Targeted mutations of bacillus anthracis dihydrofolate reductase condense complex structure-activity relationships

Article abstract of DOI:10.1021/jm100727t

Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.

Full text of DOI:10.1021/jm100727t

J. Med. Chem. 2010, 53, 7327–7336 7327
DOI: 10.1021/jm100727t
Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex
Structure-Activity Relationships†
Jennifer M. Beierlein, Nanda G. Karri, and Amy C. Anderson*
Department of Pharmaceutical Sciences, University of Connecticut, 69 N. Eagleville Road, Storrs, Connecticut 06269
Received June 16, 2010
Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind
dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other
bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to deter-
mine structure-activity relationships whereby the enzyme is altered and the analogues remain con-
stant, essentially reversing the standard experimental design. Active site mutants of the enzyme,
Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays
and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant,
suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes
bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the
influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors
test and validate these conclusions and show the value of the technique for providing new directions
during lead optimization.
Introduction
In recent years, Bacillus anthracis dihydrofolate reductase
(BaDHFRa) has been investigated as a potential target in the
treatment of the bacterial infection, anthrax.1 Dihydrofolate
reductase (DHFR), an essential enzyme in the folate biosyn-
thetic pathway, has been pursued as a therapeutic target
for several infectious diseases. DHFR is responsible for the
reduction of dihydrofolate to tetrahydrofolate through a
catalytic cycle involving nicotinamide adenine dinucleotide
phosphate (NADPH) as a cofactor.2 The DHFR active site
is highly conserved across species, containing an acidic residue
(typically Asp or Glu) at one end of a large hydrophobic
pocket.3 Potent antifolates contain a diaminopyrimidine or
s-triazine pharmacophore that forms hydrogen bonds with
the acidic residue and anchors the molecule in the pocket.3
Key hydrophobic residues in the active site, including Leu 29,
Val 32, Ile 51, and Phe 96 (Figure 1), form van der Waals
interactions with the substrate and inhibitors.
BaDHFR, unlike DHFR from many bacteria, has been
shown to be naturally insensitive to the broad spectrum anti-
biotic trimethoprim (TMP), whichtargetsthe DHFR enzyme.
Studies have shown this lack of sensitivity to TMP results
from low affinity for the enzyme.1c,4 We have previously
Figure 1. Compound 9 bound to BaDHFR (WT). The original
conformation of 9 from PDB ID 3E0B is shown in green; the
alternate conformation stabilized by F96I is shown in blue. The
two red circles indicate the positions of the 20 and 50methoxy groups
in the two alternate conformations (figure prepared in PyMol).
†Coordinates have been deposited in the Protein Data Bank with
accession codes 3JVX, 3JWM, 3JWC, 3JWK, 3JWF, 3JW5, and 3JW3.
*To whom correspondence should be addressed. Phone: (860)486-
6145. Fax: (860)486-6857. Email: amy.anderson@uconn.edu.
a Abbreviations: DHFR, dihydrofolate reductase; BaDHFR, dihy-
drofolate reductase from Bacillus anthracis; TMP, trimethoprim; DHF,
dihydrofolate; SaDHFR, dihydrofolate reductase from Staphylococcus
aureus; EcDHFR, dihydrofolate reductase from Escherichia coli; SpDHFR,
dihydrofolate reductase from Streptococcus pneumoniae; hDHFR, human
dihydrofolate reductase; NADPH, nicotinamide adenine dinucleotide phos-
phate, reduced form.
described a series of DHFR inhibitors featuring a propargyl
linker that shows an increased sensitivity to BaDHFR com-
paredto TMP.1a The most potent inhibitor inthis series shows
a hundred-fold improvement over TMP. Crystal and solution
structures of BaDHFR bound to this inhibitor have offered new
insights into protein-ligand interactions critical to potency.1a,5
However, despite this improvement in potency over TMP,
r
2010 American Chemical Society
Published on Web 09/30/2010
pubs.acs.org/jmc

7328 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Beierlein et al.
Figure 2. Structural Alignment of BaDHFR, SaDHFR, EcDHFR, and SpDHFR. * indicates active site residues.
Table 1. KM and kcat Values for Wild Type and Mutant Enzymes
the propargyl-linked compounds generally exhibit affinities
for BaDHFR that are ∼100-fold less than other bacterial
species such as Staphylococcus aureus.6
K
M (μM)
kcat (s-1
)
kcat/KM
BaDHFR
Ba(F96I)DHFR
Ba(Y102F)DHFR
33.7 ( 3.2
230.1 ( 33.97
14.44 ( 1.736
9.3
4.8
1.7
0.28
0.02
0.12
The BaDHFR residues Phe 96 and Tyr 102, which are
frequently isoleucine and phenylalanine in other TMP-sensitive
bacterial species, may play an important role in the natural
resistance of BaDHFR to TMP and the series of propargyl-
linked analogues.1 To further investigate the interactions of
antifolates with these two residues, two mutant BaDHFR
enzymes, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created
by site-directed mutagenesis. Enzyme inhibition assays show
that TMP and several of the propargyl-linked compounds
have significantly greater affinity for the mutant Ba(Y102F)-
DHFR andapproximatelyequalaffinityfor Ba(F96I)DHFR,
suggesting that substitutions that interact with Tyr 102 are
critical to determining potency. Structural analysis of seven
wild type and mutant crystal structures bound to TMP and
three propargyl-linked inhibitors reveal the basis of TMP resis-
tanceinthe wild type enzyme and shedlighton the influence of
Tyr 102 on the lipophilic linker between the pyrimidine and
aryl rings. The synthesis of two new propargyl-linked inhibitors
validates these conclusions. Overall, these mutational studies
aided in simplifying complex SAR data by revealing the
critical impact of repulsive van der Waals’ forces and electro-
statics at key positions of the propargyl-linked antifolates.
