Synthesis of bi-substrate state mimics of dihydropteroate synthase as potential inhibitors and molecular probes

Article abstract of DOI:10.1016/j.bmc.2010.12.003

The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class o

Full text of DOI:10.1016/j.bmc.2010.12.003

Bioorganic & Medicinal Chemistry 19 (2011) 1298–1305
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of bi-substrate state mimics of dihydropteroate synthase
as potential inhibitors and molecular probes
Jianjun Qi a, Kristopher G. Virga a, Sourav Das b, Ying Zhao b, Mi-Kyung Yun c, Stephen W. White c,
Richard E. Lee a,b,
⇑
a Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 847 Monroe Ave., Rm327, Memphis, TN 38613, United States
b Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail stop 1000, Memphis, TN 38105-3678, United States
c Department of Structural Biology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents.
Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as
the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural
information, a new class of transition state mimics has been designed and synthesized that have the
capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evalu-
ation of these compounds as inhibitors of Bacillus anthracis dihydropteroate synthase is described herein.
Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose
activity displayed slow binding inhibition. The most active compounds in this series contained an
oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase
active site and demonstrated a good correlation with the observed bioassay data, as well as provided
important insight for the future design of higher affinity transition state mimics.
Received 21 September 2010
Revised 25 November 2010
Accepted 1 December 2010
Available online 15 December 2010
Keywords:
Dihydropteroate synthase
Antimicrobial agent
Sulfonamide
Transition state analog
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
thus explaining the rapid development of sulfonamide resistance
found clinically. To overcome this problem, we have focused our
Dihydropteroate synthase (DHPS) has been a validated drug tar-
get for antimicrobial therapy for over 70 years.1 DHPS is the target
enzyme of the sulfonamide class of antimicrobial agents. Currently,
sulfonamides are most commonly used in combination with the
dihydrofolate reductase (DHFR) inhibitor, Trimethoprim. These
combination therapies are typically reserved for the treatment of
complicated urinary tract infections, Pneumocystis carinii infections
in immune-compromised patients, and as one of the few orally
available treatments for community acquired methicillin resistant
Staphylococcus aureus (MRSA). DHPS (FolP) catalyzes the fifth step
in the folate biosynthesis pathway and is also the point of conver-
gence of the two enzyme pathways making it an excellent target
for therapeutic intervention. DHPS is responsible for the formation
of the folate intermediate 7,8-dihydropteroic acid from p-amino-
benzoic acid (pABA) and dihydropterin-6-hydroxymethyl pyro-
phosphate (DHPP) (Fig. 1). The sulfonamides act as competitive
inhibitors with respect to the p-amino benzoic acid (pABA) sub-
strate within the DHPS enzyme active site.2–4 The pABA/sulfon-
amide binding site is formed close to the protein surface by
flexible protein loops that are amenable and tolerant to mutations,
efforts towards investigating inhibitors designed to target the con-
served central pterin binding site within DHPS.3,5,6
In this study, we have further expanded our approach to exam-
ine the potential of generating transition state inhibitors that
simultaneously contact the pterin, pABA and pyrophosphate bind-
ing sites. The use of transition state analogs is a well-established
medicinal chemistry method to develop potent enzyme inhibitors
and has lead to the development of such drugs as the HIV protease
inhibitors and the anti-influenza neuraminidase inhibitors.7,8 It is
also hoped that the development of DHPS transition state analogs
may generate biological probes capable of trapping the flexible
loops, 1 and 2, which harbors mutational sites associated with
sulfadrug resistance and form the pABA binding site such that they
may be crystallized in their active conformation. Resolution of the
active conformation is a highly significant outcome, as it will facil-
itate future structure-based drug design efforts of novel DHPS
inhibitors, but also to further elucidate the mode of sulfonamide
resistance. The belief that transition state analogs can be success-
fully developed lies in the fact that although the enzymatic cata-
lytic mechanism of DHPS is poorly understood, the actual
chemical reaction that takes place is relatively straightforward.
The reaction catalyzed by DHPS involves a nucleophilic attack
by the amino nitrogen of pABA on the exocyclic methyl carbon of
DHPP. This results in the formation of the N–C bond and the
⇑
Corresponding author. Tel.: +1 901 595 6617; fax: +1 901 595 5715.
E-mail address: Richard.lee@stjude.org (R.E. Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.003

J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 1298–1305
1299
O
OH
O
N
O
P
O
P
H2N
δ+
N
HN
H2N
O
O
O-
O- OH
O
N
N
N
H
HN
H2N
O
OH OH
O
O
O-
δ
6-hydroxymethyl-7,8-dihydropterin-
pyrophosphate
P
O
P
O
N
H
OH
O
N
N
N
H
HN
H2N
Transition state
N
H
O
O
O
Dihydropteroate
H2N
OH
+ PPi
OH
O
p-aminobenzoic acid
NH+
HN
H2N
n
P
O-
HO
n=1,2
Proposed transition state mimics
N
N
Figure 1. DHPS catalytic reaction, proposed transition state intermediate and designed transition state mimics.
elimination of the pyrophosphate moiety as a leaving group
(Fig. 1). As seen in Figure 1 (the proposed transition state) a partial
carbocation intermediate is believed to form at the extra-cyclic
methylene in the transition state, which is stabilized by delocaliza-
tion of the charge in the adjacent double bond of the pterin ring.
