Active site directed inhibitors of replication-specific bacterial DNA polymerases

Article abstract of DOI:10.1016/j.bmcl.2004.11.016

7-Substituted-N²-(3,4-dichlorobenzyl)guanines potently and competitively inhibit DNA polymerases IIIC and IIIE from Gram+ bacteria. Certain derivatives are also competitive inhibitors of DNA polymerase IIIE from Gram- bacteria.

Full text of DOI:10.1016/j.bmcl.2004.11.016

Bioorganic & Medicinal Chemistry Letters 15 (2005) 729–732
Active site directed inhibitors of replication-specific bacterial
DNA polymerases
George E. Wright,a,* Neal C. Brown,a Wei-Chu Xu,a Zheng-yu Long,a Chengxin Zhi,a
Joseph J. Gambino,a Marjorie H. Barnesb and Michelle M. Butlerb
aGLSynthesis Inc., One Innovation Drive, Worcester, MA 01605, USA
bMicrobiotix Inc., One Innovation Drive, Worcester, MA 01605, USA
Received 12 October 2004; revised 4 November 2004; accepted 4 November 2004
Available online 25 November 2004
Abstract—7-Substituted-N2-(3,4-dichlorobenzyl)guanines potently and competitively inhibit DNA polymerases IIIC and IIIE from
Gram+ bacteria. Certain derivatives are also competitive inhibitors of DNA polymerase IIIE from GramÀ bacteria.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The replicative DNA polymerases of eubacteria—the
pol IIIs—have evolved from a common ancestor into
two distinct types with respect to primary structure—a
ÔCÕ type encoded by the gene polC, and an ÔEÕ type,
encoded by dnaE.1 Significantly, dnaE-like genes are
ubiquitous, present in all classes of eubacteria, while
polC is confined to a single group—the low G:C
Gram-positives (Gram+).2 In those organisms in which
dnaE is the sole pol III gene, that is in Gram-negatives
(GramÀ) and high G:C Gram-positives, pol IIIE is the
sole replication-specific polymerase.3 In its narrow, low
G:C Gram+ domain, pol IIIC is well established as a
replication-specific enzyme.3 However, it is now clear
from recent work in B. subtilis,4,5 E. faecalis6 and
S. aureus1 that the low G:C Gram+ pol IIIE is also an
essential replication protein.
IIIE, and a subset of that class that also inhibits GramÀ
pol IIIE.
6-Anilinouracils and 6-benzylaminouracils are well
established as active-site directed inhibitors of pol IIIC.9
The active compounds have two molecular ÔdomainsÕ re-
quired for inhibition: a Ôbase-pairing domainÕ responsi-
ble for hydrogen bonding of the molecule with
cytosine bases in the DNA template, and an Ôaryl
domainÕ responsible for selectivity and affinity to the
enzyme. The phenyl ring substitution patterns in the 6-
anilinouracils, for example, ÔEMAUÕ (1), and the 6-benz-
ylaminouracils, for example, ÔDCBAUÕ (2) have been
identified as optimal for binding of the respective fami-
lies to pol IIIC. Base pairing with cytosines explains the
competitive inhibition by these compounds with respect
to dGTP, and inhibition is caused by the formation of
an inactive inhibitor/DNA/enzyme complex.9 Indeed,
The replication of chromosomal DNA is required for
eubacterial cell division, and inhibition of this process
is rapidly bactericidal.7,8 We have expanded our study
of potential antibiotics based on pol IIIC inhibition to
include assays of recombinant pol IIIE from the Gram+
B. subtilis and, in addition, a commercial preparation of
pol IIIE from the GramÀ E. coli. As reported below, we
have discovered a class of compounds that potently and
non-selectively inhibits both Gram+ pol IIIC and pol
O
O
3
3
Cl
HN
HN
O
N
H
N
H
O
N
H
N
H
CH2
Cl
EMAU, 1
DCBAU, 2
9
NH
9
NH
7
7
N
N
Cl
N
N
Keywords: DNA polymerase; Replication; Competitive inhibitors;
Antibacterial; Broad spectrum.
