Structure-based approach to the development of potent and selective inhibitors of dihydrofolate reductase from Cryptosporidium

Article abstract of DOI:10.1021/jm8009124

Cryptosporidiosis is an emerging infectious disease that can be life-threatening in an immune-compromised individual and causes gastrointestinal distress lasting up to 2 weeks in an immune-competent individual. There are few therapeutics available for eff

Full text of DOI:10.1021/jm8009124

J. Med. Chem. 2008, 51, 6839–6852
6839
Structure-Based Approach to the Development of Potent and Selective Inhibitors of
Dihydrofolate Reductase from Cryptosporidium
David B. Bolstad,#,† Erin S. D. Bolstad,#,† Kathleen M. Frey,# Dennis L. Wright,*,#,| and Amy C. Anderson*,#
Department of Pharmaceutical Sciences and Department of Chemistry, UniVersity of Connecticut, Storrs, Connecticut 06269
ReceiVed July 22, 2008
Cryptosporidiosis is an emerging infectious disease that can be life-threatening in an immune-compromised
individual and causes gastrointestinal distress lasting up to 2 weeks in an immune-competent individual.
There are few therapeutics available for effectively treating this disease. We have been exploring dihydrofolate
reductase (DHFR) as a potential target in Cryptosporidium. On the basis of the structure of the DHFR
enzyme from C. hominis, we have developed a novel scaffold that led to the discovery of potent (38 nM)
and efficient inhibitors of this enzyme. Recently, we have advanced these inhibitors to the next stage of
development. Using the structures of both the protozoal and human enzymes, we have developed inhibitors
with nanomolar potency (1.1 nM) against the pathogenic enzyme and high levels (1273-fold) of selectivity
over the human enzyme.
Introduction
Cryptosporidiosis is now regarded as an emerging infectious
disease,1 and the causative agent, Cryptosporidium hominis, has
recently been classified as a class B bioterrorism threat. Both
immune-competent and immune-compromised individuals can
Figure 1. Compound 1, a potent propargyl-based inhibitor.
be affected by cryptosporidiosis, usually through a contaminated
water supply.1,2 Cryptosporidiosis causes gastrointestinal distress
In Cryptosporidium, DHFR and TS are present as a bifunctional
enzyme, DHFR-TS (ChDHFR-TS).
lasting 1-2 weeks in an immune-competent individual and can
be life-threatening in an immune-compromised individual, such
as those with HIV, the elderly, or young children. The incidence
of cryptosporidiosis continues to rise, and outbreaks are cited
across the world.3 At this time, there is a single approved
therapeutic agent, nitazoxanide, against Cryptosporidium,4
although the application of this drug is limited to immune-
competent patients5 and effects have not been studied in children
under 12 years of age. Since the disease is more devastating
and sometimes fatal to immune-compromised patients and young
children, new drug discovery is critical.
We have been interested in exploring dihydrofolate reductase
(DHFRa) as a potential drug target in Cryptosporidium hominis
(ChDHFR). The success of DHFR inhibitors in the related
Apicomplexan parasite, Plasmodium falciparum, the causative
agent of malaria, suggests that this could be a highly effective
strategy. DHFR is an essential enzyme and plays a key role in
the folate biosynthetic pathway where it catalyzes the NADPH-
dependent reduction of dihydrofolate to tetrahydrofolate. Tet-
rahydrofolate is converted to 5,10-methylene tetrahydrofolate
by serine hydroxymethyltransferase; 5,10-methylene tetrahy-
drofolate is a key cofactor in deoxythymidine monophosphate
(dTMP) production, catalyzed by thymidylate synthase (TS).
Since DHFR is an essential enzyme not only to the parasite
but to human cells as well, it is critical that an inhibitor be able
to discriminate between the two forms of the enzymes. The
difficulty of achieving both potency and selectivity for Cryptospo-
ridium DHFR was underscored by a seminal study from Nelson
and Rosowsky6 in which they examined 96 structurally diverse
DHFR inhibitors and were unable to identify compounds that
were both potent and selective for ChDHFR. Given the difficulty
of the problem, we recognized that utilization of structures of
both the parasitic and human enzymes could provide us with a
significant advantage in the design of effective inhibitors.
Our pursuit of a structure-based drug design approach began
with the determination of crystal structures of ChDHFR-TS7,8
to 2.7 Å resolution. With this structure in hand, we envisioned
a two-stage approach to the development of effective inhibitors.
In the first stage, we would focus on developing a lead series
that would show high levels of potency against ChDHFR while
maintaining good druglike characteristics and synthetic acces-
sibility. On the basis of the structure of ChDHFR-TS, we
developed a novel series of DHFR inhibitors defined by a
propargyl linker between a 2,4-diaminopyrimidine ring and aryl
ring.9 Through these efforts, we synthesized a highly efficient
ligand (Figure 1, compound 1) with a 50% inhibition concentra-
tion (IC50) of 38 nM and molecular weight of 342 Da. After
the first stage was realized, our attention now turned to achieving
high degrees of selectivity while maintaining or increasing the
potency we already established. In this manuscript, we describe
a series of second generation propargyl analogues inspired by
structural analysis that not only maintain high levels of potency
* To whom correspondence should be addressed. For D.L.W.: phone,
(860) 486-9451; fax, (860) 486-6857; e-mail, Dennis.Wright@uconn.edu.
For A.C.A.: phone, (860) 486-6145; fax, (860) 486-6857; e-mail,
Amy.Anderson@uconn.edu.
#
Department of Pharmaceutical Sciences.
These authors contributed equally.
Department of Chemistry.
†
|
a Abbreviations: DHFR, dihydrofolate reductase; ChDHFR, dihydrofolate
reductase from Cryptosporidium hominis; dTMP, deoxythymidine mono-
phosphate; TS, thymidylate synthase; ChDHFR-TS, dihydrofolate reductase-
thymidylate synthase from Cryptosporidium hominis; hDHFR, human
dihydrofolate reductase; IC50, 50% inhibition concentration; NADPH,
nicotinamide adenine dinucleotide phosphate, reduced form.
10.1021/jm8009124 CCC: $40.75
2008 American Chemical Society
Published on Web 10/04/2008

6840 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bolstad et al.
loop in hDHFR (Scheme 1). Accordingly, we could tune the
steric interaction to destabilize binding to the human enzyme
while maintaining high potency against the protozoal form.
Clearly, the extent of these interactions would be highly
dependent on the degree of rigidity in this PEKN loop region.
Supporting the idea that the loop is rigid, a solution structure
of hDHFR determined by NMR shows little variance in this
region.10 The newly designed analogues (2 and 3) maintained
three moieties: (1) the methyl at C6 of the pyrimidine, which
is predicted to interact with Phe 36, Leu 33, and Leu 25; (2)
the propargyl methyl, which is predicted to interact favorably
with residues Thr 58 and Ile 62 and Cys 113; and (3) the 3′
methoxy group, predicted to interact with both the backbone
and side chain of Leu 25 in the active site.9
Figure 2. ChDHFR (green, PDB code 1SEJ) and hDHFR (blue, PDB
code 1KMV) seen from the same view with cocrystallized ligands in
the active site, demonstrating the substantial difference in active site
opening. The PEKN loop residues are labeled on hDHFR, with Asn
64 indicated on the underside of the loop.
A direct method for introducing steric bulk onto the aromatic
core region was required for synthesis of the second generation
inhibitors. These considerations led us to consider a biaryl type
of scaffold where the steric bulk would be introduced in the
form of an additional aromatic ring. The selection of such a
scaffold allowed us to take advantage of the flexibility and
versatility offered by Suzuki cross-coupling for the preparation
of a wide range of potential inhibitors.
against the parasitic enzyme but also exhibit extremely high
levels of selectivity.
Modeling, Chemistry, and Biological Evaluation
Structural Analysis of ChDHFR and hDHFR. Inspection
of the ChDHFR and human DHFR (hDHFR) structures reveals
that the active sites are highly homologous and residue
differences that exist maintain the same chemical properties.
The most striking difference between these two enzymes is
located at the opening to the active site. In hDHFR, access to
the active site is effectively restricted by a four-residue loop
(Pro 61, Glu 62, Lys 63, Asn 64; or PEKN loop) that is notably
absent in ChDHFR (Figure 2).7 We envisioned that this
structural difference could be exploited to design ligands with
selectivity for ChDHFR.
Initial docking analysis with our first generation propargyl
inhibitors showed that our lead compound 1 did not appear to
exploit these differences. Indeed, 1 showed only a modest 36-
fold preference for the parasitic enzyme over the human enzyme
(Table 1). It was therefore obvious that additional elements
would need to be incorporated into the initial lead structure to
develop a highly selective compound.
The two analogue families based on compounds 2 and 3 with
variable substitution patterns on the distal aryl ring were created
virtually and docked into both ChDHFR and hDHFR using the
program Surflex (Tripos) in the Sybyl environment. Surflex
flexibly docks ligands to a protomol representation of the active
site, created by probing the active site with small molecular
fragments. Ligands are fragmented and built into the protomol
based on an empirical scoring function that includes hydropho-
bic, polar, repulsive, entropic, and solvation terms. In order to
explore protein flexibility, ensembles of receptor structures were
created on the basis of minimized conformational snapshots
across a molecular dynamics time course. While there are several
methods to generate an averaged score for a given ensemble
such as weighting docking scores or conformational energy
using a Boltzmann distribution, using K*, a Boltzmann-weighted
partition function,11 or selecting ensemble members a priori
based on pharmacophoric restraints,12 we chose to use simple
arithmetic averaging for this study. Arithmetically averaging
docking scores from the ensemble provided a means to simply,
effectively and equally, weight complexes over a narrow energy
Inspection of docked complexes of the lead in ChDHFR and
hDHFR suggested that functionality projecting from the meta
or para position on the aromatic core of 1 would be correctly
poised to interact with a region of space filled by the PEKN
Table 1. Inhibitory Potency and Selectivity of DHFR Ligands (IC50 Values in nM)
IC50 (nM)
ChDHFR
selectivity ratio
IC50(ChDHFR)/(IC50)hDHFR
compd
scaffold
R1
R2
hDHFR
R-1
A
B
B
B
B
C
C
C
C
B
B
38 ( 1
1380 ( 20
1700 ( 10
1360 ( 50
1250 ( 6
7200 ( 150
1420 ( 12
2770 ( 59
3370 ( 15
4200 ( 0.1
1360 ( 26
1380 ( 26
36
944
648
595
720
75
10a (()
10b (()
10c (()
10d (()
17a (()
17b (()
17c (()
17d (()
R-10a
H
H
CH3
i-Pr
H
H
1.8 ( 0.2
2.1 ( 0.3
2.1 ( 0.5
10 ( 2
CH3
CH3
i-Pr
H
CH3
CH3
i-Pr
H
19 ( 3
H
36 ( 0.6
7.4 ( 1.9
16 ( 2
77
CH3
i-Pr
H
455
262
1273
46
1.1 ( 0.1
30 ( 1.5
S-10a
H
H

Potent and SelectiVe Cryptosporidium DHFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6841
Scheme 1. Overall Strategy for Adding Steric Substitutions to the Meta or Para Position of the Initial Lead Compounda
a GS is a sterically bulky group. The docked complex of compound 1 (magenta) is bound to the active site of ChDHFR (green).
Scheme 2. Synthesis of Meta-Linked Biphenyl Inhibitorsa
a (a) n-BuLi, -78 °C, then CH3C(O)N(CH3)2, 71%; (b) Pd(PPh3)2Cl2, Cs2CO3, dioxane; (c) Ph3PdCHOMe, THF; (d) concentrated HCl, THF, reflux,
57-82% for two steps; (e) dimethyl (1-diazo-2-oxopropyl)phosphonate, K2CO3, MeOH, 64-99%; (f) Pd(PPh3)2Cl2, CuI, Et3N, DMF, 67-94%.
range. Docking scores are returned with an associated “crash
value” that largely approximates the penetration of the ligand
into the receptor (values for crash that are closer to 0 are
preferred). Because we are attempting to exploit a steric
interaction, the crash value score within Surflex appeared as an
attractive metric to evaluate these inhibitors.
was cross-coupled with the four arylboronic acids 6a-d to give
the corresponding biphenyl derivatives 7a-d. The diisopropy-
lboronic acid 6d was not previously known but was easily
prepared from 2,6-diisopropylaniline by diazotization, iodina-
tion, and final conversion to the boronic acid via lithiation/
boronation (see Supporting Information). Homologation of the
acetyl moiety by condensation with a methoxy substituted
phosphorus ylide and hydrolysis of the resulting enolether gave
aldehydes 8a-d in very good yield. Condensation with the
Ohira-Bestmann reagent gave the corresponding terminal
acetylenes 9a-d that were converted to the inhibitors 10a-d
by a final Sonogashira coupling with iodopyrimidine 11.
The para-linked biphenyl analogues were accessed through
a similar route from the commercially available benzoic acid
derivative 12 (Scheme 3). Conversion of the acid to the acyl
chloride and treatment of the crude material with the Gilman
reagent produced an excellent yield of the acetophenone 13.
Suzuki cross-coupling proceeded as before with high levels of
efficiency to give the key biphenyl intermediates 14a-d. The
acetyl group was homologated through a series of identical
reactions as outlined previously to arrive at the inhibitor series
17a-d.
By employment of these computational methods, a series of
eight potential inhibitors was selected. Four of the inhibitors
were in the meta series of inhibitors, and four were in the para
series (see Scheme 1). Each series comprised derivatives that
incorporated increasing steric bulk at the ortho positions of the
distal aryl ring. The ortho positions were chosen for substitution
to ensure that the two aryl rings are not coplanar, thus increasing
the steric bulk. The computational analysis predicted that the
crash values against hDHFR for these inhibitors became
increasingly negative as the steric volume of the distal aryl ring
increased with minimal change in ChDHFR. For example, a
compound with an unsubstituted aryl ring yielded a crash value
of -1.31 in ChDHFR and -2.79 in hDHFR. An analogous
compound with isopropyl groups in both ortho positions yielded
increased crash values of -4.08 and -12.76 in ChDHFR and
hDHFR, respectively. On the basis of these predictors, we were
confident that this new series of inhibitors was effectively
exploring differences between these two enzymes.
The eight inhibitors produced by the routes outlined in
Schemes 2 and 3 are racemic with regard to the stereogenic
center at the propargylic carbon. Upon evaluation of these
compounds (see below) we selected the most selective racemic
inhibitor, 10a, and generated both enantiomers to probe the
stereochemical impact of this center upon affinity and selectivity.
This asymmetric route is analogous to the one described
previously9 for the generation of enantiopure 1 and relies on
Synthesis. We developed a modular and flexible approach
to these inhibitors by relying on two subsequent palladium-
catalyzed coupling reactions. The meta-linked biphenyl ana-
logues were accessed by selective functionalization of com-
mercially available 3,5-dibromoanisole 4 (Scheme 2).
Metal-halogen exchange followed by addition of N,N-
dimethylacetamide gave the acetophenone derivative 5, which