Evidence supporting transient carbocation intermediate is shown
by the inability of the oxidized form of DHPP (pterin-6-hydroxy-
methyl pyrophosphate) to act as a DHPS substrate. The absolute
geometry of the enzyme catalyzed reaction is still a matter of spec-
ulation, as it has not been defined in the available crystal struc-
tures. However, it is reasonable to assume nucleophilic attack by
pABA is probably in-line with respect to the pyrophosphate moiety
to facilitate its removal. The pyrophosphate is presumably an-
chored by a divalent cation that occupies the phosphate binding
site.9 Based on this proposed transition state, four stable transition
state analogs were designed to have the ability and conformational
flexibility to bind the pterin, pABA, and phosphate sites within
DHPS simultaneously (Fig. 1). Integral to the design of these ana-
logs was the ability to mimic the intermediate transient carbocat-
ion by the incorporation of a basic amine at the 6-position of the
pterin ring, which is a similar approach that has successfully been
applied to design of transition state inhibitors of purine nucleoside
phorphorylase and to various glycosidase enzymes.10,11 The inhib-
itors were also designed with three additional changes from the
transition state theorized. For reasons of chemical stability the 5
position nitrogen in the pterin ring was removed. A methylene
spacer was introduced between the pABA mimic and the 6-posi-
tion exocyclic nitrogen allowing the pABA mimic greater flexibility
to better fit our design model. Finally methylene spaced phospho-
nates of two lengths were designed to introduce a stable phosphate
mimic capable of interacting with the pyrophosphate binding
pocket.
quired the production of sodium nitromalonaldehyde monohy-
drate (1) from the reaction of sodium nitrite and mucobromic
acid.12 This material is considered unstable and potentially explo-
sive requiring its immediate use in the following step. The second
step is a condensation reaction of the nitromalonaldehyde onto the
existing heterocyclic ring scaffold of 2,4-diamino-6-hydroxypyri-
midine performed in the presence of aqueous base.13 This reaction
produced the fused 2-ring heterocyclic, modified 6-nitro-5-deaza
pterin as a sodium salt that was converted to free amide by boiling
the salt in hydrochloric acid, which afforded the product 2 as fine
orange-tan needles upon cooling.
Our synthetic strategy next required the 2-amino to be
protected as an amide. Initially, we explored the use of an acetyl
protection scheme. However, difficulties with the organic solubil-
ity of some of the subsequent intermediates were encountered so
we switched to the use of bulkier and more lipophilic pivaloyl pro-
tecting group. This had the desired effect of increasing solubility
and allowed the scheme to advance. Thus, 2 was protected as a
pivaloyl amide by reaction with pivaloyl anhydride to afford 3
(Scheme 2). The nitro group in the pyrimidine ring of 3 was then
reduced by catalytic hydrogenation or with iron in acetic acid to
afford 6-amino-compound 4. The next step introduced the pABA
mimic by reductive amination of 4 with 4-formyl-benzoic acid
methyl ester in the presence of borane-tert-butylamine complex,
which allowed the reaction to proceeded smoothly affording 5.14
Next, the addition of the phosphate mimic was addressed. This
was first attempted by direct alkylation of 5 with (2-bromo-ethyl,
or propyl)-phosphonic acid diethyl ester according to the proce-
dure of Singh.15 However, problems were encountered with this
procedure. In the absence of base, the coupling reaction resulted
in rapid depivaloylation of the starting material, which then pre-
cipitated out of solution blocking further reaction. A second prob-
lem was encountered when this reaction was attempted in the
presence of base (Et3N) to block the aforementioned pivaloyl
hydrolysis; alkylation of the 4-oxo occurred as a competing reac-
tion creating a mixture of products. Therefore, the alternative
approach of reductive amination of 5 with (2-oxo-ethyl, or pro-
pyl)-phosphonic acid diethyl ester was attempted (scheme 3). In
this reaction, the phosphonate aldehydes were reacted with com-
pound 5 in AcOH overnight at rt followed by reduction of the imine
intermediate by borane-tert-butylamine complex. Notable differ-
ences in the final yields of the desired products were obtained
2. Chemistry
The initial stage of the synthesis required the actual construc-
tion of the stabilized 5-deaza pterin ring lacking the nitrogen atom
at the 5 position to allow for the introduction of an amino group at
the 6 position. This core 2-amino-3,4-dihydro-4-oxo-6-nitropyri-
do[2,3-d]pyrimidine (2) was synthesized (Scheme 1) in three steps.
The first step in the construction of the heterocyclic scaffold re-

1300
J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 1298–1305
OH
N
ONa
O
N
H2N
N
NH2
CHO
NO2
CHO
NaNO2
NO2
NO2
Br
Br
CHO
COOH
N
HN
H2N
Na
H2O
a
c
H2N
N
N
N
b
2
1
Scheme 1. Reagents and conditions: (a) NaNO2, H2O, 54 °C, 1.5 h; (b) NaOH 1% aq, refluxed, 1 h; (c) 20% HCl.
