Inhibition of Bacterial Dihydrofolate Reductase by 6-Alkyl-2,4-diaminopyrimidines

Article abstract of DOI:10.1002/cmdc.201200291

(±)-6-Alkyl-2,4-diaminopyrimidine-based inhibitors of bacterial dihydrofolate reductase (DHFR) have been prepared and evaluated for biological potency against Bacillus anthracis and Staphylococcus aureus. Biological studies revealed attenuated activity relative to earlier structures lacking substitution at C6 of the diaminopyrimidine moiety, though minimum inhibitory concentration (MIC) values are in the 0.125-8μgmL^-1 range for both organisms. This effect was rationalized from three- dimensional X-ray structure studies that indicate the presence of a side pocket containing two water molecules adjacent to the main binding pocket. Because of the hydrophobic nature of the substitutions at C6, the main interactions are with protein residues Leu20 and Leu28. These interactions lead to a minor conformational change in the protein, which opens the pocket containing these water molecules such that it becomes continuous with the main binding pocket. These water molecules are reported to play a critical role in the catalytic reaction, highlighting a new area for inhibitor expansion within the limited architectural variation at the catalytic site of bacterial DHFR.

Full text of DOI:10.1002/cmdc.201200291

MED
DOI: 10.1002/cmdc.201200291
Inhibition of Bacterial Dihydrofolate Reductase by 6-Alkyl-
2,4-diaminopyrimidines
Baskar Nammalwar,[a] Christina R. Bourne,*[b] Richard A. Bunce,*[a] Nancy Wakeham,[b]
Philip C. Bourne,[b] Kal Ramnarayan,[c] Shankari Mylvaganam,[c] K. Darrell Berlin,[a]
Esther W. Barrow,[b] and William W. Barrow[b]
(Æ)-6-Alkyl-2,4-diaminopyrimidine-based inhibitors of bacterial
dihydrofolate reductase (DHFR) have been prepared and evalu-
ated for biological potency against Bacillus anthracis and
Staphylococcus aureus. Biological studies revealed attenuated
activity relative to earlier structures lacking substitution at C6
of the diaminopyrimidine moiety, though minimum inhibitory
concentration (MIC) values are in the 0.125–8 mgmLÀ1 range
for both organisms. This effect was rationalized from three-
dimensional X-ray structure studies that indicate the presence
of a side pocket containing two water molecules adjacent to
the main binding pocket. Because of the hydrophobic nature
of the substitutions at C6, the main interactions are with pro-
tein residues Leu20 and Leu28. These interactions lead to
a minor conformational change in the protein, which opens
the pocket containing these water molecules such that it be-
comes continuous with the main binding pocket. These water
molecules are reported to play a critical role in the catalytic re-
action, highlighting a new area for inhibitor expansion within
the limited architectural variation at the catalytic site of bacte-
rial DHFR.
Introduction
It is well known that the arsenal of available antibiotics is not
sufficient to meet the growing burden of bacterial resistance
and that fewer efforts are being made at a pharmaceutical
level to pursue new substances with antibiotic properties.[1–3]
We have combined this need with that of biodefense in repur-
posing the antifolate target dihydrofolate reductase (DHFR,
EC.1.5.1.3). Our recently reported library of dihydrophthalazine-
containing compounds has demonstrated potency against
DHFR in Bacillus anthracis.[4–6] We are also monitoring the
broad-spectrum potency of this series, and in particular, look-
ing for anti-staphylococcal activity. Staphylococcus aureus is
a prominent human pathogen with increasing antibiotic resist-
ance, such as methillicin- and vancomycin-resistant forms
(MRSA and VRSA, respectively).[7,8] Previous work from our
group demonstrated a conserved potency of our initial com-
pound RAB1 (1a) against S. aureus 29213, four MRSA strains
and three VRSA strains.[9] Compound 1b was also found to be
highly active. We have now attempted to enhance the broad-
spectrum potency of this series by making use of molecular
modeling techniques interfaced with chemistry, microbiology
and biochemistry, and X-ray crystallography.
The previously determined B. anthracis DHFR co-crystal
structure in complex with 1a revealed a preference for the
S-enantiomer at the single chiral center bearing a propyl
moiety.[5] This is in contrast to the co-crystal structure of
S. aureus DHFR with 1a, which revealed an alternate position
for the dihydrophthalazine portion of the inhibitor containing
the chiral center. Binding limited one position to the S-enantio-
mer, while the alternate position residing in a shallow surface
pocket could accommodate either enantiomer.[9] The binding
site residues in contact with RAB1 (1a) show little variation in
sequence identity between the S. aureus and B. anthracis spe-
cies.[5,10] However, the shape of the binding site in S. aureus
DHFR is generally wider than that of B. anthracis DHFR, allow-
ing formation of the secondary binding surface, which we ob-
served to be influenced by residue changes on the periphery
of the site. These changes include a loss of an aromatic stack-
ing interaction with Phe151 due to a His residue at position 30
in contrast to a Phe or Tyr, as well as loss of electrostatic stabi-
lization arising from Tyr to Phe changes at residues 47 and
68.[10] These observations highlighted how residues from out-
[a] Dr. B. Nammalwar, Prof. R. A. Bunce, Prof. K. D. Berlin
Department of Chemistry, Oklahoma State University
Stillwater, OK 74078 (USA)
E-mail: rab@okstate.edu
[b] Dr. C. R. Bourne, Dr. N. Wakeham, Dr. P. C. Bourne, Dr. E. W. Barrow,
Prof. W. W. Barrow
Department of Veterinary Pathobiology, Oklahoma State University
Stillwater, OK 74078 (USA)
E-mail: christina.bourne@okstate.edu
[c] Dr. K. Ramnarayan, Dr. S. Mylvaganam
Sapient Discovery Technologies
10929 Technology Place, San Diego, CA 92127 (USA)
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side of the binding pocket could modulate interactions and, in
this case, allow either enantiomer to be bound in S. aureus
DHFR versus just the S-enantiomer as complexed with B. an-
thracis DHFR.[5]
Despite these differences between the bacterial species, the
diaminopyrimidine (DAP) moiety, which is well conserved
among anti-DHFR inhibitors, complexes the equivalent protein
side chains within the binding site.[5,10] The arrangement of hy-
drogen-bond participants around the DAP ring allows for pre-
cise placement amid surrounding amino acid side chains and
requires participation of both the side chain and the main
chain from the protein, regardless of species. Examination of
the structures of B. anthracis and S. aureus DHFR highlighted
a small unoccupied pocket of ~50 ꢁ3 adjacent to the C6 posi-
tion of the DAP ring (Figure 1). In efforts to explore the poten-
tial occupancy of this pocket, a series of DAP-based antifolates
bearing methyl, ethyl, and propyl groups at C6 of the DAP
moiety were designed, synthesized, and evaluated for potency
against bacterial DHFR. A similar approach has been reported
for the antimalarial compound pyrimethamine, which has poor
antibacterial activity due to differences in the plasmodial and
bacterial DHFR protein sequences,[10,11] as well as a series of
antibacterial propargyl-linked DHFR inhibitors.[12]
Results and Discussion
Chemistry
The synthetic strategy started with 5-iodovanillin (2), which led
to compounds 3–8, and with 9, which gave (Æ)-10 (Scheme 1).
