Design, synthesis, and structure - Activity relationship studies of ATP analogues as DNA gyrase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00109-8

We report herein the design and synthesis of ATP-analogues, namely 4-amino-pyrazolo[3,4-d]pyrimidines and 4-amino-pyrazolo[1,5-a][1,3,5]triazines, with DNA gyrase inhibitory activity. Among these series, some compounds exhibited promising antibacterial activity. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00109-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 821±826
Design, Synthesis, and Structure±Activity Relationship Studies of
ATP Analogues as DNA Gyrase Inhibitors
Thomas Lubbers,* Peter Angehrn, Hans Gmunder, Silvia Herzig and Josef Kulhanek
Department of Infectious Diseases, F. Ho€mann-La Roche Ltd., CH-4070 Basel, Switzerland
Received 29 October 1999; accepted 11 February 2000
AbstractÐWe report herein the design and synthesis of ATP-analogues, namely 4-amino-pyrazolo[3,4-d]pyrimidines and 4-amino-
pyrazolo[1,5-a][1,3,5]triazines, with DNA gyrase inhibitory activity. Among these series, some compounds exhibited promising
antibacterial activity. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Modelling
In recent years emergence and spread of resistance have
caused serious problems in the e€ective treatment of
infectious diseases.1 Multiresistant pathogens were
isolated especially in the clinics. We therefore searched
for new and e€ective antibiotics, which overcome
resistance.
Investigation of the X-ray structure of the complex of
the nonhydrolyzable analogue of ATP, ADPNP, with
the 43 kDa fragment of DNA gyrase B5 reveals that the
aminopyrimidine ring of ATP is recognised by the
enzyme via a H-bond network to asp73 and a tightly
bound water molecule, which is held in addition by two
amino acids (Fig. 1). The other two water molecules
depicted ®ll the empty space between ATP and the
lipophilic cavity of the enzyme. An inhibitor, which
mimics ATP but could eventually replace these two
water molecules, should bind speci®cally to gyrase B
with higher anity than ATP. Selectivity of the
designed inhibitors against other ATP-binding enzymes
is probably guaranteed by the fact that gyrase shows an
unusual binding mode for ATP, which is shared by few
other enzymes.6 In addition high and selective anity to
gyrase B should be achieved by replacing the sugar
moiety with substituents which can interact with ile94,
and by placing substituents at the free position of the
pyrimidine ring which are capable of interacting with
arg136 and a salt bridge located in this region. As
shown by X-ray crystallography,7 the 4-hydroxy-
coumarin moiety of the antibiotic novobiocin (3) binds
to these residues (Fig. 1).
DNA topoisomerases control the topological state of
DNA. They are involved in e.g. DNA transcription and
replication and therefore are essential for cell viability.2
Like eucaryotic topoisomerases II bacterial DNA gy-
rase can relax positive supercoiled DNA, but it is unique
in its ability to introduce negative supercoils into DNA,
processes which consume ATP. The active DNA gyrase
complex consists out of a tetramer A2B2 of the subunits
A and B each of which of about 90 kDa. Quinolones,
which are successfully used as broad spectrum anti-
biotics in the clinic, target DNA gyrase A by interfering
with DNA cleavage and religation reaction. The cou-
marin containing antibiotics such as novobiocin (3),
known for more than 40 years, bind to DNA gyrase B
competing with ATP. They demonstrated in vivo e-
cacy against several bacterial pathogens. However, the
coumarins su€er from several disadvantages4 such as
toxicity and fast emergence of resistance. We are
designing inhibitors for the ATP-binding-pocket of
subunit B.3
By superpositioning ATP (1) with aminotriazine 4, a
recently described DNA gyrase B inhibitor,8 we gener-
ated 4-amino-pyrazolo[3,4-d]pyrimidines 5 and 4-amino-
pyrazolo[1,5-a][1,3,5]triazines 6 as potential DNA gyr-
ase inhibitors (Fig. 2). These would preferentially bear
an aryl-group in the 1- and 8-position of 5 and 6,
respectively, and an aryl-group linked via a heteroatom
X at position 6 and 2. Residues R1 and R4 should be
*Corresponding author. Fax: +41-61-68-86459; e-mail: thomas.
luebbers@roche.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00109-8

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T. Lubbers et al. / Bioorg. Med. Chem. Lett. 10 (2000) 821±826
È
hydrogen or small substituents in order to maximise
lipophilic interactions with the enzyme and eventually
replace the water molecules mentioned above.
cyclized onto the pyrazole with sodium methoxide.