6842 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Scheme 3. Synthesis of Para-Linked Biphenyl Inhibitorsa
Bolstad et al.
a (a) (COCl)2, cat. DMF, CH2Cl2; (b) Li(CH3)2Cu, THF, 90% for two steps; (c) Pd(PPh3)2Cl2, Cs2CO3, dioxane; (d) Ph3PdCHOMe, THF; (e) concentrated
HCl, THF, reflux, 80-92% for two steps; (f) dimethyl (1-diazo-2-oxopropyl)phosphonate, K2CO3, MeOH, 51-75%; (g) Pd(PPh3)2Cl2, CuI, Et3N, DMF,
53-97%.
Figure 3. Compounds 1 (pink), 10a (yellow), and 17a (white) docked into ChDHFR in their computationally preferred orientations. ChDHFR is
shown as a surface mapped with lipophilicity in a gradient from lipophilic (red) to neutral (green) to hydrophilic (blue).
the use of an Evans’ asymmetric alkylation strategy to set the
single stereogenic center in the inhibitor (Scheme 4).
selective (1273-fold) of all known compounds tested against
the Cryptosporidium DHFR enzyme.
Lithiation of dibromoanisole 4 as previously described,
followed by condensation with dimethylformamide and a
subsequent Suzuki coupling, gave biphenylaldehyde 18 in
excellent overall yield. The aldehyde was homologated through
the intermediacy of an enol ether and subsequently oxidized to
produce the arylacetic acid 19. The acid was converted to the
two enantiomerically pure oxazolidinones 20 and 21 under
standard conditions. Methylation of the derived enolates pro-
ceeded with high levels of diastereoselection to give oxazoli-
dinones 22 and 23. These compounds were converted to the
dibromoalkenes 24 and 25 through a three-step sequence of
reduction, oxidation, and olefination without purification of the
intermediate aldehyde. Treatment of the dibromoalkenes with
elemental magnesium delivered the terminal alkynes in excellent
yield. The two enantiomerically pure (greater than 97% ee)
inhibitors R-10a and S-10a were prepared by a final cross-
coupling reaction.
Discussion
Here, we report the design and synthesis of very potent and
selective inhibitors of the Cryptosporidium DHFR enzyme. Our
initial lead compound, 1, exhibited good potency (38 nM) but
only modest selectivity (36-fold) toward the pathogenic enzyme.
Examination of the structures of ChDHFR and hDHFR led us
to explore two biphenyl series of derivatives in which the second
aryl ring was installed at the meta or para position of the
proximal aryl ring. Computational analysis of these series led
to the synthesis of 10 new inhibitors, all of which exhibit
improved potency and selectivity. The racemic m-biphenyl
analogue 10a was the most potent and selective of the racemic
compounds, with the single R enantiomer (R-10a) being the most
potent and selective compound overall.
Inspection of the inhibition assay data in Table 1 shows
several important relationships. Relative to compound 1, all of
the newly synthesized inhibitors display increased potency
against ChDHFR while showing similar or slightly lower activity
against the human enzyme, driving the increase in selectivity.
Within the two series there are additional trends. The meta series
is generally more potent than the para series for both enzymes.
The effect of increasing methylation on the distal ring seems
negligible for the meta series in both enzymes but does appear
to decrease affinity in the para series for hDHFR. The more
sterically demanding isopropyl substitution significantly lowers
affinity in all cases. In order to re-evaluate our original
hypothesis, we returned to computational modeling.
Biological Evaluation. All compounds were evaluated in
spectrophotometric enzyme inhibition assays using ChDHFR-
TS and hDHFR. Inhibition concentrations (IC50) were measured
and are reported in Table 1. The lead compound, 1, has an IC50
value of 38 nM and modest selectivity (36-fold). All of the
biphenyl compounds are more potent than the initial lead
compound, 1, and exhibit greater selectivity for the pathogenic
enzyme. The most potent racemic compound, 10a, a m-biphenyl
derivative, is also the most selective of the racemic compounds
(944-fold). To our knowledge, the single R enantiomer of this
m-biphenyl analogue is the most potent (1.1 nM) and most

Potent and SelectiVe Cryptosporidium DHFR Inhibitors
Scheme 4. Synthesis of Nonracemic DHFR Inhibitorsa
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6843
a (a) n-BuLi, -78 °C, then DMF, 98%; (b) Pd(PPh3)2Cl2, Cs2CO3, dioxane, 82%; (c) Ph3PdCHOMe, THF; (d) concentrated HCl, THF, reflux; (e) Jones
reagent, 72% for three steps; (f) (CH3)3CCOCl, Et3N, then lithiooxazolidinone; (g) LHMDS, then MeI; (h) LiAlH4, THF; (i) Dess-Martin periodinane,
CH2Cl2; (j) CBr4, PPh3; (k) Mg, THF; (l) iodopyrimidine (11), Pd(PPh3)2Cl2, CuI, Et3N, DMF, 93-97%.
Figure 4. Compounds 1 (pink), 10a-c (yellow), and 17a-c docked into the hDHFR ensemble member with the widest opening. hDHFR surface
is colored based on a lipophilicity gradient from lipophilic (red) to neutral (green) to hydrophilic (blue).
Analysis of the Interactions of the Biphenyl Compounds
with ChDHFR. The docked conformations of both the meta
and para series of biphenyl compounds appear to increase
lipophilic contacts between the ligand and ChDHFR active site
relative to those observed with compound 1. Computational
analysis of the compounds docked into ChDHFR also explains
the clear preference for the meta-substituted compounds as
opposed to the para-substituted compounds. With the 3′-OMe
anchored in a small hydrophobic pocket, the distal phenyl ring
is projected toward the lipophilic opening of the active site in
the meta series, generating favorable contacts. In contrast, in
the para series, the distal ring is projected directly out of the
active site into solution, thus providing minimal ChDHFR
contacts (Figure 3).
this trend. In order to better understand the divergent trend
between these two families, we conducted a similar detailed
analysis with the human enzyme.
As shown in Figure 4, the active site opening in hDHFR is
substantially narrower and less lipophilic than in ChDHFR. The
PEKN loop projects perpendicularly from the wall of the active
site, creating a cleft below the loop. Our initial modeling
suggested that substitutions on the distal ring would produce
unfavorable interactions in this region. However, evaluation of
the compounds shows that inclusion of the second ring did little
to change the potency of compound 1. We have attributed this
discrepancy to a greater degree of flexibility in this loop than
originally anticipated. This increased flexibility may allow the
meta series of compounds to access the cleft below the loop in
a more productive manner, thus compensating for some of the
destabilizing interactions introduced by the second ring. In the
larger isopropyl compound, 10d, there does not appear to be
enough flexibility to compensate.
Analysis of the Interactions of the Biphenyl Compounds
with hDHFR. Although our initial computational studies
predicted that both the meta and para series would display
decreased affinity for hDHFR, only the para series followed

6844 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bolstad et al.
to residual CHCl3 solvent; 7.26 and 77.0 ppm for 1H and 13C,
respectively. Melting points were recorded on Mel-Temp 3.0
apparatus and are uncorrected. High-resolution mass spectrometry
was provided by the Notre Dame Mass Spectrometry Laboratory.
IR data were obtained using a Shimadzu 8400-s FTIR spectrometer.
TLC analyses were performed on Whatman Partisil K6F silica gel
60 plates. All glassware was oven-dried and allowed to cool under
an argon atmosphere. Anhydrous dichlormethane, ether, and
tetrahydrofuran were used directly from Baker cycletainers. An-
hydrous dimethylformamide was purchased from Acros and de-
gassed by purging with argon. Anhydrous triethylamine was
purchased from Aldrich and degassed by purging with argon. All
reagents were used directly from commercial sources unless
otherwise stated. Boronic acids 6a-c were purchased from Aldrich.
Boronic acid 6d was synthesized in two steps from commercial
2,6-diisopropylaniline (see Supporting Information). The Ohira-
Bestmann reagent18 and Dess-Martin periodinane19 were synthe-
sized according to literature procedures. Dibromoanisole 4 was
purchased from Aldrich or synthesized from commercially available
1,3,5-tribromobenzene.20 2,4-Diamino-5-iodo-6-methylpyrimidine
11 was synthesized according to literature procedures.9
In contrast, the para series of compounds project into an
alternative pocket that is severely hindered by the PEKN loop
and the opposing wall of the active site. As expected, increased
steric volume occupied by the distal aryl ring directly correlates
with a decrease in affinity.
In conclusion, these studies demonstrate that taking advantage
of small differences between these enzymes combined with
detailed structural analysis provides a significant advantage in
the design of more efficacious inhibitors. Utilizing the structural
information, we have been able to increase the selectivity of a
lead compound from 36-fold to 1273-fold (a 35-fold gain) with
a concomitant 35-fold improvement in affinity for the target
enzyme while synthesizing only 10 new analogues.
Experimental Section
Enzyme Expression, Purification, and Assays. ChDHFR-TS
was expressed in E. coli and purified using a methotrexate agarose
column.9 The gene for hDHFR was amplified using PCR from
cDNA obtained from ATCC. The gene was inserted in a pET41
vector with a C-terminal histidine tag for affinity chromatography.
The resulting construct was verified by sequencing. The hDHFR
protein was expressed in E. coli and purified using a nickel affinity
column. Enzyme activity assays were performed by monitoring the
change in UV absorbance at 340 nm as previously described.9
Enzyme assays were performed at least four times. IC50 values and
their standard deviations were calculated in the presence of varying
ligand concentration.
1-(3-Bromo-5-methoxyphenyl)ethanone (5). To a -78 °C solu-
tion of n-BuLi (1.75 M in hexanes, 10.75 mL, 18.8 mmol) in dry
THF (24 mL) under argon was added a solution of dibromide 4
(5.0 g, 18.8 mmol) in dry THF (15 mL) dropwise. White solids
formed, and the mixture was stirred for 20 min at -78 °C. Then
anhydrous dimethylacetamide (1.92 mL, 20.7 mmol) was added
slowly over ∼10 min. After the mixture was stirred an additional
20 min at -78 °C, the reaction was quenched with 30 mL of 1 M
HCl and the mixture was warmed to ambient temperature. The
mixture was diluted with ether (30 mL) and the organic phase
separated. The aqueous phase was extracted with ether (2 × 20
mL), and the combined extracts were washed with saturated
NaHCO3 and brine (30 mL each), dried over sodium sulfate, and
concentrated. The crude oil was purified by flash chromatography
(100 g SiO2, 5% EtOAc/hexanes) to provide ketone 5 as a white
solid (3.06 g, 71%): TLC Rf ) 0.19 (5% EtOAc/hexanes); mp
39.1-39.7 °C; 1H NMR (300 MHz, CDCl3) δ 7.62 (m, 1H), 7.38
(dd, J ) 2.4, 1.4 Hz, 1H), 7.22 (dd, J ) 2.4, 1.7 Hz, 1H), 3.83 (s,
3H), 2.56 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 196.3, 160.4,
Computational Modeling. All ligands were drawn in Sybyl13
in an analogous fashion to make the starting conformations as
similar as possible. Ligands were then brought to their local energy
minima using the Tripos force field. The resulting structures were
checked for proper geometries and selectively protonated at N1 of
the 2,4-diaminopyrimidine ring.
Ensembles of receptors were used to model protein flexibility.
Receptors were prepared by adding hydrogens, removing waters,
and calculating formal charges. Ensemble sets were created by
taking 500 fs conformational snapshots across a 10 000 fs molecular
dynamics run at 300 K using the Amber force field in Sybyl. All
structures were then brought to their local energy minima with a
1000 iteration energy change gradient, under the assumption that
high-energy conformations are not likely to be biologically active.
Mobility was limited to a specific active site, as defined by all atoms
falling within a sphere of a specific radius around the cocrystallized
ligand. For ChDHFR, the structure 1SEJ7 was used with a 6 Å
sphere around the tricyclic core of the ligand defining the active
site. 1KMV14 was used as the hDHFR structure. The unresolved
sidechains of Asp 21 and Lys 62 were constructed using the most
common side chain angles from a Lovell dictionary.15 Motion of
1KMV was confined to a 6 Å sphere around the ligand and PEKN
loop region residues 59-68. The conserved acidic residue (Glu
30) was held rigid throughout the MD to preserve the essential
N1H hydrogen bonding contact. It was not necessary to hold the
conserved acidic residue rigid in the case of ChDHFR, as the contact
was preserved across the time course.
139.5, 123.9, 123.0, 122.0, 112.0, 55.7, 26.6; IR (neat, KBr, cm-1
)
3084, 2939, 2837, 1693, 1454, 1277, 1043, 739; HRMS (FAB,
MH+) m/z 228.9881 (calculated for C9H10BrO2, 228.9864).
General Procedure for the Suzuki Coupling: 1-(3-Methoxy-5-
(2,6-dimethylphenyl)phenyl)ethanone (7c). To a 15 mL screw cap
pressure vessel was added ketone 5 (0.517 g, 2.26 mmol), 2,6-
dimethylphenylboronic acid 6c (0.680 g, 4.53 mmol), Cs2CO3 (2.20
g, 6.75 mmol), Pd(PPh3)2Cl2 (0.150 g, 0.21 mmol, 9% Pd), and
anhydrous dioxane (4.5 mL). The mixture was stirred and then
degassed once using the freeze/pump/thaw method. Once the
mixture warmed to room temperature, the vessel was sealed under
argon and placed in an 80 °C oil bath for 14 h (overnight). An
aliquot was analyzed by 1H NMR to confirm the disappearance of
starting material (loss of -OCH3 singlet). The dark colored mixture
was cooled, diluted with ether (8 mL), and filtered through a pad
of silica gel (∼15 g), rinsing with ether. The filtrate was
concentrated and the residue purified by flash chromatography (SiO2
25 g, 2-5% EtOAc/hexanes) to afford biphenyl ketone 7c as a
white solid (0.505 g, 88%): TLC Rf ) 0.17 (5% EtOAc/hexanes);
1H NMR (300 MHz, CDCl3) δ 7.49 (dd, J ) 2.5, 1.5 Hz, 1H),
7.35 (m, 1H), 7.20 (m, 1H), 7.13 (m, 2H), 6.94 (dd, 2.5, 1.4 Hz,
1H), 3.89 (s, 3H), 2.60 (s, 3H), 2.06 (s, 6H); 13C NMR (75 MHz,
CDCl3) δ 197.9, 160.0, 142.8, 140.6, 138.7, 135.8, 127.5, 127.4,
122.0, 120.3, 110.6, 55.5, 26.8, 20.7; IR (neat, KBr, cm-1); HRMS
(FAB, M+) m/z 254.1315 (calculated for C17H18O2, 254.1307).
1-(3-Methoxy-5-phenyl)phenylethanone (7a). According to the
general Suzuki coupling procedure, ketone 5 (0.343 g, 1.5 mmol)
was reacted with phenylboronic acid 6a (0.366 g, 3.0 mmol),
Cs2CO3 (1.48 g, 4.54 mmol), and Pd(PPh3)2Cl2 (0.044 g, 0.063
mmol, 4% Pd) in 3 mL of dioxane for 2 h. Following the general
reaction workup, flash chromatography (SiO2 25 g, 5% EtOAc/
Docking was carried out using Surflex-Dock16 as implemented
by Sybyl 7.3. Surflex-Dock includes a solvation function that
captures the difference between the potential and actual numbers
of hydrogen bond equivalents. An in-house script was used to dock
against ensembles of receptors. Because of the conserved orientation
of the 2,4-diaminopyrimidine moiety in the active site, the correct
placement of the ligand could be determined. The top scoring poses
with the conserved orientation, the N1H located within hydrogen
bond distance of the conserved acidic residue, and the C2 NH2
located within 4.3 Å of the conserved Thr oxygen were analyzed.
Scores were averaged across the ensemble because we have
previously determined this to be the most effective.17
Synthesis. General. The 1H and 13C NMR spectra were recorded
on Bruker instruments at 500 and 125 MHz or 300 and 75 MHz,
respectively. Chemical shifts are reported in ppm and are referenced

