Structure-Based Design of Novel Pyrimido[4,5-c]pyridazine Derivatives as Dihydropteroate Synthase Inhibitors with Increased Affinity

Article abstract of DOI:10.1002/cmdc.201200049

Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS is highly conserved and is distinct from the sulfonamide binding site. It therefore represents an attractive alternative target for the design of novel antibacterial agents. We previously carried out the structural characterization of a known pyridazine inhibitor in the Bacillus anthracis DHPS pterin site and identified a number of unfavorable interactions that appear to compromise binding. With this structural information, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazines were designed to improve binding affinity. Most importantly, the N-methyl ring substitution was removed to improve binding within the pterin pocket, and the length of the side chain carboxylic acid was optimized to fully engage the pyrophosphate binding site. These inhibitors were synthesized and evaluated by an enzyme activity assay, X-ray crystallography, isothermal calorimetry, and surface plasmon resonance to obtain a comprehensive understanding of the binding interactions from structural, kinetic, and thermodynamic perspectives. This study clearly demonstrates that compounds lacking the N-methyl substitution exhibit increased inhibition of DHPS, but the beneficial effects of optimizing the side chain length are less apparent.

Full text of DOI:10.1002/cmdc.201200049

MED
DOI: 10.1002/cmdc.201200049
Structure-Based Design of Novel Pyrimido[4,5-c]pyridazine
Derivatives as Dihydropteroate Synthase Inhibitors with
Increased Affinity
Ying Zhao,[a] Dalia Hammoudeh,[b] Mi-Kyung Yun,[b] Jianjun Qi,[c] Stephen W. White,[b, d] and
Richard E. Lee*[a, c]
Dihydropteroate synthase (DHPS) is the validated drug target
for sulfonamide antimicrobial therapy. However, due to wide-
spread drug resistance and poor tolerance, the use of sulfona-
mide antibiotics is now limited. The pterin binding pocket in
DHPS is highly conserved and is distinct from the sulfonamide
binding site. It therefore represents an attractive alternative
target for the design of novel antibacterial agents. We previ-
ously carried out the structural characterization of a known
pyridazine inhibitor in the Bacillus anthracis DHPS pterin site
and identified a number of unfavorable interactions that
appear to compromise binding. With this structural informa-
tion, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyri-
dazines were designed to improve binding affinity. Most im-
portantly, the N-methyl ring substitution was removed to im-
prove binding within the pterin pocket, and the length of the
side chain carboxylic acid was optimized to fully engage the
pyrophosphate binding site. These inhibitors were synthesized
and evaluated by an enzyme activity assay, X-ray crystallogra-
phy, isothermal calorimetry, and surface plasmon resonance to
obtain a comprehensive understanding of the binding interac-
tions from structural, kinetic, and thermodynamic perspectives.
This study clearly demonstrates that compounds lacking the N-
methyl substitution exhibit increased inhibition of DHPS, but
the beneficial effects of optimizing the side chain length are
less apparent.
Introduction
Since their discovery in the 1930s, the sulfonamide class of
drugs (sulfa drugs) has been widely used for the treatment of
a broad spectrum of infectious diseases.[1,2] These drugs target
the essential folate pathway in microorganisms, and combina-
tions with dihydrofolate reductase (DHFR) inhibitors such as tri-
methoprim have proven highly effective in treating E. coli uri-
nary infections, Pneumocystis carinii infections in immune-com-
promised patients and community-acquired methicillin-resist-
ant Staphylococcus aureus (MRSA).[3,4] Sulfa drugs target dihy-
dropteroate synthase (DHPS), an enzyme encoded by the folP
gene that acts at a key convergent point in folate biosynthe-
sis,[5] and mutations in the folP gene are associated with sulfa
drug resistance. Resistance mutations have now been reported
and characterized in many organisms, including drug-resistant
forms of S. aureus and P. carinii; this phenomenon, together
with frequent severe side effects associated with allergenici-
ty,[6,7] have become critical issues for the continued use of sulfa
drugs as antimicrobials. Therefore, to continue to take advant-
age of this valuable drug target, there is an urgent need for
the development of alternatives to sulfa drugs that avoid re-
sistance and overcome their poor tolerability.
dropterin pyrophosphate (DHPP; Figure 1) binds in the 7,8-di-
hydropterin binding pocket, deep within the highly conserved
DHPS b barrel, in which sulfa drug resistance mutations have
never been observed. The pterin binding site is therefore
a very attractive alternative target for the design and develop-
ment of novel antimicrobial agents, and our research group
has been pursuing this goal.[17,18]
One of the more potent pterin-pocket-targeted inhibitors
that we have encountered is a pyridazine compound that was
originally discovered by researchers at Burroughs Wellcome in
[a] Dr. Y. Zhao,+ Prof. R. E. Lee
Department of Chemical Biology and Therapeutics
St. Jude Children’s Research Hospital
262 Danny Thomas Place, Mail Stop 1000
Memphis, TN 38105 (USA)
E-mail: Richard.lee@stjude.org
[b] Dr. D. Hammoudeh,+ M.-K. Yun, Prof. S. W. White
Department of Structural Biology
St. Jude Children’s Research Hospital
262 Danny Thomas Place, Mail Stop 311
Memphis, TN 38105 (USA)
[c] Dr. J. Qi, Prof. R. E. Lee
Department of Pharmaceutical Sciences
University of Tennessee Health Science Center, Memphis, TN 38163 (USA)
To date, crystal structures of DHPS from many microbial
sources have been resolved, including complexes with sub-
strate and product analogues.[8–15] The sulfa drugs mimic the
substrate para-aminobenzoic acid (pABA) and bind at the
pABA binding site, composed of two flexible loop regions
which can readily accommodate mutations that confer sulfa
drug resistance.[11,16] In contrast, the second substrate dihy-
[d] Prof. S. W. White
Department of Microbiology, Immunology and Biochemistry
University of Tennessee Health Science Center, Memphis, TN 38163 (USA)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200049.
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which was condensed with diethyl 2-oxomalonate to give bicy-
clic compound 4. Simultaneous C4 deamination and saponifi-
cation of 4 in hot aqueous sodium hydroxide followed by
acidification afforded the target compound 5 (Scheme 1).