O
N
O
N
O
N
NO2
NO2
NH2
HN
H2N
O
HN
O
HN
N
N
N
b
N
N
a
H
H
2
4
3
COOCH3
COOCH3
H
O
N
H
N
O
O
HN
c
N
N
H
5
Scheme 2. Reagents and conditions: (a) DMAP, pivaloyl anhydride, refluxing 6 h; (b) H2 50 Psi/5% Pd–C, 2-methoxy-ethanol, rt, 3 h or Fe-AcOH, EtOH refluxing 2 h; (c) (1)
AcOH, 4-formyl-benzoic acid methyl ester, rt, overnight; (2) borane-tert-butylamine complex.
O
O
O
P
COOCH3
C2H5O P OC2H5
O
O
N
H
H
N
O
n
COOCH3
n
O
HN
O
N
+
O
HN
N
N
a
O
H
N
N
N
5
H
O
O
C2H5O P OC2H5
COOCH3
O
n
N
O
HN
b
N
N
N
H
6
7
n= 1,
n= 2,
Scheme 3. Reagents and conditions: (a) AcOH, rt, overnight; (b) borane-tert-butylamine complex.
between the two aldehydes (6 56% and 7 25% yield). This was
attributed to the ability of ethyl rather than the propyl phospho-
nate to stabilize the intermediate b-imine.
2.1. Enzyme inhibition and molecular modeling
Compounds 10–13 were all tested for inhibition of B. anthracis
DHPS using an endpoint assay and radiometric detection of the
product (Table 1).6 The results showed that oxidized analogs 10
and 11 demonstrated significant DHPS inhibition, whereas the re-
duced analogs 12 and 13 were inactive. The lack of inhibition of the
reduced analogs indicates that sp2 centers are required at the pter-
in 5–6 positions for inhibition. This is likely attributable to result-
ing steric clashes with the protein when the pterin ring at the 5–6
position is reduced and sp3 centers are introduced. The inhibition
of 10 and 11 could be improved significantly by pre-incubation of
the inhibitor before the addition of the DHPP substrate suggesting
slow binding kinetics. As could be expected from the level of
inhibition, none of the compounds demonstrated significant anti-
microbial activity when tested against a panel of gram positive
organisms including B. anthracis.
Phosphonate deprotection of 6 and 7 with excess TMSBr in DCM
at rt for 24 h gave corresponding free phosphonates 8 and 9 which
were subjected to base hydrolysis to give target compounds 10 and
11. In addition to the oxidized analogs 10 and 11, transition state
mimics in which the outer pterin-like ring was reduced were also
explored, as this ring is only partially desaturated in the natural
substrate. To synthetically achieve such analogs, selective hydroge-
nation of the outer ring of compounds 8 and 9 was achieved using
compressed hydrogen and catalytic platinum oxide in TFA to give a
mixture of desired outer ring saturated products. These acidic reac-
tion conditions also produced some of the depivaloylation product.
Rather than attempt purification at this stage, the product mixture
was advanced directly onto the next synthetic step; removal of the
remaining pivaloyl and ester functionalities via base hydrolysis to
afford target compounds 12 and 13 as their respective racemic
mixtures (Scheme 4).
The poor solubility and low affinity of these compounds in our
crystallization conditions prevented the soaking or co-crystalliza-

J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 1298–1305
1301
O
HO
P
OH
n
COOH
O
N
N
HN
H2N
N
10
11
: n=1
c
: n=2
O
P
O
P
C2H5O
OC2H5
n
HO
OH
COOCH3
COOCH3
O
O
n
N
N
O
HN
HN
O
a
N
N
N
N
N
N
H
H
6
: n=1
8
9
: n=1
: n=2
7: n=2
b
O
P
O
P
HO
O
OH
HO
OH
COOCH3
n
COOCH3
O
N
N
n
+
HN
O
N
HN
N
N
N
H
H
H2N
N
H
O
P
HO
OH
n
COOH
O
d
N
HN
H2N
N
N
H
12: n=1
13
: n=2
Scheme 4. Reagents and conditions: (a) TMSBr, dry DCM, rt 24 h; (b) H2–PtO2/50 Psi, TFA, rt, 18 h; (c) 1.5 M KOH/MeOH (1/1), rt, 24 h; (d) (1) 1 N NaOH, rt, overnight; (2)
1 N HCl.
tion these molecules with BaDHPS. Therefore, molecular modeling
experiments were performed using our established and validated
model of the BaDHPS active site in an effort to rationalize our res-
utls.5 All the inhibitors were docked into the active site using Gli-
deXP module of Maestro from Schrodinger Inc.16 The scores
obtained are shown in Table 2. As anticipated, the docking scores
followed a similar trend to the observed inhibition of the inhibi-
tors. The reduced analog 13 had poor docking scores mirroring
its observed poor enzyme inhibition. Compound 11 had the highest
docking score, also mirroring the observed biological activity. Con-
sequently, 11 was redocked into BaDHPS for exploring the possibil-
ity of alternate binding modes. The best docked complex, which
was similar to that obtained in the previous docking exercise with
the exception of the phosphonate–protein interactions, was further
subjected to a molecular dynamics simulation of 1 ns duration in
order to probe the interaction stabilities. Figure 2c, which is a snap-
shot of the system at 1 ns, shows a set of stable interactions be-
tween the inhibitor and the protein. These interactions, as well
as their differences from the binding modes of the substrate DHPP
and product pteroic acid, are discussed below.
of the 5-position nitrogen prevents the formation of a key hydro-
gen bond interaction between the 5-position nitrogen, the 3-posi-
tion keto, and Lys220. Lys220 instead forms a hydrogen bond with
the phosphonate group of 11. The pterin mimic portion of the ana-
log retains its interaction with Asn120. Although, only one of the
two interactions of Asn120 is visible in the snapshot in Figure 2c,
both interactions are observed during the course of the simulation.