Potent and SelectiVe Cryptosporidium DHFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6845
hexanes) afforded biphenyl ketone 7a as a clear oil (0.287 g, 85%):
TLC Rf ) 0.20 (5% EtOAc/hexanes); 1H NMR (300 MHz, CDCl3)
δ 7.76 (t, J ) 1.5 Hz, 1H), 7.60 (m, 2H), 7.50-7.42 (m, 3H), 7.38
(m, 1H), 7.32 (dd, J ) 2.5, 1.5 Hz, 1H), 3.90 (s, 3H), 2.64 (s, 3H);
13C NMR (75 MHz, CDCl3) δ 197.7, 160.1, 142.9, 140.0, 138.8,
128.8, 127.8 127.1, 119.9, 118.1, 111.2, 55.4, 26.7; IR (neat, KBr,
cm-1) 3082, 2939, 1684, 1591, 1358, 1211, 1045, 763; HRMS
(FAB, MH+) m/z 227.1078 (calculated for C15H15O2, 227.1072).
1-(3-Methoxy-5-(2-methylphenyl)phenyl)ethanone (7b). Accord-
ing to the general Suzuki coupling procedure, ketone 5 (0.175 g,
0.764 mmol) was reacted with 2-methylphenylboronic acid 6b
(0.207 g, 1.52 mmol), Cs2CO3 (0.750 g, 2.30 mmol), and
Pd(PPh3)2Cl2 (0.054 g, 0.076 mmol, 10% Pd) in 1.5 mL of dioxane
for 4.5 h. Following the general reaction workup, flash chroma-
tography (SiO2 7 g, 2-5% EtOAc/hexanes) afforded biphenyl
ketone 7b as a clear oil (0.159 g, 87%): TLC Rf ) 0.14 (5% EtOAc/
13C NMR (75 MHz, CDCl3) δ 200.8, 160.2, 143.2, 141.2, 139.3,
135.8, 127.3, 127.2, 121.3, 113.4, 112.3, 55.3, 52.9, 20.7. 14.5; IR
(neat, KBr, cm-1) 3063, 2974, 2716, 1722, 1589, 1458, 1207, 1029,
773; HRMS (FAB, M+) m/z 268.1466 (calculated for C18H20O2,
268.1463).
2-(3-Methoxy-5-phenylphenyl)propanal (8a). According to the
general Wittig reaction procedure, the reaction of phosphonium
chloride (0.76 g, 2.2 mmol), NaOtBu (0.279 g, 2.9 mmol) and
biphenyl ketone 7a (0.287 g, 1.3 mmol) gave, following filtration
(SiO2 27 g, 2% EtOAc/hexanes), 0.267 g of a mixture (E/Z ≈ 55:
45) of enol ethers that were immediately hydrolyzed in the
subsequent step: TLC Rf ) 0.43 (5% EtOAc/hexanes).
According to the general hydrolysis procedure, the crude enol
ether (0.265 g) was reacted with concentrated HCl (0.5 mL) in THF
(10 mL) for 1 h. Following the general reaction workup, flash
chromatography (SiO2 15 g, 5% EtOAc/hexanes) afforded aldehyde
8a as a clear oil (0.187 g, 61% from 7a): TLC Rf ) 0.20 (5%
EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 9.74 (m, 1H), 7.60
(m, 2H), 7.46 (m, 2H), 7.38 (m, 1H), 7.09 (m, 1H), 7.04 (m, 1H),
6.76 (m, 1H), 3.88 (s, 3H), 3.68 (m, 1H), 1.5 (d, J ) 7.1 Hz, 3H);
13C NMR (125 MHz, CDCl3) δ 200.7, 160.4, 143.5, 140.6, 139.5,
128.7, 127.6, 127.1, 119.6, 112.8, 111.8, 55.3, 53.1, 14.5; IR (neat,
KBr, cm-1) 2974, 2717, 1720, 1593, 1213, 1053; HRMS (FAB,
M+) m/z 240.1158 (calculated for C16H16O2, 240.1150).
2-(3-Methoxy-5-(2-methylphenyl)phenyl)propanal (8b). Accord-
ing to the general Wittig reaction procedure, the reaction of
phosphonium chloride (1.10 g, 3.21 mmol), NaOtBu (0.432 g, 4.5
mmol) and biphenyl ketone 7b (0.540 g, 2.25 mmol) gave,
following filtration (SiO2 25 g, 1% EtOAc/hexanes), 0.383 g of a
mixture (E/Z ≈ 60:40) of enol ethers that were immediately
hydrolyzed in the subsequent step: TLC Rf ) 0.44 (5% EtOAc/
hexanes).
According to the general hydrolysis procedure, the crude enol
ether (0.383 g) was reacted with concentrated HCl (0.5 mL) in THF
(8 mL) for 1.5 h. Following the general reaction workup, flash
chromatography (SiO2 12 g, 2-5% EtOAc/hexanes) afforded
aldehyde 8b as a clear oil (0.327 g, 57% from 7b): TLC Rf ) 0.19
(5% EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 9.75 (d, J )
1.4 Hz, 1H), 7.32-7.24 (m, 4H), 6.85 (m, 1H), 6.80 (m, 1H), 6.77
(m, 1H), 3.86 (s, 3H), 3.67 (dq, J ) 7.0, 1.4 Hz, 1H), 2.31 (s, 3H),
1.50 (d, J ) 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 200.7,
159.8, 144.0, 141.3, 138.8, 135.1, 130.3, 129.5, 127.5, 125.7, 121.6,
113.7, 112.5, 55.2, 52.9, 20.3, 14.4; IR (neat, KBr, cm-1) 3059,
2934, 2716, 1724, 1591, 1456, 1213, 1030, 761; HRMS (FAB, M+)
m/z 254.1312 (calculated for C17H18O2, 254.1307).
2-(3-Methoxy-5-(2,6-diisopropylphenyl)phenyl)propanal (8d). Ac-
cording to the general Wittig reaction procedure, the reaction of
phosphonium chloride (0.683 g, 2.0 mmol), NaOtBu (0.248 g, 2.6
mmol), and biphenyl ketone 7d (0.354 g, 1.14 mmol) gave,
following filtration (SiO2 20 g, 1% EtOAc/hexanes), 0.364 g of
crude material that was immediately hydrolyzed in the subsequent
step: TLC Rf ) 0.42 (5% EtOAc/hexanes).
According to the general hydrolysis procedure, the crude enol
ether (0.364 g) was reacted with concentrated HCl (0.5 mL) in THF
(8 mL) for 2 h. Following the general reaction workup, flash
chromatography (SiO2 18 g, 2-5% EtOAc/hexanes) afforded
aldehyde 8d as a clear oil (0.272 g, 73% from 7d): TLC Rf ) 0.26
(5% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 9.76 (d, J )
1.4 Hz, 1H), 7.39 (m, 1H), 7.26 (d, J ) 7.8 Hz, 2H), 6.82 (m, 1H),
6.74 (dd, J ) 2.4, 1.4 Hz, 1H), 6.69 (m, 1H), 3.86 (s, 3H), 3.68
(dq, J ) 7.0, 1.4 Hz, 1H), 2.68 (m, 2H), 1.50 (d, J ) 7.0 Hz, 3H),
1.17 (d, J ) 6.9 Hz, 6H), 1.14 (d, J ) 6.9 Hz, 3H), 1.13 (d, J )
6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 200.6, 159.7, 146.5,
142.8, 138.8, 138.7, 127.9, 122.5, 121.9, 114.0, 112.3, 55.2, 52.8,
30.2, 24.3, 24.2, 24.12, 24.08, 14.4; IR (neat, KBr, cm-1) 3059,
2961, 2868, 2719, 1726, 1587, 1454, 1202, 1053, 760; HRMS
(FAB, M+) m/z 324.2085 (calculated for C22H28O2, 324.2089).
General Procedure for the Ohira-Bestmann Homologation:
3-(3-Methoxy-5-(2,6-dimethylphenyl)phenyl)butyne (9c). To a 0 °C
solution of aldehyde 8c (0.131 g, 0.488 mmol) in MeOH (2 mL)
was added the Ohira-Bestmann reagent (0.143 g, 0.744 mmol)
1
hexanes); H NMR (300 MHz, CDCl3) δ 7.50 (m, 2H), 7.32 -
7.22 (m, 4H), 7.09 (dd, J ) 2.5, 1.4 Hz, 1H), 3.90 (s, 3H), 2.62 (s,
3H), 2.29 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 198.0, 159.6,
143.6, 140.7, 138.3, 135.2, 130.4, 129.5, 127.7, 125.9, 122.1, 120.3,
110.8, 55.5, 26.8, 20.3; IR (neat, KBr, cm-1) 3003, 2957, 1686,
1589, 1333, 1229, 762; HRMS (FAB, M+) m/z 240.1154 (calculated
for C16H16O2, 240.1150).
1-(3-(2,6-Diisopropylphenyl)-5-methoxyphenyl)ethanone (7d). Ac-
cording to the general Suzuki coupling procedure, ketone 5 (0.251
g, 1.09 mmol) was reacted with boronic acid 6d (0.450 g, 2.18
mmol), Cs2CO3 (1.07 g, 3.28 mmol), and Pd(PPh3)2Cl2 (0.039 g,
0.056 mmol, 5% Pd) in 2 mL of dioxane for 14.5 h. Following the
general reaction workup, flash chromatography (SiO2 12 g, 2%
EtOAc/hexanes) afforded biphenyl ketone 7d as a clear, viscous
oil (0.331 g, 97%): TLC Rf ) 0.19 (5% EtOAc/hexanes); 1H NMR
(500 MHz, CDCl3) δ 7.52 (dd, J ) 2.5, 1.5 Hz, 1H), 7.39 (m, 2H),
7.24 (d, J ) 7.8 Hz, 2H), 6.98 (dd, J ) 2.5, 1.3 Hz, 1H), 3.90 (s,
3H), 2.61 (sep, J ) 6.9 Hz, 2H), 2.60 (s, 3H), 1.12 (d, J ) 6.9 Hz,
12H); 13C NMR (125 MHz, CDCl3) δ 197.9, 159.5, 146.6, 142.3,
138.14, 138.09, 128.2, 122.7, 122.5, 120.8, 110.4, 55.5, 30.2, 26.8,
24.2, 24.1; IR (neat, KBr, cm-1) 3065, 2962, 1688, 1585, 1360,
1204, 735; HRMS (FAB, M+) m/z 310.1937 (calculated for
C21H26O2, 310.1933).
General Procedure for the Wittig Homologation/Enol Ether
Hydrolysis: 2-(3-Methoxy-5-(2,6-dimethylphenyl)phenyl)propanal
(8c). To a 0 °C suspension of methoxymethyltriphenylphosphonium
chloride (0.758 g, 2.21 mmol) in dry THF (6 mL) under an argon
atmosphere was added NaOtBu (0.260 g, 2.70 mmol) in one portion.
The red-orange suspension was stirred for a further 0.5 h at 0 °C.
Then a solution of biphenyl ketone 7c (0.284 g, 1.12 mmol) in
THF (1 mL) was added dropwise. Following 20 min, the reaction
was quenched with water (15 mL) and diluted with ether (15 mL).
The organic phase was separated and the aqueous phase extracted
with additional ether (2 × 10 mL). The combined extracts were
washed with brine (15 mL), dried over sodium sulfate, and
concentrated to afford the crude product that was filtered through
a column of silica (SiO2 21 g, 1% EtOAc/hexanes) to afford 0.288
g of a mixture of enol ethers (E/Z ≈ 60:40) that were immediately
hydrolyzed in the subsequent step: TLC Rf ) 0.50 (5% EtOAc/
hexanes).
To a solution of enol ether (0.288 g, 1.02 mmol) in THF (6 mL)
was added concentrated HCl (0.4 mL). The solution was warmed
in an oil bath to between 55 and 65 °C and monitored by TLC.
Once the starting material had been consumed (∼1 h), the mixture
was cooled and diluted with water and ether (15 mL each). The
organic phase was separated and the aqueous phase extracted with
additional ether (2 × 15 mL). The combined extracts were washed
with saturated NaHCO3 (20 mL) and brine (20 mL), dried over
sodium sulfate, and concentrated to afford the crude product that
was purified by flash chromatography (SiO2 11 g, 2-5% EtOAc/
hexanes) to afford aldehyde 8c as a colorless oil (0.248 g, 82%
from 7c): TLC Rf ) 0.26 (5% EtOAc/hexanes); 1H NMR (300
MHz, CDCl3) δ 9.72 (d, J ) 1.4 Hz, 1H), 7.18 (m, 1H), 7.11 (m,
2H), 6.74 (m, 1H), 6.66 (m, 1H), 6.62 (m, 1H), 3.83 (s, 3H), 3.64
(dq, J ) 7.1, 1.4 Hz, 1H), 2.06 (s, 6H), 1.47 (d, J ) 7.1 Hz, 3H);