O
N
H
NH CH2
Cl
O
N
H
N
H
*
Corresponding author. Tel.: +1 508 7546700; fax: +1 508 7547075;
e-mail: george.wright@glsynthesis.com
EMPG, 3
DCBG, 4
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.016

730
G. E. Wright et al. / Bioorg. Med. Chem. Lett. 15 (2005) 729–732
Table 1. Inhibition of pol III species by substituted 6-aminouracils and guanines
No.
Compd/substituent
Ki (lM)a
B.s. pol IIIC
B.s. pol IIIE
E.c. pol IIIE
1
2
EMAU
DCBAU
0.31
0.42
>315b
1.5
>323
>280
EMPGs:
3
EMPG
2.7
In
>248
In
In
Inc
In
In
In
3a
3b
3c
9-(CH2)4OH
7-(CH2)4OH
7-(CH2)5OH
0.47
0.28
318
DCBGs:
4
DCBG
7-(CH2)4OH
0.72
0.19
712
0.32
0.063
1.6
95
86
4a
4b
4c
9-(CH2)4OH
7-(CH2)2OEt
9-(CH2)2OEt
In
56
0.33
116
0.072
19.4
4d
4e
>209
16.9
>189
15.3
101
33
7-(CH2)3CO2Et
9-(CH2)3CO2Et
7-(CH2)3CO2H
9-(CH2)3CO2H
7-(CH2)4OAc
0.15
825
0.047
64
0.13
4f
4g
4h
4i
0.17
28
0.24
0.12
0.066
0.051
0.052
0.052
0.08
0.25
0.052
0.37
0.07
0.23
0.093
0.28
4j
7-(CH2)4-N[(CH2)4]O
7-(CH2)5OAc
0.047
0.058
0.035
0.031
0.071
0.091
0.11
26
4k
4l
13.8
41
7-(CH2)4CH(CH3)OH (R)
7-(CH2)4CH(CH3)OAc (R)
7-(CH2)5O(CO)iPr
7-(CH2)5OH
4m
4n
4o
4p
4q
4r
20.3
27.2
27
7-(CH2)2O(CH2)2OH
7-(CH2)5OMe
7-(CH2)5SMe
45
47
0.088
0.18
128
36
4s
7-(CH2)5SOMe
7-(CH2)5SO2Me
0.068
0.09
4t
51
a Assayed under truncated (-dGTP) conditions.12 Results are averages of duplicate assays (SD ꢀ30%).
b Lower limit of estimated Ki.
c <10% inhibition at highest concn. tested.
analogous N2-phenylguanines such as ÔEMPGÕ (3)
and the N2-benzylguanines such as ÔDCBGÕ (4) appear
to inhibit pol IIIC via the same mechanism.10,11
for pol IIIC and 25–100lM for pol IIIE (data not
shown).
The results of studies with 3-substituted-6-anilinoura-
cils7 prompted us to prepare N-substituted inhibitor
analogs in the guanine series. The general synthetic
strategy for these derivatives is illustrated in Scheme 1.
DCBG (or EMPG, by analogous chemistry) is alkylated
to give 9 and 7 isomers, in ratios of ꢀ2–3:1.13 Although
favoring the 9 isomers, this direct alkylation increases
the overall yield of 7 isomers compared with classical
alkylation of the 6-chloropurines via the sodium salt
method.14,15 Modification of the N-substituents pro-
vided additional derivatives, as illustrated in Scheme 1.