Compounds 8 and (Æ)-10a–b were then coupled to generate
racemic 11a–c and 12a–c (Scheme 2).
Since both 1a and 1b demonstrated excellent inhibitory
properties in our previous studies,[5,6] the propyl- and isobuten-
yl-substituted compounds 11a–c and 12a–c were targeted.
The synthesis of DAP derivatives 8a–c involved a seven-step
sequence from commercially available 5-iodovanillin (2). Meth-
ylation of the phenolic hydroxy in 2, using potassium carbon-
ate and dimethyl sulfate in N,N-dimethylformamide (DMF), fur-
Scheme 1. Synthesis of 8a–c and 10a–b. Reagents and conditions:
a) (CH3O)2SO2, K2CO3, DMF, 1008C, 96% yield; b) NaBH4, THF, 238C, 99%
yield; c) PBr3, Et2O, 238C, 96% yield; d) RCOCH2CO2Et, NaOMe, EtOH, 788C,
[a: R’=CH3, b: R’=CH2CH3, c: R’=CH2CH2CH3], 60–68% yield; e) [H2N=
C(NH2)2]2CO3, EtOH, 788C, 75–80% yield; f) POCl3, 1068C; g) NH3, EtOH,
1658C, 72–75% yield (two steps); h) RMgBr, THF, 508C [a: R=CH2CH2CH3,
b: R=CH=C(CH3)2]; i) CH2=CHCOCl, Et3N, THF, 08C, 40–50% yield (two steps).
Figure 1. A pocket adjacent to the diaminopyrimidine (DAP) moiety is present in bacterial dihydrofolate reductase (DHFR) proteins and is occupied by two
water molecules. Surface renderings of the DHFR protein from a) B. anthracis and b) S. aureus, both with compound 1a,[5,10] and c) S. aureus complexed with
compound 12c. Surfaces are colored based on Kyte–Doolittle hydrophobicity calculations, where orange is hydrophobic and green is polar. The pocket of
interest is continuous with the substrate site only at the DAP ring position; above this, it is gated by opposing Leu residues (20 and 28). In panel c, the move-
ment of Leu28 results in a complete opening of this pocket with the substrate binding site. In panel b, the alternate position for the dihydrophthalazine
moiety of 1a is shown. In all panels, the position of the co-factor NADPH can be seen at the bottom of the substrate site and, in this view, to the right of it;
NADPH is not involved in the pocket or interactions with the alkyl substituents.
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nished iododimethoxybenzaldehyde 3. Further conversion of 3
by reduction with sodium borohydride gave alcohol 4, and
treatment with phosphorus tribromide produced bromide 5.
Reaction of 5 with a series of b-keto esters, in the presence of
sodium methoxide in ethanol, led to the benzyl-substituted
keto esters 6a–c. Cyclization of 6a–c was accomplished in
good yields using guanidine carbonate in boiling ethanol to
give hydroxypyrimidines 7a–c, which were used directly in the
next sequence. These 4-hydroxypyrimidines were converted to
diaminopyrimidines 8a–c in two steps by refluxing the former
in phosphorus oxychloride, followed by treatment with etha-
nolic ammonia at 1658C. A similar strategy has been reported
previously[13] but did not involve an iodine-substituted sub-
strate. In the current work, this sequence proceeded cleanly to
give 8a–c in 80–91% yields (see Scheme 1).
value,[14] allowing for direct comparison despite the different
enzymatic efficiencies of B. anthracis DHFR and S. aureus DHFR
(Table 1). A general trend of more potent anti-staphylococcal
activity was maintained with Ki values generally four-times
lower for S. aureus than for B. anthracis. Substitutions at the C6
position of DAP increased the Ki value for each species, mark-
ing them as less effective inhibitors than the parent com-
pounds, and a decrease in potency (higher Ki) was progressive
with substitution length. In some instances, the limits of solu-
bility precluded determination of the IC50 value (such as with
11 b, 11 c and 12c), and values are listed as greater than the
highest concentration tested.
Table 1. Biological activity of 6-alkyl-DAP inhibitors against bacterial
strains B. anthracis Sterne and S. aureus 29213.
The dihydrophthalazine coupling partners (Æ)-10a–b were
obtained by reacting phthalazine (9) with the propyl and iso-
butenyl Grignard reagents, followed by N-acylation with acrylo-
yl chloride in the presence of triethylamine as described in ear-
lier reports.[5,6]
[b]
Ki [nm] (SEM)[a]
MIC [mgmLÀ1
]
B. anthracis
Compd
B. anthracis
S. aureus
S. aureus
29213
Sterne
29213
Sterne
1a
11 a
11 b
11 c
1b
12a
12b
12c
9.4 (0.2)
72.4 (0.6)
>2400
1.2 (0.1)
4.5 (0.1)
3.6 (0.1)
4.0 (0.1)
1.8 (0.1)
3.4 (0.1)
2.9 (0.1)
4.1 (0.1)
1
8
4
1
0.125–0.25
0.5
0.5–2
Final coupling reactions involving 8a–c and 10a–b were ac-
complished using a palladium acetate-promoted Heck cou-
pling in the presence of N-ethylpiperidine in anhydrous DMF
at 1408C (see Scheme 2). Purification of the products by flash
chromatography, followed by recrystallization, afforded the
final racemic compounds 11a–c and 12a–c in yields ranging
from 78–85%. Spectral and elemental analyses of these materi-
als revealed that each existed as a solvate of water and/or
ethanol.
>2400
1–2
8.3 (0.2)
14.3 (0.2)
25.4 (0.8)
>2500
0.5
4
0.125–0.25
0.25–0.5
0.25–0.5
2
4
2
[a] The Ki value is calculated from IC50 measurements with compensatory
weight based on the Km value for the dihydrofolate substrate. These
values are listed with the standard error of the mean (SEM) and represent
the mean of three or more experiments. [b] The minimum inhibitory con-
centration (MIC) is the concentration of inhibitor needed to stop all bac-
terial growth under the defined conditions (range of values, n=4).
These compounds were also incubated in cultures of the
two bacterial organisms to determine the minimum inhibitory
concentration (MIC) required to inhibit bacterial growth
(Table 1), which is a useful parameter for consideration in clini-
cal applications. The MIC values for S. aureus progressively in-
creased 10-fold between parent (1a and 1b) versus the C6
propyl substitution (11 c and 12c), indicating decreased poten-
cy within the cellular system. Interestingly, for B. anthracis, the
MIC values follow the same trend of progressively decreasing
potency only for the compounds bearing an isobutenyl at the
chiral center (1b, 12a–c), which is 9 ꢁ from the C6 position.
The other series, containing a propyl at the chiral center, dis-
plays the same MIC value and thus potent inhibition for the
parent (1a) as for the C6 propyl substitution 11 c. However,
the two intermediate substitutions, 11 a and 11 b, show an in-
crease in MIC value in excess of any expected experimental
variation. This is difficult to rationalize in terms of structural el-
ements as the Ki values do not reflect the same trend. Since
the MIC measurements are in the context of whole cells, they
require the compounds to cross cell membranes, and there are
additional factors in the MIC assay that are absent in the Ki
measurements. These factors clearly influence the resulting ac-
tivity, and consequently another explanation could involve
cross-reactivity with other member enzymes of the folate path-
way, although this has not been demonstrated.
