d, J
= 1.7 Hz, 1H), 6.90 (d, J = 8.6 Hz, 2H), 5.31 (s, 2H), 4.99 (s, 2H),
4.40 (q, J = 7.0 Hz, 1H), 3.89 (s, 3H), 2.72 (q, J = 7.6 Hz, 2H), 1.53
(d, J = 7.0 Hz, 3H), 1.24 (t, J = 7.6 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 173.2, 164.4, 160.3, 156.5, 156.5, 141.4, 133.2, 129.9, 128.6,
128.0, 119.4, 116.1, 109.4, 102.9, 91.4, 73.9, 55.7, 29.7, 26.9, 22.9, 12.9;
IR (neat cm−1) 3470, 3371, 3337, 3173, 2970, 2930, 2871, 2341, 1726,
1547, 1438, 1217, 1028, 813; HRMS (ESI, M+ + H) m/z 389.1963
(calculated for C23H25N4O2, 389.1972). HPLC (a) tR = 6.8 min, 99%;
(b) tR = 8.23 min, 99%.
steps): TLC Rf = 0.3 (5% EtOAc/hexanes); mp 75.4−76.7 °C; H
NMR (500 MHz, CDCl3) δ 8.82 (s, 2H), 8.60−8.21 (m, 2H), 7.48−
7.46 (m, 3H), 3.82 (qd, J = 7.1, 2.5 Hz, 1H), 2.34 (d, J = 2.5 Hz, 1H),
1.57 (d, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 163.7, 156.1,
137.6, 133.3, 130.9, 128.8, 128.3, 84.6, 71.9, 27.4, 23.8; IR (neat cm−1)
3205, 3059, 2978, 2934, 1584, 1547, 1425, 1296, 1175, 1094, 1069,
749, 692, 651; HRMS (DART, M+ + H) m/z 209.1103 (calculated for
C14H13N2, 209.1079).
General Procedure for Sonogashira Coupling. 6-Ethyl-5-[3-
(3-methoxy-biphenyl-4-yl)-but-1-ynyl]-pyrimidine-2,4-diamine (28).
To a 30 mL screw-cap vial was added ethyl-iodopyrimidine (0.347 g,
0.13 mmol), freshly purified CuI (0.005 g, 0.03 mmol), and
Pd(PPh3)2Cl2 (0.009 g, 0.01 mmol). Argon-purged anhydrous DMF
(0.3 mL) was added followed by alkyne 18 (0.0342 g, 0.14 mmol).
The reaction mixture was stirred under argon for 5 min followed by
argon-purged anhydrous triethylamine. The reaction mixture was
degassed once using the freeze/pump/thaw method. The vial was
sealed under argon and heated at 60 °C for 14 h (overnight). At the
end of the reaction, the dark reddish brown solution was concentrated
and the product purified by flash column chromatography (SiO2, 10g,
2% MeOH/CH2Cl2)) to afford the coupled pyrimidine 28 as a pale
yellow oil (0.035 g, 72% yield). The small amount of yellow oil was
subjected to reverse phase chromatography (NH2 capped SiO2, 2g,
100% CH2Cl2) to afford a colorless hygroscopic solid for biological
evaluation. TLC Rf = 0.1 (5% MeOH/CH2Cl2); 1H NMR (500 MHz,
CDCl3) δ 7.59−7.56 (m, 3H), 7.42 (dd, J = 7.6, 7.6 Hz, 1H), 7.42 (dd,
J = 7.6, 7.6 Hz, 1H), 7.37−7.30 (m, 1H), 7.18 (dd, J = 1.5, 7.8 Hz,
6-Ethyl-5-[3-(3-methoxy-4′-methyl-biphenyl-4-yl)-but-1-ynyl]-
pyrimidine-2,4-diamine (31). According to the general Sonogahisra
coupling procedure, ethyl-iodopyrimidine (0.026 g, 0.10 mmol), CuI
(0.004 g, 0.021 mmol, 21 mol %), Pd(PPh3)2Cl2 (0.007 g, 0.010