Among numerous N2-phenylguanines and N2-benzyl-
guanines, the 7-isomers, not the expected 9-isomers,
were the most active pol III inhibitors. 7-(4-Hydroxybu-
tyl)-EMPG (3b) potently and selectively inhibited B.
subtilis pol IIIC, but, surprisingly, the 9-isomer 3a was
inactive (Table 1). Among N-alkylated derivatives
of 4, the 7-substituted compounds, for example, 7-(4-
hydroxybutyl)-DCBG (4a), were considerably more
active than the 9-substituted compounds, for example,
4b, against both B. subtilis pol IIIC and pol IIIE.
The observation that 4a inhibited both Gram+ pol III
species prompted synthesis and assay of additional
DCBG derivatives (Table 1). With few exceptions,
Screening of an extensive library of related compounds
has shown that 6-anilinouracils such as 1 are potent
and selective inhibitors of pol IIIC, while certain 6-benz-
ylaminouracils inhibit both B. subtilis pol IIIC and pol
IIIE (Table 1).12 Among an extensive series of 6-(substi-
tuted)amino-uracils, halo-substituted 6-benzylaminor-
acils were the only derivatives that inhibited pol IIIC
and pol IIIE, and, among these, compounds bearing
the 3,4-dichloro substitution pattern of 2 were the most
potent (Table 1).
In the guanine inhibitor series, 3 was a moderately po-
tent but selective inhibitor of B. subtilis pol IIIC (Table
1), while N2-benzyl guanines showed significant activity
against both B. subtilis pol IIIs. Among these com-
pounds, the 3,4-dichlorobenzyl derivative 4 emerged as
the most potent inhibitor of the enzymes, with Ki values
of <1lM (Table 1). The results of further screening of
other N2-phenyl, benzyl and variously substituted gua-
nines showed that only halo-substituted N2-benzylgua-
nines had pol III inhibitory activity. These included
3-CF3–4-Cl, 3-Cl–4-CF3, 4-Cl, 4-CF3, and 3,4-OCH2O
benzyl compounds, but their Ki values were >10lM

G. E. Wright et al. / Bioorg. Med. Chem. Lett. 15 (2005) 729–732
731
O
N
N
Cl
HN
CH2 HN
9-AcOB-DCBG
Cl
Cl
N
O
N
(CH2)4OAc
Br-(CH2)4-OAc
K2CO3 ,DMF
N
Cl
HN
CH2 HN
+
N
H
Cl
O
N
(CH2)4OAc
N
NH4OH
MeOH
Cl
HN
CH2 HN
DCBG (3)
N
7-AcOB-DCBG, 4i
O
O
(CH2)4OH
N
O
N
(CH2)4I
N
(CH2)4
N
N
O
Cl
Cl
HN
O
HN
Cl
HN
CH2 HN
HN
CH2 HN
TMSI
N
Cl
CH2 HN
N
N
Cl
N
N
Cl
MeCN
CHCl3
4j
4a
Scheme 1.
7 isomers are more active than the corresponding 9 iso-
mers. A notable exception is 9-carboxypropyl-DCBG
(4h), which is 100-fold more active against Gram+ pol
IIIE than pol IIIC. Active EMPG derivatives are highly
selective for inhibition of pol IIIC, whereas DCBG
derivatives are potent but non-selective inhibitors of
Gram+ pol IIIC and pol IIIE. Certain 7-substituted
DCBGs, for example, 4e, 4i, 4j, 4m, 4o, inhibited E. coli
pol IIIE, a hitherto resistant bacterial pol III, although
the best compounds in this limited collection display
Ki values some 100-fold greater than the analogous
Gram+ pol III inhibitors.
competitive kinetics with respect to dGTP (Fig. 1, panel
A), and the calculated Ki 0.28lM, is essentially the same
as that derived directly from the ÔtruncatedÕ assay,
0.32lM (Table 1). We next asked if inhibition of pol
IIIC by a 7-substituted DCBG compound was also com-
petitive with dGTP, and if inhibition of the pol IIIEs
was also competitive. Assays using 7-(5-hydroxypentyl)-
DCBG (4o) and varying dGTP concentrations gave the
results displayed in panels B–D of Figure 1. In each
case, the double reciprocal plot suggested competitive
inhibition. Ki values for B. subtilis pol IIIC (0.091lM)
and pol IIIE (0.21lM) and for E. coli pol IIIE
(21lM) were essentially identical with those from the di-
rect assay of 4o (Table 1). We thus confirmed the active
site-directed mechanism of the DCBG compounds
against the Gram+ pol IIIs, and demonstrate in this
paper the first reported active site-directed inhibitors
of a GramÀ pol IIIE.