Scheme 2. Synthesis of 11a–c and 12a–c. Reagents and conditions: a) (Æ)-
10a, Pd(OAc)2, N-ethylpiperidine, DMF, 1408C, 81–84% yield; b) (Æ)-10b,
Pd(OAc)2, N-ethylpiperidine, DMF, 1408C, 78–84% yield
Biological Activity
Assessment of the parent compounds and inhibitors with alkyl
substituents at C6 of the DAP moiety were carried out with pu-
rified DHFR in an enzyme assay to gauge the level of effective-
ness at competing with the endogenous substrate, dihydrofol-
ic acid. The resulting IC50 values were incorporated with the
enzyme Km value for the substrate in order to calculate a Ki
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X-ray Data
sites” in protein–ligand binding, analogous to the currently
identified water molecules.[15] Calculated energetics indicate
the liberation of these two water molecules would require
~4 kcalmolÀ1; this inhibitor series generally displays binding
energies of 42–44 kcalmolÀ1 and are derived largely from
van der Waals interactions. These highly conserved water mole-
cules have been seen in previous DHFR structures and are well
characterized within the E. coli system. Such, water molecules
are required for positioning the folate substrate relative to the
nicotinamide co-factor, in addition to coordinating the critical
catalytic residue Asp27 to maintain optimum reaction geome-
try.[16,17] Trp22 is a secondary residue that also coordinates the
water molecules, and it is not clear whether its role is strictly
geometric or if it assists in perturbation of the local pKa as
needed to promote protonation of the substrate via Asp27. It
is also suggested that these two conserved waters might pro-
vide the catalytic proton, supporting their invariant positions
among all DHFR structures known to date and their impor-
tance to the protein structure.
X-ray crystallography was used to visualize the DAP modifica-
tions within the binding site of S. aureus DHFR for complexes
with 12b and 12c (Table 2). Similar experiments with B. anthra-
cis DHFR were not successful, as this protein has been ob-
served to crystallize poorly or not at all in the absence of
a tightly bound compound in the substrate site (unpublished
observation). Due to the high degree of similarity of residues
for each DHFR involved in binding, it is feasible to analyze the
S. aureus DHFR co-crystal structure to draw generalizations
about the mode of binding. The resulting electron density
maps, obtained prior to crystallographic refinement with the
inhibitor, clearly show the positions of the appended atoms on
the DAP ring within the S. aureus DHFR binding site (Fig-
ure 2a,b). For either 12b or 12c, the placement within the
pocket is as expected, and contacts are conserved from those
previously visualized with 1a.[9] However, the substitutions at
C6 complex along the hydrophobic edge of the identified
pocket (Figure 1c). These did not protrude into the adjacent
50 ꢁ3 pocket, where two highly conserved water molecules are
located.
The C6 alkyl group on each structure is sandwiched be-
tween the face of the central dimethoxybenzene ring of the in-
hibitor, residues Leu20, Asp27 and Leu28, and the two highly
conserved water molecules (Figure 2c,d). In addition, for the
12c complex, a rotameric change in side chain position of
Leu28 is necessary to accommodate the inhibitor (Figure 1c).
As a consequence, this movement alters the position of the
distal end of the inhibitor by ~0.7 ꢁ. The energetic costs of
this different arrangement likely play a role in the decreased
potency. Differences in biological activity between the two
species probably arise due to structural elements that influ-
ence positioning at the opposite end of the inhibitor (the dihy-
drophthalazine containing the chiral center, ~9 ꢁ away). There-
fore, when relating these observations to the B. anthracis DHFR
protein, the same mode of binding is expected, including the
maintenance of the two water molecules within the pocket.
Overall, these observations highlight the relative ease in reor-
dering hydrophobic interactions, which are not dependent on
the direction of atomic interactions, rather than displacing the
specific hydrogen bonds mediated by the water molecules.
Furthermore, the rotameric movement of Leu28 in the com-
plex with 12c revealed a continuous opening of the pocket
adjacent to the inhibitor binding pocket (Figure 1c). This could
prove very advantageous for further inhibitor design and de-
fines a minimum alkyl chain length to achieve this result.
Other computational and experimental studies have demon-
strated the important role of water at so-called “hydration
Table 2. Statistics for macromolecular X-ray data collection and refine-
ment of S. aureus DHFR+NADPH co-crystallized with compounds (Æ)-
12b,c.
(Æ)-12b
4FGH
(Æ)-12c
4FGG
PDB code[a]
Data Collection
Space Group
Cell dimensions
a, b, c [ꢁ]
a, b, g [8]
Resolution [ꢁ]
Rint
P6122
P6122
78.9, 78.9, 106.2
90, 90, 120
Inf - 2.50 (2.55–2.50)
16.0 (48.4)
19.6 (5.5)
79.0, 79.0, 107.5
90, 90, 120
Inf - 2.30 (2.36–2.30)
14.4 (50.4)
22.8 (4.8)
I/sI
Completeness [%]
Redundancy
Mosaicity
100 (100)
36.0 (26.0)
0.74
100 (100)
34.6 (23.6)
0.66
Wilson B-factor
34.0
24.8
Refinement
Resolution [ꢁ]
Rwork/Rfree
68.4–2.5
19.2/24.6
68.4–2.3
19.7/24.5
Conclusions
Protein atoms
Ligand/NADPH atoms
Water atoms
1324
39/48
69
1330
40/48
143
Alkyl extensions have been appended to the DAP moiety of
propyl- and isobutenyl-substituted, dihydrophthalazine-based
DHFR inhibitors, and the activities of the resultant compounds
have been evaluated. Testing the potency against B. anthracis,
a known bioterrorism agent, and S. aureus, a prominent
human pathogen, revealed modestly decreased potency
versus systems lacking substitution at C6 on the pyrimidine.
This effect is rationalized based on the three-dimensional struc-
tures, which highlight the conservation of two water molecules
within a small protein cavity adjacent to the main binding site.
B factor, protein
B factor, Ligand/NADPH
B factor, water
23.5
32.7/24.2
30.1
17.4
26.6/14.4
24.1
R.M.S.D. bond lengths [ꢁ]
R.M.S.D. bond angles [8]
0.008
1.184
0.008
1.225
[a] The structures have been deposited in the RCSB Protein Data Bank
(PDB) and are available from www.rcsb.org.