to TMP and the propargyl-linked analogues. BaDHFR and
Staphylococcus aureus DHFR (SaDHFR) are 85% homologous
(66% identical), yet there are striking differences between the
two enzymes. Wild-type SaDHFR is sensitive to TMP, with a
Ki of 0.0033 μM, while BaDHFR has a Ki of 24.1 μM. The
series of propargyl-linked inhibitors have IC50 values as low as
5 nM6a in SaDHFR, yet the same compounds are consistently
at least 10-fold less potent in BaDHFR. The most potent
inhibitor for BaDHFR is compound 9, with a Ki value of
300 nM, whilein SaDHFR, the samecompound hasaKi value
of 9.9 nM.1a,6a
Resistance to TMP has developed in several bacterial
species. In SaDHFR, a single mutation from Phe 98 to a
tyrosine causes a 74-fold decrease in sensitivity;6a,8 recent
studies indicate that this mutation may confer resistance by
inducing an alternate conformation of the bound cofactor,
NADPH.6a In Escherichia coli DHFR (EcDHFR) and Strep-
tococcus pneumoniae DHFR (SpDHFR), resistance has been
observed when an isoleucine positioned near the methylene
linker of TMP is mutated to a leucine.9 A structural alignment
(Figure2) of BaDHFR tothese otherspecies reveals a tyrosine
(Y102) in the same position where the mutation from a
phenylalanine to tyrosine occurs in SaDHFR and a phenyl-
alanine (F96) in the same position as the isoleucine that
mutates in EcDHFR and SpDHFR. To understand the
effects of residues F96 and Y102 on the inhibition of TMP
and the propargyl-linked analogues and to guide the optimal
substitution of future analogues, mutant enzymes were pre-
pared by site-directed mutation and evaluated with enzyme
inhibition assays and structural studies.
Results
Previous structure-activity relationship (SAR) studies1a,5,7
have generated almost 50 propargyl-linked analogues that
have been evaluated with BaDHFR. Though increasingly
potent inhibitors targeting BaDHFR have been designed,1a,4
it has been difficult to obtain inhibitors with low nanomolar
inhibition concentrations. Inhibition data indicates compound
9 (Ki = 0.33 μM) is a viable lead compound,1a however,
modifications suggested by SAR and crystal structures have
not improveditspotency. For example, the crystalstructure of
compound 9 bound to BaDHFR1a (Figure 1) shows a large
hydrophobic pocket enclosed primarily by Leu 55 and Leu 29,
yet the addition of a second phenyl ring in place of the methoxy
group (compound 11) decreased potency (Ki = 1.65 μM).
Comparisons of sequence, structure, and inhibition potency
of selected analogues probed the differences between the
active sites of BaDHFR and other bacterial species sensitive
Enzyme Activity. To begin, kinetic parameters for the enzy-
mes were compared (Table 1). Interestingly, the Ba(Y102F)-
DHFR shows a slight improvement in KM and only a 2-fold loss
in kcat/KM relative to the wild-type enzyme. The Ba(F96I)-
DHFR mutant, however, shows a much greater loss in KM
(∼7-fold) and kcat/KM (∼10-fold). This reduction is rationalized
by the direct interaction of this residue with the substrate.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7329
Table 2. Ki Values of Inhibitors against Wild Type and Mutant Enzymes
ligand
BaDHFR Ki (μM)
BaF96IDHFR Ki (μM)
fold difference
BaY102FDHFR Ki (μM)
fold difference
TMP
1
24.1 ( 0.03
1.25 ( 0.07
0.32 ( 0.02
10.2 ( 0.13
3.37 ( 0.17
4.89 ( 0.17
9.81 ( 0.02
4.28 ( 0.34
0.36 ( 0.03
0.30 ( 0.005
0.37 ( 0.02
1.21 ( 0.03
2.59 ( 0.13
0.33 ( 0.003
2.66 ( 0.27
5.16 ( 0.20
5.83 ( 0.47
11.7 ( 0.05
13.7 ( 0.51
1.15 ( 0.03
1.19 ( 0.06
34.1 ( 2.30
3.91 ( 0.09
4.39 ( 0.30
6.35 ( 0.67
4.81 ( 0.30
4.51 ( 0.02
20.5 ( 1.33
1.31 ( 0.07
1.70 ( 0.07
1.04 ( 0.07
0.49 ( 0.012
1.10 ( 0.05
7.52 ( 0.31
9.20 ( 0.69
10.4 ( 0.75
1.8
1.1
0.48 ( 0.02
0.049 ( 0.001
0.064 ( 0.0002
0.17 ( 0.006
1.19 ( 0.07
0.39 ( 0.03
2.00 ( 0.05
2.94 ( 0.23
0.20 ( 0.004
0.69 ( 0.04
0.083 ( 0.003
0.23 ( 0.02
0.43 ( 0.009
0.097 ( 0.003
1.03 ( 0.07
1.83 ( 0.09
1.60 ( 0.12
1.64 ( 0.14
50.2
25.5
5.0
2
-3.7
-3.3
-1.2
1.1
3
60.0
2.8
4
5
12.5
4.9
6
1.5
7
-1.1
-12.5
-68.3
-3.5
-1.4
2.5
1.5
1.8
8
9
-2.3
4.5
10
11
12
13
14
15
16
17
5.3
6.0
-1.5
-2.4
-1.5
1.6
3.4
2.6
2.8
3.6
1.1
7.1
Enzyme Inhibition. Ki values for a representative sample of
propargyl-linked inhibitors, reported in Table 2, were calcu-
lated using the Cheng-Prusoff method10 from the experimen-
tal IC50 values obtained from competitive inhibition assays
with a substrate concentration of 100 μM and the experimental
KM values (experimental IC50 values are reported in Supporting
Information). TMP was 50-fold more potent against Ba-
(Y102F)DHFR than the wild type enzyme. Interestingly, the
Ba(F96I)DHFR mutation resulted in only a 2-fold improve-
ment for TMP affinity. In fact, overall, the F96I mutation
causes little to no difference in activity for the propargyl-linked
compounds. Compound 9 stands out as the only ligand to lose
significant activity against the Ba(F96I)DHFR mutant.
The mutation from a polar Tyr to a hydrophobic Phe at
position 102 resulted in a 4-fold improvement in binding for
several of the propargyl-linked analogues, with some inhibi-
tors, such as 1 and 3, showing significantly greater (25- and
60-fold, respectively) improvement. The Ba(Y102F)DHFR
mutant enzyme appears to accommodate substituents on
the propargyl linker more readily than the wild type enzyme.
Compounds 1, 3, and 5 all show a 10-fold or greater
improvement in enzyme inhibition as a result of this mutation.
