Synthetic analogs of indole-containing natural products as inhibitors of sortase A and isocitrate lyase

Article abstract of DOI:10.1016/j.bmcl.2010.10.029

Guided by the inhibitory activities of indole-containing natural products against isocitrate lyase (ICL) from Candida albicans and sortase A (SrtA) from Staphylococcus aureus, a series of compounds structurally analogous to natural products were synthesized. Eight SrtA inhibitors and an ICL inhibitor having higher activities than the natural products were discovered by screening the enzyme inhibitory activities of synthesized compounds. Among the SrtA inhibitors discovered, six exhibited higher activities than p-hydroxymercuribenzoic acid, which suggests that these compounds have great potential as alternative antibacterial agents.

Full text of DOI:10.1016/j.bmcl.2010.10.029

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6882–6885
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic analogs of indole-containing natural products as inhibitors
of sortase A and isocitrate lyase
Yeon-Ju Lee a, Yu-Ri Han a, Wanki Park b, Seo-Hee Nam b, Ki-Bong Oh b, , Hyi-Seung Lee a,
⇑
⇑
a Marine Natural Products Laboratory, Korea Ocean Research and Development Institute, Ansan 426-744, Republic of Korea
b Department of Agricultural Biotechnology, Seoul National University, San 56-1, Shillim-dong, Gwanak-gu, Seoul 151-921, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Guided by the inhibitory activities of indole-containing natural products against isocitrate lyase (ICL)
from Candida albicans and sortase A (SrtA) from Staphylococcus aureus, a series of compounds structurally
analogous to natural products were synthesized. Eight SrtA inhibitors and an ICL inhibitor having higher
activities than the natural products were discovered by screening the enzyme inhibitory activities of
synthesized compounds. Among the SrtA inhibitors discovered, six exhibited higher activities than
p-hydroxymercuribenzoic acid, which suggests that these compounds have great potential as alternative
antibacterial agents.
Received 17 September 2010
Revised 1 October 2010
Accepted 6 October 2010
Available online 27 October 2010
Keywords:
Indole
b-Carboline
Isocitrate lyase
Sortase A
Ó 2010 Elsevier Ltd. All rights reserved.
Chemical library
Natural products have been regarded as a major source of phar-
maceutical leads and therapeutic agents.1 They are mostly second-
ary metabolites obtained by the action of various enzymes, which
implies that they have great potential of efficiently interacting
with enzymes. Nevertheless, natural products have attracted little
research interest in recent years due to the limited quantities that
can be obtained from nature, lengthy and low-yielding syntheses,
and non-druggable physicochemical properties.2 To overcome
these disadvantages, efforts have been made to construct chemical
libraries based on natural products.3 Compounds bearing the struc-
tural features of natural products as well as druggable properties
have been synthesized and constituted into chemical libraries. In
continuation of our researches on the discovery of inhibitors of en-
zymes related to antimicrobial activities,4 we have built a small
chemical library with indole-type natural product scaffolds and
examined the enzyme inhibitory activities of the library com-
pounds. Sortase A (SrtA) and isocitrate lyase (ICL) were selected
as target enzymes since they play crucial roles in the survival or
virulence of various pathogenic bacteria and fungi.5–8
target in the development of efficient antibacterial agent.7 The
development of SrtA inhibitors has been attempted by examining
natural products or high-throughput screening of chemical
libraries; however, efficient SrtA inhibitors have not been discov-
ered yet.
ICL is an enzyme that transforms isocitrate into glyoxylate in
the glyoxylate cycle. The glyoxylate cycle is a reaction sequence
in which acetates are converted to succinates during the energy
production and biosynthesis of cell constituents; this cycle enables
bacteria and fungi to grow on acetate in a hostile environment in-
side the macrophage where glucose is not available.6 It has been
discovered that the microbial virulence of Candida albicans signifi-
cantly decreased in the case of mutant strains lacking the ICL.