mmol, 10 mol %), and alkyne 21 (0.031 g, 0.10 mmol) were reacted in
DMF/Et3N (0.5 mL each) at 60 °C for 14 h. After the mixture was
cooled, the dark reddish brown solution was concentrated, and the
product was purified by flash chromatography (SiO2, 5 g, 2% MeOH/
CHCl3) to afford coupled pyrimidine 31 as a pale white powder (0.030
g, 77%) followed by reverse phase flash chromatography (NH2 capped
SiO2, 3 g, 100% CH2Cl2) for biological evaluation: TLC Rf = 0.08 (5%
MeOH/CH2Cl2); mp 112.8−114.3 °C; 1H NMR (500 MHz, CDCl3)
δ 7.57 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.23−7.22 (m,
2H), 7.16 (dd, J = 7.8, 1.6 Hz, 1H), 7.05 (d, J = 1.4 Hz, 1H), 5.13 (s,
2H), 4.79 (s, 2H), 4.42 (q, J = 6.9 Hz, 1H), 3.91 (s, 3H), 2.70 (q, J =
7.6 Hz, 2H), 2.38 (s, 3H), 1.54 (d, J = 7.0 Hz, 3H), 1.24 (t, J = 7.6 Hz,
3H); 13C NMR (125 MHz, CDCl3) δ 173.4, 164.5, 160.7, 156.5,
2652
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141.4, 138.4, 137.3, 130.5, 129.6, 128.1, 127.1, 119.6, 109.6, 102.4,
91.1, 74.5, 55.7, 29.8, 26.9, 23.1, 21.3, 12.7; IR (neat cm−1) 3460, 3387,
3306, 3158, 2969, 2929, 2870, 1727, 1546, 1434, 1221, 805; HRMS
(ESI, M+ + H) m/z 387.2176 (calculated for C24H27N4O, 387.2179).
HPLC (a) tR = 36.2 min, 94.8%; (b) tR = 31.4 min, 96.9%.
1282, 771, 698, 505; HRMS (ESI, M+ + H) m/z 431.2081 (calculated
for C25H27N4O3, 431.2078). HPLC (a) tR = 20.5 min, 99.4%; (b) tR =
18.1 min, 99.1%.
5-[3-(4′-Dimethylamino-3-methoxy-biphenyl-4-yl)-but-1-ynyl]-6-
ethyl-pyrimidine-2,4-diamine (35). According to the general
Sonogahisra coupling procedure, ethyl-iodopyrimidine (0.014 g, 0.05
mmol), CuI (0.002 g, 0.010 mmol, 20 mol %), Pd(PPh3)2Cl2 (0.004 g,
0.005 mmol, 10 mol %), and alkyne (0.015 g, 0.050 mmol) were
reacted in DMF/Et3N (0.5 mL/0.5 mL) at 60 °C for 6 h. After the
mixture was cooled, the dark reddish brown solution was concentrated,
and the product was purified by flash chromatography (SiO2, 10g,
100% EtOAc followed by 2% MeOH/CH2Cl2) followed by reverse
phase flash chromatography (NH2 capped SiO2, 5g, 100% CH2Cl2) to
afford pyrimidine 35 as a off-white solid (9 mg, 43%); TLC Rf = 0.22
4′-[3-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-1-methyl-prop-2-
ynyl]-3′-methoxy-biphenyl-4-carbonitrile (32). According to the
general Sonogahisra coupling procedure, ethyl-iodopyrimidine (0.056
g, 0.21 mmol), CuI (0.006 g, 0.031 mmol, 15 mol %), Pd(PPh3)2Cl2
(0.015 g, 0.021 mmol, 10 mol %), and alkyne 22 (0.084 g, 0.318
mmol) were reacted in DMF/Et3N (1 mL each) at 70 °C for 12 h.