Compound 3 with a hydrophobic methyl substituent shows
the greatest improvement, while compound 5 with a polar
hydroxyl group shows the least improvement. The addition
of a second phenyl ring, such as with compounds 14 and 15,
generally had little effect on activity against this mutant. The
second phenyl ring is distant from the 102 residue and is
probably minimally affected by any steric or electrostatic
effects caused by this mutation.
the ligands, the structures of BaDHFR bound to compounds
2 and 6, Ba(F96I)DHFR bound to TMP, and Ba(Y102F)-
DHFR bound to compounds 2, 5, 6, and TMP were deter-
mined by molecular replacement. All crystals belong to space
group P42 and contain two monomers in the asymmetric
unit. Relevant data collection and refinement statistics are
reported for each crystal in Table 3. The structures show that
both mutant enzymes conserve the canonical eight-stranded
twisted β-sheet and four flanking R helices observed in
the wild type enzyme. Conserved hydrogen bonds between
the side chain of Glu 28, an essential acidic residue, and the
backbone carbonyls of Met 6 and Phe 96 (or Ile 96 in the F96I
mutant) with the pyrimidine ring of TMP and the propargyl-
linked analogues are maintained in the mutant enzymes.
TMP Binding to Mutant BaDHFR Enzymes. Structures of
TMP bound to both mutant DHFR enzymes reveal the
factors involved in the natural resistance of the wild type
enzyme to TMP. Because TMP was bound to the protein by
soaking rather than cocrystallization, TMP occupancy was
verified by lower Rfree and Roverall values and a decrease in
difference density relative to refinement with the original
ligand. Omit maps (shown in Supporting Information) calcu-
lated for the two TMP structures according the method
described by Bhat11 also verify the presence of the TMP
ligand. The two TMP-bound structures superimpose with an
˚
rmsd of 0.246 A.
There is a dramatic change in active site volume caused by
the F96I mutation, as shown by the electrochemical surfaces
(Figure 3a). To best utilize the space gained by the presence
of a smaller isoleucine rather than a bulky phenylalanine,
there is a significant torsion angle difference between the pyrim-
idine and trimethoxyphenyl rings in the two mutants: the
Structural Evaluation. For further evaluation of the effects
of these mutations on the enzymes and their interactions with

7330 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Table 3. Crystallographic Statistics
Beierlein et al.
crystallography statistics
BaWT/2
3JVX
P42
BaWT/6
3JWM
P42
BaY102F/2
BaY102F/6
BaY102F/5
BaY102F/TMP
BaF96I/TMP
PDB code
space group
3JWC
P42
2
3JWK
P42
2
3JWF
P42
2
3JW5
P42
2
3JW3
P42
2
no. monomers in
asymmetric unit
unit cell (a, b, c in A)
2
2
˚
a, b = 78.24;
c = 66.98
a, b = 78.88;
c = 67.43
a, b = 77.85;
c = 67.10
a, b = 77.86;
c = 67.53
a, b = 77.89;
c = 67.06
a, b = 74.99;
c = 67.41
a, b = 75.43;
c = 67.46
˚
resolution (A)
28.59-2.25
99.9 (99.4)
38.95-2.57
98.8 (98.1)
42.56-2.57
99.7 (96.5)
42.68-2.08
100.0 (100.0)
38.95-2.57
98.7 (92.4)
37.50-2.89
98.2 (86.0)
41.85-2.57
99.7 (96.2)
completeness %
(last shell*, %)
unique reflections
redundancy (last shell)
18299
6.6 (6.5)
12154
7.6 (7.7)
12210
7.0 (4.7)
23117
7.3 (7.3)
12126
5.4 (3.4)
7952
6.6 (3.7)
11538
7.0 (4.7)
R
sym, % (last shell, %)
0.142 (0.512)
12.6 (3.0)
0.057 (0.154)
41.9 (14.9)
0.198/0.243
2766, 51, 193
0.114 (0.391)
19.9 (3.3)
0.191/0.230
2758, 49, 116
0.161 (0.463)
26.1 (4.9)
0.183/0.213
2769, 51, 361
0.122 (0.232)
22.8 (4.6)
0.208/0.257
2758, 49, 118
0.179 (0.443)
11.6 (2.3)
0.212/0.261
2766, 41, 37
0.110 (0.407)
22.1 (3.2)
0.219/0.262
2768, 41, 37
ÆI/σæ (last shell)
R-factor/Rfree
0.182/0.2098
2805, 49, 233
no. of atoms (protein,
ligands, solvent)
rms deviation bond
˚
lengths (A), angles (deg)
average B factor for
0.009, 1.323
27.18
24.98
34.01
90.3
0.008, 1.264
19.70
29.82
22.17
90.6
0.008, 1.296
24.29
24.23
22.75
91.3
0.010, 1.398
24.96
28.53
33.31
91.0
0.007, 1.185
30.57
31.27
28.17
90.6
0.008, 1.294
33.56
0.010, 1.371
33.99
2
protein (A )
˚
average B factor for
2
ligand (A )
38.07
43.56
˚
average B factor for
solvent molecules (A )
20.71
28.189
88.5
2
˚
residues in most favored
regions (%)
87.5
residues in additional
allowed regions (%)
9.7
9.4
8.7
9.0
9.4
12.5
11.5
Figure 3. (A) Lipophilic potential surface diagrams of Ba(F96I)DHFR (white sticks and solid surface) and Ba(Y102F)DHFR (purple sticks
and mesh surface) (created using Sybyl 8.0). (B) Active site stereo view of TMP bound to Ba(F96I)DHFR (green) and to Ba(Y102F)DHFR
(blue) (created using PyMol).
torsion angle, as defined by atoms C8, C9, C10, and C11
(Figure 3b), is 41.8ꢀ for TMP in Ba(F96I)DHFR and 24.0ꢀ
for TMP in Ba(Y102F)DHFR. Energy calculations with Sybyl
8.112 using the Tripos force field and Gasteiger-Marsili
charges show that the conformation of TMP adopted in the
F96I mutant structure suffers an energy penalty of approxi-
mately 14 kCal/mol relative to the conformation observed in
the Y102F mutant. This energy difference aids in explaining
the 26-fold difference in Ki values for the two enzymes.