Methylation of the thiol with methyl iodide yielded
hydroxytriazines 10. Chlorination in re¯uxing phos-
phorus oxychloride in the presence of a base, sub-
sequent substitution with ammonia in DMF, oxidation
with MCPBA yielded the corresponding sulfones, which
could be substituted easily with phenolates and thiolates
in DMF to yield pyrazolo[1,5-a][1,3,5]triazines 6.
Chemistry
3-Aminopyrazoles 8 were prepared by reacting alkoxy-
methylene malononitriles 9 or formylated benzylnitriles
prepared from 7 with hydrazine in alcohol (Scheme 1).
They were converted with ethoxycarbonylisothiocyanate
to the corresponding thiourea derivatives, which were
We investigated the reaction of aminopyrazoles 8 with
the commercially available dimethyl N-cyanodithio-
imino carbonate 11 to yield in one-step the triazine
Figure 1. Schematic representation of ATP bound to DNA gyrase B; superposition of ADPNP (2, blue) with novobiocin (3, grey).
Figure 2.
Scheme 1. (a) PBr3, Et2O, 0 ꢀC to rt, 6h, 96%; (b) KCN, EtOH, H2O, re¯., 4 h, 78%; (c) NaOMe, HCO2Et, benzene, rt, 2 h, 87%; (d) N2H4, EtOH,
HOAc, re¯., 6 h, 50%; (e) N2H4, EtOH, re¯., 2±5 h, 92±94%; (f) EtOCONCS, acetone, rt, 4 h, 68±89%; (g) NaOMe, MeOH, rt, 4±12 h, 66±90%; (h)
MeI, NaOH, MeOH, H2O, rt, 4±12 h, 66±90%; (i) POCl3, NEt2iPr, re¯., 2±4 h, 78±94%; (j) concd aq NH3, DMF, rt, 3 h, 50±93%; (k) MCPBA,
THF, rt, 4 h, 48±100%; (l) NaH, HXR3, DMF, rt±90 ꢀC, 3±12 h, 35±87%.

T. Lubbers et al. / Bioorg. Med. Chem. Lett. 10 (2000) 821±826
È
823
derivatives 12 (Scheme 2). This would shorten the
synthesis of 6 by four steps (Scheme 1, steps f±j). Results
from similar reactions described in the literature9 were
not conclusive as to the outcome of regioselectivity.
With aryl substituents at the pyrazole ring, we obtained
the desired product 12. In contrast, a nitrile or ester
group at the same position a€orded the opposite regio-
chemistry (13). Whereas 12 was easily oxidizable with
MCPBA, compound 13 resisted oxidation with
MCPBA even under forcing conditions.
novobiocin (3).10 We synthesised these coumarins 17±19
in order to incorporate them in our new inhibitors as
substituents at the aminopyrimidine ring.
Biology
The compounds synthesised were tested in a coupled,
spectrophotometic ATPase assay (using DNA gyrase,
pyruvate kinase and lactate dehydrogenase), which
monitored the disappearance of NADH as a function of
ATP-consumption, and for validation in the super-
coiling assay.11 The e€ect was measured as maximum
non-e€ective concentration (MNEC) de®ned as the
highest inhibitor concentration, which showed no DNA
gyrase inhibition. The in vitro antibiotic activity was
measured as minimal inhibitory concentration (MIC) in
2-fold agar dilutions (Muller-Hinton agar, inoculum 104
CFU/spot).