After the mixture was cooled, the dark reddish brown solution was
concentrated, and the product was purified by flash chromatography
(SiO2, 5 g, 2% MeOH/CHCl3) followed by reverse phase flash
chromatography (NH2 capped SiO2, 3 g, 100% CH2Cl2, 1% MeOH/
CH2Cl2) to afford coupled pyrimidine 32 as a pale white powder
(0.065 g, 78%); TLC Rf = 0.2 (5% MeOH/CH2Cl2); mp 130.9−133.1
1
(5% MeOH/CH2Cl2); mp 135.2−136.1 °C; H NMR (500 MHz,
chloroform-d) δ 7.51 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 8.6 Hz, 2H),
7.14 (d, J = 5.0 Hz, 1H), 7.03 (s, 1H), 6.78(d, J = 10.0 Hz, 2H), 5.24
(s, 2H), 5.01 (s, 2H), 4.40 (q, J = 7.0 Hz, 1H), 3.90 (s, 3H), 2.98 (s,
6H), 2.71 (q, J = 7.6 Hz, 2H), 1.53 (d, J = 7.0 Hz, 3H), 1.25 (t, J = 6.9
Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 164.7, 157.9, 156.6, 150.3,
141.9, 129.1, 128.7, 127.9, 119.0, 112.9, 109.2, 103.9, 92.0, 72.3, 55.7,
40.8, 28.1, 26.9, 22.7, 12.6; IR (neat cm−1) 3310, 3172, 2925, 2873,
1603, 1570, 807; HRMS (ES, M+ + H) m/z 416.2442 (calculated for
C25H30N5O, 416.2445).
1
°C; H NMR (500 MHz, CDCl3) δ 7.73−7.70 (m, 2H), 7.69−7.63
(m, 3H), 7.19 (dd, J = 7.8, 1.7 Hz, 1H), 7.05 (d, J = 1.7 Hz, 1H), 5.24
(s, 2H), 4.98 (s, 2H), 4.45 (q, J = 7.0 Hz, 1H), 3.94 (s, 3H), 2.71 (q, J
= 7.6 Hz, 2H), 1.55 (d, J = 7.0 Hz, 3H), 1.24 (t, J = 7.6 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 173.4, 164.5, 160.8, 156.8, 145.7, 139.3,
132.8, 132.5, 128.5, 127.9, 119.9, 119.1, 111.1, 109.6, 101.9, 90.8, 74.8,
55.6, 29.8, 26.9, 23.0, 12.7; IR (neat cm−1) 3464, 3428, 3332, 3188,
3029, 2925, 2775, 2546, 1651, 1548, 1445, 1286, 1008, 735, 557;
HRMS (DART, M+ + H) m/z 398.1983, (calculated for C24H24N5O,
398.1981). HPLC (a) tR = 19.2 min, 99.6%; (b) tR = 17.5 min, 99.5%.
Carbamic Acid 4′-[3-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-1-
methyl-prop-2-ynyl]-3′-methoxy-biphenyl-4-yl Ester (33). According
to the general Sonogahisra coupling procedure, ethyl-iodopyrimidine
(0.055 g, 0.21 mmol), CuI (0.008 g, 0.04 mmol, 21 mol %),
Pd(PPh3)2Cl2 (0.015 g, 0.021 mmol, 10 mol %), and alkyne 23 (0.092
g, 0.31 mmol) were reacted in DMF/Et3N (1 mL each) at 60 °C for
12 h. After the mixture was cooled, the dark reddish brown solution
was concentrated, and the product was purified by flash chromatog-
raphy (SiO2, 5 g, 2% MeOH/CHCl3) to afford coupled pyrimidine 33
as a pale white powder (0.076 g, 84%) followed by reverse phase flash
chromatography (NH2 capped SiO2, 3 g, 100% CH2Cl2, 1% MeOH/
CH2Cl2) for biological evaluation: TLC Rf = 0.07 (5% MeOH/
6-Ethyl-5-[3-(4′-fluoro-3-methoxy-biphenyl-4-yl)-but-1-ynyl]-pyr-
imidine-2,4-diamine (36). According to the general Sonogahisra
coupling procedure ethyl-iodopyrimidine (0.10 g, 0.39 mmol), CuI
(0.02 g, 0.08 mmol, 20 mol %), Pd(PPh3)2Cl2 (0.03 g, 0.04 mmol, 10
mol %), and alkyne (0.10 g, 0.39 mmol) were reacted in DMF/Et3N
(1 mL/1 mL) at 60 °C for 12 h. After the mixture was cooled, the dark
reddish brown solution was concentrated, and the product was purified