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Figure 2. Position and interactions of 6-alkyl substitutions within the binding site of S. aureus DHFR. The electron densities for inhibitors a) 12b and b) 12c,
prior to crystallographic refinement with ligand in place, is depicted. Protein and ligand are shown as ball-and-stick models. Electron density is rendered as
mesh with a 2Fo–Fc (blue) map at 1.2 s and an Fo–Fc (green) map at 3.0 s. These maps clearly indicate the position of the alkyl extensions in the absence of
any model bias. Panels c and d display distance measurements between atoms of inhibitor 12c (yellow carbon atoms, blue nitrogen atoms and red oxygen
atoms) and protein side chains Leu20, Trp22, Asp27, and Leu28 (colored as 12c but with grey carbon atoms). The two water molecules discussed in the text
are also depicted. The NADPH co-factor is visible behind the inhibitor at the top of the figure; NADPH is not involved in binding at the site of the 6-alkyl
extensions.
wise specified, all reactions were run under dry N2 in oven-dried
glassware. Reactions were monitored by thin layer chromatogra-
phy (TLC) on silica gel GF plates (Analtech; cat. no. 21521). Prepara-
tive separations were performed by flash column chromatogra-
phy[18] on silica gel (Davisil grade 62, 60–200 mesh) mixed with UV-
active phosphor (Sorbent Technologies; cat. no. UV-05). Band elu-
tion for all chromatographic separations was monitored using
a hand-held UV lamp. Melting points were recording on a Mel-
temp apparatus and are uncorrected. IR spectra were run as thin
films on NaCl disks using a Varian 800 FT-IR instrument. 1H and
13C NMR spectra were measured on a Varian GEMINI 300 instru-
ment at 300 MHz (1H) and 75 MHz (13C), and chemical shifts (d) are
referenced to internal tetramethylsilane (TMS). Elemental analyses
were performed by Atlantic Microlab, Inc. (Norcross, USA).
These water molecules are believed to play a critical role in the
catalytic reaction and are not displaced by these inhibitors.
The longest extension tested, a propyl group, was the mini-
mum length needed to completely open this side pocket to
the main binding site, which may be advantageous in the
design of more efficacious agents targeting bacterial DHFR.
Experimental Section
Chemistry
General: Commercial anhydrous N,N-dimethylformamide (DMF)
and dimethyl sulfoxide (DMSO) were stored under dry N2 and
transferred by syringe into reactions when required. Tetrahydrofur-
an (THF) was dried over KOH pellets and distilled from LiAlH4 prior
to use. K2CO3 was heated at 1208C under high vacuum for
a period of 16 h and stored in an oven at 908C before use. All
other commercial reagents were used as received. Unless other-
5-Iodo-3,4-dimethoxybenzaldehyde (3): This compound was pre-
pared from 2 on a 0.27 mol scale using the method of Nimgira-
wath.[19] The crude product was recrystallized (4:1 EtOH/water) to
give 3 as a white solid (25.2 g, 96%): mp: 71–728C (lit. mp: 71–
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728C[19]); 1H NMR (300 MHz, CDCl3): d=9.83 (s, 1H), 7.85 (d, J=
1.7 Hz, 1H), 7.41 (d, J=1.7 Hz, 1H), 3.93 (s, 3H), 3.92 ppm (s, 3H);
13C NMR (75 MHz, CDCl3): d=189.7, 154.2, 153.0, 134.7, 133.9, 111.0,
92.1, 60.7, 56.1 ppm; IR (NaCl): n˜ =2832, 2730, 2693 cmÀ1; To the
best of our knowledge, no IR or 13C NMR data for 3 have been
reported previously.
60.1, 55.9, 36.1, 33.2, 14.0, 7.5 ppm; IR (NaCl): n˜ =2823, 1740,
1714 cmÀ1
.
Ethyl 2-(3-iodo-4,5-dimethoxybenzyl)-3-oxohexanoate (6c): This
compound was prepared as above using ethyl butyrylacetate
(10.6 g, 67.0 mmol) in dry EtOH (70 mL), powdered NaOMe (3.63 g,
67.0 mmol), and 5 (20.0 g, 56.0 mmol, 0.84 equiv) to give 6c as
1
a colorless liquid (15.6 g, 64%): H NMR (300 MHz, CDCl3): d=7.15
(3-Iodo-4,5-dimethoxyphenyl)methanol (4): The method of
Chowdhury and co-workers was modified.[20] A solution of 3
(25.0 g, 85.0 mmol) in THF (100 mL) was treated portion-wise over
5 min with NaBH4 (1.91 g, 50.0 mmol), and the reaction was stirred
at RT for 45 min. The reaction mixture was quenched with saturat-
ed aq NH4Cl (100 mL) and extracted with EtOAc (3ꢂ125 mL). The
combined organic layers were washed with saturated aq NaCl
(100 mL), dried (MgSO4), filtered and concentrated under vacuum
to yield 4 as a thick colorless liquid (24.8 g, 99%): 1H NMR
(300 MHz, CDCl3): d=7.27 (d, J=1.6 Hz, 1H), 6.86 (d, J=1.6 Hz,
1H), 4.53 (s, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 2.83 ppm (br s, 1H);
13C NMR (75 MHz, CDCl3): d=152.4, 147.7, 139.0, 128.3, 111.2, 92.1,
(d, J=1.6 Hz, 1H), 6.70 (d, J=1.6 Hz, 1H), 4.16 (q, J=7.3 Hz, 2H),
3.82 (s, 3H), 3.79 (s, 3H), 3.74 (t, J=7.3 Hz, 1H), 3.06 (m, 2H), 2.55
(dt, J=17.6, 7.3 Hz, 1H), 2.36 (dt, J=17.6, 7.3 Hz, 1H), 1.56 (sextet,
J=7.3 Hz, 2H), 1.23 (t, J=7.3 Hz, 3H), 0.86 ppm (t, J=7.3 Hz, 3H);
13C NMR (75 MHz, CDCl3): d=204.2, 168.7, 152.2, 147.5, 136.3,
130.2, 113.5, 92.2, 61.4, 60.3 (2C), 55.8, 44.5, 33.0, 16.7, 14.0,
13.4 ppm; IR (NaCl): n˜ =2824, 1740, 1714 cmÀ1
.
2-Amino-5-(3-iodo-4,5-dimethoxybenzyl)-6-methylpyrimidin-4-ol
(7a): A solution of 6a (10.0 g, 24.6 mmol) in dry EtOH (30 mL) was
treated with guanidine carbonate (17.7 g, 98.5 mmol, 4.00 equiv),
and the reaction mixture was heated at reflux for 18 h. The EtOH
was evaporated to a minimal volume, ice-cold water (50 mL) was
added, and the reaction mixture was kept at 08C for 30 min to
give a white precipitate. The solid was filtered and washed thor-
oughly with water (100 mL) and Et2O (50 mL), and then dried
under high vacuum for 6 h to give 7a as a white solid (7.40 g,
63.9, 60.3, 55.8 ppm; IR (NaCl): n˜ =3392, 2824 cmÀ1
.
5-(Bromomethyl)-1-iodo-2,3-dimethoxybenzene (5): A stirred so-
lution of 4 (20.0 g, 68.0 mmol) in dry Et2O (100 mL) was cooled to
08C, and PBr3 (20.2 g, 7.0 mL, 74.8 mmol, 1.1 equiv) was added
dropwise over 20 min. After the addition, stirring was continued
with warming to 238C for an additional 30 min to ensure complete
conversion. The reaction mixture was quenched by dropwise addi-
tion of saturated aq NaHCO3 (200 mL) over a period of 45 min.