In the next set of side chain variants, the carboxylate group
was progressively extended away from the ring system by the
addition of methyl branched or unbranched methylene
spacers. These compounds 16–18 were obtained from 2-
amino-6-chloropyrimidin-4(3H)-one 6 with the treatment of
methylhydrazine in boiling water followed by condensation
with the appropriate diethyl a-keto ester to give compounds
9–11, respectively. Saponification of 9–11 in aqueous sodium
hydroxide followed by acidification afforded 16–18
(Scheme 2).[20]
Figure 1. Structure of pyridazine inhibitor 1 and substrate DHPP.
the late 1970s.[19,20] However, improvement was not possible
due to the lack of structural information on the complex be-
tween this compound and DHPS. We subsequently determined
the crystal structure of Bacillus anthracis DHPS (BaDHPS) in
complex with this molecule 1 (Figure 1) and confirmed that it
does engage the pterin pocket.[21] The pyridazine pterin-like
scaffold binds within the actual pocket as predicted, and the
carboxyl side chain partially occupies the anion pocket that
normally accommodates the pyrophosphate moiety of the
substrate.
The synthesis of the N8-demethyl analogues proved to be
more demanding. Initial attempts to directly demethylate the
N8 position of 16 and 18 using b-(trimethylsilyl)ethyl chlorofor-
mate were unsuccessful.[22] We therefore adopted a benzyl pro-
tecting group strategy for N8. Benzylated 7-amino-1-
benzylpyrimido[4,5-c]pyridazine-4,5(1H,6H)-diones 12–15 were
synthesized from 6 with benzylhydrazine by using a similar ap-
proach as described for 9–11.
Direct debenzylation and de-esterification of 13 was carried
out by reaction with aluminum trichloride in boiling toluene
(Scheme 3) to afford 19 in moderate yield. However, attempts
to extend this methodology to synthesize the other targeted
analogues failed due to poor reaction yields and difficulty in
separating the product from aluminum salt by-products.
This led us to apply selective hydrogenolysis as an alterna-
tive strategy to perform debenzylation (Scheme 4).[23] Inter-
mediates 12, 14, and 15 were subject to debenzylation using
formic acid and 10% palladium on carbon to give demethylat-
ed esters 20–22,[23] and these esters were subsequently saponi-
fied with aqueous sodium hydroxide to afford the target com-
pounds 23–25, respectively.
The crystal structure revealed that the A ring of 1 engages
the pterin pocket in a similar fashion to the actual pterin sub-
strate DHPP, through key interactions with conserved residues
Asp184 and Asn120 (Figure 2a).[9,21] Although the second
B ring is more structurally divergent from the pterin substrate,
the 5-carbonyl group of 1 mimics the hydrogen-bond-accept-
ing N5 nitrogen of DHPP to form a strong hydrogen bond
with the conserved Lys220. Elsewhere, the interactions are less
ideal with two notable examples: the exocyclic carboxyl group
attached to the pyridazine 6-position forms an apparently
strained salt bridge with Arg254, and the methyl group at the
8-position prevents the N8 nitrogen atom from making a favor-
able hydrogen bonding interaction with the conserved
Asp101, as observed in the pterin pyrophosphate (PtPP) and
DHPP complex structures.[9,16] Using a ligand-docking-based
approach to design and explore further analogues, we demon-
strated that removal of the N8-methyl group and optimization
of the C6 carboxylate side chain should facilitate an optimal
binding mode that more accurately mimics the binding of
DHPP.[13,21] In the current study we designed and synthesized
new 2-aminopyrimido[4,5-c]pyridazines and tested whether
they do indeed maximize these interactions, resulting in im-
proved DHPS inhibitors.
Enzyme inhibition studies
Compounds 5, 16–19, and 22–25 were all tested for the inhibi-
tion of BaDHPS by using a previously developed endpoint ra-
diometric product detection assay (Table 1).[21] Compounds 16–
18 and 5 directly probe the side chain moiety relative to the
parent compound 1. Removal of the branched methyl side
chain from 1 resulted in compound 16 with a ~50% poorer
IC50 value, mirroring the effect of methyl branch substitution in
the N8-demethylated pair 19/23. Compounds 17/18 and 24/
25 have an extra methylene side chain spacer relative to the
respective parent compounds 1 and 19, and this was observed
to give rise to decreased inhibitory activity. For both the 17/18
and 24/25 pairs, the inhibitors that are methyl branched (18
and 25) were weaker than the unbranched compounds 17 and
24; this stands in contrast to the effects observed for the
shorter chain branching pair of 1 and 16. The inhibitory activi-
ty of compound 5, in which the carboxylic acid is directly con-
nected to the bicyclic ring, dropped dramatically relative to
the initial compound 1 and showed no detectable IC50 value.
Results
Chemistry
In the first compound series the exocyclic C6 carboxylic acid
side chain was systematically varied; this group was chemically
accessible via simple modifications of existing synthetic
schemes. The first molecule targeted was compound 5, in
which the carboxylate was placed directly adjacent to the ring
system. Direct cyclization of 2-amino-6-chloropyrimidin-4(3H)-
one 6 with diethyl 2-oxomalonate did not give the desired 4,5-
dioxopyridazine, but instead produced the 3,5-dioxo ana-
logue.[20] Therefore, compound 5 was obtained by an alternate
route in which 2,4-diamino-6-chloropyrimidine 2 was treated
with methylhydrazine in boiling water to give compound 3,
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Figure 2. Crystal structures of complexes between BaDHPS and pyridazine-derived inhibitors. a) Details of the interactions of compound 1 with key residues
within the pterin binding site of BaDHPS.[21] Similar representations for compounds b) 19, c) 23, d) 25*, e) 17, and f) 24. *Compound 25 binds in two confor-
mations within the BaDHPS dimer, and these are superimposed in the figure.
Therefore, from this section of the study, the side chain substi-
Compounds 19 and 23–25 probe the removal of the N8-
methyl group relative to 1 and 16–18, respectively. All of the
demethylated compounds showed improved inhibition over
tution of 1 appears optimal.
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a benzyl group. The preference for a free terminal carboxylic
acid was also confirmed by comparing the inhibitory activity of
ester 22 with its free acid 25.