6846 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bolstad et al.
dissolved in MeOH (1 mL) followed by powdered, anhydrous
K2CO3 (0.141 g, 1.02 mmol). The mixture was stirred at 0 °C until
the starting material had been consumed by TLC (1 h). The mixture
was diluted with water (20 mL), and the aqueous phase was
extracted with ether (3 × 15 mL). The combined extracts were
washed with brine (20 mL), dried over sodium sulfate, and
concentrated to afford the crude product that was purified by flash
chromatography (SiO2 7 g, 2% EtOAc/hexanes) to afford biphe-
nylacetylene 9c as a colorless oil (0.117 g, 91%): TLC Rf ) 0.55
(5% EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.21 (m, 1H),
7.14 (m, 2H), 7.00 (m, 1H), 6.82 (m, 1H), 6.63 (m, 1H), 3.86 (s,
3H), 3.81 (dq, J ) 7.1, 2.5 Hz, 1H), 2.30 (d, J ) 2.5 Hz, 1H), 2.11
(s, 6H), 1.57 (d, J ) 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
159.8, 144.3, 142.5, 141.7, 135.9, 127.2, 127.0, 120.1, 112.6, 110.9,
86.9, 70.3, 55.2, 31.6, 24.3, 20.7; IR (neat, KBr, cm-1) 3292, 2975,
1593, 1456, 1207, 1045, 771; HRMS (FAB, M+) m/z 264.1526
(calculated for C19H20O, 264.1514).
added (1 mL), and the mixture was degassed once using the freeze/
pump/thaw method. The vial was sealed under argon and heated
at 50 °C for 3 h. After the mixture was cooled, the orange solution
was diluted with EtOAc (20 mL) and washed twice with a water/
saturated NaHCO3 solution (1:2, 20 mL) and brine (20 mL). The
organic phase was dried over sodium sulfate and concentrated to
afford the crude product that was purified by flash chromatography
(SiO2 8 g, 2% MeOH/CHCl3) to afford coupled pyrimidine 10c as
a pale solid (0.107 g, 94%). An analytical sample was generated
by crystallization from cyclohexane. TLC Rf ) 0.27 (EtOAc); mp
1
145-147 °C; H NMR (300 MHz, CDCl3) δ 7.16 (m, 1H), 7.10
(m, 2H), 6.97 (m, 1H), 6.80 (m, 1H), 6.59 (dd, J ) 2.4, 1.4 Hz,
1H), 5.14 (bs, 2H), 4.88 (bs, 2H), 4.04 (q, J ) 7.1 Hz, 1H), 3.82
(s, 3H), 2.35 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.60 (d, J ) 7.1
Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 168.5, 164.1, 160.4, 159.9,
145.0, 142.8, 141.6, 135.89, 135.87, 127.3, 127.1, 120.0, 112.6,
110.9, 101.9, 91.5, 75.5, 55.3, 33.0, 24.6, 22.8, 20.74, 20.72; HRMS
(FAB, M+) m/z 386.2110 (calculated for C24H26N4O, 386.2107);
HPLC (a) tR ) 5.19 min, 98.9%, (b) tR ) 4.94 min, 98.2%.
2,4-Diamino-5-[3-(3-methoxy-5-phenylphenyl)but-1-ynyl]-6-me-
thylpyrimidine (10a). According to the general Sonagashira cou-
pling procedure, iodopyrimidine 11 (0.080 g, 0.32 mmol),
Pd(PPh3)2Cl2 (0.016 g, 0.023 mmol, 7%), CuI (0.0010 g, 0.026
mmol, 16%), and alkyne 9a (0.131 g, 0.554 mmol) were reacted
in DMF/Et3N (1.25 mL each) at 50 °C for 4 h. Following the general
workup procedure, flash chromatography (SiO2 17 g, EtOAc)
afforded coupled pyrimidine 10a as a white foam (0.095 g, 82%):
TLC Rf ) 0.22 (EtOAc); 1H NMR (500 MHz, CDCl3) δ 7.58 (m,
2H), 7.43 (m, 2H), 7.35 (m, 1H), 7.24 (m, 1H), 7.02 (m, 1H), 6.99
(m, 1H), 5.19 (bs, 2H), 4.95 (bs, 2H), 4.08 (q, J ) 7.1 Hz, 1H),
3.87 (s, 3H), 2.39 (s, 3H), 1.63 (d, J ) 7.1 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 168.5, 164.1, 160.4, 160.2, 145.3, 143.1,
141.0, 128.8, 127.5, 127.2, 118.3, 111.5, 111.1, 101.7, 91.4, 75.7,
55.4, 33.1, 24.7, 22.8; HRMS (FAB, M+) m/z 358.1805 (calculated
for C22H22N4O, 358.1794); HPLC (a) tR ) 6.94 min, 100%, (b) tR
) 6.99 min, 100%.
2,4-Diamino-5-[3-(3-methoxy-5-(2-methylphenyl)phenyl)but-1-
ynyl]-6-methylpyrimidine (10b). According to the general Son-
agashira coupling procedure, iodopyrimidine 11 (0.091 g, 0.364
mmol), Pd(PPh3)2Cl2 (0.022 g, 0.031 mmol), CuI (0.011 g, 0.058
mmol), and alkyne 9b (0.138 g, 0.551 mmol) were reacted in DMF/
Et3N (1.25 mL each) at 50 °C for 3 h. Following the general workup
procedure, flash chromatography (SiO2 8 g, 2% MeOH/CHCl3)
afforded coupled pyrimidine 10b as a pale solid (0.118 g, 86%).
An analytical sample was generated by crystallization from ether/
hexanes. TLC Rf ) 0.24 (EtOAc); mp 121.0-123.0 °C; 1H NMR
(500 MHz, CDCl3) δ 7.28-7.23 (m, 4H), 6.98 (m, 1H), 6.96 (m,
1H), 6.76 (dd, J ) 2.3, 1.5 Hz, 1H), 5.12 (bs, 2H), 4.83 (bs, 2H),
4.05 (q, J ) 7.1 Hz, 1H), 3.84 (s, 3H), 2.37 (s, 3H), 2.28 (s, 3H),
1.62 (d, J ) 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 168.5,
164.0, 160.4, 159.5, 144.5, 143.6, 141.6, 135.2, 130.3, 129.5, 127.4,
125.7, 120.3, 113.0, 111.1, 101.8, 91.4, 75.5, 55.3, 33.0, 24.7, 22.8,
20.5; HRMS (FAB, M+) m/z 372.1956 (calculated for C23H24N4O,
372.1950); HPLC (a) tR ) 7.70 min, 99.6%, (b) tR ) 7.59 min,
99.4%.
3-(3-Methoxy-5-phenylphenyl)butyne (9a). According to the
general Ohira homologation procedure, aldehyde 8a (0.187 g, 0.78
mmol), Ohira-Bestmann reagent (0.224 g, 1.16 mmol), and K2CO3
(0.215 g, 1.55 mmol) were reacted in methanol (4 mL) for 1.25 h.
Following the general reaction workup, flash chromatography (SiO2
8 g, 2% EtOAc/hexanes) afforded alkyne 9a as a clear oil (0.220
g, 64%): TLC Rf ) 0.44 (5% EtOAc/hexanes); 1H NMR (300 MHz,
CDCl3) δ 7.62 (m, 2H), 7.47 (m, 2H), 7.38 (m, 1H), 7.24 (m, 1H),
7.04 (m, 1H), 6.99 (m, 1H), 3.90 (s, 3H), 3.84 (dq, J ) 7.1, 2.5
Hz, 1H), 2.32 (d, J ) 2.5 Hz, 1H), 1.59 (d, J ) 7.1 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 160.2, 144.6, 143.0, 141.1, 128.7, 127.4,
127.2, 118.4, 111.6, 111.2, 86.9, 70.4, 55.3, 31.8, 24.2; IR (neat,
KBr, cm-1) 3290, 3034, 2976, 1595, 1421, 1213, 1049, 702; HRMS
(FAB, M+) m/z 236.1225 (calculated for C17H16O, 236.1201).
3-(3-Methoxy-5-(2-methylphenyl)phenyl)butyne (9b). According
to the general Ohira homologation procedure, aldehyde 8b (0.175
g, 0.688 mmol), Ohira-Bestmann reagent (0.202 g, 1.05 mmol),
and K2CO3 (0.192 g, 1.39 mmol) were reacted in methanol (4 mL)
for 1.5 h. Following the general reaction workup, flash chroma-
tography (SiO2 8 g, 2% EtOAc/hexanes) afforded the bipheny-
lacetylene 9b as a clear oil (0.172 g, 99%): TLC Rf ) 0.44 (5%
1
EtOAc/hexanes); H NMR (300 MHz, CDCl3) δ 7.38-7.29 (m,
4H), 7.05 (m, 2H), 6.85 (m, 1H), 3.91 (s, 3H), 3.87 (dq, J ) 7.1,
2.5 Hz, 1H), 2.39 (s, 3H), 2.35 (d, J ) 2.5 Hz, 1H), 1.64 (d, J )
7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 159.5, 143.8, 143.4,
141.7, 135.2, 130.3, 129.6, 127.3, 125.7, 120.3, 113.0, 111.1, 86.8,
70.3, 55.2, 31.6, 24.2, 20.4; IR (neat, KBr, cm-1) 3290, 2976, 1593,
1454, 1211, 710; HRMS (FAB, M+) m/z 250.1369 (calculated for
C18H18O, 250.1358).
3-(3-Methoxy-5-(2,6-diisopropylphenyl)phenyl)butyne (9d). Ac-
cording to the general Ohira homologation procedure, aldehyde 8d
(0.272 g, 0.838 mmol), Ohira-Bestmann reagent (0.251 g, 1.31
mmol), and K2CO3 (0.236 g, 1.71 mmol) were reacted in methanol
(3.5 mL) for 1.5 h. Following the general reaction workup, flash
chromatography (SiO2 15 g, 1% EtOAc/hexanes) afforded biphe-
nylacetylene 9d as a clear oil (0.214 g, 80%): TLC Rf ) 0.39 (5%
EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.36 (m, 1H), 7.23
(d, J ) 7.2 Hz, 2H), 6.99 (m, 1H), 6.82 (m, 1H), 6.63 (dd, J ) 2.4,
1.3 Hz, 1H), 3.84 (s, 3H), 3.79 (dq, J ) 7.1, 2.4 Hz, 1H), 2.66 (m,
2H), 2.27 (d, J ) 2.4 Hz, 1H), 1.54 (d, J ) 7.1 Hz, 3H), 1.13 (d,
J ) 6.9 Hz, 6H), 1.11 (d, J ) 6.9 Hz, 3H), 1.10 (d, J ) 6.9 Hz,
3H); 13C NMR (125 MHz, CDCl3) δ 159.4, 146.71, 146.66, 143.8,
142.1, 139.3, 127.8, 120.8, 113.2, 110.8, 86.9, 70.2, 55.2, 31.6,
30.2, 24.34, 24.29, 24.2, 24.1; IR (neat, KBr, cm-1) 3308, 3059,
2961, 2835, 2245, 1587, 1454, 1202, 1055, 739; HRMS (FAB, M+)
m/z 320.2140 (calculated for C23H28O, 320.2140).
General Procedure for the Sonogashira Coupling: 2,4-Diamino-
5-[3-(3-methoxy-5-(2,6-dimethylphenyl)phenyl)but-1-ynyl]-6-meth-
ylpyrimidine (10c). To an oven-dried 8 mL screw cap vial was
added iodopyrimidine 11 (0.074 g, 0.296 mmol), CuI (0.009 g,
0.047 mmol, ∼15%), and Pd(PPh3)2Cl2 (18 mg, 0.026 mmol, ∼8%).
Degassed (argon purge) anhydrous DMF (0.5 mL) was added
followed by alkyne 9c (0.117 g, 0.442 mmol) as a solution in DMF
(0.5 mL). Degassed (argon purge) anhydrous triethylamine was
2,4-Diamino-5-[3-(3-methoxy-5-(2,6-diisopropylphenyl)phenyl)-
but-1-ynyl]-6-methylpyrimidine (10d). According to the general
Sonagashira coupling procedure iodopyrimidine 11 (0.110 g, 0.44
mmol), Pd(PPh3)2Cl2 (0.026 g, 0.037 mmol), CuI (0.011 g, 0.058
mmol), and alkyne 9d (0.210 g, 0.655 mmol) were reacted in DMF/
Et3N (1.25 mL each) at 50 °C for 3.5 h. Following the general
workup procedure flash chromatography (SiO2 15 g, 2% MeOH/
CHCl3) afforded coupled pyrimidine 10d as a pale solid (0.131 g,
67%). An analytical sample was generated by crystallization from
5% EtOAc/hexanes. TLC Rf ) 0.28 (EtOAc); 1H NMR (500 MHz,
CDCl3) δ 7.34 (m, 1H), 7.20 (m, 2H), 6.99 (m, 1H), 6.82 (m, 1H),
6.62 (dd, J ) 2.5, 1.3 Hz, 1H), 5.16 (bs, 2H), 4.94 (bs, 2H), 4.04
(q, J ) 7.2 Hz, 1H), 3.82 (s, 3H), 2.64 (m, 2H), 2.35 (s, 3H), 1.60
(d, J ) 7.2 Hz, 3H), 1.13-1.04 (m, 12H); 13C NMR (125 MHz,
CDCl3) δ 168.5, 164.0, 160.4, 159.4, 146.63, 146.61, 144.4, 142.3,
139.2, 127.9, 122.53, 122.51, 120.7, 113.2, 110.9, 101.7, 91.4, 75.5,