[Note: Quenching was done slowly to minimize frothing.] The
layers were separated, and the aqueous layer was extracted with
EtOAc (3ꢂ150 mL). The combined EtOAC and Et2O layers were
washed with saturated aq NaCl, dried (MgSO4), filtered and concen-
trated under vacuum to yield 5 as a pale yellow solid (23.2 g,
96%): mp: 64–658C; 1H NMR (300 MHz, CDCl3): d=7.37 (d, J=
1.6 Hz, 1H), 6.91 (d, J=1.6 Hz, 1H), 4.40 (s, 2H), 3.87 (s, 3H),
3.83 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=152.5, 149.0, 135.4,
1
75%): mp: 185–1868C; H NMR (300 MHz, DMSO-d6): d=7.54 (br s,
1H), 7.08 (s, 1H), 6.97 (s, 1H), 6.58 (br s, 2H), 3.76 (s, 3H), 3.65 (s,
3H), 3.59 (s, 2H), 1.98 ppm (s, 3H); 13C NMR (75 MHz, DMSO-d6):
d=169.6, 161.0, 158.4, 151.9, 145.9, 141.0, 128.6, 113.3, 108.6, 92.2,
59.7, 55.7, 29.8, 21.3 ppm; IR (NaCl): n˜ =3516–2358, 1654 cmÀ1
.
2-Amino-5-(3-iodo-4,5-dimethoxybenzyl)-6-ethylpyrimidin-4-ol
(7b): This compound was prepared as above using 6b (10.0 g,
23.8 mmol) and guanidine carbonate (17.7 g, 95.2 mmol,
4.00 equiv) in dry EtOH (30 mL) to give 7b as a white solid (7.70 g,
1
78%): mp: 190–1918C; H NMR (300 MHz, DMSO-d6): d=11.0 (br s,
1H), 7.07 (s, 1H), 6.94 (s, 1H), 6.54 (br s, 2H), 3.76 (s, 3H), 3.65 (s,
3H), 3.61 (s, 2H), 2.35 (q, J=7.1 Hz, 2H), 1.01 ppm (t, J=7.1 Hz,
3H); 13C NMR (75 MHz, DMSO-d6): d=167.2 (br), 163.8 (br), 153.9,
152.0, 146.2, 140.1, 128.6, 113.3, 109.0, 92.3, 59.8, 55.8, 28.8, 27.2
130.7, 113.4, 92.2, 60.4, 56.0, 32.3 ppm; IR (NaCl): n˜ =2827 cmÀ1
.
Ethyl 2-(3-iodo-4,5-dimethoxybenzyl)-3-oxobutanoate (6a): The
method of Chowdhury and co-workers was modified.[20] A solution
of ethyl acetoacetate (8.75 g, 8.57 mL, 67.0 mmol) in dry EtOH
(70 mL) was treated with powdered NaOMe (3.63 g, 67.0 mmol),
and the reaction mixture was warmed to 508C over 30 min. The
warm mixture was treated dropwise with 5 (20.0 g, 56.0 mmol,
0.84 equiv), and the reaction was heated at reflux for 18 h. After
cooling, the crude product was concentrated under vacuum and
purified on a 100 cm ꢂ 3 cm silica gel column (1:3 EtOAc/hexane)
to give 6a as a colorless liquid (15.4 g, 68%): 1H NMR (300 MHz,
CDCl3): d=7.16 (d, J=1.6 Hz, 1H), 6.71 (d, J=1.6 Hz, 1H), 4.17 (q,
J=7.3 Hz, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 3.74 (t, J=7.3 Hz, 1H), 3.06
(m, 2H), 2.23 (s, 3H), 1.25 ppm (t, J=7.3 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=201.9, 168.8, 152.3, 147.5, 136.2, 130.2, 113.5, 92.3, 61.6,
61.1, 60.3, 55.8, 32.9, 29.5, 14.0 ppm; IR (NaCl): n˜ =2828, 1736,
(br), 12.6 ppm; IR (NaCl): n˜ =3405–2390, 1666 cmÀ1
.
2-Amino-5-(3-iodo-4,5-dimethoxybenzyl)-6-propylpyrimidin-4-ol
(7c): This compound was prepared as above using 6c (10.0 g,
23.0 mmol) and guanidine carbonate (16.6 g, 92.2 mmol,
4.00 equiv) in dry EtOH (30 mL) to give 7c as a white solid (7.90 g,
1
80%): mp: 194–1958C; H NMR (300 MHz, DMSO-d6): d=7.16 (br s,
1H), 7.08 (s, 1H), 6.95 (s, 1H), 6.71 (br s, 2H), 3.76 (s, 3H), 3.65 (s,
3H), 3.61 (s, 2H), 2.29 (t, J=7.3 Hz, 2H), 1.45 (sextet, J=7.3 Hz,
2H), 0.84 ppm (t, J=7.3 Hz, 3H); 13C NMR (75 MHz, DMSO-d6): d=
169.0, 164.2, 157.8, 151.9, 145.9, 141.3, 128.7, 113.3, 108.6, 92.2,
59.8, 55.7, 35.9, 29.4, 21.4, 14.0 ppm; IR (NaCl): n˜ =3520–2320,
1654 cmÀ1
.
1716 cmÀ1
.
5-(3-Iodo-4,5-dimethoxybenzyl)-6-methylpyrimidine-2,4-diamine
(8a): A mixture of 7a (6.00 g, 15.0 mmol) in POCl3 (15 mL) was
heated at reflux for 2 h. During this time, the suspension gradually
became a light brown solution. The solution was cooled in an ice
bath for 20 min and was then slowly added dropwise to crushed
ice (150 g) with vigorous stirring to give a white precipitate. The
solid was filtered under vacuum and washed thoroughly with
water (100 mL), 20% EtOH/water (50 mL), and finally with Et2O
(50 mL) to give the corresponding chloride as a white solid (5.70 g,
91%). This product was contaminated with several minor impuri-
ties and proved difficult to purify. Thus, it was used directly in the
next step.
Ethyl 2-(3-iodo-4,5-dimethoxybenzyl)-3-oxopentanoate (6b): This
compound was prepared as above using ethyl 3-oxovalerate
(9.69 g, 67.0 mmol) in dry EtOH (70 mL), powdered NaOMe (3.63 g,
67.0 mmol), and 5 (20.0 g, 56.0 mmol, 0.84 equiv) to give 6b as
1
a colorless liquid (14.1 g, 60%): H NMR (300 MHz, CDCl3): d=7.15
(d, J=2.0 Hz, 1H), 6.69 (d, J=2.0 Hz, 1H), 4.16 (q, J=7.3 Hz, 2H),
3.82 (s, 3H), 3.79 (s, 3H), 3.74 (t, J=7.3 Hz, 1H), 3.06 (m, 2H), 2.62
(dq, J=18.1, 7.3 Hz, 1H), 2.39 (dq, J=18.1, 7.3 Hz, 1H), 1.23 (t, J=
7.3 Hz, 3H), 1.03 ppm (t, J=7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d=204.9, 168.9, 152.3, 147.6, 136.4, 130.3, 113.6, 92.3, 61.5, 60.3,
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Inhibition of Bacterial Dihydrofolate Reductase
MED
A stirred suspension of the chloride (5.50 g, 13.1 mmol) in dry
EtOH (80 mL) was cooled to 08C, and NH3 gas was bubbled
through the solution for 15–20 min. The resulting solution was
transferred to a glass-lined 450 mL stainless steel pressure reactor
(Paar; no. 4760) and heated to 1658C for 16 h. The reaction was
cooled, and the solvent was evaporated under vacuum. The crude
product was purified by flash chromatography on a 70 cm ꢂ 3 cm
silica gel column (CH2Cl2/MeOH/Et3N 95:5:1) to give 8a as a white
15.9 Hz, 1H), 7.57–7.37 (complex m, 4H), 7.12 (s, 1H), 6.87 (s, 1H),
6.76 (br s, 2H), 6.28 (br s, 2H), 5.84 (t, J=6.6 Hz, 1H), 3.78 (s, 3H),
3.76 (s, 2H), 3.74 (s, 3H), 2.17 (s, 3H), 1.54 (m, 2H), 1.19 (m, 2H),
1.16 ppm (t, J=7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6): d=165.5,
163.5, 158.5, 152.5, 146.0, 142.9, 136.7, 135.9, 133.6, 131.7, 128.3,
127.8, 126.5, 126.1, 123.6, 117.9 (2C), 114.0, 103.1, 60.7, 55.7, 50.3,
22.6, 19.7, 17.8, 15.2, 13.6 ppm (one aromatic C was unresolved); IR
(NaCl): n˜ =3329, 3185, 1651, 1616 cmÀ1
;
Anal. calcd for
1
solid (4.21 g, 80%): mp: 236–2378C; H NMR (300 MHz, DMSO-d6):
C28H32N6O3·3.5H2O·0.5EtOH: C 57.60, H 6.33, N 13.88, found: C
57.75, H 6.43, N 13.54.
