Pterin–sulfa conjugates as dihydropteroate synthase inhibitors and antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2016.07.006

The sulfonamide class of antibiotics has been in continuous use for over 70?years. They are thought to act by directly inhibiting dihydropteroate synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin–sulfonamide conjugates. In this study, eight pterin–sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin–sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.

Full text of DOI:10.1016/j.bmcl.2016.07.006

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3950–3954
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pterin–sulfa conjugates as dihydropteroate synthase inhibitors
and antibacterial agents
Ying Zhao a,y, William R. Shadrick a,y, Miranda J. Wallace a, Yinan Wu c,d, Elizabeth C. Griffith c, Jianjun Qi b,
Mi-Kyung Yun c, Stephen W. White c,d, Richard E. Lee a,b,
⇑
a Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 1000, Memphis, TN 38105, United States
b Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, United States
c Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 311, Memphis, TN 38105, United States
d Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The sulfonamide class of antibiotics has been in continuous use for over 70 years. They are thought to act
by directly inhibiting dihydropteroate synthase (DHPS), and also acting as prodrugs that sequester pterin
pools by forming dead end pterin–sulfonamide conjugates. In this study, eight pterin–sulfonamide con-
jugates were synthesized using a novel synthetic strategy and their biochemical and microbiological
properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition
was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote
an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of
the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product
analog pteroic acid. The antimicrobial activities of the pterin–sulfonamide conjugates were measured
against Escherichia coli in the presence and absence of folate precursors and dependent metabolites.
These results show that the conjugates have appreciable antibacterial activity and act by an on target,
anti-folate pathway mechanism rather than as simple dead end products.
Received 4 May 2016
Revised 29 June 2016
Accepted 2 July 2016
Available online 4 July 2016
Keywords:
Sulfonamide
Antibiotics
Antifolate
Dihydropteroate synthase
Pterin
Ó 2016 Elsevier Ltd. All rights reserved.
Sulfonamides were the some of the first successful antimicro-
bial agents and have been used continuously since the 1940s to
treat a wide variety of Gram-positive, Gram-negative and proto-
zoal infections.1–3 Sulfonamides are usually prescribed in combina-
tion with the dihydrofolate reductase inhibitor trimethoprim to
maximize therapeutic response and reduce the development of
resistance.4,5 Today, this drug combination is relied upon as a valu-
able therapeutic option in two important areas: (1) as an inexpen-
sive oral treatment for community acquired bacterial infections
resistant to frontline agents such as penicillins and fluoro-
quinolones; and (2) as a prophylaxis treatment for the prevention
of Pneumocystis jirovecii pneumonia in immuno-compromised can-
cer and HIV patients.6,7 Over time, sulfonamide use has been com-
promised by the emergence of resistance and the poor tolerance of
patients to long-term treatment.6,8
catalyzes the conjugation of 6-hydroxymethyl-7,8-dihydropterin
pyrophosphate (DHPPP) with pABA to form 7,8-dihydropteroate,
an intermediate in the folate biosynthesis pathway (Fig. 1A).10 In
addition to inhibiting DHPS, sulfonamides can also act as alterna-
tive substrates by forming dead end pterin–sulfonamide products,
which may also inhibit enzymes later in the pathway (Fig. 1B).10–12
In this role, the contribution to the overall antimicrobial potency of
this class of antibiotics is poorly defined. For the purpose of contin-
uing our studies on the inhibition of DHPS and its therapeutic
potential, we sought to chemically synthesize a panel of pterin–
sulfonamide conjugates.
Repeating the previously published alkylation based route13 to
these conjugates, we obtained only incorrect isomeric products,
challenging the previously ascribed pharmacological proper-
ties.14,15 Consequently, a new synthetic route to rapidly and accu-
rately generate pterin–sulfonamide conjugates was developed. The
new conjugates were tested for inhibition of DHPS and co-crystal-
lized with the enzyme to characterize their mode of binding.