The synthesis of the pyrazolo[3,4-d]pyrimidines
5
(Scheme 3) started with the preparation of 1-aryl-5-
aminopyrazoles 15 from ethoxymethylene malononitrile
derivatives 8 and arylhydrazines 14. Condensation with
benzoylisothiocyanate prepared in situ, subsequent
cyclization with sodium hydroxide and methylation
with methyl iodide yielded the pyrimidones 16. Re¯ux-
ing with phosphorus oxychloride in the presence of a
base followed by substitution with an amine or ammo-
nia and ®nally oxidation with MCPBA a€orded the
sulfones, which could be easily substituted with pheno-
lates and thiolates to yield the products 5.
Results and Discussion
The pyrazolotriazines with a 4-hydroxy-coumarin (6b,c)
or a 3-mercapto benzoic acid substituent (6d,e) were
good enzyme inhibitors (Table 1) proving our modelling
hypothesis.
As discussed above we were hoping to reach the cou-
marin-binding site of novobiocin (3) with our new inhi-
bitors to gain anity through p-stacking to a salt bridge
and interactions with the basic arg136. The benzamide
residue on the coumarin ring is necessary for the good
antibacterial activity of novobiocin (3) but is fairly large
and lipophilic. Recently novobiocin-derivatives with
smaller and more hydrophobic coumarin-residues 17±19
(Fig. 3) were described that were equally active to
The pyrazolopyrimidines 5 followed a similar trend to
the pyrazolotriazines 6 (Table 2). Again, the 4-hydroxy-
coumarin (5m±q) and 3-mercaptobenzoic acid deriva-
tives (5g±5l) were the most active enzyme inhibitors.
The residue R1 (H, Me, Et) had a strong in¯uence on
Scheme 2. (a) CH2Cl2, NEt2iPr, 50 ꢀC, 3d.
Scheme 3. (a) R2NHNH2ÃHCl 14, NEt3, MeOH, re¯., 3 h, 58±97%; (b) PhCOCl, KSCN, acetone, re¯., 3±12h, 78±100%; (c) NaOH, MeOH, re¯., 2
h, 54±100%; (d) MeI, NaOH, MeOH, H2O, rt, 2±12h, 63±100%; (e) POCl3, NEt2iPr, re¯., 1 h, 78±99%; (f) NH3gas or NH2R4, DMF or THF, rt,
12h, 59±99%; (g) MCPBA, THF, 0 ꢀC to rt, 12 h, 64±92%; (h) NaH, HXR3, DMF, 70 ꢀC, 11±97%.
Figure 3.

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T. Lubbers et al. / Bioorg. Med. Chem. Lett. 10 (2000) 821±826
È
Table 1. In vitro E. coli DNA gyrase supercoiling activity (MNEC (mg/mL))
R1
R2a
-X-R3 (compound no.)
Me
H
-CN
3,4-(MeO)2-Ph-
3,4-(Cl)2-Ph-
8
0.1
6b
6c
0.5
1
6d
6e
>63 (<16b) 6a
aThe numbering refers to the position of the substituent on the benzene ring.
bThe activity was determined with the ATPase assay.
Table 2. In vitro E. coli DNA gyrase supercoiling activity (MNEC (mg/mL))
R2 a
R1
R4
-X-R3 (compound no.)
3,5-(Cl)2-Ph-
3-F-Ph-
H
Me
Et
H
H
H
c-Pr
Et
H
63±125b
31±63b
63±125b
5a
5b
5c
>125
63±125b
>16 (</b)
5d
5e
5f
1
1
0.25
8
8
8
5g
5h
5i
5j
5k
5l
0.5
0.25
0.06
4
5m
5n
5o
5p
Et
1
5q
aThe numbering refers to the position of the substituent on the benzene ring.
bThe activity was determined with the ATPase assay.
the activity. The ethyl group was obviously best capable
of optimising the lipophilic interactions with the enzyme
(e.g. 5m,n,o).
bacterials than 6. From the compounds listed in Table 2
only 5i was moderately antibacterial active. All unsub-
stituted hydroxycoumarins; although being highly
potent enzyme inhibitors (5m±o); were inactive against
bacteria. As we anticipated 3-substituted 4-hydroxy
coumarin substituentsÐespecially with amide and urea
residues (5v,w)Ðled to inhibitors with improved cell
permeability and promising activity against Gram-positive
bacteria including novobicin resistant strains (Table 4).