Potent and SelectiVe Cryptosporidium DHFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6847
55.3, 33.0, 30.23, 30.21, 24.7, 24.31, 24.30, 24.22, 24.16, 22.8;
HRMS (FAB, M+) m/z 442.2730 (calculated for C28H34N4O,
442.2733); HPLC (a) tR ) 8.79 min, 98.7%, (b) tR ) 7.42 min,
98.4%.
raphy (SiO2 11 g, 5% EtOAc/hexanes) afforded biphenyl ketone
14c as a very viscous oil that slowly formed a solid (0.325 g, 93%):
TLC Rf ) 0.45 (15% EtOAc/hexanes); 1H NMR (300 MHz, CDCl3)
δ 7.66 (m, 2H), 7.25-7.12 (m, 4H), 3.83 (s, 3H), 2.68 (s, 3H),
2.04 (s, 6H); 13C NMR (75 MHz, CDCl3) δ 197.7, 156.8, 137.5,
137.1, 136.0, 135.1, 130.7, 127.4, 127.1, 121.6, 109.5, 55.5, 26.5,
20.2; IR (neat, KBr, cm-1); HRMS (FAB, MH+) m/z 255.1396
(calculated for C17H19O2, 255.1385).
1-(4-Bromo-3-methoxyphenyl)ethanone (13). To a 0 °C suspen-
sion of benzoic acid 12 (2.00 g, 8.66 mmol) in dry CH2Cl2 (42
mL) and dry benzene (17 mL) with 8 drops (from 18G needle) of
anhydrous DMF was added oxalyl chloride (0.84 mL, 9.62 mmol)
dropwise. Evolution of gas was observed during the addition, and
suspended solids began to go into solution. The mixture was
warmed to ambient temperature and stirred until all of solids had
gone into solution (2.5 h). The volatile components were evaporated,
and the crude white solid was dried under high vacuum for 10 h
and then used directly in the next step. To a -78 °C suspension of
powdered CuI (6.59 g, 34.6 mmol) in anhydrous THF (70 mL)
was added MeLi (3 M in DME, 11.6 mL, 34.8 mmol) dropwise,
quickly. The mixture was stirred for 5 min at -78 °C and then for
15 min at 0 °C. The now clear solution containing yellow solids
was cooled to -78 °C, and a cooled (-78 °C) solution of the crude
acid chloride in THF (30 mL) was added dropwise via cannula.
The mixture was stirred for 40 min at -78 °C. Then 3 mL of
methanol was added and the mixture warmed to ambient temper-
ature. The reaction contents were poured into saturated NH4Cl (200
mL), diluted with ether (50 mL), and stirred vigorously for 1 h.
The organic phase was separated, and the aqueous phase was
extracted with ether (4 × 30 mL). The combined extracts were
washed with saturated NH4Cl, water, and brine (75 mL each), dried
over sodium sulfate, and concentrated. The crude oil was purified
by flash chromatography (60 g SiO2, 5% EtOAc/hexanes) to provide
ketone 13 as an amber oil that solidified very slowly in the freezer
(1.78 g, 90%): TLC Rf ) 0.20 (5% EtOAc/hexanes); mp 31.5-32.0
°C; 1H NMR (300 MHz, CDCl3) δ 7.46 (d, J ) 8.1 Hz, 1H), 7.50
(d, J ) 1.9 Hz, 1H), 7.40 (dd, J ) 8.1, 1.9 Hz, 1H), 3.96 (s, 3H),
2.60 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 196.9, 156.2, 137.5,
133.3, 122.1, 117.9, 110.4, 56.3, 26.5; IR (neat, KBr, cm-1) 3072,
2941, 1682, 1583, 1402, 1283, 1024, 704; HRMS (FAB, MH+)
m/z 228.9848 (calculated for C9H10BrO2, 228.9864).
1-(4-(2,6-Diisopropylphenyl)-3-methoxyphenyl)ethanone (14d).
According to the general Suzuki coupling procedure, ketone 13
(0.251 g, 1.09 mmol) was reacted with boronic acid 6d (0.451 g,
2.19 mmol), Cs2CO3 (1.07 g, 3.28 mmol), and Pd(PPh3)2Cl2 (0.040
g, 0.056 mmol, 5% Pd) in 2 mL of dioxane for 17.5 h. Following
the general reaction workup, flash chromatography (SiO2 13 g, 2%
EtOAc/hexanes) afforded biphenyl ketone 14d as a viscous, clear
oil (0.328 g, 96%): TLC Rf ) 0.19 (5% EtOAc/hexanes); 1H NMR
(500 MHz, CDCl3) δ 7.65 (m, 2H), 7.41 (t, J ) 7.7 Hz, 1H), 7.27
(d, J ) 7.7 Hz, 2H), 7.21 (m, 1H), 3.82 (s, 3H), 2.69 (s, 3H), 2.53
(sep, J ) 6.9 Hz, 2H), 1.12 (m, 12H); 13C NMR (125 MHz, CDCl3)
δ 197.7, 157.5, 146.6, 137.5, 135.0, 134.5, 131.1, 128.2, 122.4,
121.2, 109.1, 55.3, 30.6, 26.5, 24.1, 23.7; IR (neat, KBr, cm-1
)
3063, 2962, 1684, 1603, 1081, 1034, 762; HRMS (FAB, M+) m/z
310.1925 (calculated for C21H26O2, 310.1933).
2-(3-Methoxy-4-phenylphenyl)propanal (15a). According to the
general Wittig reaction procedure, the reaction of phosphonium
chloride (1.20 g, 3.5 mmol), NaOtBu (0.433 g, 4.5 mmol), and
biphenyl ketone 14a (0.457 g, 2.0 mmol) gave, following filtration
(SiO2 21 g, 1% EtOAc/hexanes), 0.504 g of a mixture (E/Z ≈ 1:1)
of enol ethers that were immediately hydrolyzed in the subsequent
step: TLC Rf ) 0.43 (5% EtOAc/hexanes).
According to the general hydrolysis procedure, the crude enol
ether (0.504 g) was reacted with concentrated HCl (0.5 mL) in THF
(10 mL) for 1 h. Following the general reaction workup, flash
chromatography (SiO2 22 g, 2-5% EtOAc/hexanes) afforded
aldehyde 15a as a clear oil (0.437 g, 90% from 14a): TLC Rf )
1
0.19 (5% EtOAc/hexanes); H NMR (300 MHz, CDCl3) δ 9.76
(d, J ) 1.4 Hz, 1H), 7.56 (m, 2H), 7.45 (m, 2H), 7.36 (m, 2H),
6.93 (dd, J ) 7.7, 1.7 Hz, 1H), 6.83 (d, J ) 1.7 Hz, 1H), 3.84 (s,
1-(3-Methoxy-4-phenyl)phenylethanone (14a). According to the
general Suzuki coupling procedure, ketone 13 (0.470 g, 2.05 mmol)
was reacted with phenylboronic acid 6a (0.502 g, 4.12 mmol),
Cs2CO3 (2.01 g, 6.17 mmol), and Pd(PPh3)2Cl2 (0.081 g, 0.12 mmol,
6% Pd) in 4 mL of dioxane for 22 h. Following the general reaction
workup, flash chromatography (SiO2 21 g, 2-5% EtOAc/hexanes)
afforded biphenyl ketone 14a as a white solid (0.457 g, 98%): TLC
3H), 3.70 (dq, J ) 7.0, 1.4 Hz, 1H), 1.53 (d, J ) 7.0 Hz, 3H); 13
C
NMR (75 MHz, CDCl3) δ 200.8, 156.9, 138.2, 137.9, 131.3, 129.9,
129.4, 128.0, 127.0 120.6 111.1, 55.5, 53.0, 14.5; IR (neat, KBr,
cm-1) 3028, 2974, 2719, 1728, 1485, 1273, 1034; HRMS (FAB,
M+) m/z 240.1166 (calculated for C16H16O2, 240.1150).
2-(3-Methoxy-4-(2-methylphenyl)phenyl)propanal (15b). Ac-
cording to the general Wittig reaction procedure, the reaction of
phosphonium chloride (1.23 g, 3.6 mmol), NaOtBu (0.440 g, 4.6
mmol), and biphenyl ketone 14b (0.490 g, 2.04 mmol) gave,
following filtration (SiO2 28 g, 1% EtOAc/hexanes), 0.520 g of a
mixture (E/Z ≈ 60:40) of enol ethers that were immediately
hydrolyzed in the subsequent step: TLC Rf ) 0.38 (5% EtOAc/
hexanes).
1
Rf ) 0.43 (15% EtOAc/hexanes); H NMR (300 MHz, CDCl3) δ
7.65-7.56 (m, 4H), 7.49-7.35 (m, 4H), 3.88 (s, 3H), 2.65 (s, 3H);
13C NMR (75 MHz, CDCl3) δ 197.5, 156.5, 137.23, 137.17, 135.5,
130.6, 129.3, 128.0, 127.5 121.6, 109.8, 55.5, 26.5; IR (neat, KBr,
cm-1) 3074, 3003, 1675, 1599, 1398, 1223, 1033, 873; HRMS
(FAB, MH+) m/z 227.1073 (calculated for C15H15O2, 227.1072).
1-(3-Methoxy-4-(2-methylphenyl)phenyl)ethanone (14b). Ac-
cording to the general Suzuki coupling procedure, ketone 13 (0.491
g, 2.14 mmol) was reacted with 2-methylphenylboronic acid 6b
(0.582 g, 4.28 mmol), Cs2CO3 (2.09 g, 6.42 mmol), and
Pd(PPh3)2Cl2 (0.080 g, 0.11 mmol, 5% Pd) in 4.3 mL of dioxane
for 21 h. Following the general reaction workup, flash chromatog-
raphy (SiO2 16 g, 2-5% EtOAc/hexanes) afforded biphenyl ketone
14b as a clear oil (0.490 g, 95%): TLC Rf ) 0.44 (15% EtOAc/
hexanes); 1H NMR (300 MHz, CDCl3) δ 7.67 (m, 2H), 7.36-7.26
According to the general hydrolysis procedure, the crude enol
ether (0.520 g) was reacted with concentrated HCl (0.5 mL) in THF
(10 mL) for 1 h. Following the general reaction workup, flash
chromatography (SiO2 23 g, 2-5% EtOAc/hexanes) afforded
aldehyde 15b as a clear oil (0.425 g, 82% from 14b): TLC Rf )
1
0.19 (5% EtOAc/hexanes); H NMR (300 MHz, CDCl3) δ 9.77
(d, J ) 1.4 Hz, 1H), 7.30-7.15 (m, 5H), 6.89 (dd, J ) 7.6, 1.6
Hz, 1H), 6.79 (d, J ) 1.6 Hz, 1H), 3.78 (s, 3H), 3.71 (dd, J ) 7.0,
1.4 Hz, 1H), 2.16 (s, 3H), 1.52 (d, J ) 7.0 Hz, 3H); 13C NMR (75
MHz, CDCl3) δ 201.0, 157.1, 138.1, 138.0, 136.8, 131.5, 130.1,
130.0, 129.6, 127.4, 125.4, 120.2, 110.6, 55.4, 53.1, 19.9, 14.5; IR
(neat, KBr, cm-1) 3016, 2974, 2719, 1728, 1610, 1456, 1269, 1034;
HRMS (FAB, M+) m/z 254.1326 (calculated for C17H18O2,
254.1307).
(m, 4H), 7.23 (m, 1H), 3.87 (s, 3H), 2.70 (s, 3H), 2.20 (s, 3H); 13
C
NMR (75 MHz, CDCl3) δ 197.6, 156.8, 137.4, 136.3, 136.1, 130.9,
129.6, 129.4, 127.6, 125.4, 121.3, 109.2, 55.4, 26.4, 19.7 (one
aromatic carbon unresolved); IR (neat, KBr, cm-1) 3063, 2961,
1680, 1288, 1218, 1034; HRMS (FAB, M+) m/z 240.1148
(calculated for C16H16O2, 240.1150).
1-(3-Methoxy-4-(2,6-dimethylphenyl)phenyl)ethanone (14c). Ac-
cording to the general Suzuki coupling procedure, ketone 13 (0.312
g, 1.36 mmol) was reacted with 2,6-dimethylphenylboronic acid
6c (0.410 g, 2.73 mmol), Cs2CO3 (1.33 g, 4.08 mmol), and
Pd(PPh3)2Cl2 (0.080 g, 0.11 mmol, 5% Pd) in 2.8 mL of dioxane
for 23 h. Following the general reaction workup, flash chromatog-
2-(3-Methoxy-4-(2,6-dimethylphenyl)phenyl)propanal (15c). Ac-
cording to the general Wittig reaction procedure, the reaction of
phosphonium chloride (0.78 g, 2.3 mmol), NaOtBu (0.287 g, 3.0
mmol), and biphenyl ketone 14c (0.304 g, 1.2 mmol) gave,
following filtration (SiO2 12 g, 2% EtOAc/hexanes), 0.356 g of a

