Novel pyridobenzimidazole derivatives exhibiting antifungal activity by the inhibition of β-1,6-glucan synthesis

Article abstract of DOI:10.1016/j.bmcl.2010.05.024

Based on the HTS hit compound 1a, an inhibitor of β-1,6-glucan synthesis, we synthesized novel pyridobenzimidazole derivatives and evaluated their antifungal activity. Among the compounds synthesized, we identified the potent compound 15e, which exhibits

Full text of DOI:10.1016/j.bmcl.2010.05.024

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3893–3896
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Bioorganic & Medicinal Chemistry Letters
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Novel pyridobenzimidazole derivatives exhibiting antifungal activity by the
inhibition of b-1,6-glucan synthesis
a
b
a
a
Hiroshi Takeshita a, , Jun Watanabe , Yoichi Kimura , Katsuhiro Kawakami , Hisashi Takahashi ,
*
Makoto Takemura a, Akihiro Kitamura c, Kazuhiko Someya d, Ryohei Nakajima e
a Medicinal Chemistry Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b Exploratory Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
c R&D Planning Department, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
e Vaccine Business Planning Department, Daiichi Sankyo Co., Ltd, 3-5-1, Nihonbashihoncho, Chuo-ku, Tokyo 103-8426, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the HTS hit compound 1a, an inhibitor of b-1,6-glucan synthesis, we synthesized novel pyrido-
benzimidazole derivatives and evaluated their antifungal activity. Among the compounds synthesized,
we identified the potent compound 15e, which exhibits excellent activity superior to fluconazole against
both Candida glabrata and Candida krusei. From the SAR study, we revealed essential moieties for anti-
fungal activity.
Received 1 March 2010
Revised 6 May 2010
Accepted 11 May 2010
Available online 15 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Antifungal
b-1,6-Glucan
Cell wall
Pyridobenzimidazole
Candida glabrata
Recently, the threat of fungal infection has increased due to a
growing number of immunocompromised patients, which is
caused by the development of cancer chemotherapy and organ
transplant, and the growth of human immunodeficiency virus
(HIV) infection.1 There are several antifungal agents,1b,2 but these
agents are not considered to satisfy medical needs because of their
toxicity,2b limited routes,2b and the emergence of drug-resistant3
and drug-low-susceptible4 strains. Given these observations, the
development of new antifungal agents having a novel mode of ac-
tion has been keenly desired.
In the course of our study, we discovered pyridobenzimidazole
derivative 1a5 (Fig. 1) inhibiting the synthesis of fungal cell wall,
which is absent from mammalian cells. Compound 1a was the
high-throughput screening (HTS) hit compound discovered from
our chemical library, and we reported that 1a exhibited antifungal
activity by inhibiting the synthesis of b-1,6-glucan, which is a com-
ponent of fungal cell wall. To our knowledge, 1a is the first com-
pound bearing antifungal activity by inhibiting b-1,6-glucan
synthesis. These results prompted us to evaluate the potency of
pyridobenzoimidazole scaffold as an antifungal agent. In this re-
port, we describe the synthesis and the structure–activity relation-
ships (SAR) of a series of pyridobenzimidazole derivatives with
modification at the C-1 to C-4 positions.
As outlined in Scheme 1, chloropyridobenzimidazole 5, the pre-
cursor of target compounds, was prepared according to a known
method6 for the synthesis of pyridobenzimidazole 4-carbonitriles.
Thus, benzimidazole-2-acetonitrile 2 and corresponding b-ketoes-
ter 3 were heated in the presence of ammonium acetate to give tri-
cyclic compound 4, and chlorination of 4 in POCl3 gave 5. Compound
4f without any substituent at the C-3 position was synthesized from
2 and b-methoxyacrylate 6 obtained by the Wittig reaction of ethyl
phenylglyoxylate 7. b-Ketoester 3g was obtained by benzoylation of
ethyl butyrate 9. As shown in Schemes 2 and 3, chloride 5 was trea-
ted with various amines to afford target compounds.
The synthesis of 15i and 15h possessing oxy functional groups
at the C-2 position was started from the known compound 4h6a
NC
N
N
N
H
N
1a
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8344.
E-mail address: takeshita.hiroshi.km@daiichisankyo.co.jp (H. Takeshita).
Figure 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.024