Inhibition data showing that the activity of TMP against
Ba(Y102F)DHFR increases 50-fold compared to the wild-
type enzyme indicates that Y102 may play a major role in the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7331
Figure 4. (A) Compound 2 bound to BaDHFR (green) and to Ba(Y102F)DHFR (blue). (B) Compound 6 bound to BaDHFR (green) and to
Ba(Y102F)DHFR (blue). (C) Compound 6 bound to Ba(Y102F)DHFR (green) and compound 5 bound to Ba(Y102F)DHFR (blue). Figures
generated using PyMol.
resistance of BaDHFR to TMP. Structural evidence suggests
the change from a tyrosine to a phenylalanine increases the
hydrophobicity of the pocket near the methylene linker of
TMP, improving hydrophobic interactions (Figure 3). Crystal
structuresandenzyme inhibition data withBa(Y102F)DHFR
and propargyl-linked compounds further clarified the effects
of this mutation in greater detail.
both the electrostatic and steric properties of the pocket. The
rmsd between the structure of BaDHFR (WT) and the
Y102F mutants bound to compounds 2 and 6 is only
˚
0.17 A, suggesting that the structure is only minimally
perturbed by the removal of the hydroxyl group and that
the influence of steric differences is limited. Furthermore,
because the corresponding SaDHFR mutant (TMP resistant
F98Y) reflects a change in electrostatics, not in steric
differences,6a we investigated electrostatics in more depth.
Comparison of enzyme inhibition data within an analogous
series of propargyl-linked compounds and energy calculations
suggest that change in polarity from a tyrosine to a phenylala-
nine may have an advantageous effect on the lipophilic pro-
pargyl linker, particularly with a hydrophobic substituent. By
comparing compounds 1, 3, and 5, which have a trimethoxy-
phenyl ring and various substituents on the propargyl linker, it
is apparent that a propargylic hydrogen is preferred over a
hydroxyl by 4-fold and by 8-fold over a methyl (H > OH>
Me) with the wild type enzyme. This trend is reversed for the
Y102F mutant, with the propargylic hydrogen being preferred
over a methyl by 4-fold and by 8-fold over a hydroxyl (H>
Me > OH). Further supporting the fact that electrostatics most
likely plays a role in determining activity, energy calculations
performed with the structures of compound 6, containing a
methoxy propargylic substituent and either F102 or Y102
(from the respective crystal structures), show that the former
complex is 18.5 kcal/mol more favorable than the latter. In
these calculations, the electrostatic energy changes by twice the
extent that the van der Waals energy changes.
Analysis of Ba(Y102F)DHFR Structures. Structures with
compounds 2, 6 and 5, possessing hydrogens, a methoxy
group, and a hydroxyl group, respectively, on the propargyl
linker were determined to investigate the effect of the Y102F
mutation (Figure 4). Racemic mixtures of compounds 5 and 6
were used in enzyme assays and protein crystallization. A clear
preference for the (S) enantiomer of compound 6 and the (R)
enantiomer of compound 5 appear in the electron density and
yield lower Rfree values in the refinement. The (S) enantiomer
of compound 6 allows the methyl group of the methoxy
substituent to interact with Leu 21 (not shown in Figure 4b
for clarity) as well as to be oriented away from the polar pocket
containing NADPH. The oxygen of the methoxy group is
˚
oriented to be within hydrogen bonding distance (2.80 A) of
Asn 47 (Figure 4B). The enzyme prefers the (R) enantiomer of
5 as the optimal configuration to form a hydrogen bond
between the hydroxyl substituent and the side chain of Asn
˚
47 (2.60 A). The propargyl linker and trimethoxyphenyl ring
of 5 are positioned closer to NADPH and the flanking helix
than is observed with compound 6 (Figure 4C).
Clearly, the presence or absence of the hydroxyl group in
BaDHFR (WT) or Ba(Y102F)DHFR, respectively, influences

7332 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Scheme 1. Synthesis of Compounds 12a and 13b
Beierlein et al.
a (a) Trifluoroacetic acid, pyridine, DCM, RT, 1 h, 92%; (b) PhB(OH)2, Pd(P(Ph)3)4, K3PO4, dioxane, S5 ꢀC, 48 h, 99%; (c) OsO4, NalO4, dioxane,
H2O, RT, 1 h; (d) dimethyl (1-diazo-2-oxopropyl)phosphonate, K2CO3, MeOH, 0 ꢀC, 2 h, 38%; (2 steps); (e) 2,4-diamino-6-ethyl-5-iodopyrimidine,
Pd(P(Ph)3)2CI2, Cul, DMF, triethylamine, 60 ꢀC, 18 h, 51.4%. b (a) PH3PCH2OMeCI, NaO’Bu, THF, 0 ꢀC; (b) concentrated HCI, THF, reflux, 1 h,
26.8% (2 steps); (c) dimethyl (1-diazo-2-oxopropyl)phosphonate, K2CO3, MeOH, 0 ꢀC, 2 h, 61%; (d) 2,4-diamino-6-ethyl-5-iodopyrimidine,
Pd(P(Ph)3)2CI2, Cul, DMF, triethylamine, 60 ꢀC, 64%.
Understanding an Outlier: Compound 9 Activity. Com-
pound 9 suffers a 68-fold loss in affinity with the F96I
mutation. It incurs the largest loss in potency of any pro-
pargyl-linked analogue for either mutant enzyme. Previous
structures of 9 bound to BaDHFR1a,5 placed the 20 methoxy
group near the NADPH cofactor and the 50 methoxy group
facing toward Phe 96 (Figure 1 green conformation). The
decrease in potency for other 20,50-substituted analogues
(specifically such as compound 11) confounded the apparent
SAR and led to the theory that the aryl ring may be freely
alternating between this conformation captured in the
crystal structure and an inverted conformation that directs
the 20 methoxy group near Phe 96 and places the 50 methoxy
group near Asn 47, in a position analogous to the 30 position
of the trimethoxyphenyl ring of TMP (Figure 1 blue conforma-
tion). Mutation of Phe 96 to the smaller Ile reduces the van
der Waals interactions that could favor this inverted con-
formation. The theory that compound 9 may be in two
alternate conformations is supported by data from com-
pound 8, which has the same aryl ring as 9. Compound 8
shows a 13-fold decrease in activity for the F96I mutant and
is the only other compound to lose significant potency with
the mutant enzyme. Inhibition data for compounds 11 and
10 also support the idea of a rotating phenyl ring. Compound
11 (Ki = 1.65 μM), with lower affinity than 9, has a phenyl
ring in place of the 50 methoxy group of 9, sterically restrict-
ing it from adopting the alternate conformation. Compound
10 (Ki = 0.41) lacks the 20 methoxy group of 11, yet exhibits
increased affinity, showing that the 20 methoxy group is not
critical for binding. Examination of the electron density for
the crystal of BaDHFR with compound 9 reveals that while
the conformation with the 20 methoxy group facing toward
NADPH is preferred (as determined by minimal difference
density), the alternate conformation is tolerated with some
difference density present.