by flash chromatography (SiO2, 10 g, 100% EtOAc followed by 1%
MeOH/CH2Cl2) followed by reverse phase flash chromatography
(NH2 capped SiO2, 3 g, 100% CH2Cl2) to afford pyrimidine 36 as a
off-white solid (0.12 g, 79%); TLC Rf = 0.26 (5% MeOH/CH2Cl2);
mp 129.6−130.0 °C; 1H NMR (500 MHz, chloroform-d) δ 7.56−7.50
(m, 3H), 7.13−7.09 (m, 3H), 7.01(s, 1H), 5.30 (br s, 2H), 5.19 (br s,
2H) 4.41 (q, J = 6.9 Hz, 1H), 3.91 (s, 3H), 2.73 (q, J = 7.6 Hz, 2H),
1.54 (d, J = 7.0 Hz, 3H), 1.27 (t, J = 7.6 Hz, 3H). 13C NMR (125
MHz, CDCl3) δ 164.5, 162.6 (d, J = 245.0 Hz, 1C), 159.5, 156.6,
140.6, 137.3, 130.5, 128.8 (d, J = 7.5 Hz, 2C), 128.1, 119.7, 115.8 (d, J
= 21.2 Hz, 2C), 109.6, 102.8, 91.4, 73.7, 55.7, 29.1, 26.9, 22.9, 12.7; IR
(neat cm−1) 3463, 3418, 3309, 3164, 2964, 2927, 1621, 1547, 1432,
1219, 1018; HRMS (DART, M++H) m/z 391.1921 (calculated for
1
CH2Cl2); H NMR (500 MHz, MeOD) δ 7.53 (d, J = 7.8 Hz, 1H),
7.46 (d, J = 8.6 Hz, 2H), 7.13 (dd, J = 7.8,1.60, 1H), 7.11 (d, J = 1.3
Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 4.41 (q, J = 6.9 Hz, 1H), 3.93 (s,
3H), 2.67 (q, J = 7.6 Hz, 2H), 1.52 (d, J = 7.0 Hz, 3H), 1.22 (t, J = 7.6
Hz, 3H); 13C NMR (125 MHz, MeOD) δ 173.5, 166.1, 162.2, 158.3,
157.9, 142.7, 133.8, 130.9, 129.1, 128.9, 119.9, 116.7, 110.1, 103.2,
91.4, 74.9, 56.2, 30.4, 27.9, 23.4, 13.3; δ IR (neat cm−1) 3477, 3386,
3336, 3195, 2970, 2929, 2873, 2361, 2023, 1603, 1437, 1217, 1027,
813. HRMS (ESI, M+ + Na) m/z 455.1947 (calculated for
C23H24FN4O, 391.1934). HPLC (a) tR = 25.5 min, 96.8%; (b) tR
=
19.8 min, 97.5%.
5-(3-Biphenyl-4-yl-but-1-ynyl)-6-ethyl-pyrimidine-2,4-diamine
(37). According to the general Sonogahisra coupling procedure ethyl-
iodopyrimidine (0.040 g, 0.15 mmol), CuI (0.006 g, 0.03 mmol, 21
mol %), Pd(PPh3)2Cl2 (0.011 g, 0.02 mmol, 10 mol %), and alkyne 27
(0.034 g, 0.17 mmol) were reacted in DMF/Et3N (1 mL each) at 60
°C for 12 h. After the mixture was cooled, the dark reddish brown
solution was concentrated, and the product was purified by flash
chromatography (SiO2, 5 g, 2% MeOH/CHCl3) to afford coupled
pyrimidine 37 as a pale white powder (0.040 g, 77%) followed by
reverse phase flash chromatography (NH2 capped SiO2, 3 g, 100%
CH2Cl2) for biological evaluation: TLC Rf = 0.1 (5% MeOH/
CH2Cl2); mp 129.3−131.1 °C; 1H NMR (500 MHz, CDCl3) δ 7.59−
7.55 (m, 4H), 7.48 (d, J = 8. Hz, 2H), 7.42 (dd, J = 7.6, 7.6 Hz, 2H),
7.42 (dd, J = 7.6, 7.6 Hz, 1H), 7.36−7.30 (m, 1H), 5.13 (s, 2H), 4.87
(s, 2H), 4.07 (q, J = 7.1 Hz, 1H), 2.69 (q, J = 7.6 Hz, 2H), 1.61 (d, J =
7.1 Hz, 3H), 1.23 (t, J = 7.6 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
173.7, 164.5, 160.9, 142.6, 140.9, 140.0, 128.9, 127.6, 127.5, 127.4,
127.3, 101.8, 90.8, 75.6, 32.9, 29.9, 24.9, 12.8; IR (neat cm−1) 3415,
3304, 3162, 2973, 2927, 2871, 1618, 1547, 1436, 1281, 761, 692, 479;
HRMS (ESI, M+ + H) m/z 343.1907 (calculated for C22H23N4,
343.1917). HPLC (a) tR = 19.1 min, 98.9%; (b) tR = 17.3 min, 98.5%.