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H. Takeshita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3893–3896
R3
R3
N
R3
O
NC
N
R2
Cl
NC
HN
R2
O
NC
N
b
R2
a
O
NH
+
N
OEt
3
2
5a, R2 = Et, R3 = Me
5b, R2 = H, R3 = Me
5c, R2 = F, R3 = Me
5d, R2 = nBu, R3 = Me
5e, R2 = Ph, R3 = Me
5f, R2 = Ph, R3 = H
5g, R2 = Et, R3 = Ph
4a, R2 = Et, R3 = Me
4b, R2 = H, R3 = Me
4c, R2 = F, R3 = Me
a
NC
N
for 4f
4d, R2 = nBu, R3 = Me
4e, R2 = Ph, R3 = Me
4f, R2 = Ph, R3 = H
4g, R2 = Et, R3 = Ph
O
NH
+
O
OEt
6
2
O
O
d
c
O
O
+
O
O
OEt
O
OEt
OEt
OEt
O
OEt
9
8
7
6
3g
Scheme 1. Reagents and conditions: (a) AcONH4, 140–150 °C (83% for 4a, 85% for 4b, 64% for 4c, 90% for 4d, 85% for 4e, 19% for 4f, 34% for 4g); (b) POCl3, reflux (90% for 5a,
64% for 5b, 82% for 5c, 81% for 5d, 64% for 5e, 74% for 5f, 33% for 5g); (c) MeOCH2P+Ph3Clꢀ, NaH, THF (76%); (d) NaH, DME (48%).
15a, R1 =
16a, R1 =
17a, R1 =
12a, R1 =
13a, R1 =
1a, R1 =
N
N
N
N
H
N
H
NC
N
NC
N
amine
a
N
N
N
R1
10a, R1 =
N
Cl
N
NH2
N
H
N
14a, R1 =
N
11a, R1 =
N
H
N
5a
N
N
Scheme 2. Reagents and condition: (a) Et3N, DMF, 80 °C (45% for 1a, 57% for 10a, 56% for 11a, 55% for 12a, 54% for 13a, 53% for 14a, 67% for 15a, 52% for 16a, 51% for 17a).
R3
N
R3
N
a
R2
N
NC
N
R2
Cl
As illustrated in Scheme 5, compound 20 possessing a carbam-
oyl moiety at the C-4 position instead of a cyano group was
obtained by treating with concd H2SO4 in low yield (16%).
NC
N
HN
+
N
The compounds synthesized were assayed for their in vitro
antifungal activity against Candida albicans (C. albicans), Candida
glabrata (C. glabrata), Candida krusei (C. krusei), Candida tropicalis
(C. tropicalis) and Aspergillus fumigatus (A. fumigatus) by using the
method described in our previous report.5 The results are shown
as the minimum inhibitory concentration (MIC) (the lowest drug
concentration producing an optically clear well) against each fun-
gus for our compounds and as the lowest drug concentration pro-
ducing a prominent reduction in turbidity for fluconazole.
N
5
18
15b, R2 = H, R3 = Me
15c, R2 = F, R3 = Me
15d, R2 = Bu, R3 = Me
n
15e, R2 = Ph, R3 = Me
15f, R2 = Ph, R3 = H
15g, R2 = Et, R3 = Ph
The activity of compounds substituted with various amines at
the C-1 position was shown in Table 1. HTS hit compound 1a
Scheme 3. Reagents and condition: (a) Et3N, DMF, 80 °C (57% for 15b, 65% for 15c,
57% for 15d, 56% for 15e, 73% for 15f, 71% for 15g).
exhibited moderate activity of MIC values that were 1
lg/ml
against C. glabrata and 4 g/ml against C. krusei, respectively. The
l
according to the method in Scheme 4. Acetylation of the hydroxyl
group of 4h and successive chlorination afforded chloride 5i, which
was treated with (3S)-(dimethylamino)pyrrolidine 18 to furnish
2-(acetoxyethyl) compound 15i. Deacetylation of 15i with 1 N
NaOH afforded tetracyclic compound 19 as a main product and
only a low amount of the target compound 15h. To avoid this intra-
molecular cyclization, more weakly basic LiOH was used and then
2-(hydroxyethyl) compound 15h was obtained in 83% yield.
activity of compound 10a with a terminal primary amine instead
of a tertiary amine decreased. Propylamino compound 12a without
a terminal amino moiety exhibited no inhibitory activity. It was
considered that a terminal amine was essential for antifungal
activity. Additionally, (dimethylamino)propylamino compound
11a whose methylene carbon was extended was also inactive. This
result indicated that the length between the terminal amine and
pyridobenzimidazole scaffold was important for antifungal