Both compounds incorporate a hydrogen at the propargyl
position, as dictated by the Y102F mutant data. Additionally,
compound 12 was synthesized to explore the effect of a bulky
substitution at the 20 position on the F96 residue, while
compound 13 was synthesized to test the hypothesis that a
30 methoxy group may be a more favorable substitution
when compared to a 20 methoxy group. Both compounds
were accessed through a convergent route, employing a key
Sonagashira coupling that has been used successfully to build
the current library of propargyl-linked analogues1a,7 to join
the arene and pyrimidine fragments of the molecule. The two
required propargylarenes were obtained through a series of
reactions shown in Scheme 1. Phenol 18 was obtained in two
steps from the commercially available 4-methoxyphenol.13
Triflate addition to 18 afforded aryl triflate 19. Suzuki cross-
coupling to 19 gave the olefin 20 in excellent yield. Cleavage of
20 and subsequent reaction with the Ohira-Bestmann reac-
tion gave alkyne 21, which underwent Sonagashira coupling
with 2,4-diamino-6-ethyl-5-iodo pyrimidine to give the final
product 12. Synthesis of 13 followed a similar route. Benzal-
dehyde 22 was obtained in two steps from commercially
available 2,4-dibromoanisole.7b The Wittig reaction of 22
afforded the enol ether followed by direct hydrolysis to give
the aldehyde 23. Condensation of 22 with the Ohira-
Bestmannreagent gave the terminal acetylene24. Sonagashira
coupling afforded the final compound 13.
Compounds 12 and 13 were tested against the wild-type
and mutant enzymes (Table 2). As conclusions drawn from
mutant data predicted, compound 12 lost activity with the
increased steric bulk at the 20 position, when compared to its
two closest analogues, compounds 9 and 11, due to its
proximity to Phe 96. Validating the method of using targeted
site directed mutations to aid ligand design, compound 13
proved to be the most potent biphenyl analogue synthesized
to date (Ki = 0.33 μM) and further supported the hypothesis
that a 30 methoxy group is more favorable than a 20 methoxy
group because it is 4-fold more potent than the related 20
analogue, compound 11. Inhibition data from compound 13
also supports the Y102F data by showing that an unsubsti-
tuted propargyl linker is preferable to a methyl substitution,
Synthesis and Evaluation of New Lead Compounds. To
evaluate the ability of targeted site-directed mutagenesis to
simplify complex SAR data and guide further analogue design,
two new propargyl-linked analogues were synthesized and
tested against the wild type and mutant BaDHFR enzymes.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7333
because the potency of 13 improves on the activity of its
methyl-substituted analogue, compound 14, by 8-fold.
Recombinant Protein Expression and Purification. BaDHFR
recombinant protein was expressed and purified according to
reported procedures.1a Ba(F96I)DHFR recombinant protein
was expressed in M15 cells upon induction with 1 mM IPTG
at midlog phase. Protein expression was extended for an addi-
tional 6 h at 37 ꢀC after induction. Cells were harvested by
centrifugation. Pellets were lysed with 1ꢀ Bugbuster (Novagen)
and DNase for 30 min at room temperature and then centri-
fuged at 4 ꢀC under high speed to collect the supernatant. The
supernatant was loaded onto a nickel affinity column and was
washed with 20 mM Tris, 1 mM DTT, and 300 mM KCl
(pH 8.0). Bound protein was eluted across a linear gradient with
20 mM Tris, pH 8.0, 1 mM DTT, 100 mM KCl, and 250 mM
imidazole. Fractions with pure protein were concentrated to
∼1 mL and loaded onto a size exclusion column (S200) for
desalting. Protein was eluted into a final buffer of 20 mM Tris,
50 mM KCl, 5 mM DTT, and 0.5 mM EDTA. Fractions were
analyzed by SDS-PAGE. Protein was concentrated to ∼5
mg/mL, flash frozen, and stored at -80 ꢀC until use. Expression
and purification of Ba(Y102F)DHFR was carried out in an
identical manner.
Enzyme Assays. Enzyme activity assays were performed at
25 ꢀC by monitoring the rate of enzyme-dependent NADPH
oxidation at an absorbance of 340 nm over several minutes.1a
Reactions were performed in a buffer containing 20 mM TES,
pH 7.0, 50 mM KCl, 10 mM 2-mercaptoethanol, 0.5 mM EDTA,
and 1 mg/mL BSA. All inhibition assays were performed with a
single, limiting concentration of enzyme and saturating concen-
trations of NADPH and dihydrofolate (DHF). IC50 values were
calculated as the average of three independent experiments.
Standard reactions to determine the KM of DHF were carried
out using DHF concentrations of 25, 50, 100, 150, and 200 μM.
Activity at each concentration was calculated as the average of
three independent experiments. All supporting data for enzyme
activity is reported in Supporting Information.
Discussion
Site-directed mutagenesis has often been used to explore
enzyme function, enzyme folding, and to assist with structural
determination. Alanine scanning mutagenesis has been a
particularlyusefultechnique forstudyingthe effectsof specific
amino acid side chains on protein function and binding.14
While structural-activity relationship studies employing ala-
nine scanning have largely focused on receptor interactions
with small peptides,15 this technique has also been applied to
investigate small molecule interactions.16 To the best of our
knowledge, however, rational site-directed mutagenesis has
not been extensively applied to the interpretation of structure-
activity relationships. These studies of mutant BaDHFR
enzymes have shown a more directed approach to be a useful
tool in understanding trends observed in the SAR data. For
example, mutations in the BaDHFR active site revealed that
the polar character of Y102 negatively impacts the enzyme’s
affinity with TMP and the propargyl-linked compounds, thus
aiding in elucidating one factor of the enzyme’s innate resis-
tance to TMP. While polar propargylic substitutions may
appear appealing, such substitutions have been shown in other
species tohavea negativeimpact on cell permeability.17 Muta-
tional studies also helped further interpret information gath-
ered from structure-based drug design studies. Classical SAR
suggested that a 20 methoxy group was a beneficial substitu-
tion for the BaDHFR active site, however, traditional lead
optimization has failed to improve on the activity of compound
9, a relatively simple molecule. Now, with the data from the
mutants, it is clear that the asymmetry of compound 9 and its
ability to adopt alternate conformations enhances its entropic
contribution to binding. Unfortunately, the ability of com-
pound 9 to adopt these conformations is unique, and viable
lead optimization will most likely have to follow an indepen-
dent path. The studies of mutant BaDHFR enzymes also
helped further analyze the complex SAR data to under-
stand the role of substituents on the propargyl linker of the lead
compounds as well as the effect of substitution patterns around
the aryl ring. Currently, inhibitor development has been directed
toward a methyl substituent on the propargyl bridge due to
the efficacy seen in other DHFR species such as SaDHFR,
Cryptosporidium hominis DHFR, and Candida glabrata
DHFR.6a,7b,17,18 These mutational studies indicate that a
propargylic hydrogen would be a more favorable substituent
for BaDHFR. This technique of targeted site-directed muta-
tions derived from mutations observed in other species repre-
sents an interesting approach to reducing large amounts of
complex SARdatafor interpretation and canbeused tobetter
direct structure-based lead optimization.