6-Ethyl-5-[3-(6-phenyl-pyridin-3-yl)-but-1-ynyl]-pyrimidine-2,4-
diamine (46). According to the general Sonogahisra coupling
procedure, ethyl-iodopyrimidine (0.071 g, 0.27 mmol), CuI (0.011
C24H26N5NaO3, 455.1928). HPLC (a) tR = 6.8 min, 98%; (b) tR
=
8.2 min, 98.7%.
4′-[3-(2,4-Diamino-6-ethyl-pyrimidin-5-yl)-1-methyl-prop-2-
ynyl]-3′-methoxy-biphenyl-4-carboxylic Acid Methyl Ester (34).
According to the general Sonogahisra coupling procedure, ethyl-
iodopyrimidine (0.061g, 0.23 mmol), CuI (0.009 g, 0.05 mmol, 21 mol
%), Pd(PPh3)2Cl2 (0.016 g, 0.023 mmol, 10 mol %), and alkyne 24
(0.100 g, 0.34 mmol) were reacted in DMF/Et3N (1 mL each) at 60
°C for 12 h. After the mixture was cooled, the dark reddish brown
solution was concentrated, and the product was purified by flash
chromatography (SiO2, 5g, 2% MeOH/CHCl3) to afford coupled
pyrimidine 34 as a pale white powder (0.077 g, 77%) followed by
reverse phase flash chromatography (NH2 capped SiO2, 3 g, 100%
CH2Cl2, 1% MeOH/CH2Cl2): TLC Rf = 0.1 (5% MeOH/CH2Cl2);
mp 168.2−170.8 °C; 1H NMR (500 MHz, CDCl3) δ 8.08 (d, J = 8.55
Hz, 2H), 7.64−7.60 (m, 3H), 7.21 (dd, J = 7.8, 1.6 Hz, 1H), 7.08 (d, J
= 1.5 Hz, 1H), 5.15 (s, 2H), 4.84 (s, 2H), 4.43 (q, J = 7.0 Hz, 1H),
3.93 (s, 3H), 3.92 (s, 3H), 2.70 (q, J = 7.6 Hz, 2H), 1.54 (d, J = 7.0
Hz, 3H), 1.23 (t, J = 7.6 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ
173.5, 167.2, 164.5, 160.8, 156.7, 145.7, 140.2, 131.9, 130.3, 129.2,
128.3, 127.2, 120.0, 109.7, 102.1, 90.9, 74.7, 55.8, 52.4, 29.9, 26.9, 23.1,
12.8; IR (neat cm−1) 3427, 3302, 3163, 2925, 2851, 2150, 1699, 1548,
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g, 0.06 mmol, 21 mol %), Pd(PPh3)2Cl2 (0.019 g, 0.03 mmol, 10 mol
%), and alkyne 43 (0.061 g, 0.3 mmol) were reacted in DMF/Et3N (1
mL each) at 60 °C for 12 h. After the mixture was cooled, the dark
reddish brown solution was concentrated, and the product was purified
by flash chromatography (SiO2, 5 g, 2% MeOH/CHCl3) to afford
coupled pyrimidine 46 as a pale white hygroscopic solid (0.070 g,
75%), followed by reverse phase flash chromatography (NH2 capped
SiO2, 3 g, 100% CH2Cl2, 1% MeOH/CH2Cl2) for biological
evaluation: TLC Rf = 0.1 (5% MeOH/CH2Cl2); 1H NMR (500
MHz, CDCl3) δ 8.72 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 7.2 Hz, 2H),
7.81 (dd, J = 8.2, 2.3 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.46 (dd, J =
7.5, 7.5 Hz, 1H), 7.46 (dd, J = 7.5, 7.5 Hz, 1H), 7.41−7.38 (m, 1H),
5.09 (s, 2H), 4.84 (s, 2H), 4.11 (q, J = 7.1 Hz, 1H), 2.68 (q, J = 7.6
Hz, 2H), 1.63 (d, J = 7.1 Hz, 3H), 1.22 (t, J = 7.6 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 173.9, 164.4, 160.9, 156.4, 148.6, 139.3, 137.3,
135.3, 129.1, 128.9, 127.1, 120.6, 100.6, 90.4, 76.2, 30.6, 29.9, 24.7,
12.7; IR (neat cm−1) 3469, 3308, 3166, 2972, 2931,1730, 1542, 1435,
1238, 1018, 739, 692; HRMS (ESI, M+ + H) m/z 344.1865 (calculated
for C21H21N5, 344.1875). HPLC (a) tR = 6.9 min, 99.5%; (b) tR = 7.1
min, 99.2%.