H. Takeshita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3893–3896
3895
OAc
OR
OH
a, b
c
NC
N
NC
N
NC
N
NC
HN
O
N
Cl
N
N
N
N
O
N
19
5i
15i, R = Ac
15h, R = H
4h
d
Scheme 4. Reagents and conditions: (a) Ac2O, DMAP, pyridine (77%); (b) POCl3, reflux (83%); (c) 18, Et3N, DMF, 80 °C (41%); (d) LiOH, THF–H2O (83%).
activity. In regards to cyclic amines, though methylpiperazinyl
compound 13a and (dimethylamino)piperidinyl compound 14a
exhibited no activity, (3S)-(dimethylamino)pyrrolidinyl compound
15a showed more potent activity than 1a. The configuration of the
dimethylamino moiety affected activity as shown in the less potent
activity of (R)-form 16a as compared to that of (S)-form 15a. As in
the case of acyclic amines, pyrroridinyl compound 17a without a
terminal amine exhibited no activity.
NC
N
H2NOC
N
a
N
N
N
N
H
N
N
H
20
1a
Scheme 5. Reagents and condition: (a) concd H2SO4, EtOH, reflux (16%).
Table
2 shows the MIC values of the compounds with
(3S)-(dimethylamino)pyrrolidinyl moiety at the C-1 position and
Table 1
In vitro antifungal activity of pyridobenzimidazole derivatives possessing various substituents at the C-1 position
15a, R1 =
16a, R1 =
17a, R1 =
12a, R1 =
13a, R1 =
1a, R1 =
N
N
N
N
H
N
H
NC
N
N
N
N
R1
10a, R1 =
N
NH2
N
H
N
14a, R1 =
N
11a, R1 =
N
H
N
N
N
Organism
MIC (lg/ml)
Fluconazolea
1a
10a
11a
12a
13a
14a
15a
16a
17a
Candida albicans ATCC24433
Candida glabrata IFO0622
Candida krusei TIMM0269
Candida tropicalis TIMM0313
Aspergillus fumigatus ATCC36607
0.25
2
16
0.25
>64
>128
1
4
>128
>128
>128
4
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>32
>32
>32
>32
>32
>128
>128
>128
>128
>128
>128
0.5
2
32
>128
>128
1
16
64
>128
>64
>64
>64
>64
>64
a
The lowest drug concentration producing a prominent reduction in turbidity was employed as end point.
Table 2
In vitro antifungal activity of pyridobenzimidazole derivatives possessing various substituents at the C-2 position
NC
N
R2
N
N
N
Organism
MIC (lg/ml)
Fluconazolea
R2=
15a
Et
15b
H
15c
F
15d
nBu
15e
Ph
15h
(CH2)2OH
15i
(CH2)2OAc
Candida albicans ATCC24433
Candida glabrata IFO0622
Candida krusei TIMM0269
Candida tropicalis TIMM0313
Aspergillus fumigatus ATCC36607
0.25
2
16
0.25
>64
>128
0.5
2
32
>128
>128
8
32
>128
>128
>128
4
16
>128
>128
>128
60.125
0.5
0.5
>128
>128
>128
32
16
>128
>128
>128
1
4
16
>128
60.125
60.125
0.5
>128
a
The lowest drug concentration producing a prominent reduction in turbidity was employed as end point.