Crystallization. Protein (5 mg/mL) was incubated with 2 mM
NADPH and 2 mM ligand for 1 h at 4 ꢀC. After incubation, the
protein-ligand complex was concentrated to ∼15 mg/mL using
a microcon (Amicon). Initial crystal hits were grown at room
temperature by hanging drop diffusion in 22.5% (w/v) PEG
10,000, 0.1 M MES, pH 6.75, at an equal ratio of protein to
crystallization solution. Microseeding was used to obtain iso-
lated crystals in 12% (w/v) PEG 10000 and 0.1 MES, pH 6.75, at
a protein concentration of 12 mg/mL. TMP-bound protein
crystals were obtained by soaking good quality crystals, cocrys-
tallized with compound 6, with 10 mM TMP in DMSO at room
temperature for two days. Crystals were cryoprotected in 15%
ethylene glycol and flash frozen with liquid nitrogen. Data were
collected at Brookhaven National Synchrotron Light Source on
beamlines X29A and X25. All data sets were collected at 100 K.
Structure Determination. All structures were solved by molec-
ular replacement. The program Phaser19 and a model of BaDHFR
(PDB code 3E0B1a) were used to determine initial phase in-
formation. The program Coot20 was used to visualize the electron
density map and build the model. The model was refined with the
program Refmac5.21 Models were validated using Ramachandran
plots and Procheck.22
1
Synthesis. The H and 13C NMR spectra were recorded on
Experimental Section
Bruker instruments at 500 and 125 MHz. Chemical shifts are
reported in ppm and are referenced to residual CHCl3 solvent:
7.26 and 77.0 ppm for 1H and13C, respectively. Melting points were
recorded on Mel-Temp 3.0 apparatus and are uncorrected. High-
resolution mass spectroscopy was provided by the Notre Dame
Mass Spectrometry Laboratory. 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
dichloromethane, ether, and tetrahydrofuran were used directly
from Baker cycletainers. Anhydrous dimethylformamide was pur-
chased from Acros and degassed by purging with argon. Anhy-
drous triethylamine was purchased from Aldrich and degassed by
Enzyme Cloning. Site-directed mutagenesis was used to change
the earlier reported construct for BaDHFR-pET411a to Ba-
(F96I)DHFR by a single point mutation. The Ba(F96I)-pET41
construct was amplified using PCR and inserted into vector
pQE2 (Qiagen). Ba(F96I)-pQE2 clones were verified by sequenc-
ing. The resulting construct contained the Ba(F96I)DHFR
gene with an N-terminal histidine and a DAPase stop point
(Ile 2 was mutated to Arg) for future His-tag removal. Site-
directed mutagenesis, amplification, and insertion in vector
pQE2 was carried out in the same manner to change BaDHFR
to Ba(Y102F)DHFR.

7334 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Beierlein et al.
purging with argon. All reagents were used directly from commer-
cial sources unless otherwise stated. 2-Allyl-4-methoxyphenol was
synthesized according to literature procedures.13 3-Methoxy-5-
phenylbenzaldehyde was synthesized according to literature proce-
dures.7b The Ohira-Bestmann reagent was synthesized according
to literature procedures.23 2,4-Diamino-5-iodo-6-ethylpyrimidine
was synthesized according to literature procedures.7a Both final
compounds had a purity of >95% as determined by HPLC analy-
sis in two mobile phase conditions (please see Supporting Informa-
tion for more details).
2-Allyl-4-methoxyphenyl trifluoromethanesulfonate (19). To a
round-bottom flask under argon, 2-allyl-4-methoxyphenol 17
(2.00 g, 12.2 mmol) and pyridine (2.93 mL, 36.6 mmol) were
dissolved in 67 mL of dichloromethane. A solution of trifluoro-
acetic acid (2.25 mL, 13.4 mmol) in 34 mL of dichloromethane
was added dropwise over several minutes. The reaction mixture
was then stirred at room temperature for 1 h. After completion,
the reaction was quenched with 67 mL of H2O, and the layers
were separated. The aqueous layer was extracted with dichloro-
methane (48 mL, 3ꢀ). The combined organics were washed
with H2O (60 mL), 10% HCl (60 mL), and brine (60 mL), dried
over anhydrous Na2SO4, and concentrated to afford 19 as a red
oil (3.33 g, 92%). The product was taken onto the next step
without further purification: TLC Rf = 0.76 (9:1 Hex:EtOAc).
1H NMR (CDCl3) δ 7.20 (d, J = 3.3,1 H), 6.5 (d, J = 6.7, 1H),
6.82-6.80 (dq, J = 6.7, 1 H), 5.97-5.91 (m, 1H), 5.21 -5.16 (m,
2H), 3.83 (s, 3H), 3.48 (d, J = 5.0, 2H). 13C NMR (CDCl3) δ
159.0, 141.4, 134.3, 122.3, 119.9, 117.6, 117.4, 116.2, 114.8,
112.8, 55.6, 34.2. HRMS (FAB, Mþ) m/z 297.0403 (calculated
for C11H11F3O4S, 297.0364).
2-Allyl-4methoxybiphenyl (20). To an oven-dried sealed tube
was added triflate 19 (1.50 g, 5.0 mmol), phenylboronic acid
(778.5 mg, 5.6 mmol), Pd(P(Ph)3)4 (151.8 mg, 0.13 mmol),
K3PO4 (1.61 g, 7.6 mmol), and anhydrous dioxane (25.2 mL).