However, Asp184 only interacts with one of the two nitrogens. The
conserved water molecule interacts with the other nitrogen dur-
ing the course of the simulation, although this interaction is not
visible in Figure 2c. The second difference noted was with respect
to the pyrophosphate binding site that hydrogen bonded via the
Arg68 and Asn27 in Figure 2c. Equivalent interactions are not
present in either Figure 2a or b. On the other hand, Arg254
forms hydrogen bonding interactions with the phosphonate in
Figure 2c, similar to those in Figure 2a and b. The third difference
relates to carboxylate binding site of the pABA group. The carbox-
ylate group of the pABA-mimic portion of 11 hydrogen bonds with
Phe222, Ala190 and Phe189. It is close to 90° to the pterin-mimic
portion of 11. This conformation is different from the slightly
stretched conformation adopted by pteroic acid in 1tx0 (Fig. 2b),
in which the carboxylate group hydrogen bonds with Ser221
instead.
The transition state analog 11 lacks the nitrogen in the 5 posi-
tion of the pterin ring, which has been removed to maintain
chemical stability. As evidenced in Figure 2a and b, the absence

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J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 1298–1305
Table 1
transition-state mimics that are found in the substrate, the product
BaDHPS inhibition by the designed inhibitors
and presumably, in the transition state with DHPS. If these interac-
tions can be addressed, most notably the inclusion of a hydrogen
bond acceptor at the 5 position of the pterin ring and an increase
in the distance between the pterin and pABA mimic rings, then
we would predict higher affinity inhibitors will be obtained. A fur-
ther consideration in the design that needs to be incorporated is
the degree of SN2 versus SN1 character in the enzyme reaction.
Finally, as satisfactory aqueous solubility will be required for the
development of such inhibitors towards drugs, future inhibitor
designs must address the poor solubility in aqueous and organic
media of such pterin like systems resulting from their high crystal
lattice energies.
Structure
%
% Inhibition
activity
(with
10 min
pre-
Inhibition
activity
(without
pre-
incubation) incubationa)
O
P OH
HO
N
COOH
COOH
16.3 at
200
30.5 at
200 lM
O
N
10
11
l
M
N
HN
H2N
4. Experimental section
O
OH
P
HO
O
The reactions were monitored by thin layer chromatography
(TLC) on pre-coated Merck 60 F254 silica gel plates and visualized
by UV detection. Horizon HPFC flash chromatography system was
performed with Biotage silica gel cartridges, Biotage Inc. (Lake
Forest, VA). HPLC on a Gilson 215 HPLC (Middleton, WI) equipped
32.9 at
200 M
44.3 at
200 M
N
l
l
HN
H2N
N
N
O
P
with an XterraÒ Prep MS C18 5
MA); gradient MeCN/H2O 0.1% TFA, 0–100% (30 min) with dual
wavelength detection at 215 and 254 nm. Analytical HPLC analysis
were performed on a Bruker Esquire LC/MS equipped with an All-
l
m, 19 Â 100 mm column (Milford,
HO
OH
COOH
COOH
O
N
3.6 at
200
5.7 at
200 lM
12
lM
N
HN
H2N
tech Platinum EPS 5
l
m C18, 4.6 Â 150 mm column (Deerfield, IL);
gradient MeCN/H2O 0.1% TFA, 0–100% (22 min) with detection at
254 nm. All compounds were determined to be of acceptable pur-
ity (>95%) using this method. Melting points were obtained on a
Thomas Scientific Uni-melt capillary mp apparatus (Swedesboro,
NJ) and were uncorrected. All 1H, 13C NMR and 2-D NMR spectra
were recorded on a Bruker ARX-300 or Varian INOVA-500 spec-
trometers. Chemical shifts are reported in ppm (d) relative to resid-
ual solvent peak or internal standard (tetramethylsilane) and
coupling constants (J) are reported in hertz (Hz). Mass spectra were
recorded on a Bruker Esquire LC/MS using ESI (Billerica, MA).
N
H
O
OH
P
HO
O
3.9 at
200
6.3 at
200
13
N
lM
l
M
HN
H2N
N
N
H
a
For pre-incubation, enzyme was incubated with inhibitor at 37 °C for 10 min.
Reaction was started by adding substrates.
4.1. Sodium nitromalonaldehyde monohydrate (1)
To a solution of NaNO2 (33.00 g, 0.48 mol) in water (33 mL) was
dropwise added a solution of mucobromic acid (33.00 g, 0.13 mol)
in 95% ethanol at 53–55 °C about 1 h and kept stirring for 15 min at
the same temperature. The reaction was cooled to 0 °C and filtered.