Thermodynamic and kinetic analyses
The binding affinities of the most potent derivatives compared
with that of 1 were further examined with respect to thermo-
dynamics and binding kinetics using isothermal titration calo-
rimetry (ITC) and surface plasmon resonance (SPR). Both tech-
niques showed that all six compounds bind to BaDHPS at a 1:1
molar ratio with respect to the DHPS monomer (Table 2; see
Supporting Information figures S1–S3 for the specific ITC bind-
ing isotherms and SPR kinetic
Scheme 1. Reagents and conditions: a) CH3NHNH2, H2O, reflux, 5 h, 27%;
b) diethyl 2-oxomalonate, CH3OH (anhyd), reflux, 3 days, 44%; c) 1. 1n
NaOH, RT, overnight, 2. HCl, 47%.
binding profiles). Comparing the
N-methylated and demethylated
pairs 1/19, both the ITC and SPR
data show an approximate two-
fold increase in affinity upon de-
methylation. ITC reveals that the
increase in free energy of binding
of 19 over 1 results from an in-
creased entropic contribution,
which overcompensates for some
loss to the enthalpy of binding.[24]
With regard to the 16/23 pair, it
was not possible to determine
binding by ITC, but SPR shows
a
similar twofold level of im-
proved binding upon removal of
the N8-methyl group. ITC and SPR
both show that increasing the
side chain length in compounds
Scheme 2. Reagents and conditions: a) CH3NHNH2, H2O, reflux, 5 h, or BnNHNH2·2HCl, Et3N, H2O, reflux, overnight;
b) H2O or CH3OH, reflux; c) 1. 1n NaOH, RT, overnight, 2. HCl.
24 and 25 substantially decreases binding affinity, and SPR re-
veals that there is a pronounced and undesirable increase in
the disassociation rate. A comparison between methyl-substi-
tuted 24 and straight-chain 25 analogues shows that the
methyl substitution is disfavored overall; it is more entropically
favored, but significantly penalized in enthalpic terms. Overall,
the data from these experiments agree well with the results
obtained from the enzyme assays
Scheme 3. Reagents and conditions: a) AlCl3, PhCH3, 1008C, 3 h.
(Table 1).
Structural analyses
In the published crystal structure
of the 1–DHPS complex, a network
of hydrogen bonding interactions
between Asn120, Asp184, a struc-
tured water molecule (W1) and
Scheme 4. Reagents and conditions: a) HCOOH, 10% Pd/C; b) 1. 1n NaOH, RT, overnight, 2. HCl.
their corresponding methylated analogues, and compounds 19
and 23 showed greater inhibition than the parent compound
1. Finally, to expand the SAR of the pyridazine analogues,
a number of synthetic intermediates were also tested for inhib-
itory activity. The saponification products of N8-benzylated an-
alogues 12–15 lacked significant enzyme inhibition (data not
shown), consistent with the binding pocket at the N8 position
being unable to accommodate a bulky substituent such as
Lys220 anchors the A ring to the pterin binding pocket in
a similar fashion to PtPP and DHPP (Figure 2a; see Supporting
Information figure S5 for the complex between the DHPP ana-
logue PtPP and BaDHPS).[9,16] The B ring is bound via hydrogen
bonding interactions with Lys220 and a second structured
water molecule (W2), a salt bridge between the acetate group
of the small molecule and Arg254, van der Waals interactions
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nate group results in
two conformations
for this moiety in the
two active sites of
the crystallographic
Table 1. BaDHPS inhibitory activities of pyridazines.
Compd
Structure
Inh. [%][a] IC50 [mm][b] Compd
Structure
Inh. [%][a] IC50 [mm]
1
97.0
96.0
52.5
65.0
32.4
22
32
19
23
24
25
22
95.3
91.0
75.8
91.8
33.0
11
18
dimer
(Figure 2d).
The two conforma-
tions appear to be
the result of alter-
nate but equally ac-
cessible salt bridge
16
17
18
5
interactions
with
Arg254 and Lys220.
Note that each con-
formation does not
145
ND
ND
57
affect
interactions
within the pterin
pocket, but does
slightly impact the
36
position
of
the
pterin-like
(Figure 2d).
scaffold
260
Compound 17 is
identical to 1 save
for an extra methyl-
ene group in the
side chain. The com-
plex structures are
[a] Enzyme inhibitory activity determined at a test compound concentration of 250 mm. [b] ND: not determined.
nearly
identical
between the methyl group of the linker and Phe189, and the
methyl group at position 8 with the b carbon atom of Asp101
(Figure 2a).
except that the carboxylate group more intimately engages
the anion pocket and Arg254 (Figure 2e). Likewise, compound
24 is identical to 19 apart from the additional methylene
group, and the structures are again very similar apart from the
improved binding within the anion pocket (Figure 2 f). This
effect of adding the extra methylene group was anticipated in
the design of these compounds.[21] Note that in both com-
plexes, although the carboxylate moiety interacts more favora-
bly with the anion pocket, alternate hydrogen bonds to
Ser218 and Lys220 are lost.
We first structurally characterized the complex with 19 to di-
rectly observe the effect of removing the N8-methyl group
(Figure 2b). The A ring is bound in the pterin pocket by an
identical constellation of hydrogen bonding interactions,
except that the hydrogen bond between the 4-oxo group and
Lys220 is missing. As predicted from the substrate analogue
complex,[21] the effect of removing the N8-methyl group from
the B ring is to allow the resulting NH group to form a hydro-
gen bond with the side chain of Asp101, which rotates to opti-
mize this interaction. Regarding the carboxylate side chain, its
interactions with Arg254 within the anion binding pocket and
with Phe189 via the branching methyl group are largely un-
changed. A small but significant effect of removing the N8-
methyl group is that the pterin-like ring structure rotates and
moves to accommodate the new interaction with Asp101. Su-
perimposing the complexes of compounds 1 and 19 with re-
spect to the protein backbone shows this subtle movement
and the rotation of Asp101 (Figure 3a).
Discussion
Compound 1 was identified as a rather potent inhibitor of
DHPS some 30 years ago in a study by researchers at Bur-
roughs Wellcome that was designed to generate pterin-like an-
tibacterial agents as alternatives to the pABA-like sulfa
drugs.[19–21] Although the study generated impressive SAR infor-
mation, it ultimately suffered from a lack of structural data. We
addressed this problem a couple years ago and structurally
characterized 1 in the pterin binding pocket of DHPS from
B. anthracis.[21] The structure suggested a number of ways in
which 1 could be further optimized, and we have reported
herein the results of this optimization. Specifically, we designed
Compared with 19, compound 23 only lacks the methyl
group in the carboxylate linker, and the crystal structures are
unsurprisingly very similar (Figure 2c). The van der Waals inter-
action with Phe189 is missing, and the 4-oxo group no longer
interacts with the W1 structured water or Lys220. Compared
with 23, compound 25 has one extra methylene group in the
side chain linker, and the additional flexibility of the propio-
and synthesized
a series of 4,5-dioxo-1,4,5,6-tetrahydro-
pyrimido[4,5-c]pyridazines and characterized their DHPS inhibi-
tory properties by using a direct enzyme assay, ITC and SPR
measurements, and crystallography.
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Table 2. Summary of ITC and SPR binding data for the most potent inhibitors of BaDHPS.