d=6.99 (s, 1H), 6.91 (s, 1H), 6.51 (br s, 2H), 6.14 (br s, 2H), 3.77 (s,
3H), 3.69 (s, 2H), 3.65 (s, 3H), 2.08 ppm (s, 3H); 13C NMR (75 MHz,
DMSO-d6): d=163.3, 159.6, 159.4, 152.0, 146.4, 138.3, 128.3, 113.4,
102.4, 92.6, 59.8, 55.8, 29.2, 20.1 ppm; IR (NaCl): n˜ =3420–2200,
(Æ)-(E)-3-{5-[(2,4-Diamino-6-ethyl-5-pyrimidinyl)methyl]-2,3-di-
methoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one
(11b): This compound was prepared as above using 8b (1.00 g,
2.42 mmol), (Æ)-10a (606 mg, 2.66 mmol, 1.10 equiv),[6] N-ethylpi-
peridine (300 mg, 0.36 mL, 2.66 mmol, 1.10 equiv), and Pd(OAc)2
(20 mg, 0.089 mmol) dissolved in dry DMF (9 mL) under N2 to give
1637 cmÀ1
.
5-(3-Iodo-4,5-dimethoxybenzyl)-6-ethylpyrimidine-2,4-diamine
(8b): This compound was prepared as above using 7b (6.00 g,
14.4 mmol) and 15 mL of POCl3 to give the expected chloride as
a tan solid (5.61 g, 90%). A stirred suspension of the chloride
(5.50 g, 12.6 mmol) in dry EtOH (80 mL) was cooled to 08C, treated
with NH3, and then heated to 1658C for 16 h. Purification by flash
chromatography (CH2Cl2/MeOH/Et3N 95:5:1) gave 8b as an off-
white solid (4.20 g, 80%): mp: 242–2438C; 1H NMR (300 MHz,
DMSO-d6): d=6.98 (d, J=1.6 Hz, 1H), 6.91 (d, J=1.6 Hz, 1H), 6.57
(br s, 2H), 6.23 (br s, 2H), 3.77 (s, 3H), 3.72 (s, 2H), 3.65 (s, 3H),
2.41 (q, J=7.7 Hz, 2H), 1.04 ppm (t, J=7.7 Hz, 3H); 13C NMR
(75 MHz, DMSO-d6): d=164.1, 163.6, 159.6, 152.0, 146.4, 138.5,
128.3, 113.4, 101.6, 92.5, 59.8, 55.8, 28.8, 25.8, 12.9 ppm; IR (NaCl):
1
11 b as a pale yellow solid (1.06 g, 85%): mp: 192–1938C; H NMR
(300 MHz, DMSO-d6): d=7.94 (s, 1H), 7.84 (d, J=15.9 Hz, 1H), 7.61
(d, J=15.9 Hz, 1H), 7.57–7.36 (complex m, 4H), 7.12 (overlapping s,
2H and 1H), 6.88 (s, 1H), 6.60 (br s, 2H), 5.84 (t, J=6.6 Hz, 1H),
3.78 (overlapping s, 3H and 2H), 3.74 (s, 3H), 2.52 (obscured, 2H),
1.53 (m, 2H), 1.18 (t, J=7.1 Hz, 3H), 1.16 (obscured, 2H), 1.10 ppm
(t, J=7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6): d=165.5, 164.1,
157.4, 152.5, 146.1, 142.9, 136.6, 135.6, 133.6, 131.7, 128.3, 127.9,
126.5, 126.1, 123.6, 118.0, 117.9, 114.0, 102.7, 60.8, 55.8, 50.3, 36.8,
29.0, 24.8, 17.8, 13.6, 12.8 ppm (one aromatic C was unresolved); IR
(NaCl): n˜ =3329, 3185, 1651, 1616 cmÀ1
;
Anal. calcd for
n˜ =3460–2200, 1633 cmÀ1
.
C29H34N6O3·5.0H2O: C 57.60, H 6.33, N 13.90, found: C 57.67, H 6.33,
N 13.61.
5-(3-Iodo-4,5-dimethoxybenzyl)-6-propylpyrimidine-2,4-diamine
(8c): This compound was prepared as above using 7c (6.00 g,
14.0 mmol) and POCl3 (15 mL) to give the expected chloride as
a tan solid (5.70 g, 91%). A stirred suspension of the chloride
(5.50 g, 12.3 mmol) in dry EtOH (80 mL) was cooled to 08C, treated
with NH3, and then heated to 1658C for 16 h. Purification by flash
chromatography (CH2Cl2/MeOH/Et3N 95:5:1) gave 8c as an off-
white solid (4.31 g, 82%): mp: 233–2348C; 1H NMR (300 MHz,
DMSO-d6): d=6.98 (s, 1H), 6.91 (s, 1H), 6.39 (br s, 2H), 6.06 (br s,
2H), 3.77 (s, 3H), 3.72 (s, 2H), 3.66 (s, 3H), 2.36 (t, J=7.1 Hz, 2H),
1.49 (sextet, J=7.1 Hz, 2H), 0.84 ppm (t, J=7.1 Hz, 3H); 13C NMR
(75 MHz, DMSO-d6): d=164.2, 163.4, 160.2, 151.9, 146.3, 138.8,
128.4, 113.4, 101.8, 92.5, 59.8, 55.8, 34.9, 29.0, 21.5, 13.9 ppm; IR
(Æ)-(E)-3-{5-[(2,4-Diamino-6-propyl-5-pyrimidinyl)methyl]-2,3-di-
methoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one
(11c): This compound was prepared as above using 8c (1.00 g,
2.34 mmol), (Æ)-10a (587 g, 2.57 mmol, 1.10 equiv),[6] N-ethylpiperi-
dine (290 mg, 0.35 mL, 2.57 mmol, 1.10 equiv), and Pd(OAc)2
(20 mg, 0.089 mmol) dissolved in dry DMF (9 mL) under N2 to give
11 c as an off-white solid (1.00 g, 81%): mp: 140–1418C; 1H NMR
(300 MHz, DMSO-d6): d=7.93 (s, 1H), 7.85 (d, J=15.9 Hz, 1H), 7.58
(d, J=15.9 Hz, 1H), 7.57–7.35 (complex m, 4H), 7.09 (s, 1H), 6.88 (s,
1H), 6.50 (br s, 2H), 6.09 (br s, 2H), 5.84 (t, J=6.6 Hz, 1H), 3.77
(overlapping s, 3H and 2H), 3.74 (s, 3H), 2.44 (t, J=7.1 Hz, 2H),
1.53 (m, 2H), 1.16 (m, 4H), 0.87 (t, J=7.1 Hz, 3H), 0.82 ppm (t, J=
7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6): d=165.5, 163.6 (2C),
159.6, 152.4, 145.9, 142.8, 136.6, 136.4, 133.6, 131.7, 128.2, 127.8,
126.5, 126.1, 123.6, 117.9, 117.7, 114.1, 102.4, 60.8, 55.7, 50.3, 36.8,
34.7, 29.5, 21.5, 17.8, 13.9, 13.6 ppm; IR (NaCl): n˜ =3335, 3190,
1650, 1615 cmÀ1; Anal. calcd for C30H36N6O3·2.75H2O: C 62.32, H
6.92, N 14.32, found: C 62.26, H 6.69, N 14.06.