The crude product was recrystallized from a solution of 95% etha-
nol (50 mL) and water (12 mL) to give 1 (7.25 g, 36.3%).
Table 2
Glide docking scores for the synthesized inhibitors
Compound
Chirality at C
Chirality at N
Docking score
11
12
10
12
13
13
12
13
13
12
À6.93
À6.85
À6.55
À6.43
À6.27
À5.90
À5.70
À5.40
À5.33
À4.59
S
R
S
R
S
R
R
S
S
R
S
S
S
R
R
4.2. 2-Amino-6-nitro-3H-pyrido[2,3-d]pyrimidin-4-one (2)
A
mixture of 2,4-diamino-6-hydroxypyrimidine (5.78 g,
45.86 mmol) and 1 (7.20 g, 45.86 mmol) in 46 mL of NaOH (1%
aq) was refluxed vigorously for 1 h. On chilling to 50 °C, a volumi-
nous mass of yellow needles separated. The solid was recrystal-
lized from 400 mL of 20% hydrochloric acid to give 2 (5.40 g,
57%) as green-tan needles. Mp >300 °C; 1H NMR (300 MHz,
DMSO-d6) d 9.39 (d, 1H, J = 2.7 Hz), 8.78 (d, 1H, J = 2.7 Hz),
8.60–6.80 (br, 2H). 13C NMR (75 MHz, DMSO-d6) d 163.5, 161.6,
156.1, 150.6, 138.0, 130.9, 110.5; m/z 206 (MÀ).
R
3. Conclusions
Herein we describe the design and synthesis of the first known
trivalent DHPS inhibitors. The inhibitors appear to demonstrate
slow binding inhibition, which is consistent with the rearrange-
ment of the active site to bind these large transition-state mimics.
This may also bode well for future inhibitor design as slow Koff
rates have been shown to be important in the action of several
inhibitor classes.17 If designed well, transition-state mimic inhibi-
tors should have nanomolar inhibition. Thus, there is still much
room to improve the design of these inhibitors. From the modeling
experiments, it is clear that there are 3 interactions missing in our
4.3. 2,2-Dimethyl-N-(6-nitro-4-oxo-3,4-dihydro-pyrido-
[2,3-d]pyrimidin-2-yl)-propionamide (3)
A mixture of 2 (2.00 g, 9.66 mmol), 4-dimethylaminopyridine
(DMAP, 0.20 g) and trimethylacetic anhydride (10 mL) was heated
at reflux vigorously for 6 h. The mixture was cooled to 0 °C, diluted
with ether and then filtered. The crude product was dissolved in

J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 1298–1305
1303
4.4. N-(6-Amino-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-
2-yl)-2,2-dimethyl-propionamide (4)
Method A. A solution of 3 (0.1 g, 0.34 mmol) in 2-methoxy-eth-
anol (20 mL) was charged with 5% Pd–C (0.02 g) and hydrogenated
at 50 Psi for 3 h at room temperature. The reaction solution was fil-
tered and the filtrate was evaporated to give 4 as a yellow solid
(0.081 g, yield 90%). TLC (ethyl acetate) Rf = 0.1; 1H NMR
(500 MHz, DMSO-d6) d 12.11 (s, 1H), 11.06 (s, 1H), 8.32 (d, 1H,
J = 3.0 Hz), 7.46 (d, 1H, J = 3.0 Hz), 5.69 (s, 2H), 1.24 (s, 9H). 13C
NMR (75 MHz, DMSO-d6) d 181.4, 161.3, 145.8, 144.5, 142.9,
115.0, 114.5, 79.1, 48.5, 26.3; m/z 284 (M++Na).
Method B. Fe-powder (0.5 g, 8.93 mmol) was added to a flask,
followed by water (5 mL) and EtOH (10 mL) at about 50–55 °C,
then 2–3 drop of HOAc was added and kept stirring for 5 min. A
mixture of 3 (0.5 g, 1.72 mmol) in EtOH (5 mL) was added to the
reaction flask. The mixture was stirred at 50–55 °C for 1 h and
cooled to room temperature, extracted with chloroform and fil-
tered. The organic phase was dried with anhydrous Na2SO4, and
then evaporated to yield 4 (0.42 g, yield 94%).
4.5. 4-{[2-(2,2-Dimethyl-propionylamino)-4-oxo-3,4-dihydro-
pyrido[2,3-d]pyrimidin-6-ylamino]-methyl}-benzoic acid methyl
ester (5)
A mixture of 4 (1.075 g, 4.12 mmol), 4-formyl-benzoic acid
methyl ester (0.67 g, 4.12 mmol) in AcOH (100 mL) was stirred
overnight at room temperature. To the reaction solution was added
borane-tert-butylamine complex (0.179 g, 2.0 mmol). The resulting
mixture was stirred for 2 h at room temperature and evaporated.
The residue was purified by chromatography using 4% MeOH in
EtOAc as eluent to give 5 as a yellow solid (0.401 g, yield 24%).