Compd
Structure
ITC data
DH
SPR data[b]
]
KD [mm]
N[a]
ÀTDS
DG
ka [mÀ1 sÀ1
]
kd [sÀ1
KD [nm]
KD(eq) [nm]
[kcalmolÀ1
]
1
0.124
0.8
À9.8
0.39
ND
À9.41
2.84(1)ꢁ105
3.42(3)ꢁ105
7.95(4)ꢁ10À2
280(2)
267(2)
16
ND[c]
ND
ND
ND
6.94(6)ꢁ10À2
203(2)
190(2)
19
23
24
25
0.076
ND
0.9
ND
0.8
0.7
À8.7
ND
À0.95
ND
À9.65
ND
3.66(2)ꢁ105
4.19(3)ꢁ105
6.43(2)ꢁ105
4.42(6)ꢁ105
4.39(3)ꢁ10À2
4.35(3)ꢁ10À2
3.39(1)ꢁ10À1
3.14(1)ꢁ10À1
120(1)
104(1)
528(3)
710(10)
123(1)
110(1)
535(2)
713(6)
0.51
0.273
À7.5
À10.4
À1.1
1.46
À8.6
À8.94
[a] Stoichiometry of interaction, experimental error: Æ0.01. [b] Values in parentheses represent the standard error of last reported decimal place. [c] ND:
not determined.
Two apparently non-ideal features of 1 were systematically
analyzed: the length and branched nature of the carboxylate
side chain at the 6-position of the two-ring scaffold, and the
presence or absence of the methyl group at the N8 position.
In the crystal structure of the complex with compound 1, the
carboxylate group does not optimally fit into the anion pocket,
and the N8-methyl group prevents a hydrogen bonding inter-
action with Asp101. In addition, the methyl group on the car-
boxylate side chain of 1 was considered favorable due to
a van der Waals interaction with the conserved Phe189 that
creates part of the pterin binding pocket. Based on our initial
SAR analysis,[21] we would have expected that 24 would display
the highest potency, but this is not the case. Although the
crystal structure fully supports our prediction, the assay and
the physical measurements consistently rank 24 below 1 in po-
tency. Although this is a disappointing and unexpected result,
a deeper analysis of the data does reveal possible explanations
that will prove invaluable as we move forward with our goal of
developing a new DHPS-based lead compound.
methyl group in going from 1 to 16 and 19 to 23 generates
less potency. Consistent with these data, compound 16, which
contains the N8 methyl and lacks the side chain methyl group
is the least potent inhibitor of the quartet. Inconsistencies
appear if the carboxylate side chain is extended by one meth-
ylene group. Although the crystal structures clearly show that
the extension allows more optimal docking within the anion
pocket, this is not reflected by the potency values. Thus, in the
pairings 1/17, 16/18, 19/24, and 23/25, the additional methyl-
ene group consistently decreases potency. In the context of
the longer side chain, the beneficial effects of the branching
methyl group are equivocal; in the 24/25 pairing, SPR suggests
tighter binding, whereas ITC and the enzyme assay suggest
lower potency. However, it should be noted that the methyl
group introduces chirality into the side chain, and measure-
ments with these molecules involve both enantiomers, where-
as the crystal structures confirm that only one enantiomer (R)
binds. Thus, the potency of the chiral compounds is likely to
be higher than is apparent from the measurements. Another
factor to consider in the loss of the methyl group from the ex-
tended side chain is the increased flexibility and the greater
entropic penalty associated with its binding and consequent
effects on structured water molecules near the site. Figure 2d
shows that 25 can indeed adopt two side chain conformations,
Considering only derivatives with one methylene group in
the carboxylate side chain, our SAR predictions are fully sup-
ported by the data. Thus, removing the N8-methyl group in
going from 1 to 19 and from 16 to 23 results in a better inhib-
itor by all three metrics. Likewise, removing the side chain
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analysis, this movement and the subtle changes in the hydro-
gen bonding patterns are clearly apparent. These small
changes are likely to have significant effects on binding affinity.
The described molecules are essentially two linked fragments
or pharmacophores that engage adjacent pterin and pyro-
phosphate binding pockets in DHPS, and the difficulty in con-
necting these units and capturing the maximum affinity gain is
well documented because of subtle but important steric and
conformational problems.[25]
Conclusions
Results from this study suggest that the demethylated pyrida-
zine core is an optimized pterin mimic DHPS inhibitor, but the
carboxylate side chain remains an area for further optimization
to generate higher-affinity inhibitors. Our recent structural
studies have shown that correct occupancy of the pyrophos-
phate pocket is required for stabilization of the outer loop
structure of DHPS,[16] and hence further efforts are currently
underway to generate inhibitors that more fully contact this
area.
Experimental Section
Chemistry
Starting materials were purchased from commercial sources except
dimethyl 3-methyl-2-oxopentanedioate and were used without fur-
ther purification. Dimethyl 3-methyl-2-oxopentanedioate was syn-
thesized as previously reported.[26] The reactions were monitored
by thin-layer chromatography (TLC) on pre-coated Merck 60 F254
silica gel plates and visualized by UV detection. The purity of final
compounds was determined by UPLC/UV/ELSD/MS (see Support-
ing Information for UPLC/UV/ELSD/MS method). The average UV
and ELSD purity is >95% for all final compounds.[27] Melting points
were obtained on a Thomas Scientific Uni-Melt capillary melting
point apparatus (Swedesboro, NJ, USA) and are uncorrected. All
1H NMR spectra were recorded on a Bruker Ultrashield 400 Plus in-
strument. Chemical shift values (d) are expressed in ppm relative
to tetramethylsilane as internal standard; s=singlet, d=doublet,
t=triplet, q=quartet, m=multiplet, br=broad signal. Coupling
constants (J) are reported in hertz (Hz).
Figure 3. Superimposed crystal structures of BaDHPS bound to compounds
a) 1 (yellow) and 19 (grey); b) 17 (yellow) and 19 (grey).
and the ITC data reveal that the binding of this compound is
associated with the highest entropic penalty.
Notably, the side chain and the terminal carboxylate are
clearly beneficial, as evident in comparing 16 with 5 and 25
with 22. During the course of our studies, we also analyzed
compound 21, which is the ester analogue of compound 24.
The crystal structure of the 21 complex (see Supporting Infor-
mation figure S4) shows that the ester prevents interaction
with Arg254 in the anion pocket, and this is reflected by a sig-
nificant decrease in inhibitory potency (21, IC50: 376 mm).