Microbiological profiling using Escherichia coli indicated that
pterin–sulfonamides are not only good inhibitors of DHPS but also
have substantial antimicrobial activity that can be antagonized by
folate precursors.
Sulfonamides target the enzyme dihydropteroate synthase
(DHPS) and mimic the substrate p-aminobenzoic acid (pABA).1,9
We previously reported detailed structural, biochemical and com-
putational studies on the catalytic mechanism of DHPS.10 DHPS
⇑
Corresponding author.
y
Co-first authors.
http://dx.doi.org/10.1016/j.bmcl.2016.07.006
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

Y. Zhao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3950–3954
3951
Figure 1. (A) Reaction catalyzed by dihydropteroate synthase; (B) general structure of pterin–sulfonamide conjugates.
Scheme 1. Initial synthetic strategy.
K2CO3, N,N-
dimethyl-4-
Cl
N
nitrosoaniline,
NC
N
N
NC
N
N
6
POCl3
81%
H
pyridine NC
N
N
EtOH, rt, 30 min
Cl
N
Cl
N
NC
N
N
91%
H2N
89%
N
O
H2N
H2N
H2N
O
5
7
8
1.DOWEX 50WX8-
100 ion-exchange
resin, methanol
CHO
O
N
AcOH,
100 o
NH2
C
HCl
5% NaOH
N
CH(OCH3)2
NC
N
CH(OCH3)2
HN
N
97%
2.NaOCH3,CH3OH,
guanidine carbonate
79%
88%
H2N
N
H2N
N
H2N
N
N
66%
9
10
11
O
S
O
1) AcOH, sufonamides;
2) borane tert-
O
O
R
O
N
HCOOH,
butylamine complex, rt,
2 h
CHO
rt, 30 min
N
N
N
N
CH(OCH3)2
HN
AcHN
HN
N
H
HN
AcHN
67%
N
N
AcHN
N
N
12
13
14 a-h
O
S
O
N
O
N
O
N
1) 0.1 N NaOH;
2) HCl
O
N
R
N
H
N
H
N
N
H
N
HN
N
H
15
16
17
H2N
N
O
N
N
N
15-22
N
H
OCH3
OCH3
N
N
N
O
N
H
H
18
19
20
NH2
O
N
H
CH3
22
21
Scheme 2. Synthetic method for production of pterin–sulfonamide conjugates.
Our initial strategy to synthesize the conjugates was based on
the method detailed by Piper and Montgomery.13 Briefly, commer-
cially available (2,4-diaminopteridin-6-yl)methanol 1 was treated
with triphenyl phosphine and bromine to afford the 6-bro-
momethyl pterin analog 2,13 which was expected to couple with
the appropriate sulfonamide, followed by 4-deamination, to give
target compounds in three steps (Scheme 1). However, when com-
pound 2 reacted with sulfamethoxazole 3 in the presence of N,N-
diisopropylethylamine (DIEA), only isomeric sulfonamide linked
compound 4 was isolated as the main product, instead of the
desired aniline linked pterin–sulfamethoxazole 15. Attempts to
modify the synthesis by protection of the sulfonamide or alter
the alkylating conditions failed.16–18
An alternative synthetic route that provided the necessary
flexibility to conjugate a large variety of sulfonamides to pterin
was developed around the key acyl protected formyl-pterin

3952
Y. Zhao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3950–3954
Table 1
Inhibitory activities of pterin–sulfa conjugates
O
S
O
R
O
N
N
N
HN
H2N
N
H
Compd
R
BaDHPS in vitro
FP binding inhibition
IC50 M)
Minimum inhibitory concentration E. coli K12, in l
Mb,c
(
l
(ꢀ) PPia
(+) 2 mM PPia
M9 media
10.9 (0.8)c
M9 plus 5
467 (263)c
lg/mL PABA
M9 plus 20
29 (1.5)c
lg/mL methionine
O
15
20.6 3.5
3.4 0.1
N
N
H
N
O
N
N
16
17
18
22.5 1.0
19.3 0.6
>62.5
8.1 0.7
9.4 0.7
2.5 0.2
28.3 (1.5)
17.1 (1.6)
29.5 (2.7)
>412 (249)
>470 (666)
>471 (669)
47 (5.9)
78 (6.2)
98 (6.3)
N
H
N
H
N
N
H
O
N
N
19
20