TLC (ethyl acetate) Rf = 0.18; mp >300 °C; 1H NMR (500 MHz,
DMSO-d6) d 12.12 (br, 1H), 11.08 (br, 1H), 8.44 (d, 1H, J = 2.0 Hz),
7.94 (d, 2H, J = 8.0 Hz), 7.52 (d, 2H, J = 8.0 Hz), 7.24 (d, 1H,
J = 3.5 Hz), 7.05 (t, 1H, J = 6.0 Hz), 4.49 (d, 2H, J = 6.0 Hz), 3.83 (s,
3H), 1.23 (s, 9H). 13C NMR (75 MHz, CDCl3) d 179.8, 166.2, 144.0,
142.5, 141.7, 129.6, 129.0, 126.7, 115.2, 113.5, 51.6, 47.1, 39.8,
26.4; m/z 410 (M++H), 432 (M++Na).
4.6. 4-({[2-(Diethoxy-phosphoryl)-ethyl]-[2-(2,2-dimethyl-pro-
pionylamino)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-6-yl]-
amino}-methyl)-benzoic acid methyl ester (6)
A mixture of 5 (0.19 g, 0.46 mmol), (2-oxo-ethyl)-phosphonic
acid diethyl ester (0.15 g, about 80%, 0.67 mmol) and 3 Å molecular
sieve (0.5 g) in AcOH (20 mL) was stirred overnight at room temper-
ature. Then borane-tert-butylamine complex (0.12 g) was added.
The reaction solution was stirred for additional 2 h at room temper-
ature, and then filtered. The filtrate was evaporated and then the
residue was dissolved in chloroform, washed with a solution NaH-
CO3 and brine. The organic phase was dried with anhydrous Na2SO4
and then evaporated. The resulting residue was purified by chroma-
tography using 5% MeOH in AcOEt as eluent to give 6 as a yellow so-
lid (0.15 g, yield 56%). TLC (MeOH/ethyl acetate = 1:15) Rf = 0.45. 1H
NMR (300 MHz, CDCl3) d 12.50–11.60 (1H, br), 8.45 (d, 1H,
J = 3.0 Hz), 8.00 (d, 2H, J = 8.1 Hz), 7.67 (d, 1H, J = 3.0 Hz), 7.28 (d,
2H, J = 8.4 Hz), 4.71 (s, 2H), 4.13 (m, 4H), 3.92 (s, 3H), 3.84 (m,
2H), 2.15 (m, 2H), 1.35 (t, 6H, J = 6.9 Hz), 1.34 (s, 9H); m/z 572 (MÀ).
Figure 2. Models of BaDHPS active site showing substrate, product and inhibitor
binding: (a) model of the crystal structure of DHPP bound to the DHPS active site;
(b) model of the crystal structure of product pteroic acid bound to DHPS; (c) a
snapshot taken at 1 ns of the MD simulation run on the docked complex of 11 and
BaDHPS.
chloroform and filtered. The filtrate was concentrated to a small
volume, cooled to 0 °C, filtered and washed with chloroform to give
3 as a pale yellow crystal (2.09 g, 74%). TLC (ethyl acetate) Rf = 0.71;
mp 208–210 °C; 1H NMR (500 MHz, DMSO-d6) d 12.76–12.22 (br,
1H), 12–11.5 (br, 1H), 9.56 (d, 1H, J = 3.0 Hz), 8.93 (d, 1H,
J = 3.0 Hz), 1.28 (s, 9H). 13C NMR (75 MHz, CDCl3) d 180.5, 159.0,
151.1, 150.8, 140.5, 132.0, 114.0, 40.2, 26.3; m/z 290 (MÀ).
4.7. 4-({[3-(Diethoxy-phosphoryl)-propyl]-[2-(2,2-dimethyl-
propionylamino)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-
6-yl]-amino}-methyl)-benzoic acid methyl ester (7)
A mixture of 5 (0.91 g, 2.22 mmol), (2-oxo-propyl)-phosphonic
acid diethyl ester (1.50 g, 7.73 mmol) in AcOH (15 mL) was stirred

1304
J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 1298–1305
overnight at 70–75 °C. Then borane-tert-butylamine complex
(0.024 g) was added to the reaction mixture hourly with stirring
for 10 h at 70–75 °C. The reaction was monitored by HPLC until
there was no change for the ratio of product. The reaction solution
was evaporated. The residue was dissolved with chloroform and
then washed with brine. The organic phase was dried with
anhydrous Na2SO4 and then evaporated. The resulting residue
was purified by chromatography with 5% MeOH in AcOEt as eluent
to give 7 (0.33 g, yield 25%, 600 mg of starting material was recov-
ered). TLC (MeOH/ethyl acetate = 1:10), Rf = 0.3. 1H NMR
(500 MHz,CDCl3) d 11.96 (br, 1H), 8.45 (s, 1H), 8.27 (s, 1H), 8.00
(d, 2H, J = 8.0 Hz), 7.64 (d, 1H, J = 3.5 Hz), 7.28 (d, 2H, J = 8.0 Hz),
4.70 (s, 2H), 4.15–4.06 (m, 4H), 3.91 (s, 3H), 3.62 (t, 2H,
J = 8.0 Hz), 2.05–1.96 (m, 2H), 1.80 (tt, 2H, J1 = 18.5 Hz, J2 =
7.5 Hz), 1.33 (s, 9H), 1.32 (t, 6H, J = 7.0 Hz). 13C NMR (75 MHz,
CDCl3) d 180.2, 166.0, 161.1, 149.2, 145.6, 142.1, 142.0, 141.6,
129.5, 128.8, 125.9, 114.8, 114.5, 61.1, 53.7, 51.4, 51.0, 39.7, 26.2,
22.2, 19.7, 19.6, 15.9, 15.8; m/z 588 (M++H).