However, limited examples of ICL inhibitors have been reported
thus far, and they have mostly not been successful due to their
low activities or high toxicities.8
In a previous work, we had reported the discovery of six known
5-hydroxyindole compounds (2–7) along with a novel compound
6-hydroxydihydro-b-carboline 1 from the tropical sponge Hyrtios
sp. and examined their inhibitory activities against ICL from C. albi-
cans (Fig. 1).4a Among them, bis-5-hydroxyindole containing gly-
SrtA is an enzyme that catalyzes the covalent attachment of
surface proteins to the peptidoglycan cell layer in Gram-positive
bacteria such as Staphylococcus aureus.5 Since surface proteins
promote interactions between the invading pathogen and animal
tissues, thereby providing strategies for bacterial escape from the
host’s immune response, SrtA has been regarded as a promising
oxy linker (7) showed promising activity level (IC50: 29 lg/mL,
89.0 lM). Subsequently, the inhibitory activities of these com-
pounds against SrtA from S. aureus were examined;10 however,
inhibitions were not observed with any of the compounds except
dihydro-b-carboline 1 that exhibited moderate inhibitory activity
(IC50: 67 lg/mL, 290 lM) (Table 1). Consequently, compounds 1
and 7 were considered to be attractive scaffolds for the construc-
⇑
Corresponding authors. Tel.: +82 2 880 4646; fax: +82 2 873 3112 (K.-B.O.);
tel.: +82 31 400 6179; fax: +82 31 400 6170 (H.-S.L.).
tion of our chemical library due to their promising levels of
E-mail addresses: ohkibong@snu.ac.kr (K.-B. Oh), hslee@kordi.re.kr (H.-S. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.029

Y.-J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6882–6885
6883
O
HO
HO
HO
HO
OMe
N
NH
NH2
N
H
N
H
N
H
N
H
CO2H
O
1
2
3
4
H
N
O
O
HO
OH
HO
O
H
HO
OH
O
N
H
5
N
H
6
N
H
7
Figure 1. Compounds obtained from the tropical marine sponge Hyrtios sp.
Table 1
since methods for the syntheses of indoles and b-carbolines have
been well studied and established.
Inhibitory activities of natural products 1–7 against ICL and SrtAa
b
We began our research by examining the analogs of 7. One of
the indole moieties was substituted with functionalities bearing
a hydrogen bond donor (OH, NH) adjacent to the carbonyl group.
Reactions between the corresponding indoles and oxalyl chloride
followed by the addition of aniline or water afforded desired prod-
ucts (8a–8h) with high yields (78–82%) (Scheme 1).12 Disappoint-
ingly, the synthesized analogs listed in Table 2 showed no ICL
inhibitory activities; this suggests that both the indole moieties
of 7 are required.9 In the case of SrtA inhibitory activity, however,
the compounds obtained from indole (8a and 8e; entries 1 and 5)
and 2-methylindole (8d and 8h; entries 4 and 8) exhibited potent
activities, while the compounds containing 5-hydroxy and 5-meth-
oxyindole moieties exhibited no activities (entries 2, 3, 6, and 7). In
Entry
Compound
ICL IC50
(lg/mL,
lM)
SrtA IC50 (lg/mL, lM)
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
87 (380)
>100
>100
53 (301)
61 (318)
40 (247)
29 (89)
6.0 (51)
—
67 (290)
>100
>100
>100
>100
>100
>100
—
3-NPc
p-HMBd
42 (124)
a
b
c
Enzyme inhibitory activities were measured as described in Refs. 4,10.