26.8 3.0
15.2 0.6
1.9 0.1
17.2 (1.6)
34.3 (3.8)
>412 (537)
86 (3.4)
N
H
O
N
N
11.5 0.8
>412 (>644)
172 (10.1)
N
H
OCH 3
OCH 3
NH
21
22
17.8 0.8
18.6 0.8
11.3 1.1
17.1 1.2
197 (25)
136 (22)
236 (>934)
236 (50)
2
>514 (>934)
>200 (58)
N
H
O
Ampicillin
Chloramphenicol
Trimethoprim
ND
ND
ND
ND
ND
ND
1.58
8.5
1.7
2
9.7
1.4
2.1
9.7
0.7
a
b
c
Present as sodium pyrophosphate. Average of 3 experiments, SEM.
Performed according to.24
Corresponding values for parent sulfonamides shown in parentheses.
intermediate 13 shown in Scheme 2. Treatment of commercially
available 2-amino-5-(chloromethyl)-3-cyanopyrazine 1-oxide 5
with phosphorus trichloride at room temperature in tetrahydrofu-
ran solution resulted in smooth deoxygenation to give 6,19 which
was treated with pyridine with stirring overnight at room temper-
ature to afford pyridinium salt 7.20 Salt 7 reacted with N,N-
dimethyl-4-nitrosoaniline in the presence of potassium carbonate
to give nitrone 8, which was then treated with 6 N HCl to give alde-
hyde 9. Quantitative conversion of 9 to its dimethyl acetal in the
presence of DOWEX 50WEX8-100 ion-exchange resin, followed
by condensation with guanidine carbonate in the presence of
sodium methoxide, afforded 2,4-diamino pterin 10. Treatment of
10 with hot 5% sodium hydroxide gave 6-formylpterindine
dimethyl acetal 11, which was treated with acetic anhydride at
100 °C to give 2-acetyl protected 12. The 6-formylpterine dimethyl
acetal 12 was hydrolyzed with the treatment of formic acid to
afford 6-formylpterin 13.21 Coupling of 6-formylpterin 13 with dif-
ferent sulfonamides or dapsone (4-[(4-aminobenzene)sulfonyl]
aniline) was carried out smoothly in acetic acid to give the
corresponding Schiff base compound, which was subsequently
reduced with borane tert-butylamine complex to give 14a–h.22
Deprotection of 14a–h with 0.1 N NaOH afforded target
compounds 15–22.23
Compounds 15–22 were all tested for inhibition of Bacillus
anthracis DHPS using a fluorescence polarization (FP) based com-
petition binding assay, performed in the absence or presence of
2 mM sodium pyrophosphate (Table 1 and Supplementary mate-
rial).25 Pyrophosphate (PPi) is the biochemical byproduct of DHPS
catalysis, and its inclusion in the FP assay allowed us to confirm
the site-specific binding of pterin–sulfonamide conjugates. More
specifically, we have previously observed that PPi stabilizes two
flexible loops in the DHPS active site that form the pABA–sulfon-
amide binding site.10
As a group, the pterin–sulfonamide conjugated compounds are
more efficient than SMX (FP IC50 > 62.5 lM) at displacing the probe
from the active site in the FP competition assay. These compounds
show an even more efficient displacement of probe when PPi is
added to the assay. Among all analogs, pterin–sulfadimethoxine
(pterin–SDM) conjugate 19 and pterin–sulfamethoxazole (pterin–
SMX) conjugate 15 have the highest potency against BaDHPS, with
IC50 values of 1.9 lM and 3.4 lM, respectively, in the presence of
PPi. Conjugation of pterin with other sulfa analogs resulted in com-
pounds with good inhibitory activity with the weakest inhibition
observed for the sulfanilamide conjugate 22.