beamline X4A (CaDHFR) or X4C (CgDHFR). Molecular replace-
ment was used to determine all three structures using PDB ID 3EEK15
for CgDHFR and PDB ID 1AOE34 for CaDHFR. Initial phase
information was obtained using Phaser;35 electron density and model
building were performed with Coot.36 Refinement was carried out with
Refmac 537 as part of the CCP4 package. Procheck was used to assess
model quality.38
Enzyme Activity. Enzyme inhibition was determined by
monitoring the consumption of NADPH at 340 nm for one minute.
Reactions were performed with 20 mM TES at pH 7.0, 50 mM KCl,
10 mM 2-mercaptoethanol, 0.5 mM EDTA, and 1 mg/mL bovine
serum albumin. Saturating concentrations of cofactor (100 μM
NADPH) and substrate (100 μM dihydrofolate) are used with a
limiting concentration of enzyme. The enzyme, NADPH, and inhibitor
(added as a racemic mixture) are allowed to incubate 5 min before the
addition of substrate. Inhibitors are stored as 50 mM stock solutions in
DMSO. Stock solutions are diluted in DMSO to appropriate working
concentrations so that less than 2% DMSO is added to the assay
solution, and inhibition is close to 50%. All assays are conducted in
triplicate at 25 °C.
6-Ethyl-5-[3-(6-p-tolyl-pyridin-3-yl)-but-1-ynyl]-pyrimidine-2,4-di-
amine (47). According to the general Sonogahisra coupling procedure,
ethyl-iodopyrimidine (0.059 g, 0.23 mmol), CuI (0.009 g, 0.05 mmol,
21 mol %), Pd(PPh3)2Cl2 (0.016 g, 0.022 mmol, 10 mol %), and
alkyne 44 (0.06 g, 0.27 mmol) were reacted in DMF/Et3N (1 mL
each) at 60 °C for 12 h. After the mixture was cooled, the dark reddish
brown solution was concentrated, and the product was purified by
flash chromatography (SiO2, 5g, 2% MeOH/CHCl3) to afford coupled
pyrimidine 47 as a pale white powder (0.063 g, 76%) followed by
reverse phase flash chromatography (NH2 capped SiO2, 3g, 100%
CH2Cl2, 1% MeOH/CH2Cl2) for biological evaluation: TLC Rf = 0.1
(5% MeOH/CH2Cl2); mp 144−146.1 °C; 1H NMR (500 MHz,
CDCl3) δ 8.74 (d, J = 2.2 Hz, 1H), 7.91 (d, J = 8.1 Hz, 2H), 7.82 (dd,
J = 8.2, 2.3 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.30 (d, J = 8.6 Hz, 2H),
5.25 (s, 2H), 5.07 (s, 2H), 4.13 (q, J = 7.1 Hz, 1H), 2.72 (q, J = 7.6
Hz, 2H), 2.42 (s, 3H), 1.66 (d, J = 7.1 Hz, 3H), 1.26 (t, J = 7.6 Hz,
3H); 13C NMR (125 MHz, CDCl3) δ 173.9, 164.5, 161.1, 156.4,
148.5, 139.1, 136.9, 136.5, 135.2, 129.7, 126.9, 120.3, 100.6, 90.3, 76.2,
30.6, 29.9, 24.6, 21.5, 12.7; IR (neat cm−1) 3459, 3319, 3152, 2973,
2933, 2873, 1542, 1443, 923, 819, 762; HRMS (ESI, M+ + H) m/z
358.2013 (calculated for C22H24N5, 358.2026). HPLC (a) tR = 9.7
min, 99.7%; (b) tR = 9.4 min, 99.5%.