6848 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bolstad et al.
mixture (E/Z ≈ 65:35) of enol ethers that were immediately
hydrolyzed in the subsequent step: TLC Rf ) 0.37 (5% EtOAc/
hexanes).
3-(3-Methoxy-4-(2,6-dimethylphenyl)phenyl)butyne (16c). Ac-
cording to the general Ohira homologation procedure, aldehyde 15c
(0.284 g, 1.06 mmol), Ohira-Bestmann reagent (0.307 g, 1.60
mmol), and K2CO3 (0.293 g, 2.12 mmol) were reacted in methanol
(15 mL, too much solvent used in this example) for 2 h. Following
the general reaction workup, flash chromatography (SiO2 13 g, 2%
EtOAc/hexanes) afforded biphenylacetylene 16c as a white solid
(0.210 g, 75%): TLC Rf ) 0.43 (5% EtOAc/hexanes); mp
46.3-47.6 °C; 1H NMR (300 MHz, CDCl3) δ; 13C NMR (75 MHz,
CDCl3) δ 156.5, 142.9, 137.9, 136.7, 130.6, 127.8, 126.99, 126.95,
According to the general hydrolysis procedure, the crude enol
ether (0.356 g) was reacted with concentrated HCl (0.5 mL) in THF
(7 mL) for 1.5 h. Following the general reaction workup, flash
chromatography (SiO2 15 g, 2-5% EtOAc/hexanes) afforded
aldehyde 15c as a clear oil (0.294 g, 92% from 14c): TLC Rf )
1
0.19 (5% EtOAc/hexanes); H NMR (500 MHz, CDCl3) δ 9.80
(d, J ) 1.5 Hz, 1H), 7.22 (m, 1H), 7.16 (m, 2H), 7.09 (d, J ) 7.6
Hz, 1H), 6.94 (dd, J ) 7.6, 1.5 Hz, 1H), 6.84 (d, J ) 1.5 Hz, 1H),
3.78 (s, 3H), 3.74 (dq, J ) 7.0, 1.5 Hz, 1H), 2.07 (s, 6H), 1.56 (d,
J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 201.0, 156.8,
137.7, 137.5, 136.5, 131.1, 128.6, 127.1, 126.9, 120.3, 110.6, 55.3,
53.0, 20.4, 14.4; IR (neat, KBr, cm-1) 2974, 2719, 1731, 1456,
1269, 1040, 773; HRMS (FAB, M+) m/z 268.1475 (calculated for
C18H20O2, 268.1463).
118.9, 109.5, 87.1, 70.3, 55.3, 31.7, 24.3, 20.5; IR (neat, KBr, cm-1
)
3290, 3016, 2976, 2245, 1610, 1464, 1244, 1039, 733; HRMS
(FAB, M+) m/z 264.1515 (calculated for C19H20O, 264.1514).
3-(3-Methoxy-4-(2,6-diisopropylphenyl)phenyl)butyne (16d). Ac-
cording to the general Ohira homologation procedure, aldehyde 15d
(0.270 g, 0.832 mmol), Ohira-Bestmann reagent (0.248 g, 1.29
mmol), and K2CO3 (0.231 g, 1.67 mmol) were reacted in methanol
(3.5 mL) for 1.5 h. Following the general reaction workup, flash
chromatography (SiO2 15 g, 1% EtOAc/hexanes) afforded biphe-
nylacetylene 16d as a clear oil (0.181 g, 68%): TLC Rf ) 0.39
2-(3-Methoxy-4-(2,6-diisopropylphenyl)phenyl)propanal (15d).
According to the general Wittig reaction procedure, the reaction
of phosphonium chloride (0.622 g, 1.8 mmol), NaOtBu (0.225 g,
2.3 mmol), and biphenyl ketone 14d (0.326 g, 1.05 mmol) gave,
following filtration (SiO2 20 g, 1% EtOAc/hexanes), 0.364 g of
crude material that was immediately hydrolyzed in the subsequent
step: TLC Rf ) 0.44 (5% EtOAc/hexanes).
1
(5% EtOAc/hexanes); H NMR (300 MHz, CDCl3) δ 7.38 (dd, J
) 8.3, 7.2 Hz, 1H), 7.24 (d, J ) 7.5 Hz, 2H), 7.06-6.99 (m, 3H),
3.89 (dq, J ) 7.2, 2.5 Hz, 1H), 3.76 (s, 3H), 2.60 (m, 2H), 2.53 (d,
J ) 2.5 Hz, 1H), 1.64 (d, J ) 7.2 Hz, 3H), 1.14-1.07 (m, 12H);
13C NMR (75 MHz, CDCl3) δ 157.2, 147.3, 142.8, 135.5, 131.1,
127.8, 127.4, 122.4, 118.5, 109.1, 87.2, 70.3, 55.1, 31.7, 30.4, 24.3,
23.8; IR (neat, KBr, cm-1); HRMS (FAB, M+) m/z 320.2149
(calculated for C23H28O, 320.2140).
According to the general hydrolysis procedure, the crude enol
ether (0.334 g) was reacted with concentrated HCl (0.5 mL) in THF
(8 mL) for 2 h. Following the general reaction workup, flash
chromatography (SiO2 19 g, 2-5% EtOAc/hexanes) afforded
aldehyde 15d as a viscous oil (0.272 g, 76% from 14d): TLC Rf )
2,4-Diamino-5-[3-(3-methoxy-4-phenylphenyl)but-1-ynyl]-6-me-
thylpyrimidine (17a). According to the general Sonagashira cou-
pling procedure, iodopyrimidine 11 (0.084 g, 0.336 mmol),
Pd(PPh3)2Cl2 (0.023 g, 0.033 mmol), CuI (0.012 g, 0.063 mmol),
and alkyne 16a (0.117 g, 0.495 mmol) were reacted in DMF/Et3N
(1 mL each) at 50 °C for 3 h. Following the general workup
procedure flash chromatography (SiO2 10 g, 2% MeOH/CHCl3)
afforded coupled pyrimidine 17a as a pale solid (0.103 g, 85%).
An analytical sample was generated by crystallization from ether/
hexanes. TLC Rf ) 0.23 (EtOAc); mp 163.2-165.2 °C; 1H NMR
(500 MHz, CDCl3) δ 7.53 (m, 2H), 7.40 (m, 2H), 7.34-7.29 (m,
2H), 7.10-7.07 (m, 2H), 5.17 (bs, 2H), 4.90 (bs, 2H), 4.09 (q, J )
7.1 Hz, 1H), 3.83 (s, 3H), 2.41 (s, 3H), 1.65 (d, J ) 7.1 Hz, 3H);
13C NMR (125 MHz, CDCl3) δ 168.5, 164.1, 160.5, 156.6, 144.1,
138.2, 131.0, 129.5, 129.3, 128.0, 126.9, 119.1, 109.9, 101.8, 91.4,
75.7, 55.6, 33.0, 24.7, 22.9; HRMS (FAB, M+) m/z 358.1815
(calculated for C22H22N4O, 358.1794); HPLC (a) tR ) 5.62 min,
97.7%, (b) tR ) 6.55 min, 96.9%.
1
0.24 (5% EtOAc/hexanes); H NMR (500 MHz, CDCl3) δ 9.80
(d, J ) 1.6 Hz, 1H), 7.38 (m, 1H), 7.24 (d, J ) 7.4 Hz, 2H), 7.08
(d, J ) 7.6 Hz, 1H), 6.90 (dd, J ) 7.6, 1.5 Hz, 1H), 6.80 (d, J )
1.5 Hz, 1H), 3.74 (s, 3H), 3.73 (dq, J ) 7.1, 1.6 Hz, 1H), 2.56
(sep, J ) 6.9 Hz, 2H), 1.56 (d, J ) 7.1 Hz, 3H), 1.12 (d, J ) 6.9
Hz, 6H), 1.10 (d, J ) 6.9 Hz, 6H); 13C NMR (125 MHz, CDCl3)
δ 201.1, 157.6, 147.1 137.7, 135.1, 131.6, 128.4, 127.9, 122.4,
120.0, 110.2, 55.1, 53.0, 30.5, 24.2, 23.7, 14.4; IR (neat, KBr, cm-1
)
3061, 2959, 2868, 2719, 1725, 1568, 1269, 1038, 764; HRMS
(FAB, M+) m/z 324.2103 (calculated for C22H28O2, 324.2089).
3-(3-Methoxy-4-phenylphenyl)butyne (16a). According to the
general Ohira homologation procedure, aldehyde 15a (0.437 g, 1.82
mmol), Ohira-Bestmann reagent (0.523 g, 2.72 mmol), and K2CO3
(0.505 g, 3.05 mmol) were reacted in methanol (25 mL, too much
solvent used in this example) for 1.5 h. Following the general
reaction workup, flash chromatography (SiO2 20 g, 2% EtOAc/
hexanes) afforded biphenylacetylene 16a as a clear oil (0.220 g,
51%, low yield likely because of high dilution): TLC Rf ) 0.39
(5% EtOAc/hexanes); 1H NMR (300 MHz, CDCl3) δ 7.58 (m, 2H),
7.46 (m, 2H), 7.36 (m, 2H), 7.10 (m, 2H), 3.88 (s, 3H), 3.86 (dq,
J ) 7.1, 2.5 Hz, 1H), 2.35 (d, J ) 2.5 Hz, 1H), 1.62 (d, J ) 7.1
Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 156.5, 143.3, 138.3, 130.9,
129.5, 129.2, 127.9, 126.8, 119.1, 109.9, 87.0, 70.3, 55.5, 31.6,
24.1; IR (neat, KBr, cm-1) 3288, 3028, 2975, 1614, 1409, 1274,
1040, 700; HRMS (FAB, M+) m/z 236.1219 (calculated for
C17H16O, 236.1201).
2,4-Diamino-5-[3-(3-methoxy-4-(2-methylphenyl)phenyl)but-1-
ynyl]-6-methylpyrimidine (17b). According to the general Son-
agashira coupling procedure, iodopyrimidine 11 (0.083 g, 0.331
mmol), Pd(PPh3)2Cl2 (0.023 g, 0.033 mmol), CuI (0.012 g, 0.063
mmol), and alkyne 16b (0.125 g, 0.498 mmol) were reacted in
DMF/Et3N (1 mL each) at 50 °C for 2.5 h. Following the general
workup procedure flash chromatography (SiO2 10 g, 2% MeOH/
CHCl3) afforded coupled pyrimidine 17b as a pale solid (0.103 g,
97%). An analytical sample was generated by crystallization from
ether/hexanes. TLC Rf ) 0.25 (EtOAc); mp 177.2-180.1 °C, with
3-(3-Methoxy-4-(2-methylphenyl)phenyl)butyne (16b). Accord-
ing to the general Ohira homologation procedure, aldehyde 15b
(0.425 g, 1.67 mmol), Ohira-Bestmann reagent (0.483 g, 2.51
mmol), and K2CO3 (0.465 g, 3.36 mmol) were reacted in methanol
(24 mL, too much solvent used in this example) for 1.5 h. Following
the general reaction workup, flash chromatography (SiO2 22 g, 2%
EtOAc/hexanes) afforded the biphenylacetylene 16b as a clear oil
1
dec; H NMR (500 MHz, CDCl3) δ 7.26-7.20 (m, 3H), 7.18 (m,
1H), 7.12 (m, 1H), 7.07-7.04 (m, 2H), 5.15 (bs, 2H), 4.86 (bs,
2H), 4.10 (q, J ) 7.1 Hz, 1H), 3.77 (s, 3H), 2.41 (s, 3H), 2.15 (s,
3H), 1.66 (d, J ) 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
168.5, 164.1, 160.4, 156.8, 143.9, 138.3, 136.9, 131.2, 130.1, 129.6,
129.4, 127.3, 125.4, 118.7, 109.3, 101.9, 91.5, 75.7, 55.4, 33.1,
24.8, 22.9, 20.0; HRMS (FAB, M+) m/z 372.1933 (calculated for
C23H24N4O, 372.1950); HPLC (a) tR ) 6.33 min, 98.5%, (b) tR )
7.16 min, 97.3%.
1
(0.290 g, 70%): TLC Rf ) 0.39 (5% EtOAc/hexanes); H NMR
(300 MHz, CDCl3) δ 7.36-7.24 (m, 4H), 7.20 (d, J ) 8.0 Hz,
1H), 7.11 (m, 2H), 3.92 (dq, J ) 7.1, 2.5 Hz, 1H), 3.86 (s, 3H),
2.39 (d, J ) 2.5 Hz, 1H), 2.24 (s, 3H), 1.67 (d, J ) 7.1 Hz, 3H);
13C NMR (75 MHz, CDCl3) δ 156.6, 143.2, 138.4, 136.8, 131.0,
130.1, 129.5, 129.3, 127.2, 125.4, 118.7, 109.3, 87.1, 70.3, 55.3,
31.7, 24.2, 19.9; IR (neat, KBr, cm-1) 3288, 3016, 2975, 2247,
1612, 1412, 1238, 1039, 729; HRMS (FAB, M+) m/z 250.1346
(calculated for C18H18O, 250.1358).
2,4-Diamino-5-[3-(3-methoxy-4-(2,6-dimethylphenyl)phenyl)but-
1-ynyl]-6-methylpyrimidine (17c). According to the general Son-
agashira coupling procedure, iodopyrimidine 11 (0.080 g, 0.319
mmol), Pd(PPh3)2Cl2 (0.022 g, 0.031 mmol), CuI (0.011 g, 0.058
mmol), and alkyne 16c (0.125 g, 0.473 mmol) were reacted in DMF/
Et3N (1 mL each) at 50 °C for 3.5 h. Following the general workup