To establish the mode of binding of the pterin–sulfonamide
conjugates in the DHPS active site, we soaked compound 16 into

Y. Zhao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3950–3954
3953
Figure 2. (A) Yersinia pestis DHPS (YpDHPS) with compound 16 at the active site. Also shown is the omit map (mFo-DFc) showing the electron density of 16 contoured at 2
r
(PDB ID: 5JQ9). (B) YpDHPS with the product analog pteroic acid bound at the active site (PDB ID: 3TYU). (C) Bacillus anthracis DHPS (BaDHPS) with the pterin–sulfathiazole
(DHP–STZ) catalytic product bound at the active site (PDB ID: 3TYE). (D) Superimposition of the YpDHPS 16 complex and the YpDHPS near transition state complex (PDB ID:
3TYZ). In all figures, the DHPS cartoon and carbon atoms are gray, the oxygen, nitrogen and sulfur atoms are red, blue and yellow respectively, and the compound carbons are
cyan. In (D), the transition state structure is shown as pink cartoon and carbon atoms, the ordered loop2 is shown in green, and the essential Mg2+ ion is shown in lime.
apo crystals of Yersinia pestis DHPS (YpDHPS) and determined the
structure of the complex to 2.1 Å resolution (PDB ID: 5JQ9). As
anticipated, 16 occupies both the pterin and pABA binding pocket
of DHPS (Fig. 2A). The mode of binding is very similar to those of
pteroic acid26 and the sulfathiazole–pterin adduct that was gener-
ated catalytically in crystallo10 (Fig. 2B and C). Residues D96, N115,
D185, K221 and R255 interact with the pterin moiety, and F190,
K221 and S222 interact with the sulfonamide/pABA groups
(YpDHPS residue numbering). We have also determined the struc-
ture of the DHPS near transition state structure,10 and its superpo-
sition onto the 16 complex shows how closely the compound
mimics the binding modes of pterin and pABA. Note that weak
electron density adjacent to 16 (Fig. 2A) corresponds to the DHPS
anion-binding pocket, which is occupied by sulfate and pyrophos-
phate in the DHP–STZ and transition state structures, respectively
(Fig. 2C and D). Occupancy of the anion-binding pocket by
pyrophosphate and the resulting stabilization of loop2 (Fig. 2D)
would explain our observation that pyrophosphate augments the
binding of the conjugate compounds.
The antibacterial activities of these conjugate compounds and
their corresponding parent sulfonamides were tested against
Escherichia coli K12 (Table 1) in M9 minimal media27 suitable for
testing sulfonamide activity. Under these conditions, most conju-
gates showed appreciable antibacterial activity, with the exception
of the sulfanilamide and dapsone hybrids (21, 22) mirroring the
weaker direct activity of the corresponding parent sulfonamides.24
The sulfamethoxazole hybrid (15) showed the most potency with a
activities for the conjugates were usually around 10 times weaker
than for the corresponding parent sulfonamides (compound 15
10.9 lM, vs sulfamethoxazole 0.8 lM).
We next explored whether the observed antimicrobial activities
are a result of on target biochemical inhibition. MIC activities were
examined in the presence of the added folate precursor pABA or
the dependent metabolite methionine (Table 1). Addition of pABA
completely ablated MIC activity for all conjugates,28 and a similar
but less dramatic effect was seen when growth media was supple-
mented with methionine. These data mirrored the MIC shifts we
observed with the corresponding sulfonamides, supporting the
hypothesis that the pterin conjugates function as direct antibacte-
rial compounds through inhibition of the folate pathway.