The crystal structures reveal that removal of the N8-methyl
group does allow the interaction with Asp101 as predicted,
but the now smaller pyridazine scaffold moves to optimize this
interaction. The precise positioning of the pyridazine scaffold
and its interactions within the pterin pocket also appear to be
modulated by the presence or absence of the side chain
methyl group and the manner in which the terminal carboxyl-
ate engages the anion pocket. Although the moderate resolu-
tion of our complex structures (2.2–2.5 ꢂ) precludes a detailed
6-(1-Methylhydrazinyl)pyrimidine-2,4-diamine (3): A stirred mix-
ture of 4-chloro-2,6-diaminopyrimidine (0.694 g, 4.8 mmol) and
CH3NHNH2 (0.553 g, 12 mmol) in CH3OH (30 mL) was heated at
reflux under N2 for 18 h. After cooling overnight, the white solid
was collected by filtration and dried to give 3 as a white solid
(0.198 g, 27%); mp: 215–2178C; 1H NMR (400 MHz, [D6]DMSO):
d=3.06 (s, 3H), 4.33 (brs, 2H), 5.37 (brs, 2H), 5.41 (s, 1H),
5.58 ppm (brs, 2H).
Methyl 5,7-diamino-1-methyl-4-oxo-1,4-dihydropyrimido[4,5-c]-
pyridazine-3-carboxylate (4): A mixture of 3 (0.1 g, 0.649 mmol)
and diethyl 2-oxomalonate (0.148 mL, 0.908 mmol) in anhydrous
CH3OH (10 mL) was heated at reflux for 72 h under N2. The solid
was filtered out from the hot reaction mixture and washed by fil-
tration to give 4 as a pale-green solid (0.072 g, 44%); mp: ~2748C
1
(dec.); H NMR (400 MHz, [D6]DMSO): d=3.78 (s, 3H), 3.81 (s, 3H),
7.12 (brd, 2H), 7.98 (brs, 1H), 8.82 ppm (brs, 1H).
ChemMedChem 2012, 7, 861 – 870
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R. E. Lee et al.
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7-Amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]-
pyridazine-3-carboxylic acid (5): A mixture of methyl 4 (0.25 g,
0.999 mmol) and 4n NaOH (12.5 mL) was stirred at reflux over-
night. The white solid was filtered out and then dissolved in hot
H2O. The resulted solution was acidified with dilute HCl to pH 5–6,
and the precipitate was collected by filtration and dried to give 5
as a white solid (0.112 g, 47%); mp: >3008C; 1H NMR (400 MHz,
[D6]DMSO): d=3.89 (s, 3H), 7.22 (brs, 1H), 8.37 ppm (brs, 1H);
HRMS m/z [M+H]+ calcd for C8H8N5O4: 238.0576, found: 238.0553.
two days as a light-pink solid (0.189 g, 41%); mp: >3008C;
1H NMR (400 MHz, [D6]DMSO): d=1.15 (t, J=7.2 Hz, 3H), 3.49 (s,
2H), 4.04 (q, J=7.2 Hz, 2H), 5.39 (s, 2H), 7.26–7.35 (m, 5H),
10.94 ppm (brs, 1H).
Ethyl 2-(7-amino-1-benzyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido-
[4,5-c]pyridazin-3-yl)propanoate (13): Compound 13 was ob-
tained from 8 (0.150 g, 0.649 mmol) and diethyl 2-methyl-3-oxosuc-
cinate (0.240 mL, 1.297 mmol) by following the general method de-
scribed above after 3 h as as an off-white solid (0.185 g, 77%); mp:
1
2-Amino-6-(1-methylhydrazinyl)pyrimidin-4(3H)-one (7): A stirred
mixture of 2-amino-6-chloropyrimidin-4(3H)-one (1.75 g, 12 mmol)
and CH3NHNH2 (2.76 g, 60 mmol) in H2O (90 mL) was heated at
reflux for 3 h, and the resulting solution was allowed to stand at
room temperature for 5 h before being cooled at 48C overnight.
The precipitate was collected by filtration and dried under vacuum
at 508C to give 7 as an off-white solid (1.37 g, 74%); mp: ~2758C
(dec.); 1H NMR (400 MHz, [D6]DMSO): d=3.11 (s, 3H), 4.45 (brs,
2H), 4.99 (s, 1H), 6.17 (brs, 2H), 9.84 ppm (brs, 1H).
>3008C; H NMR (400 MHz, [D6]DMSO): d=1.09 (t, J=7.2 Hz, 3H),
1.28 (d, J=7.2 Hz, 3H), 3.85 (t, J=7.2 Hz, 2H), 3.97–4.03 (m, 2H),
5.38 (q, J=14.8 Hz, 2H), 7.26–7.36 (m, 5H), 10.92 ppm (brs, 1H).
Methyl 3-(7-amino-1-benzyl-4,5-dioxo-1,4,5,6-tetrahydropyrimi-
do[4,5-c]pyridazin-3-yl)butanoate (14): Compound 14 was ob-
tained from 8 (0.29 g, 1.26 mol) and dimethyl 3-methyl-2-oxopen-
tanedioate (0.26 g, 1.38 mmol) by following the general method
described above after 3 h as a white solid (0.18 g, 39%); mp:
1
>3008C; H NMR (400 MHz, [D6]DMSO): d=1.08 (d, J=6.9 Hz, 3H),
2-Amino-6-(1-benzylhydrazinyl)pyrimidin-4(3H)-one (8): A stirred
mixture of 2-amino-6-chloropyrimidin-4(3H)-one (0.4 g, 2.75 mmol)
and BnNHNH2·2HCl (1.072 g, 5.50 mmol) along with Et3N
(2.857 mL, 20.61 mmol) in H2O (15 mL) was heated at reflux over-
night. The reaction mixture was cooled to room temperature, and
the solid was filtered out and dried over P2O5 to give 8 as an off-
2.34 (dd, J=16.1, 7.0 Hz, 1H), 2.42–2.48 (m, 1H), 2.69 (m, 1H), 3.45
(s, 3H), 5.16–5.50 (m, 2H), 7.19–7.42 (m, 5H), 10.84 (s, 1H),
12.02 ppm (s, 1H).
Methyl 3-(7-amino-1-benzyl-4,5-dioxo-1,4,5,6-tetrahydropyrimi-
do[4,5-c]pyridazin-3-yl)propanoate (15): Compound 15 was ob-
tained from 8 (0.3 g, 1.297 mmol) and dimethyl 2-oxopentane-
dioate (0.469 mL, 3.24 mmol) by following the general method de-
scribed above after 3 h as an off-white solid (0.394 g, 85%); mp:
1
white solid (220 mg, 35%); mp: ~2808C (dec.); H NMR (400 MHz,
[D6]DMSO): d=4.36 (brs, 2H), 4.86 (s, 2H), 5.05 (s, 1H), 6.22 (brs,
2H), 7.19–7.33 (m, 5H), 9.79 ppm (brs, 1H).