Potent and SelectiVe Cryptosporidium DHFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6849
procedure flash chromatography (SiO2 9 g, 2% MeOH/CHCl3)
afforded coupled pyrimidine 17c as a pale solid (0.121 g, 98%).
An analytical sample was generated by crystallization from ether/
hexanes. TLC Rf ) 0.26 (EtOAc); 1H NMR (500 MHz, CDCl3) δ
7.16 (dd, J ) 8.4, 6.5 Hz, 1H), 7.10 (bd, J ) 7.5 Hz, 2H), 7.06 (m,
2H), 7.00 (d, J ) 8.1 Hz, 1H), 5.20 (bs, 2H), 4.94 (bs, 2H), 4.11
(q, J ) 7.1 Hz, 1H), 3.74 (s, 3H), 2.41 (s, 3H), 2.02 (s, 6H), 1.67
(d, J ) 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 168.5, 164.2,
160.4, 156.7, 143.6, 137.9, 136.70, 136.67, 130.8, 128.0, 127.1,
127.0, 118.9, 109.5, 102.0, 91.5, 75.7. 55.5, 33.0, 24.7, 20.5; HRMS
(FAB, M+) m/z 386.2092 (calculated for C24H26N4O, 386.2107);
HPLC (a) tR ) 4.42 min, 98.8%, (b) tR ) 4.70 min, 98.7%.
2,4-Diamino-5-[3-(3-methoxy-4-(2,6-diisopropylphenyl)phenyl)-
but-1-ynyl]-6-methylpyrimidine (17d). According to the general
Sonagashira coupling procedure, iodopyrimidine 11 (0.086 g, 0.34
mmol), Pd(PPh3)2Cl2 (0.023 g, 0.033 mmol), CuI (0.008 g, 0.042
mmol), and alkyne 16d (0.166 g, 0.518 mmol) were reacted in
DMF/Et3N (1 mL each) at 50 °C for 3 h. Following the general
workup procedure flash chromatography (SiO2 15 g, 2% MeOH/
CHCl3) afforded coupled pyrimidine 17d as a pale solid (0.131 g,
53%). An analytical sample was generated by crystallization from
cyclohexane. TLC Rf ) 0.29 (EtOAc); 1H NMR (500 MHz, CDCl3)
δ 7.35 (t, J ) 7.7 Hz, 1H), 7.21 (d, J ) 7.7 Hz, 2H), 7.04 (m, 2H),
7.00 (d, J ) 8.0 Hz, 1H), 5.13 (bs, 2H), 4.81 (bs, 2H), 4.12 (q, J
) 7.1 Hz, 1H), 3.72 (s, 3H), 2.56 (sep, J ) 6.9 Hz, 2H), 2.41 (s,
3H), 1.69 (d, J ) 7.1 Hz, 3H), 1.10-1.04 (m, 12H); 13C NMR
(125 MHz, CDCl3) δ 168.6, 164.1, 160.4, 157.3, 147.3, 147.2,
143.5, 135.4, 131.3, 127.8, 127.5, 122.4, 118.4, 109.0, 102.1, 91.5,
75.8, 55.1, 33.0, 30.5, 24.6, 24.3, 23.8, 22.8; HRMS (FAB, M+)
m/z 442.2709 (calculated for C28H34N4O, 442.2733); HPLC (a) tR
) 7.58 min, 99.0%, (b) tR ) 7.06 min, 100%.
3-Bromo-5-methoxybenzaldehyde. To a stirring -78 °C solution
of n-BuLi (in hexanes, 1.7 M, 11.25 mL, 19.1 mmol) in dry ether
(24 mL) under argon was added a solution of dibromoanisole 4
(5.08 g, 19.1 mmol) dissolved in ether (12 mL) dropwise via
cannula. A white precipitate formed, and the mixture was stirred
at -78 °C for 20 min. Dimethylformamide (1.6 mL, 20.6 mmol)
was added dropwise generating a homogeneous solution that was
stirred an additional 20 min at -78 °C. The reaction was quenched
by the addition of 10% aqueous HCl (30 mL), and the mixture
was allowed to warm to ambient temperature. The organic phase
was separated, and the aqueous layer was extracted once with ether
(40 mL). The combined extracts were washed with saturated
NaHCO3 and brine (30 mL each), dried over sodium sulfate, and
concentrated to give 3-bromo-5-methoxybenzaldehyde as an amber
solid with sufficient purity for subsequent transformations (4.04 g,
98%). Flash chromatography of 0.5 g of material (SiO2 20 g, 5%
EtOAc/hexanes) provided an analytical sample: TLC Rf ) 0.19 (5%
EtOAc/hexanes); mp 39.7 - 40.4 °C; 1H NMR (300 MHz, CDCl3)
δ 9.88 (s, 1H), 7.54 (m, 1H), 7.29 (m, 1H), 7.28 (m, 1H), 3.85 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 190.4, 160.7, 138.5, 125.5,
123.6, 123.4, 112.1, 55.7; IR (neat, KBr, cm-1).
3-Methoxy-5-phenylbenzaldehyde (18). According to the general
Suzuki coupling procedure, 3-bromo-5-methoxybenzaldehyde (4.04
g, 18.8 mmol) was reacted with phenylboronic acid (4.58 g, 37.5
mmol), Cs2CO3 (18.0 g, 55.2 mmol), and Pd(PPh3)2Cl2 (0.263 g,
0.374 mmol, 2% Pd) in 38 mL of dioxane for 3 h. Following the
general reaction workup, flash chromatography (SiO2 140 g, 2 -
5% EtOAc/hexanes) afforded aldehyde 18 as a viscous oil (3.29 g,
82%): TLC Rf ) 0.42 (15% EtOAc/hexanes); 1H NMR (500 MHz,
CDCl3) δ 10.05 (s, 1H), 7.69 (m, 1H), 7.63 (m, 1H), 7.61 (m, 1H),
7.48 (m, 2H), 7.42-7.38 (m, 3H), 3.93 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 192.1, 160.6, 143.5, 139.6, 138.2, 129.0, 128.1,
127.1, 122.4, 120.1, 110.9, 55.7; IR (neat, KBr, cm-1) 3063, 2939,
2734, 1699, 1593, 1332, 1217, 1049; HRMS (FAB, M+) m/z
212.0826 (calculated for C14H12O2, 212.0837).
1:1) of enol ethers that were immediately hydrolyzed in the
subsequent step: TLC Rf ) 0.31 (5% EtOAc/hexanes).
The enol ether mixture previously obtained was dissolved in THF
(75 mL), and 2 N HCl was added (1 mL). The flask was fitted
with a reflux condenser and the mixture heated to reflux. The
reaction was monitored by TLC until the disappearance of starting
material was observed (∼2 h). Extended reaction times increase
the incidence of aldol products, so this should be avoided. The
mixture was cooled, diluted with ethyl acetate (100 mL), and
washed with NaHCO3 (2 × 30 mL), water (30 mL), and brine (30
mL). The organic phase was dried over anhydrous MgSO4, filtered,
and concentrated to provide the crude aldehyde (3.5 g) that was
immediately used in the following oxidation. Purification of this
aldehyde prior to oxidation resulted in a decrease in overall yield
of carboxylic acid from arylaldehyde, presumably because of
instability of the alkylaldehyde to chromatographic conditions.
To the crude aldehyde (3.5 g) in acetone (130 mL) at 0 °C was
added freshly prepared Jones’ reagent dropwise until the solution
became a dark-red color. After the mixture was stirred for 5 min,
isopropyl alcohol was added until a dark-green mixture formed.
The mixture was concentrated on a rotovap and dried under a high
vacuum for a short time (∼30 min). The dark-green crusty residue
was suspended in NaOH (1 N, 200 mL) and transferred to a
separatory funnel where it was washed with two portions of ether
(75 mL each). The aqueous phase was acidified with concentrated
HCl to pH ∼1 and extracted with CH2Cl2 (100 mL × 3). The
combined extracts were dried over anhydrous MgSO4 and concen-
trated to provide carboxylic acid 19 as a white solid (2.63 g, 72%
three steps): TLC Rf ) 0.08 (50% EtOAc/hexanes); mp 140.4 -
1
141.6 °C; H NMR (500 MHz, CDCl3) δ 10.7 (bs, 1H), 7.57 (m,
2H), 7.43 (m, 2H), 7.35 (m, 1H), 7.10 (m, 1H), 7.05 (m, 1H), 6.84
(m, 1H), 3.86 (s, 3H), 3.69 (s, 2H); 13C NMR (75 MHz, CDCl3) δ
177.4, 160.1, 143.1, 140.7, 134.9, 128.7, 127.5, 127.2, 120.9, 113.8,
112.0, 55.4, 41.2; HRMS (FAB M+) m/z 242.0943 (calculated for
C15H14O3, 242.0943).
(S)-4-Isopropyl-3-(2-(3-methoxy-5-phenylphenyl)acetyl)oxazoli-
din-2-one (20). To a solution of carboxylic acid 19 (1.24 g, 5.11
mmol) in dry THF (25 mL) was added Et3N (0.78 mL, 5.61 mmol).
The solution was cooled to -78 °C, and pivolyl chloride (0.69 mL,
5.6 mmol) was added dropwise. A precipitate formed, and the
mixture was stirred an additional 15 min at -78 °C and then at 0
°C for 1 h. In a separate flame-dried round-bottomed flask was
added the Evans (S)-isopropyloxazoldinone (0.552 g, 4.27 mmol).
Dry THF (20 mL) was added, and the solution was cooled to -78
°C under argon. A solution of n-BuLi (2.45 M in hexanes, 2.1 mL,
5.11 mmol) was added dropwise, producing a very thick mixture
that was difficult to stir. The mixture was maintained at -78 °C
for 15 min and then warmed to 0 °C for 40 min. The mixture
containing the mixed anhydride was cooled again to -78 °C, and
then the 0 °C solution of lithiated oxazolidinone was added dropwise
via cannula. The resultant mixture was stirred at -78 °C for 30
min and then at 0 °C for 2 h. The reaction was quenched with
water (20 mL) and extracted with EtOAc (4 × 15 mL). The
combined extracts were washed with brine, dried over sodium
sulfate, and concentrated to give the crude product that was purified
by flash chromatography (SiO2 53 g, 10-25% EtOAc/hexanes) to
provide 20 as a very viscous amber oil (0.829 g, 60%): TLC Rf )
0.39 (1:2 EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.59
(m, 2H), 7.43 (m, 2H), 7.34 (m, 1H), 7.16 (m, 1H), 7.05 (m, 1H),
6.89 (m, 1H), 4.45 (m, 1H), 4.40 (AB d, J ) 15.2 Hz, 1H), 4.29
(AB d, J ) 15.2 Hz, 1H), 4.25 (m, 1H), 4.19 (dd, J ) 9.2, 3.1 Hz,
1H), 3.85 (s, 3H), 2.37 (m, 1H), 0.89 (d, J ) 7.1 Hz, 3H), 0.82 (d,
J ) 6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 170.8, 159.9,
153.9, 142.7, 140.7, 135.4, 128.6, 127.4, 127.1, 121.0, 113.9, 111.7,
63.2, 58.5, 55.2, 41.5, 28.2, 17.8, 14.5; IR (neat, KBr, cm-1) 2964,
1767, 1697, 1593, 1218, 1061, 766; HRMS (FAB, M+) m/z
353.1626 (calculated for C21H23NO4, 353.1627).
(3-Methoxy-5-phenyl)phenylacetic Acid (19). According to the
general Wittig reaction procedure, the reaction of phosphonium
chloride (9.08 g, 26.5 mmol), NaOtBu (3.26 g, 33.9 mmol), and
aldehyde 18 (3.20 g, 15.1 mmol) gave, following chromatography
(SiO2 100 g, 1-5% EtOAc/hexanes), 3.88 g of a mixture (E/Z ≈
(R)-4-Isopropyl-3-(2-(3-methoxy-5-phenylphenyl)acetyl)oxazo-
lidin-2-one (21). To a solution of carboxylic acid 19 (1.0 g, 4.13
mmol) in dry THF (25 mL) was added Et3N (0.92 mL, 6.61 mmol).
The solution was cooled to -78 °C, and pivolyl chloride (0.56 mL,