(NaCl): n˜ =3480–2340, 1639 cmÀ1
.
(Æ)-(E)-3-{5-[(2,4-Diamino-6-methyl-5-pyrimidinyl)methyl]-2,3-di-
methoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one
(11a): A stirred solution of 8a (1.00 g, 2.50 mmol) in dry DMF
(8 mL) under N2 was treated with (Æ)-1-(1-propyl-2(1H)-phthalazin-
yl)-2-propen-1-one [(Æ)-10a][6] (627 mg, 2.75 mmol, 1.10 equiv) dis-
solved in DMF (1 mL), followed by N-ethylpiperidine (310 mg,
0.38 mL, 2.75 mmol, 1.10 equiv). This solution was then treated
with Pd(OAc)2 (20 mg, 0.089 mmol), and the reaction mixture was
heated at 1408C for 20 h. Isolation of the expected product was
achieved by pouring the cooled reaction mixture directly onto
a 50 cm ꢂ 2.5 cm silica gel flash chromatography column packed
in CH2Cl2. Impurities were eluted using CH2Cl2, and the final prod-
uct was collected using CH2Cl2/MeOH/Et3N (97:3:1). Evaporation of
the solvent gave a pale yellow solid, which was further purified
using a 15 cm ꢂ 2 cm silica gel column, packed with CH2Cl2 and
eluted with CH2Cl2/MeOH/Et3N (97:3:1). This second chromatogra-
phy removed yellow-colored impurities as well as several minor
contaminants. Evaporation of the solvent gave 11 a as a light
purple solid (1.05 g, 84%): mp: 137–1388C; 1H NMR (300 MHz,
DMSO-d6): d=7.94 (s, 1H), 7.84 (d, J=15.9 Hz, 1H), 7.60 (d, J=
(Æ)-(E)-3-{5-[(2,4-Diamino-6-methyl-5-pyrimidinyl)methyl]-2,3-di-
methoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-
one (12a): This compound was prepared as above using 8a
(1.00 g,
2.50 mmol),
(Æ)-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-
propen-1-one [(Æ)-10b] (660 mg, 2.75 mmol, 1.10 equiv),[6] N-ethyl-
piperidine (310 mg, 0.38 mL, 2.75 mmol, 1.10 equiv), and Pd(OAc)2
(20 mg, 0.089 mmol) dissolved in dry DMF (9 mL) under N2 to give
12a as an off-white solid (1.02 g, 80%): mp: 165–1668C; 1H NMR
(300 MHz, DMSO-d6): d=7.93 (s, 1H), 7.83 (d, J=15.9 Hz, 1H), 7.56
(d, J=15.9 Hz, 1H), 7.52 (m, 2H), 7.43 (d, J=7.1 Hz, 1H), 7.30 (d,
J=7.1 Hz, 1H), 7.11 (s, 1H), 6.96 (br s, 2H), 6.86 (s, 1H), 6.52 (br s,
2H), 6.49 (d, J=9.9 Hz, 1H), 5.24 (d, J=9.9 Hz, 1H), 3.78 (s, 3H),
3.76 (s, 2H), 3.73 (s, 3H), 2.19 (s, 3H), 1.96 (s, 3H), 1.60 ppm (s, 3H);
13C NMR (75 MHz, DMSO-d6): d=165.2, 163.7, 157.8, 152.5, 146.1,
142.1, 136.8, 135.6, 133.8, 133.5, 132.2, 128.2, 127.9, 126.3, 126.2,
ChemMedChem 0000, 00, 1 – 10
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C. R. Bourne, R. A. Bunce et al.
MED
123.1, 122.1, 118.0, 117.9, 114.0, 103.3, 60.7, 55.7, 49.2, 29.5, 25.3,
detected with the redox-sensitive tetrazolium dye 3-(4,5-dime-
thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium (MTS), which was measured by absorbance of 450 nm
light. Inhibitors were tested with at least six concentrations, and
the percent inhibition relative to a control reaction was used to
construct a four-parameter curve fit (KCjunior software package),[21]
providing a calculation of the IC50 value.
19.1, 18.4 ppm (one aromatic C was unresolved); IR (NaCl): n˜ =
3333,
3186,
1652,
1618 cmÀ1
;
Anal.
calcd
for
C28H32N6O3·2.5H2O·0.5EtOH: C 62.15, H 6.94, N 14.47, found: C
62.44, H 7.04, N 14.60.
(Æ)-(E)-3-{5-[(2,4-Diamino-6-ethyl-5-pyrimidinyl)methyl]-2,3-di-
methoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-
one (12b): This compound was prepared as above using 8b
(1.00 g, 2.42 mmol), (Æ)-10b (639 mg, 2.66 mmol, 1.10 equiv),[6] N-
ethylpiperidine (300 mg, 0.36 mL, 2.66 mmol, 1.10 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) dissolved in dry DMF (9 mL) under
N2 to give 12b as a pale yellow solid (1.06 g, 84%): mp: 192–
Protocols to determine the minimum inhibitory concentration
(MIC) were also described earlier[9] and followed the guidelines
given in the Clinical Laboratory and Standards Institute (CLSI)
manual.[22] Inhibitors were tested as a series of ten concentrations
made in twofold dilutions. The MIC value was defined as the
lowest concentration of inhibitor that prevented bacterial growth
(visually and as measured at 600 nm) after a 16 h or 20 h incuba-
tion at 378C in atmospheric CO2.