4.11. Potassium 4-{[(2-amino-4-oxo-3,4-dihydro-pyrido-[2,3-d]-
pyrimidin-6-yl)-(3-phosphono-propyl)-amino]-methyl}-benzo-
ate (11)
Compound 11 was synthesized as described for 10 from com-
pound 9 (0.06 g, 0.11 mmol) and was obtained as an orange solid
(0.002 g, 4% yield). Mp >300 °C; 1H NMR (500 MHz, CD3OD) d
8.22 (s, 1H), 8.04 (d, 2H, J = 8.3 Hz), 7.54 (d, 2H, J = 8.3 Hz), 7.43
(s, 1H), 4.65 (t, 2H, J = 7.3 Hz), 4.53 (s, 2H), 2.21 (q, 2H,
J = 6.8 Hz), 1.80–1.70 (m, 2H); m/z 472.1 (M+K)+.
4.12. 4-{[(2-Amino-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-
6-yl)-(2-phosphono-ethyl)-amino]-methyl}-benzoic acid (12)
To a solution of 8 (0.2 g, crude product) in TFA (20 mL) was
added PtO2 (0.1 g), then the reaction solution was hydrogenated
under 50 Psi at room temperature for 18 h. To the reaction mixture
was added PtO2 (0.08 g), and the mixture was hydrogenated for an
additional 6 h at 50 Psi at room temperature and monitored by LC–
MS to ensure the completion of starting material. The reaction
solution was filtered and the filtrate was evaporated. The crude
product was used in the next step without further purification.
To a solution of 1 N NaOH (30 mL) was added the above crude
product. The reaction solution was stirred at room temperature
for 12 h and then adjusted to pH ꢀ1. The reaction solution was
purified by HPLC using 0.1% TFA in water and 0.35% TFA in aceto-
nitrile as eluent to give 12 as a white solid (0.03 g, yield 27% from
6). 1H NMR (D2O + NaOD, 500 MHz) d 7.82 (d, 2H, J = 8.0 Hz), 7.46
(d, 2H, J = 8.0 Hz), 3.82 (d, 2H, J = 3.0 Hz), 3.44 (m, 1H), 3.13 (t,
1H, J = 11 Hz), 3.03 (m, 1H), 2.86 (m, 2H), 2.73 (m, 1H), 2.39 (dd,
1H, J1 = 11 Hz, J2 = 15 Hz), 1.69 (m, 2H); m/z 422 (MÀÀH).
4.8. 4-{[[2-(2,2-Dimethyl-propionylamino)-4-oxo-3,4-dihydro-
pyrido[2,3-d]pyrimidin-6-yl]-(2-phosphono-ethyl)-amino]-
methyl}-benzoic acid methyl ester (8)
A mixture of 6 (0.06 g,0.1 mmol) and bromo-trimethyl-silane
(0.153 g, 1 mmol) in DCM (10 mL) was stirred at room temperature
under nitrogen for 72 h and then quenched with MeOH (10 mL)
followed by stirring for an additional 2 h. The reaction mixture
was evaporated and the residue was purified by HPLC using 1% for-
mic acid/water solution and acetonitrile to give 8 as a yellow solid
(0.03 g, yield 58%). Mp 232–234 °C; 1H NMR (500 MHz, CD3OD) d
8.40 (s, 1H), 8.00 (d, 2H, J = 7.8 Hz), 7.52 (d, 2H, J = 7.3 Hz), 7.43
(s, 1H), 4.51 (s, 2H), 3.90 (s, 3H), 2.86–2.78 (m, 2H), 1.41–1.32
(m, 2H), 1.33 (s, 9H); m/z 518 (M++H).
4.13. 4-{[(2-Amino-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-
6-yl)-(3-phosphono-propyl)-amino]-methyl}-benzoic acid (13)
Compound 13 was synthesized as described for 12 from 7
(0.33 g, 0.56 mmol) and was obtained as a pale yellow solid prod-
uct (yield 38% from 7). 1H NMR (500 MHz, D2O + NaOD) d 7.78 (d,
2H, J = 8.5 Hz), 7.42 (d, 2H, J = 8.5 Hz), 3.80 (s, 2H), 3.40 (m, 1H),
3.30 (s, 1H), 3.10 (t, 1H, J = 11 Hz), 3.06–2.98 (m, 1H), 2.75–2.67
(m, 1H), 2.62–2.53 (m, 2H) 2.35 (dd, 1H, J1 = 15.5 Hz,
J2 = 10.5 Hz), 1.76–1.64 (m, 2H), 1.30–1.20 (m, 2H). 13C NMR
(75 MHz, D2O + NaOD) d 21.35 (d, J = 92 Hz), 26.76 (d, J = 130 Hz),
41.62, 51.02 (d, J = 21 Hz), 53.17 (d, J = 13 Hz), 85.96, 128.0,
128.40, 128.63,129.26, 134.68, 141.21, 159.85, 161.02, 173.67,
174.96; m/z 438(M++H).