Additional substituents R4 on the amino group
decreased the activity (5i±k,o,p). The 3,5-dichloro-
phenyl group proved to be the best substituent in the 1-
position (5i,l and 5o,q). The best series was found to
be 4-amino-1-(3,5-dichlorophenyl)-3-ethyl pyrazolopy-
rimidines. We were able to obtain an X-ray structure of
5i complexed with the 23 kDa fragment of DNA gyrase
B (loop-deletion mutant of S. aureus enzyme;12 Fig. 4).
Our modelling hypothesis was thus con®rmed: speci®c
H-bonds to asp73 and to a water molecule, p-stacking
to the salt bridge, ionic interactions with arg136, lipo-
philic interactions with ile94 and replacement of one
H2O by the ethyl group.
Variations on the coumarin substituent (5q±w, Table 3)
decreased the activity compared to the highly potent
coumarin 5o. Still the enzyme activities of the 3-acyl-4-
hydroxy coumarin derivatives 5t±w were good enough
to be antibacterial active if they could penetrate well
through the bacterial membrane.
All investigated pyrazolotriazines 6 were almost inactive
against bacteria; they showed at best slight activity
against S. epidermidis; except for compound 6e, which
was unfortunately inactive against S. aureus (Table 4).
In general, the pyrazolopyrimidines 5 were better anti-
Figure 4. X-ray structure12 of the 23kDa loop-deletion fragment of
DNA gyrase B with 5i (Weblab Viewer Pro 3.0 1).

T. Lubbers et al. / Bioorg. Med. Chem. Lett. 10 (2000) 821±826
È
825
Table 3. In vitro E. coli DNA gyrase supercoiling activity (MNEC (mg/mL))
R1
R2 (compound no.)
CO2Et
H
COMe
NHAc
NHCONHBu
Me
1-Me-piperazine
NMe2
>125b
5q
5ra
5sa
5oa
>125b
63±125b
0.06
OH
2
5ta
2
5ua
0.5
5v
0.5
5w
aThe compounds showed at best slight activity against S. epidermidis.
bThe activity was determined with the ATPase assay.
Table 4. In vitro E. coli DNA gyrase supercoiling activity (MNEC) and antibacterial activity (MIC)
Compound no. MNEC
(mg/mL)
MIC (mg/mL)a
S. aureus ATCC S. aureus S. aureus S. aureus S. aureus Smith S. aureus S. epidermidis S. pyogenes E. faecium
b15
25923
NovoR Coum-R Smith
QR-54
H19982 ATCC 14990
vanA E23-8
Novobiocin (3)
6e
5i
5v
5w
0.25
1
0.25
0.5
0.5
0.25
>32
16
16
4
8
>32
32
16
8
16
>32
32
16
8
0.25
>32
16
8
8
>32
2
0.25
>32
0.12
4
0.5
4
2
8
32
>3 2
>32
16
0.5
n.d.b
1
4
4
4
8
2
4
4
2
aResistance is mentioned as follows: NovoR (against novobiocin, R144-I in ATCC 25923), Coum-R (against coumermycin), QR54 (against methi-
cillin and quinolones), H19982 (against methicillin), vanA E23-8 (against ampicillin, trimethoprim, vancomycin).
bn.d.=not determined.
In summary, we have designed new ATP-analogues,
namely 4-amino-pyrazolo[3,4-d]pyrimidines and 4-amino-
pyrazolo[1,5-a][1,3,5]triazines, which exhibited potent
DNA gyrase inhibition and good antibacterial activity
against several resistant gram-positive bacteria.
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tests.
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