1
>3008C; H NMR (400 MHz, [D6]DMSO): d=2.60 (t, J=7.2 Hz, 2H),
General method for the synthesis of 9–15: A mixture of 7 (or 8)
and the appropriate keto ester in solvent (distilled H2O, expect 9
and 12, which were obtained from anhydrous CH3OH) was heated
at reflux for 1.5–24 h. The resulting precipitate was collected by fil-
tration from the hot mixture, washed with reaction solvent, and
dried under vacuum over P2O5 to give target compounds 9–11 (or
12–15).
2.78 (t, J=7.2 Hz, 2H), 3.49 (s, 3H), 5.32 (s, 2H), 7.26–7.35 (m, 5H),
10.87 ppm (brs, 1H).
General method for the synthesis of 16–18: A suspension of
compounds 9–11 in THF (10 mL) and 1n NaOH (6 mL) was stirred
at room temperature overnight. The solvent was removed by evap-
oration under vacuum to a small volume. The solution was neutral-
ized with dilute HCl to pH 5–6. The fluffy solid was filtered out and
dried over P2O5 to give target compounds 16–18.
Ethyl 2-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido-
[4,5-c]pyridazin-3-yl)acetate (9): Compound 9 was obtained from
7 (0.50 g, 3.22 mmol) and diethyl 2-oxosuccinate (1.03 g, 0.904 mL,
5.48 mmol) by following the general method described above after
2-(7-Amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-
c]pyridazin-3-yl)acetic acid (16): Compound 16 was obtained
from 9 (0.1 g, 0.358 mmol) by following the general method de-
scribed above as an off-white solid (0.065 g, 72%); mp: >3008C;
1H NMR (400 MHz, [D6]DMSO): d=3.41 (s, 2H), 3.73 (s, 3H), 10.89 (s,
1H), 12.36 ppm (s, 1H); HRMS m/z [M+H]+ calcd for C9H10N5O4:
252.0733, found: 252.0701.
3 h as
a
white solid (0.355 g, 39%); mp: >3008C; 1H NMR
(400 MHz, [D6]DMSO): d=1.18 (t, J=7.2 Hz, 3H), 3.47 (s, 2H), 3.73
(s, 3H), 4.06 (q, J=7.2 Hz,2H), 10.89 ppm (brs, 1H).
Methyl 3-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimi-
do[4,5-c]pyridazin-3-yl)butanoate (10): Compound 10 was ob-
tained from 7 (0.08 g, 0.43 mol) and dimethyl 3-methyl-2-oxopen-
tanedioate (0.07 g, 0.43 mmol) by following the general method
described above after 3 h as a light-pink solid (0.03 g, 27%); mp:
3-(7-Amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-
c]pyridazin-3-yl)butanoic acid (17): Compound 17 was obtained
from 11 (0.035 g, 0.12 mmol) by following the general method de-
scribed above as a off-white solid (0.021 g, 63%); mp: ~2958C
(dec.); 1H NMR (400 MHz, [D6]DMSO): d=1.08 (d, J=6.9 Hz, 3H),
2.33 (dd, J=16.0, 7.4 Hz, 1H), 2.64 (dd, J=16.1, 7.4 Hz, 1H), 3.50 (h,
J=7.1 Hz, 1H), 3.70 (s, 3H), 10.79 (s, 1H), 12.01 ppm (s, 1H); HRMS
m/z [M+H]+ calcd for C11H14N5O4: 280.1046, found: 280.1041.
1
>3008C; H NMR (400 MHz, [D6]DMSO): d=1.09 (d, J=6.9 Hz, 3H),
2.44 (dd, J=15.9, 7.3 Hz, 1H), 2.70 (dd, J=15.9, 7.5 Hz, 1H), 3.48–
3.55 (m, 1H), 3.56 (s, 3H), 3.69 (s, 3H), 10.80 ppm (s, 1H).
Methyl 3-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimi-
do[4,5-c]pyridazin-3-yl)propanoate (11): Compound 11 was ob-
tained from 7 (0.8 g, 5.16 mmol) and dimethyl 2-oxoglutarate
(0.896 mL, 6.19 mmol) by following the general method described
above after 1.5 h as a yellow solid (0.71 g, 49%); mp: >3008C;
1H NMR (400 MHz, [D6]DMSO): d=2.59 (t, J=7.6 Hz, 2H), 2.57 (t,
J=7.6 Hz, 2H), 3.59 (s, 3H), 3.69 (s, 3H), 10.81 ppm (brs, 1H).
3-(7-Amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-
c]pyridazin-3-yl)propanoic acid (18): Compound 18 was obtained
from 10 (0.3 g, 1.02 mmol) by following the general method de-
scribed above as a yellow solid (0.258 g, 95%); mp: >3008C;
1H NMR (400 MHz, [D6]DMSO): d=2.52 (t, J=7.6 Hz, 2H), 2.78 (t,
J=7.6 Hz, 2H), 3.49 (s, 3H), 7.30 (brs, 2H), 10.99 (brs, 1H),
12.10 ppm (brs, 1H); HRMS m/z [M+H]+ calcd for C10H12N5O4:
266.0889, found: 266.0877.
Ethyl 2-(7-amino-1-benzyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido-
[4,5-c]pyridazin-3-yl)acetate (12): Compound 12 was obtained
from 8 (0.3 g, 1.297 mmol) and diethyl 2-oxosuccinate (0.642 mL,
3.89 mmol) by following the general method described above after
868
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DHPS Inhibitors
MED
1
2-(7-Amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-
3-yl)propanoic acid (19): A mixture of 13 (0.2 g, 0.541 mmol) and
AlCl3 (0.289 g, 2.17 mmol) in PhCH3 (anhyd, 30 mL) was heated at
reflux for 3 h. The solvent was decanted, and the residue was dis-
solved in DMSO and purified by LC (Waters Prep LC System) with
H2O (0.1% HCOOH) and CH3OH (0.1% HCOOH) as eluent to give
19 as a light-yellow solid (0.035 g, 27%); mp: >3008C; 1H NMR
(400 MHz, [D6]DMSO): d=1.28 (d, J=7.2 Hz, 3H), 3.80 (q, J=7.2 Hz,
1H), 7.08 (brs, 2H), 10.76 (brs, 1H), 12.22 (brs, 1H), 12.62 ppm
(brs, 1H); HRMS m/z [MÀH]À calcd for C9H8N5O4: 250.0576, found:
250.0595.