Previously reported data.4a
3-Nitropropionate, an ICL inhibitor used as a positive control.
d
p-Hydroxymercuribenzoic acid, a SrtA inhibitor used as positive control.
particular, the SrtA inhibitory activities of compound 8d (IC50
:
activities as well as druggable physicochemical properties.11 In
addition, easy and fast syntheses of various analogs are possible
17 lg/mL, 61 lM) and 8e (IC50: 13 lg/mL, 60 lM) were twice as
high as that of p-hydroxymercuribenzoic acid (p-HMB)7d (IC50
:
O
R1
R3
R1 = H, OH, OMe
R1
R2
oxalyl chloride, THF, 0 ºC
then, DIPEA, Aniline or water, rt
78 ~ 82 %yield
N
H
R2 = H, Me
R2
O
R3 = NHPh, OH
N
H
Indole, 2-methylindole
5-hydroxyindole, 5-methoxyindole
8
Scheme 1. Synthesis of indoleglyoxamide (8a–8d) and indoleglyoxylate (8e and 8f).
Table 2
SrtA and ICL inhibitory activities of 8a
O
R3
R1
R2
O
N
H
8
b
c
Entry
Compound
R1
R2
R3
ICL IC50
(lg/mL,
lM)
SrtA IC50 (lg/mL, lM)
1
2
3
4
5
6
7
8
8a
8b
8c
8d
8e
8f
H
H
H
H
Me
H
H
H
Me
NHPh
NHPh
NHPh
NHPh
OH
OH
OH
OH
>100
>100
>100
>100
>100
>100
>100
>100
46 (174)
>100
>100
17 (61)
13 (69)
>100
OH
OMe
H
H
OH
OMe
H
8g
8h
>100
27 (133)
a
b
c
Enzyme inhibitory activities were measured as described in Refs. 4,10.
IC50 of 3-nitropropionate (positive control) = 6.0 g/mL (50 M).
IC50 of p-HMB (positive control) = 42 g/mL (142 M).
l
l
l
l

6884
Y.-J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6882–6885
42
lg/mL, 124
lM), which is a known SrtA inhibitor used as a po-
and a-oxocarbonyl moieties play critical roles in the interaction
sitive control (entries 4 and 5).
of compounds with SrtA.
To gain further insights into the effects of each functional group
of 8a and 8e on the SrtA inhibitory activity, three more analogs
were examined (Fig. 2). In the case of N1,N2-diphenyloxalamide,
wherein both the indoles of 8a were replaced with anilines, no SrtA
inhibitory activity was observed. Further, the removal of each car-
bonyl group (3-glyceroindole, 3-indoleacetic acid) also caused
complete loss of activities. It is highly probable that both indole
We then turned our attention to compound 1 whose analogs
can be prepared easily by Pictet-Spengler reactions.13a Tetrahy-
dro-b-carbolines 9 were synthesized from the corresponding tryp-
tamines and aldehydes employing well-known procedures,13 and
dihydro-b-carbolines 10 were prepared by the oxidation of 9
(Scheme 2).13d In contrast to dihydro-b-carboline 1, analogous tet-
rahydro-b-carboline 9b showed no activity against both enzymes,
O
OH
OH
H
N
O
O
N
O
N
H
H
N
H
N1,N2-diphenyloxalamide
SrtA IC50 = >100 µg/mL
3-glyceroindole
SrtA IC50 = >100 µg/mL
3-indoleacetic acid
SrtA IC50 = >100 µg/mL
Figure 2. Loss of SrtA inhibitory activities caused by the deletion of an indole or carbonyl moieties.
R4
R4
R4
R5COH
acetic acid
25 ºC or refulx
DDQ
acetonitrile
25 ºC
NH2
NH
R5
N
N
H
N
H
N
H
R5
75 ~ 81 %yield
65 ~ 73 %yield
10
9
Scheme 2. Syntheses of tetrahydro-b-carbolines (9) and dihydro-b-carbolines (10).