The mixture was stirred and then degassed once using the freeze/
pump/thaw method. Once the mixture warmed to room tem-
perature, the vessel was sealed under argon and placed in an
85 ꢀC oil bath for 48 h. The dark-colored mixture was cooled,
diluted with ether (10 mL), and filtered through a pad of silica
gel (∼20 g), rinsing with ether. The filtrate was concentrated and
the residue purified by flash chromatography (SiO2, 94 g, 1%
EtOAc/hexanes) to afford the olefin 20 as a colorless oil (1.13 g,
99%): TLC Rf = 0.68 (9:1 Hex: EtOAc). 1H NMR (CDCl3) δ
7.49 (t, J = 5.9, 2 H), 7.43 (t, J = 5.9, 3H), 7.31 (d, J = 5.9, 1H),
6.98 (d, J = 20.5, 1H), 6.94 (dd, J = 20.5, 1H), 6.01 (m, 1H), 5.16
(dd, J = 20.5, 1H), 5.10 (dd, J = 20.5, 1H), 3.90 (s, 1H), 3.45 (d,
J = 6.83, 2H). 13C NMR (CDCl3) δ 159.1, 141.5, 138.7, 137.6,
134.7, 131.2, 129.6, 128.1, 126.7, 116.0, 115.2, 111.6, 55.3, 37.8.
HRMS (FAB, Mþ) m/z 225.1274 (calculated for C16H16O,
225.1235).
4-Methoxy-2-(prop-2-ynyl)biphenyl (21). To a round-bottom
flask, olefin 20 (0.678 g, 3.0 mmol), OsO4 (21.6 mg, 0.058 mmol),
and NaIO4 (2.55 g, 11.9 mmol) were suspended in dioxane
(22 mL), and H2O (7.4 mL). The reaction mixture was stirred
for 1 h at room temperature. After 1 h, the reaction mixture was
diluted with H2O (35 mL) and Et2O (90 mL) and the layers were
separated. The aqueous layer was extracted with Et2O (45 mL,
3ꢀ). The combined organics were washed with brine (90 mL),
dried over anhydrous Na2SO4, and concentrated to afford the
crude aldehyde.
product that was purified by flash chromatography (SiO2,
75 g, 1% EtOAc/hexanes) to afford alkyne 21 as a colorless oil
1
(0.2009 g, 38%): TLC Rf = 0.69 (9:1 Hex:EtOAc). H NMR
(CDCl3) δ 7.50 (m, 2 H), 7.44 (m, 3H), 7.35 (s, 1H), 7.28 (m, 1H),
6.97 (m, 1H), 3.94 (s, 3H), 3.61 (s, 2H), 2.27 (s, 1H). 13C NMR
(CDCl3) δ 159.3, 140.8, 135.1, 134.1, 131.2, 129.4, 128.4, 128.1,
127.0, 114.5, 112.5, 82.6, 55.4, 23.4. HRMS (FAB, Mþ) m/z
223.1105 (calculated for C16H15O, 223.1117).
2,4-Diamino-5-[3-(2-phenyl-5-methoxyphenyl)prop-1-ynyl]-6-
ethylpyrimidine (12). To an oven-dried sealed tube was added
2,4-diamino-6-ethyl-5-iodopyrimidine (97.8 mg, 0.37 mmol),
Pd(PPh3)2Cl2 (29.3 mg, 0.037 mmol), CuI (15.0 mg, 0.080 mmol),
and acetylene 21 (189.2 mg, 0.74 mmol). Degassed (argon purge)
anhydrous DMF and triethylamine (0.53 mL each) were added,
the tube was sealed, and the mixture degassed by one cycle of
freeze-pump-thaw. The mixture was stirred at 60 ꢀC for 18 h
and then added to a separatory funnel containg EtOAc (16 mL).
The organic layer was washed twice with a water/saturated
NaHCO3 solution (1:2, 16 mL) and then brine (16 mL). The
organic layer was then dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by flash
chromatography (SiO2, 100 g, EtOAc) to afford the coupled
product 12 as a white powder (68.7 mg, 51.4%). An analytical
sample was obtained by crystallization from EtOAc. TLC Rf =
0.29 (EtOAc); mp =195.2-196.6 ꢀC. 1H NMR (CDCl3) δ 7.46
(t, J = 6.3, 2 H), 7.38 (t, J = 6.25, 3H), 7.22 (m, 2H), 6.90 (dd,
J = 5.0, 1H), 3.89 (s, 3H), 3.81 (s, 2H), 2.68 (q, J = 5.0, 2H), 1.24
(t, J = 5.0, 3H). 13C NMR (CDCl3) δ 164.3, 160.6, 159.2, 140.8,
135.7, 134.0, 131.2, 129.4, 128.4, 128.0, 127.0, 114.3, 112.3, 96.9,
90.7,75.5, 55.4, 29.7, 24.9, 12.6. HRMS (FAB Mþ) m/z 359.1857
(calculated for C22H23N4O, 359.1866); HPLC (a) tR = 11.32
min, 96.8%, (b) tR = 33.62, 96.9%
2-(5-Methoxybiphenyl-3-yl)acetaldehyde (23). To a 0 ꢀC
suspension of methoxymethyltriphenylphosphonium chloride
(3.50 g, 10.2 mmol) in dry THF (30.5 mL) under an argon
atmosphere was added NaOtBu (1.45 g, 15.1 mmol) in one
portion. The red-orange suspension was stirred for a further
0.5 h at 0 ꢀC. Then a solution of 3-methoxy-5-phenylbenzalde-
hyde 22 (1.23 g, 5.8 mmol) in THF (10.0 mL) was added
dropwise. Following 20 min, the reaction was quenched with
water (97.5 mL) and diluted with ether (60 mL). The organic
phase was separated and the aqueous phase extracted with
additional ether (3 ꢀ 97.5 mL). The combined extracts were
washed with brine (60 mL), dried over Na2SO4, and concen-
trated to afford the crude product that was filtered through a
column of silica (90 g, 2-5% EtOAc/hexanes) to afford 1.31 g of
a mixture of enol ethers (E/Z ≈ 60:40) that were immediately
hydrolyzed in the subsequent step.