In conclusion, we have demonstrated that pterin–sulfonamide
conjugates have antibacterial action through biochemical inhibi-
tion of DHPS and antagonism of the folate pathway. It is important
to note that the conjugates synthesized in this study are in the oxi-
dized form of pterin and not the reduced form dihydropterin that is
formed by the natural biochemical action of DHPS on DHPPP and
sulfonamides, which likely accounts for some of the reduction in
pharmacological activity. However, our structures reveal that the
oxidation state of the pterin ring has little effect on how it binds
into the pterin pocket (compare Fig. 2A and B with C and D).
The lower MIC activity of the conjugates compared to their cor-
responding sulfonamides may be attributable to differences in
penetration and efflux of the pterin sulfonamide conjugates versus
sulfonamides. Further studies are ongoing to study the susceptibil-
ity to efflux, capacity to inhibit enzymes later in the pathway and
MIC of 10.9 lM. Overall the minimal inhibitory concentration

3954
Y. Zhao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3950–3954
8. Ho, J. M.; Juurlink, D. N. CMAJ 2011, 183, 1851.
in cell metabolism of the conjugates. The pharmacological advan-
tages that pterin–sulfonamide conjugates may have over sulfon-
amides are also worthy of further study. The conjugates may
have a lower propensity for sulfonamide mediated anaphylaxis,
as the oxidatively reactive aniline group in the sulfonamides is hin-
dered by the pterin conjugation.29 Another important observation
from this study is the enhanced binding to DHPS by these conju-
gates that presumably results from the introduction of a pterin
anchor. The pterin anchor also has implications for the develop-
ment of resistance to these conjugates. As previously noted,30 the
pterin pocket is highly conserved and unlikely to tolerate muta-
tions that might decrease binding affinity. This is not true of the
isolated sulfonamides because the pABA-binding site can tolerate
such mutations.10 Thus, the activity of the conjugates against
defined sulfonamide resistant strains should be determined.
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Acknowledgements
This research was supported by funding from the National Insti-
tutes of Health (grant R01AI070721 to R.E.L. and S.W.W.) and the
American Lebanese Syrian Associated Charities (ALSAC), St. Jude
Children’s Research Hospital.
benzenesulfonamide (15):
A mixture of N-(6-((4-(N-(5-methylisoxazol-3-
yl)sulfamoyl)phenylamino)methyl)-4-oxo-3,4-dihydropteridin-2-yl)
acetamide (0.12 g, 0.255 mmol) in 0.1 M NaOH (24 mL) was stirred at room
temperature overnight. The reaction was quenched by adjusting the pH to 8
with diluted HCl. The reaction solution was concentrated to about 5 mL, then
the reaction solution was adjusted to pH 5–6 with diluted HCl and filtered,
washed with water and MeOH, then dried to give a yellow solid (0.106 g, 97%);
1H NMR (400 MHz, DMSO-d6): d 2.25 (s, 3H), 4.46 (d, 2H, J = 5.6 Hz), 6.03 (s,
1H), 6.68, 7.50 (dd, 4H, J = 8.8 Hz), 7.07 (br s, 2H, D2O exch), 7.33 (br t, 1H,
J = 5.6 Hz, D2O exch), 8.64 (s, 1H), 11.09 (br s, 1H, D2O exch), 11.57 (br s, 1H,
D2O exch); m/z 429.2 (ES+H+).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.07.
006.
24. Zlitni, S.; Ferruccio, L. F.; Brown, E. D. Nat. Chem. Biol. 2013, 9, 796.
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T.; White, S. W.; Lee, R. E. Bioconjugate Chem. 2011, 22, 2110.
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27. ATCC Medium 2511. The carbon source in this media is glucose.
28. Bock, L.; Miller, G. H.; Schaper, K. J.; Seydel, J. K. J. Med. Chem. 1974, 17, 23.
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