4.9. 4-{[[2-(2,2-Dimethyl-propionylamino)-4-oxo-3,4-dihydro-
pyrido[2,3-d]pyrimidin-6-yl]-(3-phosphono-propyl)-amino]-
methyl}-benzoic acid methyl ester (9)
A mixture of 7 (0.12 g, 0.2 mmol) and bromo-trimethyl-silane
(0.306 g, 2 mmol) in DCM (10 mL) was stirred at room temperature
under nitrogen for 72 h and then quenched with MeOH (10 mL)
followed by stirring for an additional 2 h. The reaction mixture
was evaporated and the residue was purified by HPLC using 1% for-
mic acid/water solution and acetonitrile to give 9 as a reddish solid
(0.06 g, yield 56%). 1H NMR (500 MHz, CD3OD) d 8.42 (d, 1H,
J = 2.9 Hz), 8.23 (d, 2H, J = 2.4 Hz), 8.05 (d, 2H, J = 8.1 Hz), 7.57 (d,
2H, J = 8.1 Hz), 4.81 (t, 2H, J = 7.3 Hz), 4.61 (s, 2H), 3.92 (s, 3H),
2.25 (q, 2H, J = 7.3 Hz), 1.82–1.72 (m, 2H), 1.36 (s, 9H); m/z
532(M++H).
4.13.1. Enzyme assay
DHPS activity was measured in a 30
lL reaction containing 5 lM
14C pABA, 12.5
lM 6-Hydroxymethyl-7,8-dihydropterin diphos-
phate, 10 mM magnesium chloride, 2% PEG400, 4% DMSO, 50 mM
HEPES pH 7.6, and 10 ng DHPS.6,18 After 30 min incubation at
4.10. Potassium 4-{[(2-amino-4-oxo-3,4-dihydro-pyrido[2,3-
d]pyrimidin-6-yl)-(2-phosphono-ethyl)-amino]-methyl}-ben-
zoate (10)
37 °C, the reactions were stopped by addition of 1 lL of 50% acetic
acid in an ice bath. The labeled product of the reaction, 14C dihydro-
pteroate, was separated from 14C pABA by thin layer chromatogra-
phy. Aliquots (15 lL) of the reaction mixture were spotted onto
A mixture of 8 (0.03 g, 0.06 mmol) and 1.5 N KOH (50 mL) in
MeOH (50 mL) was stirred for 72 h at room temperature. The reac-
tion mixture was concentrated to about 5 mL volume. The reaction
solution pH was adjusted to about 10 with AcOH. The solution was
purified by HPLC using 1% formic acid/water solution and acetoni-
trile to afford 10 as the monopotassium salt as a greenish-yellow
solid (0.012 g, yield 44%). Mp >300 °C; 1H NMR (500 MHz, CD3OD)
d 8.22 (s, 1H), 8.04 (d, 2H, J = 8.3 Hz), 7.50 (d, 2H, J = 8.1 Hz), 7.43 (s,
1H), 4.52 (s, 2H), 2.42–2.15 (m, 2H), 1.79–1.72 (m, 2H); m/z 458.1
(M++K).
Polygram TLC plates and developed with ascending chromatogra-
phy in 100 mM phosphate buffer pH 7.0. The plates were scanned
using a Typhoon (GE Healthcare) and analyzed with ImageQuant
TL. Inhibition was tested by adding 200 lM inhibitor to reaction
solution. In addition, the reaction was performed using a 10 min
pre-incubation of inhibitor and enzyme prior to substrate addition.
4.13.2. Molecular modeling
All molecular modeling steps utilized the Maestro Suite version
9.0 from Schrodinger Inc. The compounds 12 and 13 were proton-

J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 1298–1305
1305
ated at the central linker nitrogen at pH 7, and a chiral centre exists
at C6. Consequently, all possible stereoisomers were modeled and
docked into the structure of BaDHPS obtained from the work of
Kirk et al.5 The GlideXP module of Maestro was utilized for docking
with only the highest scored pose retained for each molecule. The
BaDHPS receptor structure for docking contained a conserved
water molecule as shown in Figure 2c. The docking grid was gen-
erated with the centre of the box set to the centroid of the bound
inhibitor in the BaDHPS structure in Kirk et al.5 and the box size
was fixed by ‘dock with ligand length’ option in Glide set to 15 Å.
Compound 11 was then redocked with the option to retain top
10 poses. The poses generated were visually inspected and best
ranked pose was found to have a plausible binding mode. This
was then subjected to 20 cycles of minimization using the Impact
module with OPLS_2005 force field and all parameters set to de-
fault, followed by a 1 ns molecular dynamics simulation in vacuo
using Impact with a time step of 1 fs, default OPLS_2005 force field
and NVT constant temperature ensemble set to 298.15 K. Snap-
shots every 0.5 ps were recorded. All other parameters were left
at their default values. For Figure 2a and b, crystal structures
1tww and 1tx0, respectively were used to reproduce Figures 5
and 6 from Babaoglu et al.3
S.W.W. and R.E.L.), Cancer Center core Grant CA21765, and the
American Lebanese Syrian Associated Charities (ALSAC). .
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Funding for this research was provided by National Institutes of
Health Grants AI060953 (to S.W.W. and R.E.L.) and AI070721 (to