Table 3
SrtA and ICL inhibitory activities of 9 and 10a
R4
R4
NH
R5
N
N
H
N
H
R5
9
10
b
c
Entry
Compound
R4
R5
ICL IC50
(lg/mL,
l
M)
SrtA IC50 (lg/mL, lM)
1
2
3
4
5
9a
9b
9c
9d
9e
H–
–CO2H
–CO2H
–CO2H
–C5H11
–C5H11
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
OH
OMe
H
OMe
6
7
8
9f
H
>100
>100
>100
>100
9g
9h
OH
OMe
28 (106)
32 (115)
9
9i
9j
H
H
41 (137)
21 (75)
>100
>100
OMe
10
11
12
13
9k
H
50 (180)
>100
>100
OMe
10a
10b
H
24 (97)
7 (25)
OMe
>100
a
b
c
Enzyme inhibitory activities were measured as described in Refs. 4,10.
IC50 of 3-nitropropionate (positive control) = 6.0 g/mL (50 M).
IC50 of p-HMB (positive control) = 42 g/mL (142 M).
l
l
l
l

Y.-J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6882–6885
6885
Table 4
p-HMB. A tetrahydro-b-carboline 9j with ICL inhibitory activities
higher than those of natural products (1–7) was also discovered.
It would be worthwhile to emphasize that all of the synthesized
compounds were predicted to have druggable physicochemical
properties like natural products 1 and 7.11 Further investigations
into the development of efficient antibacterial agents based on
these results are still in progress.
Minimum inhibitory concentrations (MICs) of selected synthesized analogs against
bacterial strainsa,b
Entry
Compound
MIC (
l
g/mL,
lM)
SA
BS
ST
PV
ML
EC
1
2
3
4
5
6
7
8
9
8a
8d
8e
8h
9g
9h
9i
9j
9k
10a
10b
Ampicillin
>200
>200
>200
>200
100
>200
12.5
6.25
12.5
>200
>200
1.56
>200
>200
>200
>200
100
>200
6.25
12.5
6.25
>200
>200
0.78
>200
>200
>200
>200
100
>200
12.5
12.5
6.25
>200
>200
1.56
>200
>200
>200
>200
100
>200
6.25
12.5
6.25
>200
>200
0.78
>200
>200
>200
>200
100
>200
12.5
12.5
6.25
>200
>200
0.78
>200
>200
>200
>200
>200
>200
50
50
50
>200
>200
50
Acknowledgments
This research was supported by Korea Ocean Research and
Development Institute (PE98416) and the Ministry of Land, Trans-
port and Maritime Affairs, Korea (PM55421).
10
11
12
References and notes
a
MIC value represents concentration giving complete inhibition relative to the
negative control. See Ref. 7a for detailed procedures.
1. (a) Li, J. W. H.; Vederas, J. C. Science 2009, 324, 161; (b) Harvey, A. L. Drug Discov.
Today 2008, 13, 894; (c) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461.
2. Lam, K. S. Trends Microbiol. 2007, 15, 279. and references cited therein.
3. (a) John, J. E. Drug Discov. Today 2010, 15, 409; (b) Bugni, T. S.; Richard, B.;
Bhoite, L.; Cimbora, D.; Harper, M. K.; Ireland, C. M. J. Nat. Prod. 2008, 71, 1095;
(c) Messer, R.; Fuhrer, C. A.; Häner, R. Curr. Opin. Chem. Biol. 2005, 9, 259; (d)
Boldi, A. M. Curr. Opin. Chem. Biol. 2004, 8, 281.
b
SA, Staphylococcus aureus ATCC 6538p; BS, Bacillus subtilis ATCC 6633; ST, Sal-
monella typhimurium ATCC 14028; PV, Proteus vulgaris ATCC 3851; ML, Micrococcus
luteus IFC 12708; EC, Escherichia coli ATCC 25922.
as given in Table 3. Neither of the derivatives of 9b (9a, 9c) exhib-
ited any activities (entries 1–3). Compounds bearing a pentyl
group instead of carboxylic acid (9b and 9e) also showed no activ-
ities (entries 4 and 5).