Antifungal Activity. Stock cultures of C. albicans (strain SC5314)
or C. glabrata (strain NCCLS84), thawed from storage in 50% glycerol
at −80 °C, were streaked on YM agar plates and grown at 37 °C for 48
h. Isolated colonies from the plate were suspended in 100 mL of
glucose−salt−biotin (GSB) media containing ammonia chloride (2 g),
potassium phosphate (0.35 g), magnesium sulfate (0.24 g), sodium
citrate (0.3 g), piperazine-N,N′-bis[2-ethanesulfonic acid] (3.4 g),
biotin (40 mg), and glucose (20 g) in 1 L of water at a final pH of 7.1.
Strain SC5314 was grown at 25 °C for 18 h (30 C for 24−36 h for
5314), and strain NCCLS84 was grown at 37 °C for 48−62 h. An
aliquot was removed from the shake flask culture, diluted to between 1
× 105 and 1 × 106 cells/mL in GSB media, and added to 96 well test
plates (100 μL per well) containing test compounds dispensed in
DMSO (1 μL). Amphotericin B and itraconazole were used as
controls.
C. albicans cell viability was determined by the addition of Alamar
Blue (10 μL) to each well after a 24 h incubation period. Antifungal
activity was determined by observing the shift of maximum absorbance
of Alamar Blue 123 from 570 to 600 nm indicating the minimum
inhibitory concentration (MIC) of the compound under investigation.
NCCLS84 has a much slower rate of metabolism than C. alicans
strains, and therefore, Alamar blue could not be used to detect cell
viability in a reasonable time frame (<24 h). The XTT Cell
Proliferation kit (ATCC) was used as an alternative. Tetrazolium
dye, XTT, along with an electron-activating reagent (50 μL), is add to
96-well plates and incubated for 24 h at 37 °C. Cell viability is
indicated by a color change from a dark orange to a bright orange
color that can be detected at 475−550 nM.
Kinetic Solubility Assay. Compounds were initially dissolved as
20 μg/mL dimethyl sulfoxide (DMSO) solutions and diluted in
filtered water in the presence or absence of 200 μg/mL
methylcellulose (METHOCEL A4M; Dow Corning, Midland, MI).
The final concentration of DMSO of all samples is 0.2%. All samples
were incubated at room temperature for 30 min and centrifuged for 10
min at 15,000 rpm. The supernatants of the samples were analyzed by
reversed phase HPLC. The mobile phase consisted of 50% acetonitrile
(ACN) and 50% potassium phosphate buffer (50 mM, pH 7.0), using
an isocratic flow rate of 1.5 mL/min. Solubility was determined as the
maximal concentration for which absorption is linearly related to the
log of the concentration.39
6-Ethyl-5-[3-(2-phenyl-pyrimidin-5-yl)-but-1-ynyl]-pyrimidine-
2,4-diamine (48). According to the general Sonogahisra coupling
procedure, ethyl-iodopyrimidine (0.105 g, 0.4 mmol), CuI (0.028 g,
0.08 mmol, 21 mol %), Pd(PPh3)2Cl2 (0.028 g, 0.04 mmol, 10 mol %),
and alkyne 45 (0.123 g, 0.6 mmol) were reacted in DMF/Et3N (1.3
mL each) at 60 °C for 12 h. After the mixture was cooled, the dark
reddish brown solution was concentrated, and the product was purified
by flash chromatography (SiO2, 5 g, 2% MeOH/CHCl3) to afford
coupled pyrimidine 48 as a pale white powder (0.099 g, 71%) followed
by reverse phase flash chromatography (NH2 capped SiO2, 3g, 100%
CH2Cl2, 1% MeOH/CH2Cl2) for biological evaluation: TLC Rf = 0.1
1
(5% MeOH/CH2Cl2); mp 161.3−162.8 °C; H NMR (500 MHz,
CDCl3) δ 8.84 (s, 2H), 8.62−8.02 (m, 2H), 7.88−7.37 (m, 3H), 5.16
(s, 2H), 4.98 (s, 2H), 4.10 (q, J = 7.1 Hz, 1H), 2.67 (q, J = 7.6 Hz,
2H), 1.65 (d, J = 7.2 Hz, 3H), 1.22 (t, J = 7.6 Hz, 3H); 13C NMR (12
MHz, CDCl3) δ 174.1, 164.4, 163.8, 161.1, 156.1, 137.5, 133.9, 130.9,
128.8, 128.3, 99.2, 89.9, 29.9, 28.7, 24.3, 12.7; IR (neat cm−1) 3401,
3312, 3159, 2970, 2933, 2871, 2222, 1623, 1563, 1427, 802, 740, 687;
HRMS (ESI, M+ + H) m/z 345.1817 (calculated for C20H21N6,
345.1822). HPLC (a) tR = 6.7 min, 99.6%; (b) tR = 7.6 min, 99.6%.