Inhibition of pol IIIC by uracil and simple guanine
inhibitors is strictly competitive with dGTP.8–10 That
this is true for the 7-substituted EMPGs was demon-
strated by variable substrate assays with 7-(5-hydroxy-
pentyl)-EMPG
(3c).
This
compound
showed
1.00
0.75
0.50
0.25
0.00
-0.25
0.75
0.50
0.25
0.00
-0.25
-0.5 -0.25
0
0.25
0.5
0.75
1
-0.5 -0.25
0
0.25
0.5
0.75
1
1/[dGTP] (µM-1)
1/[dGTP] (µM-1)
(A)
(B)
1.00
0.15
0.10
0.05
0.00
0.75
0.50
0.25
0.00
-0.05
-0.5 -0.25
-0.25
-0.5
0
0.25
0.5
0.75
1
-0.25
0
0.25
1/[dGTP] (µM-1)
1/[dGTP] (µM-1)
(C)
(D)
Figure 1. Double reciprocal (Lineweaver–Burk) plots demonstrating effect of varying dGTP concentration on inhibition of pol III species.12 Panel A,
B.s. pol IIIC + 3c. Panel B, B.s. pol IIIC + 4o. Panel C, B.s. pol IIIE + 4o. Panel D, E.c. pol IIIE + 4o. Inhibitor concentrations: (j) 3 · Ki; (m)
Ç
10 · Ki; (Â) 30 · Ki; ( ) no inhibitor.

732
G. E. Wright et al. / Bioorg. Med. Chem. Lett. 15 (2005) 729–732
10. Wright, G. E.; Brown, V. M.; Baril, E. F.; Brown, N. C.
Nucl. Acids Res. 1982, 10, 4431.
At first glance it is unusual that the 9-substituted gua-
nines are weak or inactive pol III inhibitors, given that
the polymerase substrate dGTP has the deoxyribo-
nucleotidyl group at N9. However, substituents in the
7 position of the guanines are nearly isosteric with those
in the 3 position of the uracil-based inhibitors. (Com-
pare the structures of 1 and 2 with those of 3 and 4.)
The 3 substituents in the uracil compounds enhance
pol III inhibitory activity, and, in certain cases, impart
potent antibacterial activity.16 The results of screening
the guanines for antibacterial activity in vitro will be
presented elsewhere.
11. Butler, M. M.; Dudycz, L. W.; Khan, N. N.; Wright, G.
E.; Brown, N. C. Nucl. Acids Res. 1990, 18, 7381.
12. Recombinant pol IIIC and pol IIIE of Bacillus subtilis
(B.s.) were prepared according to Hammond, R.; Barnes,
M.; Mack, S.; Mitchener, J.; Brown, N. Gene 1991, 98, 29,
and Ref. 5, respectively. E. coli (E.c.) pol IIIE was the
three subunit core enzyme purchased from Enzyco.
The enzymes were assayed with activated calf thymus
DNA according to the method described by Barnes, M.
H.; Brown, N. C. Nucl. Acids Res. 1979, 6, 1203. Apparent
inhibition constants (Ki values) were obtained directly in a
truncated assay lacking the competitor dGTP (Wright, G.
E.; Brown, N. C. Biochim. Biophys. Acta 1976, 432, 37),
or conventionally by variation of the concentration of
dGTP.