>3008C; H NMR (400 MHz, [D6]DMSO): d=2.52 (t, J=7.2 Hz, 2H),
2.72 (t, J=7.2 Hz, 2H), 6.99 (brs, 2H), 10.69 (s, 1H), 12.06 (s, 1H),
12.53 ppm (s, 1H); HRMS m/z [M+H]+ calcd for C9H11N5O4:
252.0733, found: 252.0701.
Enzyme assays
DHPS activity was measured in a 30 mL reaction containing 5 mm
[14C]pABA, 10 mm 6-hydroxymethyl-7,8-dihydropterin diphosphate,
10 mm MgCl2, 2% DMSO, 50 mm HEPES pH 7.6, and 10 ng
DHPS.[28,29] After 30 min incubation at 378C, the reactions were
stopped by the addition of 1 mL 50% acetic acid in an ice bath.
The labeled product of the reaction, [14C]dihydropteroate, was sep-
arated from [14C]pABA by TLC. Aliquots (15 mL) of the reaction mix-
ture were spotted onto Polygram TLC plates (CEL 300 PEI) pur-
chased from Macherey–Nagel and developed with ascending chro-
matography in 100 mm phosphate buffer, pH 7.0. The plates were
scanned using a Typhoon (GE Healthcare) and analyzed with Im-
ageQuant TL. Inhibitor compounds were dissolved in DMSO, and
inhibition was tested at 500 or 250 mm, depending on solubility.
The final concentration of DMSO in the reaction mixture was 2%.
To determine the 50% inhibitory concentration (IC50) values, DHPS
activities were measured in the presence of various concentrations
of the compounds using the conditions described above, but with
5 ng DHPS.
General method for the synthesis of 20–22: A solution of 12 (or
14–15) and 10% Pd/C (1:1 w/w) in HCOOH (10 mL) was stirred at
room temperature under N2 for 24 h. The reaction solution was fil-
tered through a Celite pad, and the filter cake was washed with
warm HCOOH (10 mL). The solvent was removed under reduced
pressure to the minimum volume, and the residue was purified by
LC (Waters Prep LC System) with H2O (0.1% HCOOH) and CH3OH
(0.1% HCOOH) as eluent to give compound 20 (or 21–22).
Ethyl 2-(7-amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyri-
dazin-3-yl)acetate (20): Compound 20 was obtained from 12
(0.587 g, 1.652 mmol) by following the general method described
above as a white solid (0.07 g, 16%); mp: >3008C; 1H NMR
(400 MHz, [D6]DMSO): d=1.17 (t, J=7.2 Hz, 3H), 3.47 (s, 2H), 4.06
(q, J=7.2 Hz, 2H), 7.15 (brs, 2H), 10.89 (brs, 1H), 12.70 ppm (brs,
1H).
Crystallography
Methyl 3-(7-amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]-
pyridazin-3-yl)butanoate (21): Compound 21 was obtained from
14 (0.11 g, 0.30 mmol) by following the general method described
above as a white solid (0.04 g, 48%); mp: >3008C; 1H NMR
(400 MHz, [D6]DMSO): d=1.08 (d, J=6.9 Hz, 3H), 2.44 (dd, J=15.9,
7.1 Hz, 1H), 2.71 (dd, J=15.9, 7.7 Hz, 1H), 3.54 (m, 1H), 3.55 (s,
3H), 7.02 (brs, 2H), 10.70 (s, 1H), 12.51 ppm (s, 1H).
Compounds were dissolved in the crystallization mother liquor
(1.45m Li2SO4, 0.1m Bis-Tris propane pH 9.0) until saturated. Co-
crystal structures of B. anthracis DHPS with compounds 17, 19, 21,
23, 24, and 25 were obtained by soaking the small molecules into
pre-grown crystals, which were obtained as previously described.[9]
After a soaking period of 12 h, the crystals were cryoprotected by
a brief immersion in a mixture of 50% paratone-N/50% mineral oil
and flash-frozen in liquid nitrogen. Diffraction data were collected
at the SER-CAT 22-ID and 22-BM beamlines of the Advanced
Photon Source and processed using HKL2000.[30] Structures were
refined using refmac5,[31] CNS,[32] and Phenix,[33] and model building
was performed using the programs Coot[34] and O.[35] Figures 2 and
3 (as well as figures S4 and S5 in the Supporting Information) were
rendered with PyMOL.[36] The atomic coordinates for the DHPS in-
hibitor-bound structures have been deposited into the RCSB Pro-
tein Data Bank with the following PDB IDs: 23, 4DAI; 19, 4DAF; 17,
4D9P; 24, 4D8Z; 21, 4D8A; 25, 4DB7. Table 3 lists crystallographic
refinement statistics for these.
Methyl 3-(7-amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]-
pyridazin-3-yl)propanoate (22): Compound 22 was obtained from
15 (0.3 g, 0.84 mmol) by following the general method described
above as a white solid (0.025 g, 11%); mp: >3008C; 1H NMR
(400 MHz, [D6]DMSO): d=2.61 (t, J=6.8 Hz, 2H), 2.76 (t, J=6.8 Hz,
2H), 3.58 (s, 3H), 7.11 (brs, 2H), 10.92 (brs, 1H), 12.52 ppm (brs,
1H).
2-(7-Amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-
3-yl)acetic acid (23): Compound 23 was obtained from 20
(0.025 g, 0.094 mmol) by following the general method described
for the synthesis of 16–18 as a white solid (0.02 g, 89%); mp:
1
>3008C; H NMR (400 MHz, [D6]DMSO): d=3.40 (s, 2H), 7.15 (brs,
2H), 10.74 (s, 1H), 12.31 (brs, 1H), 12.62 ppm (s, 1H); HRMS m/z
[M+H]+ calcd for C8H8N5O4: 238.0576, found: 238.0553.
Isothermal titration calorimetry
3-(7-Amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyrida-
zin-3-yl)butanoic acid (24): Compound 24 was obtained from 21
(0.013 mg, 0.05 mmol) by following the general method described
for the synthesis of 16--18 as a white solid (0.005 mg, 41%); mp:
The purified B. anthracis DHPS protein was dialyzed against 50 mm
HEPES pH 7.6, 5 mm MgCl2. ITC titrations were performed in 5%
DMSO, 40 mm HEPES pH 7.6, 4 mm MgCl2 at 258C. Nineteen injec-
tions (2 mL) of each ligand (200 mm solution) were added to 203 mL
protein solution (20 mm). ITC titrations were performed on an
Auto-iTC200 isothermal titration calorimeter (MicroCal), and data
were analyzed with MicroCal Origin 7.0 software using a one-site
binding model.