To a solution of enol ether (1.31 g, 5.44 mmol) in THF (34 mL)
was added concentrated HCl (2.3 mL). The solution was warmed
to 55 ꢀC and stirred for 1 h. After 1 h, TLC showed the starting
material had been consumed. The mixture was cooled and
diluted with water and ether (88 mL each). The organic phase
was separated and the aqueous phase extracted with additional
ether (2 ꢀ 88 mL). The combined extracts were washed with
saturated NaHCO3 (88 mL) and brine (88 mL), dried over
Na2SO4, and concentrated to afford the crude product that
was purified by flash chromatography (SiO2, 140 g, 5% EtOAc/
hexanes) to afford aldehyde 23 as a colorless oil (0.3343 g,
26.8%): TLC Rf = 0.13 (9:1 Hex:EtOAc). 1H NMR (CDCl3) δ
9.81 (s, 1H), 7.63 (d, J = 7.6, 2H), 7.49 (t, J = 7.6, 2H), 7.41 (t,
J = 7.6, 1H), 7.12 (s, 1H), 7.08 (s, 1H), 6.80 (s, 1H), 3.90 (s, 3H),
3.74 (s, 2H). 13C NMR (CDCl3) δ 199.2, 160.5, 143.5, 140.7,
133.7, 128.9, 127.7, 127.3, 121.0, 114.1, 111.9, 55.5, 50.7. HRMS
(FAB, Mþ) m/z 227.1062 (calculated for C15H15O2, 227.1067).
3-Methoxy-5-(prop-2-ynyl)biphenyl (24). To a 0 ꢀC solution of
aldehyde 23 (0.334 g, 1.46 mmol) in MeOH (2.0 mL) was added
the Ohira-Bestmann reagent (0.831 g, 4.38 mmol) dissolved in
MeOH (3.0 mL) followed by powdered, anhydrous K2CO3-
(0.340 g, 2.46 mmol). The mixture was stirred at 0 ꢀC for 2 h.
To a 0 ꢀC solution of the crude aldehyde in MeOH (3.9 mL)
was added the Ohira-Bestmann reagent (1.70 g, 9.0 mmol)
dissolved in MeOH (6.6 mL) followed by powdered, anhydrous
K2CO3(0.705 g, 5.11 mmol). The mixture was stirred at 0 ꢀC for
2 h. After 2 h, MeOH was removed under vacuum. The residue
was dissolved in water and ether (50 mL each). The organic
phase was separated and the aqueous phase extracted with
additional ether (3 ꢀ 50 mL). The combined extracts were
washed with saturated NaHCO3 (50 mL) and brine (50 mL),
dried over Na2SO4, and concentrated to afford the crude

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7335
After 2 h, MeOH was removed under vacuum. Residue was
dissolved in water and ether (23 mL each). The organic phase
was separated and the aqueous phase extracted with additional
ether (3 ꢀ 23 mL). The combined extracts were washed with
saturated NaHCO3 (23 mL) and brine (23 mL), dried over
Na2SO4, and concentrated to afford the crude product that
was purified by flash chromatography (SiO2, 30 g, 1% EtOAc/
hexanes) to afford alkyne 24 as a colorless oil (0.1987 g, 61.2%):
TLC Rf = 0.42 (9:1 Hex:EtOAc). 1H NMR (CDCl3) δ 7.71 (d,
J = 8.0, 2H), 7.55 (t, J = 8.0, 2H), 7.48 (t, J =8.0, 1H), 7.30 (s,
1H), 7.15 (s, 1H), 7.05 (s, 1H), 3.95 (s, 3H), 3.75 (s, 2H), 2.36 (s,
1H). 13C NMR (CDCl3) δ 160.3, 143.0, 141.1, 138.1, 128.9,
127.7, 127.3, 119.5, 112.5,111.4, 81.9, 71.0, 55.4, 25.0. HRMS
(FAB, Mþ) m/z 223.1115 (calculated for C16H15O, 223.1117).
2,4-Diamino-5-[3-(2-methoxy-5-phenylphenyl)prop-1-ynyl]-6-
ethylpyrimidine (13). To an oven-dried sealed tube was added
2,4-diamino-6-ethyl-5-iodopyrimidine (117.9 mg, 0.45 mmol),
Pd(PPh3)2Cl2 (31.6 mg, 0.045 mmol), CuI (8.5 mg, 0.045 mmol),
and acetylene 24 (198.7 mg, 0.89 mmol). Degassed (argon purge)
anhydrous DMF and triethylamine (2.25 mL each) were added,
and the tube was sealed and the mixture degassed by one cycle of
freeze-pump-thaw. The mixture was stirred at 60 ꢀC for 18 h
and then added to a separatory funnel containg EtOAc (17 mL).
The organic layer was washed twice with a water/saturated
NaHCO3 solution (1:2, 7 mL) and then brine (7 mL). The
organic layer was then dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by flash chromato-
graphy (SiO2, 15 g, EtOAc) to afford the coupled product 13 as a
light-yellow powder (103.9 mg, 64.4%). An analytical sample
was obtained by dissolving in chloroform and precipitation with
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1
hexane. TLC Rf = 0.23 (EtOAc); mp = 156.7-158.5 ꢀC. H
NMR (CDCl3) δ 7.61 (d, J = 6.6, 2 H), 7.47 (t, J = 6.6, 2H), 7.38
(t, J = 6.6, 1H), 7.24 (s, 1H), 7.05 (s, 1H), 6.98 (s, 1H), 5.22(s,
2H), 4.95 (s, 2H), 3.96 (s, 2H), 3.90 (s, 3H), 2.75 (q, J = 6.4, 2H),
1.26 (t, J = 6.4, 3H). 13C NMR (CDCl3) 173.4, 164.5, 160.8, 160.3,
143.0, 140.9, 138.8, 128.8, 127.6, 127.1, 119.3, 112.4, 111.2, 96.2,
90.5, 75.8, 55.4, 29.6, 26.4, 12.7. HRMS (FAB, Mþ) m/z
359.1858 (calculated for C22H23N4O, 359.1856). HPLC (a) tR =
11.28 min, 98.2%, (b) tR = 33.14, 98.2%.
(10) Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibition
constant (KI) and the concentration of inhibitor which causes 50%
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Supporting Information Available: Tables of measured IC50
values, nonlinear regression plots for determination of enzyme
kinetic parameters, omit density maps, NMR spectra, and
HPLC purity data. This material is available free of charge via
the Internet at http://pubs.acs.org.
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