Acknowledgements
1
13. All new compounds were fully characterized by H NMR
This work was supported by small business grant
AI51103 from the National Institutes of Health.
spectra, and elemental (CHN) or HRMS analyses. A
typical synthesis follows: A mixture of DCBG (4) and
1.25equiv K2CO3 was stirred in DMF for 15min. 4-
Bromobutyl acetate (1.1equiv) was added, and the mix-
ture was stirred at 45ꢁC for 18h. The mixture was poured
into water, extracted with CHCl3, and the residue from the
organic layer purified on a silica gel column. Elution with
2% MeOH in CHCl3 gave the 7 isomer 4i (25%), and
elution with 4% MeOH in CHCl3 gave the 9 isomer (62%).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2004.11.016.
1
[Characteristic H NMR patterns distinguished between 7
and 9 isomers (Kjellberg, J.; Johansson, N. Tetrahedron
1986, 42, 6541).] A solution of 4i in MeOH was stirred
with concd. NH4OH at rt for 6h, the solution concen-
trated, and the residue purified on a silica gel column.
Elution with 15% MeOH in CHCl3 gave 4a (90%). A
solution of 4a and a 5-fold excess of TMSI in CHCl3 was
heated at reflux for 6h. After coming to rt, the solution
was treated with MeOH and Na2SO3, stirred for 0.5h, and
concentrated. Water was added and the suspension was
filtered, giving the 4-iodobutyl intermediate (93%). A
solution of the 4-iodobutyl intermediate and excess
morpholine in MeCN was stirred at rt for 18h. Concen-
tration and crystallization of the residue from MeOH gave
4j (71%). Yields and properties of all new compounds are
in Supporting Information.
References and notes
1. Inoue, R.; Kaito, C.; Tanabe, M.; Kamura, K.; Akimitsu,
N.; Sekimizu, K. Mol. Genet. Genomics 2001, 266, 564.
2. Huang, Y. P.; Ito, J. Nucl. Acids Res. 1998, 26, 5300.
3. Kornberg, A.; Baker, T. DNA Replication, 2nd ed.; W. H.
Freeman: New York, 1991.
4. Dervyn, E.; Suski, C.; Daniel, R.; Bruand, C.; Chapuis, J.;
`
Errington, J.; Janniere, L.; Ehrlich, S. D. Science 2001,
294, 1716.
5. Barnes, M. H.; Miller, S. D.; Brown, N. C. J. Bacteriol.
2002, 184, 3834.
6. Foster, K. A.; Barnes, M. H.; Stephenson, R. O.; Butler,
M. M.; Skow, D. J.; LaMarr, W. A.; Brown, N. C. Protein
Purif. Expr. 2003, 27, 90.
7. Tarantino, P. M., Jr.; Zhi, C.; Gambino, J. J.; Wright, G.
E.; Brown, N. C. J. Med. Chem. 1999, 42, 2035.
8. Daly, J. S.; Giehl, T. J.; Brown, N. C.; Zhi, C.; Wright, G.
E., III.; Ellison, R. T., III. Antimicrob. Agents Chemother.
2000, 44, 2217.
14. Wright, G. E.; Dudycz, L. W.; Kazimierczuk, Z.; Brown,
N. C.; Khan, N. N. J. Med. Chem. 1987, 30, 109.
15. Xu, H.; Maga, G.; Focher, F.; Smith, E.; Spadari, S.;
Gambino, J.; Wright, G. E. J. Med. Chem. 1995, 38,
49.
16. Zhi, C.; Long, Z.-Y.; Gambino, J.; Xu, W.-C.; Brown, N.
C.; Barnes, M.; Butler, M.; LaMarr, W.; Wright, G. E. J.
Med. Chem. 2003, 46, 2731.
9. Wright, G. E.; Brown, N. C. Curr. Op. Anti-Infect. Invest.
Drugs 1999, 1, 45.