6850 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bolstad et al.
4.54 mmol) was added dropwise. A precipitate formed, and the
mixture was stirred an additional 15 min at -78 °C and then at 0
°C for 1 h. In a separate flame-dried round-bottomed flask was
added the Evans (R)-isopropyloxazoldinone (0.490 g, 3.79 mmol).
Dry THF (20 mL) was added, and the solution was cooled to -78
°C under argon. A solution of n-BuLi (2.3 M in hexanes, 1.81 mL,
4.13 mmol) was added dropwise, producing a very thick mixture
that was difficult to stir. The mixture was maintained at -78 °C
for 15 min and then warmed to 0 °C for 40 min. The mixture
containing mixed anhydride was cooled again to -78 °C, and then
the 0 °C solution of lithiated oxazolidinone was added dropwise
via cannula. The resultant mixture was stirred at -78 °C for 30
min and then at 0 °C for 2 h. The reaction was quenched with
water (20 mL) and extracted with EtOAc (4 × 15 mL). The
combined extracts were washed with brine dried over sodium sulfate
and concentrated to give the crude product that was purified by
flash chromatography (SiO2 40 g, 15% EtOAc/hexanes) to provide
21 as a very viscous amber oil (0.632 g, 47%): TLC Rf ) 0.39
(1:2 EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.58 (m, 2H),
7.43 (m, 2H), 7.34 (m, 1H), 7.15 (m, 1H), 7.04 (m, 1H), 6.88 (m,
1H), 4.45 (m, 1H), 4.39 (AB d, J ) 15.2 Hz, 1H), 4.29 (AB d, J
) 15.2 Hz, 1H), 4.26 (m, 1H), 4.20 (dd, J ) 9.2, 3.1 Hz, 1H),
3.85 (s, 3H), 2.38 (m, 1H), 0.89 (d, J ) 7.0 Hz, 3H), 0.82 (d, J )
6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 170.9, 160.0, 153.9,
142.7, 140.8, 135.4, 128.6, 127.4, 127.2, 121.1, 114.0, 111.9, 63.2,
58.5, 55.3, 41.6, 28.2, 17.9, 14.5; IR (neat, KBr, cm-1) 2964, 1768,
1695, 1593, 1215, 1059, 764; HRMS (FAB, M+) m/z 353.1628
(calculated for C21H23NO4, 353.1627).
(S)-4-Isopropyl-3-((S)-2-(3-methoxy-5-phenylphenyl)propanoyl)-
oxazolidin-2-one (22). To a -78 °C solution of biphenyloxazoli-
dinone 20 (0.829 g, 2.34 mmol) in dry THF (23 mL) under argon
was added a solution of LHMDS (1 M in THF, 3 mL, 3 mmol).
The yellow-orange solution was stirred for 1 h at -78 °C. Then
methyliodide (0.59 mL, 9.45 mmol) was added dropwise. Following
5 min the mixture was warmed to 0 °C and stirred an additional
1 h. Saturated ammonium chloride (20 mL) was added and the
mixture warmed to ambient temperature. The mixture was diluted
with ethyl acetate (20 mL), and the organic phase was separated.
The aqueous phase was extracted with additional ethyl acetate (2
× 15 mL), and the combined organic phases were washed with
brine (30 mL), dried over anhydrous Na2SO4, and concentrated to
provide the crude oil that was purified by flash chromatography
(SiO2 43 g, 10% EtOAc/hexanes) to afford alkylated product 22
as a clear viscous oil and a single diastereomer (0.657 g, 76%):
TLC Rf ) 0.63 (1:2 EtOAc/hexanes); 1H NMR (500 MHz, CDCl3)
δ 7.58 (m, 2H), 7.42 (m, 2H), 7.34 (m, 1H), 7.19 (m, 1H), 7.02
(m, 1H), 6.93 (m, 1H), 5.22 (q, J ) 7.0 Hz, 1H), 4.38 (m, 1H),
4.18 - 4.09 (m, 2H), 3.86 (s, 3H), 2.46 (m, 1H), 1.57 (d, J ) 7.0
Hz, 3H), 0.93 (d, J ) 7.1 Hz, 3H), 0.92 (d, J ) 6.9 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 174.4, 160.0, 153.6, 142.8, 142.1, 140.9,
128.7, 127.4, 127.2, 119.6, 112.7, 111.8, 63.1, 59.0, 55.3, 43.0,
28.5, 19.7, 18.0, 14.7; IR (neat, KBr, cm-1) 3034, 2966, 1776,
1697, 1593, 1204, 1055, 763; HRMS (FAB, M+) m/z 367.1796
(calculated for C22H25NO4, 367.1784).
(300 MHz, CDCl3) δ 7.58 (m, 2H), 7.43 (m, 2H), 7.34 (m, 1H),
7.19 (m, 1H), 7.02 (dd, J ) 2.4, 1.6 Hz, 1H), 6.92 (m, 1H), 5.21
(q, J ) 7.0 Hz, 1H), 4.38 (m, 1H), 4.20-4.09 (m, 2H), 3.86 (s,
3H), 2.46 (m, 1H), 1.56 (d, J ) 7.0 Hz, 3H), 0.93 (d, J ) 7.1 Hz,
3H), 0.92 (d, J ) 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
174.4, 160.0, 153.6, 142.8, 142.1, 140.9, 128.7, 127.4, 127.2, 119.6,
112.7, 111.8, 63.1, 59.0, 55.3, 43.0, 28.5, 19.7, 18.0, 14.7; IR (neat,
KBr, cm-1) 2966, 2837, 1771, 1695, 1456, 1227, 1053, 762; HRMS
(FAB, M+) m/z 367.1794 (calculated for C22H25NO4, 367.1784).
(S)-2-(3-Methoxy-5-phenylphenyl)propan-1-ol. To a 0 °C solu-
tion of methyloxazolidinone 22 (0.228 g, 0.621 mmol) in dry THF
(10 mL) under argon was added LAH (0.071 g, 1.87 mmol) in one
portion. Following 1 h, the reaction was quenched by the careful
sequential addition of 0.07 mL of water, 0.14 mL of 4 N NaOH,
and 0.07 mL of water. The mixture was stirred for 15 min at
ambient temperature, and the solids were filtered (celite) and rinsed
with ether. The filtrate was concentrated and the residue purified
by flash chromatography (SiO2 11 g, 10-25% EtOAc/hexanes) to
afford the alcohol as a clear viscous oil (0.122 g, 81%): TLC Rf )
0.3 (1:2 EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.58 (m,
2H), 7.44 (m, 2H), 7.36 (m, 1H), 7.06 (m, 1H), 7.00 (m, 1H), 6.79
(m, 1H), 3.87 (s, 3H), 3.75 (m, 2H), 3.00 (hex, J ) 6.9 Hz, 1H),
1.38 (t, J ) 6.2 Hz, 1H), 1.32 (d, J ) 6.9 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 160.2, 145.7, 143.0, 141.2, 128.7, 127.5, 127.2,
119.0, 112.3, 110.9, 68.6, 55.3, 42.7, 17.6; IR (neat, KBr, cm-1
)
3371, 3059, 2961, 1593, 1462, 1213, 1022, 764; HRMS (FAB, M+)
m/z 242.1311 (calculated for C16H18O2, 242.1307).
(R)-3-(3-Methoxy-5-phenylphenyl)-1,1-dibromobutene (24). To
a 0 °C solution of previously synthesized alcohol (0.285 g, 1.17
mmol) in dry CH2Cl2 (14 mL) was added Dess-Martin periodinate
(0.748 g, 1.76 mmol) in a single portion. The mixture was stirred
at 0 °C for 1 h, then was diluted with a 1:1 mixture of saturated
aqueous NaHCO3 and 20% Na2S2O3 (20 mL). The biphasic mixture
was stirred until all solids were dissolved. Then the organic phase
was separated and the aqueous phase was extracted with two
additional portions of CH2Cl2 (15 mL). The combined organic
phases were washed with brine (30 mL), dried over anhydrous
Na2SO4, and concentrated to afford the crude aldehyde that was
used immediately in the next step.
To a 0 °C solution of CBr4 (1.16 g, 3.5 mmol) in dry CH2Cl2
(28 mL) was added PPh3 (1.84 g, 7.0 mmol) in a single portion.
The resulting dark-yellow solution was stirred a further 5 min. Then
the crude aldehyde dissolved in CH2Cl2 (5 mL) was added dropwise.
The now wine-red solution was stirred for 30 min and then poured
into ice cold ether (150 mL), producing a white precipitate. The
mixture was filtered through a column of silica gel (20 g)
equilibrated with hexanes and rinsed with hexanes until product
elution ceased. The filtrate was concentrated and the residue purified
by flash chromatography (SiO2 30 g, 2% EtOAc/hexanes) to afford
dibromoalkene 24 as a clear viscous oil (0.409 g, 88% 2 steps):
TLC Rf ) 0.52 (5% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3)
δ 7.62 (m, 2H), 7.48 (m, 2H), 7.39 (m, 1H), 7.09 (m, 1H), 7.03
(m, 1H), 6.82 (m, 1H), 6.59 (d, J ) 9.5 Hz, 1H), 3.90 (s, 3H), 3.84
(dq, J ) 9.5, 7.0 Hz, 1H), 1.47 (d, J ) 7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 160.2, 144.9, 143.1, 142.5, 141.1, 128.7,
127.5, 127.2, 118.4, 111.9, 110.9, 88.7, 55.3, 43.6, 20.1; IR (neat,
KBr, cm-1) 2966, 1593, 1337, 1213, 1053, 762; HRMS (FAB, M+)
m/z 393.9595 (calculated for C17H16Br2O, 393.9568).
(R)-2-(3-Methoxy-5-phenylphenyl)propan-1-ol. To a 0 °C solu-
tion of methyloxazolidinone 23 (0.285 g, 0.776 mmol) in dry THF
(10 mL) under argon was added LAH (0.089 g, 2.34 mmol) in one
portion. Following 1.5 h, the reaction was quenched by the careful
sequential addition of 0.09 mL of water, 0.18 mL of 4 N NaOH,
and 0.09 mL of water. The mixture was stirred for 15 min at
ambient temperature, and the solids were filtered (Celite) and rinsed
with ether. The filtrate was concentrated and the residue purified
by flash chromatography (SiO2 14 g, 10-15% EtOAc/hexanes) to
afford the alcohol as a clear viscous oil (0.175 g, 93%): TLC Rf )
0.3 (1:2 EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.59 (m,
2H), 7.44 (m, 2H), 7.36 (m, 1H), 7.07 (m, 1H), 7.01 (m, 1H), 6.80
(m, 1H), 3.87 (s, 3H), 3.75 (m, 2H), 3.01 (h, J ) 6.9 Hz, 1H), 1.46
(R)-4-Isopropyl-3-((R)-2-(3-methoxy-5-phenylphenyl)propanoyl)-
oxazolidin-2-one (23). To a -78 °C solution of biphenyloxazoli-
dinone 21 (0.471 g, 1.33 mmol) in dry THF (13 mL) under argon
was added a solution of LHMDS (1 M in THF, 1.67 mL, 1.67
mmol). The yellow-orange solution was stirred for 1 h at -78 °C.
Then methyl iodide (0.35 mL, 5.61 mmol) was added dropwise.
Following 5 min the mixture was warmed to 0 °C and stirred an
additional 0.5 h. Saturated ammonium chloride (20 mL) was added
and the mixture warmed to ambient temperature. The mixture was
diluted with ethyl acetate (20 mL), and the organic phase was
separated. The aqueous phase was extracted with additional ethyl
acetate (2 × 15 mL), and the organic phases were combined,
washed with brine (30 mL), dried over anhydrous Na2SO4, and
concentrated to provide the crude oil that was purified by flash
chromatography (SiO2 24 g, 10% EtOAc/hexanes) to afford
alkylated product 23 as a clear viscous oil and a single diastereomer
1
(0.285 g, 58%): TLC Rf ) 0.63 (1:2 EtOAc/hexanes); H NMR

Potent and SelectiVe Cryptosporidium DHFR Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6851
(m, 1H), 1.32 (d, J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
δ 160.2, 145.7, 143.0, 141.1, 128.7, 127.4, 127.2, 119.0, 112.3,
110.9, 68.6, 55.3, 42.7, 17.6; IR (neat, KBr, cm-1) 3371, 3058,
2961, 1593, 1454, 1215, 1020, 764; HRMS (FAB, M+) m/z
242.1295 (calculated for C16H18O2, 242.1307).
(S)-3-(3-Methoxy-5-phenylphenyl)-1,1-dibromobutene (25). To
a 0 °C solution of the previously synthesized alcohol (0.175 g, 0.722
mmol) in dry CH2Cl2 (9 mL) was added Dess-Martin periodinate
(0.459 g, 1.08 mmol) in a single portion. The mixture was stirred
at 0 °C for 1.5 h, then was diluted with a 1:1 mixture of saturated
aqueous NaHCO3 and 20% Na2S2O3 (20 mL). The biphasic mixture
was stirred until all solids were dissolved. Then the organic phase
was separated and the aqueous phase was extracted with two
additional portions of CH2Cl2 (10 mL). The combined organic
phases were dried over anhydrous Na2SO4 and concentrated to
afford the crude aldehyde that was used immediately in the next
step.
2,4-Diamino-5-[(S)-3-(3-methoxy-5-phenylphenyl)but-1-ynyl]-6-
methylpyrimidine (S-10a). According to the general Sonagashira
coupling procedure, iodopyrimidine 11 (0.052 g, 0.208 mmol),
Pd(PPh3)2Cl2 (0.011 g, 0.016 mmol), CuI (0.005 g, 0.026 mmol),
and alkyne 26 (0.098 g, 0.413 mmol) were reacted in DMF/Et3N
(1 mL each) at 60 °C for 2 h. Following the general workup
procedure flash chromatography (SiO2 10 g, 2% MeOH/CHCl3)
afforded coupled pyrimidine S-10a as a pale solid (0.073 g, 97%,
98% ee). An analytical sample was generated by triturating in ether.
TLC Rf ) 0.22 (EtOAc); mp 140.2-141.5 °C; 1H NMR (500 MHz,
CDCl3) δ 7.58 (m, 2H), 7.44 (m, 2H), 7.35 (m, 1H), 7.24 (m, 1H),
7.02 (m, 1H), 6.99 (m, 1H), 5.11 (bs, 2H), 4.82 (bs, 2H), 4.08 (q,
J ) 7.1 Hz, 1H), 3.88 (s, 3H), 2.39 (s, 3H), 1.63 (d, J ) 7.1 Hz,
3H); 13C NMR (125 MHz, CDCl3) δ 168.6, 164.1, 160.4, 160.2,
145.3, 143.1, 141.0, 128.8, 127.6, 127.2, 118.3, 111.5, 111.1, 101.8,
91.5, 75.7, 55.4, 33.2, 24.7, 22.9; HRMS (FAB, M+) m/z 358.1779
(calculated for C22H22N4O, 358.1794); HPLC (a) tR ) 6.90 min,
99.2%, (b) tR ) 6.92 min, 99.4%.
To a 0 °C solution of CBr4 (0.718 g, 2.17 mmol) in dry CH2Cl2
(18 mL) was added PPh3 (1.14 g, 4.35 mmol) in a single portion.
The resulting dark-yellow solution was stirred a further 5 min, and
then the crude aldehyde dissolved in CH2Cl2 (2 mL) was added
dropwise. The now wine-red solution was stirred for 40 min and
then poured into ice cold ether (150 mL), producing a white
precipitate. The mixture was filtered through a column of silica
gel (15 g) equilibrated with hexanes and rinsed with hexanes until
product elution ceased. The filtrate was concentrated and the residue
purified by flash chromatography (SiO2 18 g, 2% EtOAc/hexanes)
to afford dibromoalkene 25 as a clear viscous oil (0.216 g, 76%
2,4-Diamino-5-[(R)-3-(3-methoxy-5-phenylphenyl)but-1-ynyl]-6-
methylpyrimidine (R-10a). According to the general Sonagashira
coupling procedure, iodopyrimidine 11 (0.071 g, 0.28 mmol),
Pd(PPh3)2Cl2 (0.014 g, 0.020 mmol), CuI (0.007 g, 0.037 mmol),
and alkyne 27 (0.132 g, 0.559 mmol) were reacted in DMF/Et3N
(1.3 mL each) at 50 °C for 2.5 h. Following the general workup
procedure flash chromatography (SiO2 14 g, 2% MeOH/CHCl3)
afforded the coupled pyrimidine R-10a as a pale solid (0.095 g,
93%, 98% ee). An analytical sample was generated by triturating
1
in ether. TLC Rf ) 0.22 (EtOAc); mp 139.3-140.4 °C; H NMR
(500 MHz, CDCl3) δ; 13C NMR (125 MHz, CDCl3) δ 168.6, 164.1,
160.4, 160.2, 145.3, 143.1, 141.0, 128.8, 127.6, 127.2, 118.3, 111.5,
111.1, 101.8, 91.5, 75.7, 55.4, 33.2, 24.7, 22.9; HRMS (FAB, M+)
m/z 358.1790 (calculated for C22H22N4O, 358.1794); HPLC (a) tR
) 6.88 min, 99.2%, (b) tR ) 6.89 min, 98.7%.
1
two steps): TLC Rf ) 0.52 (5% EtOAc/hexanes); H NMR (500
MHz, CDCl3) δ 7.59 (m, 2H), 7.46 (m, 2H), 7.37 (m, 1H), 7.07
(m, 1H), 7.01 (dd, J ) 2.3, 1.6 Hz, 1H), 6.80 (m, 1H), 6.56 (d, J
) 9.5 Hz, 1H), 3.88 (s, 3H), 3.81 (dq, J ) 9.5, 7.0 Hz, 1H), 1.45
(d, J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 160.2, 145.0,
143.2, 142.5, 141.1, 128.7, 127.5, 127.3, 118.4, 111.9, 110.9, 88.7,
55.4, 43.6, 20.1; IR (neat, KBr, cm-1) 2966, 1593, 1456, 1339,
1213, 1053, 762; HRMS (FAB, M+) m/z 393.9580 (calculated for
C17H16Br2O, 393.9568).
Acknowledgment. We gratefully acknowledge Justin Fair
(University of Connecticut) for his assistance in performing the
HPLC analyses and funding from NIH (Grant AI065143 to
D.L.W. and Grant GM067542 to A.C.A.).
(S)-3-(3-Methoxy-5-phenylphenyl)butyne (26). To the dibro-
moalkene 24 (0.099 g, 0.25 mmol) in an 8 mL screw cap vial was
added magnesium (0.012 g, 0.50 mmol) and dry THF (0.25 mL).
The vial was sealed tightly with a rubber septum and flushed with
argon. The mixture was heated in a 75 °C oil bath for 2.5 h when
a check by TLC showed consumption of the starting material. The
mixture was cooled and the residue purified by flash chromatog-
raphy (SiO2 6 g, 5% EtOAc/hexanes) to afford acetylene 26 as a
clear viscous oil (0.057 g, 96%): TLC Rf ) 0.44 (5% EtOAc/
Supporting Information Available: Details of HPLC purity
determinations for compounds 10a-d, S-10a, R-10a, 17a-d,
including instrumentation, tabulated data, and copies of chromato-
grams; details of % ee determinations for S-10a, R-10a; 1H NMR
and 13C NMR spectra of compounds 5, 7 to 10a-d, 13, 14 to
17a-d, 18-27, S-10a, R-10a, and appropriate intermediates;
synthesis of boronic acid 6d. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
hexanes); H NMR (300 MHz, CDCl3) δ 7.59 (m, 2H), 7.44 (m,
2H), 7.35 (m, 1H), 7.20 (m, 1H), 7.00 (dd, J ) 2.4, 1.6 Hz, 1H),
6.96 (m, 1H), 3.88 (s, 3H), 3.81 (dq, 7.1, 2.5 Hz, 1H), 2.29 (d, J
) 2.5 Hz, 1H), 1.56 (d, J ) 7.1 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 160.1, 144.6, 143.0, 141.1, 128.7, 127.5, 127.2, 118.4,
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