When carboxylic acid groups were replaced with the phenyl
ring, dramatic increases in SrtA inhibitory activities were observed
4. (a) Lee, H.-S.; Yoon, K.-M.; Han, Y.-R.; Lee, K.-J.; Chung, S.-C.; Kim, T.-I.; Lee, S.-
H.; Shin, J.; Oh, K.-B. Bioorg. Med. Chem. Lett. 2009, 19, 1051; (b) Lee, H.-S.; Lee,
T.-H.; Yang, S. H.; Shin, H. J.; Shin, J.; Oh, K.-B. Bioorg. Med. Chem. Lett. 2007, 17,
2483.
5. (a) Ton-That, H.; Schneewind, O. J. Biol. Chem. 1999, 274, 24316; (b)
Mazmanian, S. K.; Liu, G.; Ton-That, H.; Schneewind, O. Science 1999, 285, 760.
6. (a) Muñoz-Elías, E. J.; McKinney, J. D. Nat. Med. 2005, 11, 638; (b) Lorez, M. C.;
Fink, G. R. Nature 2001, 412, 83.
7. (a) Frankel, B. A.; Bentley, M.; Kruger, R. G.; McCafferty, D. G. J. Am. Chem. Soc.
2004, 126, 3404; (b) Kudryavtsev, K.; Bentley, M. L.; McCafferty, D. G. Bioorg.
Med. Chem. 2009, 17, 2886; (c) Suree, N.; Yi, S. W.; Thieu, W.; Marohn, M.;
Damoiseaux, R.; Chan, A.; Jung, M. E.; Clubb, R. T. Bioorg. Med. Chem. 2009, 17,
7174; (d) Kim, S.-H.; Shin, D.-S.; Oh, M.-N.; Chung, S.-C.; Lee, C.-S.; Oh, K.-B.
Biosci. Biotechnol. Biochem. 2004, 68, 421; (e) Park, B.-S.; Kim, J.-G.; Kim, M.-R.;
Lee, S.-E.; Takeoka, G. R.; Oh, K.-B.; Kim, J.-H. J. Agric. Food Chem. 2005, 53, 9005;
(f) Jang, K. H.; Chung, S.-C.; Shin, J.; Lee, S.-H.; Kim, T.-I.; Lee, H.-S.; Oh, K.-B.
Bioorg. Med. Chem. Lett. 2007, 17, 5366.
8. (a) Lee, H.-S.; Lee, T.-H.; Lee, J.-H.; Chae, C.-S.; Chung, S.-C.; Shin, D.-S.; Shin, J.;
Oh, K.-B. J. Agric. Food Chem. 2007, 55, 6923; (b) Yang, H.-C.; Yu, J.; Oh, K.-B.;
Shin, D.-S.; Cho, W.-J.; Shin, J.; Kim, S. Arch. Pharm. Res. 2007, 30, 955.
9. For the cloning, expression and purification of ICL from the genomic DNA of C.
albicans (ATCC 10231) and the procedure for the evaluation of inhibitory
activities of compounds against ICL, see: (a) Shin, D.-S.; Kim, S.; Yang, H.-C.; Oh,
K.-B. J. Microbiol. Biotechnol. 2005, 15, 652; (b) Hautzel, R.; Anke, H.; Sheldric,
W. S. J. Antibiot. 1990, 43, 1240.
in the case of 9g (IC50: 28
115 M); however, 9f remained inactive (entries 6–8). The oxida-
tion of 9f and 9h to 10a (IC50: 24 g/mL, 97 M) and 10b (IC50
g/mL, 25 M), respectively, caused further increases in activi-
lg/mL, 106 lM) and 9h (IC50: 32 lg/mL,
l
l
l
:
7
l
l
ties (entries 12 and 13), and the activities of these four compounds
were higher than that of p-HMB.