Crystallization and Structure Determination. C. glabrata and
C. albicans DHFR were expressed and purified as described
previously.14,32,33 Crystallization of the protein with ligand also
followed previously described procedures.32 Briefly, the NADPH and
inhibitor (at 1 mM concentration) and protein were incubated for 2 h
at 4 °C, concentrated to 17 mg/mL and mixed with reservoir solution
(1:1) to form a hanging drop solution. Crystals were harvested and
flash frozen. Data were collected at Brookhaven National Laboratory,
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Tabular HPLC data, H and 13C NMR spectra, statistics for
crystallographic data collection and refinement, additional
figures, and sequence alignments. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Article
echinocandins among fluconazole-resistant bloodstream isolates of
Candida glabrata. J. Clin. Microbiol. 2012, 50, 1199−1203.
(11) Kuyper, L.; Baccanari, D.; Jones, M.; Hunter, R.; Tansik, R.;
Joyner, S.; Boytos, C.; Rudolph, S.; Knick, V.; Wilson, H. R.; Caddell,
J. M.; Friedman, H.; Comley, J.; Stables, J. High-affinity inhibitors of
dihydrofolate reductase: antimicrobial and anticancer activities of 7,8-
dialkyl-1,3-diaminopyrrolo[3,2-f ]quinazolines with small molecular
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G.; Hansel, W.; Schaper, K.; Seydel, J. Folate-synthesizing enzyme
system as target for development of inhibitors and inhibitors
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Accession Codes
The Protein Data Bank accession codes are 4HOE, 4HOF, and
4HOG.
AUTHOR INFORMATION
■
Corresponding Authors
*(D.L.W.) Phone: 860-486-9451. Fax: 860-486-6857. E-mail:
dennis.wright@uconn.edu.
*(A.C.A.) Phone: 860-486-6145. Fax: 860-486-6857. E-mail:
amy.anderson@uconn.edu.
Author Contributions
§N.G.-D. and J.L.P. contributed equally to this work.
Notes
The authors declare no competing financial interest.
(14) Liu, J.; Bolstad, D.; Smith, A.; Priestley, N.; Wright, D.;
Anderson, A. Structure-guided development of efficacious antifungal
agents targeting Candida glabrata dihydrofolate reductase. Chem. Biol.
2008, 15, 990−996.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the support of the NIH
(GM067542).
(15) Liu, J.; Bolstad, D.; Smith, A.; Priestley, N.; Wright, D.;
Anderson, A. Probing the active site of Candida glabrata dihydrofolate
reductase with high resolution crystal structures and the synthesis of
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analysis of the active sites of dihydrofolate reductase from two species
of Candida uncovers ligand-induced conformational changes shared
among species. Bioorg. Med. Chem. Lett. 2013, 23, 1279−1284.
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Biochem. 1999, 63, 1246−1252.
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(19) deNobel, J.; Klis, F.; Priem, J.; Munnik, T.; van den Ende, H.
The glucanase-soluble mannoproteins limit cell wall porosity in
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ABBREVIATIONS USED
■
DHFR, dihydrofolate reductase; MIC, minimum inhibitory
concentration; BSI, bloodstream infection; IC50, 50% inhibition
concentration; CgDHFR, C. glabrata DHFR; CaDHFR, C.
albicans DHFR; NADPH, nicotinamide adenine dinucleotide
phosphate; SAR, structure−activity relationship; HPMC,
hydroxypropyl methylcellulose; TLC, thin layer chromatog-
raphy; HRMS, high-resolution mass spectrometry
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