1
>3008C; H NMR (400 MHz, [D6]DMSO): d=1.07 (d, J=6.9 Hz, 3H),
2.33 (dd, J=15.6, 7.1 Hz, 1H), 2.63 (dd, J=16.7, 7.7 Hz, 1H), 3.50 (q,
J=7.0 Hz, 1H), 10.65 (s, 1H), 11.97 (s, 1H), 12.49 ppm (s, 1H);
HRMS m/z [M+H]+ calcd for C10H12N5O4: 266.0889, found:
266.0877.
3-(7-Amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-
3-yl)propanoic acid (25): Compound 25 was obtained from 23
(0.02 g, 0.075 mmol) by following the general method described
for the synthesis of 16–18 as a white solid (0.012 g, 61%); mp:
SPR methods
Binding studies were performed at 258C using a BIACORE T100 (GE
Healthcare) surface plasmon resonance (SPR) instrument; 10ꢁ
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R. E. Lee et al.
MED
[7] D. A. Dibbern, Jr., A. Montanaro, Ann. Allergy Asthma Immunol. 2008,
100, 91–101.
Table 3. Crystallographic statistics of refinement.[a]
[8] A. Achari, D. Somers, J. N. Champness, P. K. Bryant, J. Rosemond, D. K.
Stammers, Nat. Struct. Mol. Biol. 1997, 4, 490–497.
Parameter 17
19
21
23
24
25
[9] K. Babaoglu, J. Qi, R. E. Lee, S. W. White, Structure 2004, 12, 1705–1717.
[10] A. M. Baca, R. Sirawaraporn, S. Turley, W. Sirawaraporn, W. G. J. Hol, J.
Mol. Biol. 2000, 302, 1193–1212.
[11] Y. Haasum, K. Strom, R. Wehelie, V. Luna, M. C. Roberts, J. P. Maskell,
L. M. C. Hall, G. Swedberg, Antimicrob. Agents Chemother. 2001, 45,
805–809.
[12] I. C. Hampele, A. D’Arcy, G. E. Dale, D. Kostrewa, J. Nielsen, C. Oefner,
M. G. P. Page, H.-J. Schonfeld, D. Stuber, R. L. Then, J. Mol. Biol. 1997,
268, 21–30.
Resolution 87.0–2.2 30.0–2.5 35.6–2.2 30.0–2.5 29.0–2.2 30.0–2.4
range [ꢂ]
Rwork
Rfree
0.233
0.270
0.257
0.286
0.219
0.259
0.253
0.282
0.216
0.259
0.257
0.283
[a] See Supporting Information tables S2 and S3 for further crystallo-
graphic statistics of data collection and refinement.
[13] K. E. Hevener, W. Zhao, D. M. Ball, K. Babaoglu, J. Qi, S. W. White, R. E.
Lee, J. Chem. Inf. Model. 2009, 49, 444–460.
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creadie, J. Mol. Biol. 2005, 348, 655–670.
[15] C. Levy, D. Minnis, J. P. Derrick, Biochem. J. 2008, 412, 379–388.
[16] M.-K. Yun, Y. Wu, Z. Li, Y. Zhao, M. B. Waddell, A. M. Ferreira, R. E. Lee, D.
Bashford, S. W. White, Science 2012, 335, 1110 – 1114.
[17] Y. Zhao, D. Hammoudeh, W. Lin, S. Das, M.-K. Yun, Z. Li, E. Griffith, T.
Chen, S. W. White, R. E. Lee, Bioconjugate Chem. 2011, 22, 2110–2117.
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[19] R. W. Morrison, Jr., W. R. Mallory, V. L. Styles, “Pyrimido[4,5-c]pyridazines,
Their Use in Pharmaceutical Compositions, Process and Intermediates
for Their Preparation”, EP 0000383 (A1), 1979.
[20] R. W. Morrison, W. R. Mallory, V. L. Styles, J. Org. Chem. 1978, 43, 4844–
4849.
His–BaDHPS was immobilized on a nitrilotriacetic acid-derivatized
carboxymethyldextran-coated gold surface (NTA Chip, GE Health-
care) at a level of ~5360 RU using the manufacturer’s protocol.
The kinetics of association and dissociation were monitored at
a flow rate of 100 mLminÀ1. Compounds were prepared in 20 mm
Tris pH 7.7, 150 mm NaCl, 5 mm MgCl2, 1 mm TCEP, 0.005%
Tween 20, and 5% DMSO as threefold serial dilutions decreasing
from 3 mm to 37 nm for 1, 9 mm to 111 nm for 19, and 1 mm to
12.3 nm for 22. Each compound was injected in triplicate at each
concentration. The data were processed, double-referenced, sol-
vent corrected, and analyzed by kinetic and equilibrium affinity
methods using the software package Scrubber2 (version 2.0b, Bio-
Logic Software).
[21] K. E. Hevener, M. K. Yun, J. J. Qi, I. D. Kerr, K. Babaoglu, J. G. Hurdle, K.
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[22] A. L. Campbell, D. R. Pilipauskas, I. K. Khanna, R. A. Rhodes, Tetrahedron
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[23] B. ElAmin, G. M. Anantharamaiah, G. P. Royer, G. E. Means, J. Org. Chem.
1979, 44, 3442–3444.
[24] J. E. Ladbury, G. Klebe, E. Freire, Nat. Rev. Drug Discovery 2010, 9, 23–
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Acknowledgements
Funding for this research was provided by National Institutes of
Health grant AI070721, Cancer Center core grant CA21765, and
the American Lebanese Syrian Associated Charities (ALSAC). We
thank Matthew Frank from the Department of Infectious Diseases
at St. Jude Children’s Research Hospital for help with the radio-
metric enzyme inhibition assay. We also thank Jerrod Scarbor-
ough, Drs. Lei Yang, and Bing Yan from the Department of Chem-
ical Biology and Therapeutics at St. Jude Children’s Research Hos-
pital for their help in the analysis and purification of final com-
pounds, and Brett Waddell for assistance with the SPR experi-
ments. Crystallography data were collected at Southeast
Regional Collaborative Access Team (SER-CAT) 22-ID and 22-BM
beamlines at the Advanced Photon Source, Argonne National
Laboratory. Supporting institutions may be found at http://
www.ser-cat.org/members.html. Use of the Advanced Photon
Source was supported by the US Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract No. W-
31-109-Eng-38.
Keywords: antimicrobial agents · dihydropteroate synthase ·
heterocycles · structure-based drug design
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Received: January 24, 2012
Revised: February 27, 2012
Published online on March 13, 2012
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