Increasing the size of the phenyl ring by further substitutions
resulted in significant improvements in ICL inhibitory activities
(entries 9–11). In particular, compound 9j bearing the m-methoxy-
phenyl ring exhibited higher ICL inhibitory activity (IC50: 21
lg/
mL, 75 M) higher than the natural product 7 (29 g/mL, 89 l
l
l
M),
which is the most potent ICL inhibitor among the natural products
(Table 1, entry 7).
The effects of the obtained enzyme inhibitors on the bacterial
growth were determined by employing a two fold microtiter broth
dilution method against various bacterial strains (Table 4). The
minimum inhibitory concentrations (MICs) were determined
according to a published protocol.7a SrtA inhibitors (entries 1–6,
entries 10–11) exhibited no growth inhibitory activity against all
types of strains, except in the case of 9g that insignificantly inhib-
ited the growth of most cell lines (entry 5). In the case of the SrtA
inhibitor, this result might suggest selective inhibition of SrtA or
low toxicity of these compounds as it is known that SrtA inhibitors
disrupt the pathogenesis of bacterial infections without affecting
the microbial viability.14 On the other hand, ICL inhibitors
(9i–9k; entries 7–9) caused moderate to high growth inhibitions
of most of the strains as expected.
10. For the procedure for the evaluation of inhibitory activities of compounds
against SrtA, see Ref. 7d.
11. Natural products 1 and 7 show zero violations of Lipinski’s rule of five and
Veber rules. The physicochemical properties such as log P, polar surface are,
number of hydrogen bond donors and acceptors, and number of rotational
bonds were predicted using Molinspirations (Molinspiration Cheminformatics,
Nova ulica 61, SK-900 26 Slovensky Grob, Slovak Republic. Available online at:
http://www.molinspiration.com/cgi-bin/properties). For Lipinski’s rule of five,
see: (a) Lipinski, C. A.; Lombardo, F.; Dominy, B. W. Adv. Drug Deliv. Rev. 1997,
23, 3; For Veber rules, see; (b) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B.
R.; Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615.
12. (a) Thompson, M. J.; Borsenberger, V.; Louth, J. C.; Judd, K. E.; Chen, B. J. Med.
Chem. 2009, 52, 7503; (b) Gitto, R.; Luca, D. L.; Ferro, S.; Citraro, R.; Sarro, G. D.;
Costa, L.; Ciranna, L.; Chimirri, A. Bioorg. Med. Chem. 2009, 17, 1640.
13. (a) Cox, E. D.; Cook, J. M. Chem. Rev. 1995, 95, 1797; (b) Trujillo, J. I.; Meyers, M.
J.; Anderson, D. R.; Hegde, S.; Mahoney, M. W.; Vernier, W. F.; Buchler, I. P.; Wu,
K. W.; Yang, S.; Hartman, S. J.; Reitz, D. B. Bioorg. Med. Chem. Lett. 2007, 17,
4657; (c) Kawashima, Y.; Horiguchi, A.; Taguchi, M.; Tuyuki, Y.; Karasawa, Y.;
Araki, H.; Hatayama, K. Chem. Pharm. Bull. 1995, 43, 783; (d) Shen, Y.-C.; Chen,
C.-Y.; Hsieh, P. W.; Duh, C.-Y.; Lin, Y.-M.; Ko, C.-L. Chem. Pharm. Bull. 2005, 53,
32.
In conclusion, we have prepared a series of synthesized analogs
of natural products, which exhibit ICL and SrtA inhibitory activi-
ties. Among the 21 synthesized analogs, an indoleglyoxamide 8d,
an indoleglyoxylate 8e, and four b-carbolines (9g, 9h, 10a, and
10b) exhibited SrtA inhibitory activities higher than that of
14. (a) Jonsson, I. M.; Mazmanian, S. K.; Schneewind, O.; Bremell, T.; Tarkowski, A.
Microbes Infect. 2003, 5, 775; (b) Mazmanian, S. K.; Liu, G.; Lenoy, E. R.;
Schneewind, O. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5510.