1
1938C; H NMR (300 MHz, DMSO-d6): d=7.91 (s, 1H), 7.84 (d, J=
15.9 Hz, 1H), 7.53 (d, J=15.9 Hz, 1H), 7.50 (m, 2H), 7.43 (d, J=
7.1 Hz, 1H), 7.30 (d, J=7.1 Hz, 1H), 7.07 (s, 1H), 6.88 (s, 1H), 6.49
(d, J=9.9 Hz, 1H), 6.29 (br s, 2H), 5.91 (br s, 2H), 5.24 (d, J=9.9 Hz,
1H), 3.77 (s, 3H), 3.76 (s, 2H), 3.73 (s, 3H), 2.45 (q, J=7.3 Hz, 2H),
1.96 (s, 3H), 1.60 (s, 3H), 1.07 ppm (t, J=7.3 Hz, 3H); 13C NMR
(75 MHz, DMSO-d6): d=166.0, 165.2, 163.4, 160.6, 152.4, 145.9,
142.1, 136.8, 136.7, 133.8, 133.5, 132.2, 128.2, 127.8, 126.3, 126.2,
123.1, 122.2, 117.8, 117.6, 114.1, 101.6, 60.8, 55.7, 49.2, 29.5, 26.4,
X-rax crystallography and modeling
Initial studies utilizing molecular modeling were carried out by Sa-
pient Discovery (San Diego, USA). These included calculation of
energy values upon complexation using in-house software based
on Monte-Carlo formalism.
25.3, 18.4, 13.0 ppm; IR (NaCl): n˜ =3329, 3182, 1651, 1615 cmÀ1
;
Anal. calcd for C30H34N6O3·2.0H2O: C 64.04, H 6.81, N 14.90, found:
C 64.05, H 6.72, N 14.64.
X-ray data were collected by Sapient Discovery using a Bruker Pro-
teum/R 6000 instrument suite that included a SMART6000 CCD de-
tector. Data integration and scaling were carried out with the Pro-
teum software suite.[23] Scaling statistics were obtained with XPREP
(Table 2). The value for Rint as reported from this procedure includ-
ed all data, in contrast to limiting to the strongest reflections as
used in comparable programs.[24] Crystallographic data were iso-
morphous with the previously determined structure of S. aureus
DHFR co-crystallized with (Æ)-1,[9] allowing the protein portion of
the model (PDB code: 3M08) to be used directly in the refinement
program Phenix[25] with visualization and model building using
Coot.[26] Figures were generated using the program Chimera.[27] Co-
ordinates and structure factors have been deposited with the Pro-
tein Data Bank[28] and were given the identifier codes 4FGH
[(Æ)-12b] and 4FGG [(Æ)-12c].
(Æ)-(E)-3-{5-[(2,4-Diamino-6-propyl-5-pyrimidinyl)methyl]-2,3-di-
methoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-
one (12c): This compound was prepared as above using 8c
(1.00 g, 2.34 mmol), (Æ)-10b (617 mg, 2.57 mmol, 1.10 equiv),[6]
N-ethylpiperidine (290 mg, 0.35 mL, 2.57 mmol, 1.10 equiv), and
Pd(OAc)2 (20 mg, 0.089 mmol) dissolved in dry DMF (9 mL) under
N2 to give 12c as an off-white solid (980 mg, 78%): mp: 138–
1
1398C; H NMR (300 MHz, DMSO-d6): d=7.90 (s, 1H), 7.84 (d, J=
15.9 Hz, 1H), 7.50 (m, 3H), 7.43 (d, J=7.1 Hz, 1H), 7.30 (d, J=
7.1 Hz, 1H), 7.05 (s, 1H), 6.88 (s, 1H), 6.48 (d, J=9.9 Hz, 1H), 6.03
(br s, 2H), 5.69 (br s, 2H), 5.24 (d, J=9.9 Hz, 1H), 3.76 (s, 3H), 3.73
(overlapping s, 2H and 3H), 2.39 (t, J=7.1 Hz, 2H), 1.95 (s, 3H),
1.59 (s, 3H), 1.52 (sextet, J=7.1 Hz, 2H), 0.86 ppm (t, J=7.1 Hz,
3H); 13C NMR (75 MHz, DMSO-d6): d=166.4, 165.2, 163.2, 161.4,
152.4, 145.9, 142.1, 137.0, 136.8, 133.8, 133.5, 132.2, 128.2, 127.8,
126.24, 126.17, 123.1, 122.2, 117.8, 117.6, 11.42, 101.8, 60.8, 55.7,
49.2, 35.7, 29.8, 25.3, 21.6, 18.4, 14.1 ppm; IR (NaCl): n˜ =3340, 3177,
1656, 1606 cmÀ1; Anal. calcd for C31H36N6O3·0.75H2O: C 66.41, H
7.18, N 14.99, found: C 66.26, H 6.91, N 14.74.
Acknowledgements
We gratefully acknowledge support of this work by the US Na-
tional Institute of Allergy and Infectious Diseases (NIAID) [1-R01-
AI090685-01] of the US National Institutes of Health (NIH) to
W.W.B. We are also pleased to acknowledge funding for the Okla-
homa Statewide NMR Facility by the National Science Founda-
tion (USA) (BIR-9512269), the Oklahoma State Regents for Higher
Education (USA), the W. M. Keck Foundation (Los Angeles, USA),
and Conoco, Inc. Molecular graphic images were produced using
the UCSF Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco [supported by NIH P41 RR001081].
Biology
All biological studies utilized stocks of racemic inhibitor dissolved
in DMSO; the concentration of which was kept ꢀ1% during all
assay procedures. Enzyme preparation, crystallization, activity (IC50
and Ki), and structure solution and refinements were carried out as
in previous studies.[6,9]
Briefly, recombinant DHFR protein was expressed in BL21 (DE3)
E. coli cultures and purified by IMAC using a C-terminal 6-His tag.
For crystallization, the 6-His tag was cleaved using an introduced
thrombin site, and protein was further purified by size-exclusion
chromatography.[9] Crystallization conditions were as previously
published: 15% polyethylene glycol 6000 in a 0.1m 2-(N-morpho-
lino)ethanesulfonic acid (MES) buffer (pH 6.5) with 0.15m NaOAc.
The enzymatic activity was assayed using a 0.05m phosphate buf-
fered system (pH 7.0) also containing 5% glycerol, 10 mm EDTA,
1 mm b-mercaptoethanol at 308C with saturating NADPH and di-
hydrofolate. Conversion of dihydrofolate to tetrahydrofolate was
Keywords: 6-alkylpyrimidines · antibiotics · Bacillus anthracis ·
dihydrofolate reductases · inhibitors · Staphylococcus aureus
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Received: June 8, 2012
Published online on && &&, 0000
ChemMedChem 0000, 00, 1 – 10
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&
9
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These are not the final page numbers!

FULL PAPERS
B. Nammalwar, C. R. Bourne,*
From resistance to terrorism: A series
of (Æ)-6-alkyl-2,4-diaminopyrimidines
was synthesized and evaluated for in-
hibition of bacterial dihydrofolate reduc-
tase (DHFR). Biological studies revealed
slightly attenuated activity relative to
structures lacking C6 alkyl substitution.
This arises from a conformational
R. A. Bunce,* N. Wakeham, P. C. Bourne,
K. Ramnarayan, S. Mylvaganam,
K. D. Berlin, E. W. Barrow, W. W. Barrow
&& – &&
Inhibition of Bacterial Dihydrofolate
Reductase by 6-Alkyl-2,4-
diaminopyrimidines
change of the protein resulting in expo-
sure of a hydrated pocket contiguous
with the existing binding site.
&10
&
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
ÝÝ
These are not the final page numbers!