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H. Takeshita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3893–3896
Table 3
understood, therefore further work is needed to understand the ef-
fect of Kre6p inhibition in Aspergillus species.
In vitro antifungal activity of pyridobenzimidazole derivatives possessing various
substituents at the C-3 and C-4 positions
In summary, we have demonstrated that pyridobenzimidazole
scaffold can be utilized for novel antifungal agents inhibiting b-
1,6-glucan synthesis and we provide the following findings. (1)
The basic substituent at the C-1 position and the cyano group at
the C-4 position were essential for antifungal activity, (2) the lipo-
philic substituent at the C-2 position and the methyl moiety at the
C-3 position were of importance to potent activity. Further chem-
ical modifications and investigations of in vivo efficacy are cur-
rently under way.
Organism
MIC (lg/ml)
Fluconazolea
15f
15g
20
Candida albicans ATCC24433
Candida glabrata IFO0622
Candida krusei TIMM0269
Candida tropicalis TIMM0313
Aspergillus fumigatus ATCC36607
0.25
2
16
0.25
>64
>128
60.125
2
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
a
The lowest drug concentration producing a prominent reduction in turbidity
was employed as end point.
Acknowledgment
The authors would like to thank members of the analysis group
of Daiichi Sankyo R&D Associe Co., Ltd for their analytical
determinations.
various substituents at the C-2 position. The activity of compound
15b without any substituent at the C-2 position decreased, and the
not sterically bulky fluoro compound 15c also showed less potent
activity than ethyl compound 15a. Additionally, acetoxyethyl com-
pound 15i and hydroxyethyl compound 15h showed less potent
activity. In contrast, compound 15d possessing a n-butyl moiety
longer than an ethyl moiety, exhibited more potent activity against
C. glabrata, C. krusei and C. tropicalis. Moreover, phenyl compound
15e7 also exhibited excellent activity of MIC values that were
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.024.
References and notes
60.125 lg/ml against both C. glabrata and C. krusei, and 0.5 lg/
ml against C. tropicalis. These results suggested that the introduc-
tion of lipophilic substituents to the C-2 position enhanced anti-
fungal activity.
1. (a) Drew, R. Int. J. Antimicrob. Agents 2006, 27S, S36; (b) Enoch, D. A.; Ludlam, H.
A.; Brown, N. M. J. Med. Microbiol. 2006, 55, 809.
2. (a) Blot, S.; Vandewoude, K. Drugs 2004, 64, 2159; (b) Wiebe, V.; Karriker, M.
Clin. Tech. Small Anim. Pract. 2005, 20, 250.
Next, the results of the substitution at the C-3 and C-4 positions
were shown in Table 3. The activity of 15f without a methyl group
at the C-3 position was less potent than that of corresponding 15e.
Meanwhile, 15g possessing a phenyl substituent did not show any
activity. Therefore it was considered to be favorable that the sub-
stituent at the C-3 position was a measurably bulky lipophilic moi-
ety. Compound 20 bearing a carbamoyl group instead of a cyano
group at the C-4 position showed no activity.
Compound 15e exhibited the most potent activity among the
compounds synthesized and it was confirmed that 15e inhibited
b-1,6-glucan synthesis selectively.8 Compared to fluconazole, 15e
was more active against both C. glabrata and C. krusei and showed
almost equal activity against C. tropicalis. Although the MIC values
of all the compounds synthesized against C. albicans were
3. (a) Hakki, M.; Staab, J. F.; Marr, K. F. Antimicrob. Agents Chemother. 2006, 50,
2522; (b) Moudgal, V.; Little, T.; Boikov, D.; Vazquez, J. A. Antimicrob. Agents
Chemother. 2005, 49, 767.
4. (a) Arendrup, M. C.; Fuursted, K.; Gahrn-Hansen, B.; Jensen, I. M.; Knudsen, J. D.;
Lundgren, B.; Schonheyder, H. C.; Tvede, M. J. Clin. Microbiol. 2005, 43, 4434; (b)
Hobson, R. P. J. Hosp. Infect. 2003, 55, 159.
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Agents Chemother. 2009, 53, 670.
6. (a) Badawey, E.; Kappe, T. Eur. J. Med. Chem. 1995, 30, 327; (b) El-Hawash, S. A.
M.; Badawey, E. A. M.; Kappe, T. Pharmazie 1999, 54, 341; (c) Badawey, E. A. M.;
Kappe, T. Eur. J. Med. Chem. 1999, 34, 663; (d) Rida, S. M.; Soliman, F. S. G.;
Badawey, E. A. M.; Kappe, T. J. Heterocycl. Chem. 1988, 25, 1725; (e) Russell, R. K.;
Van Nievelt, C. E. J. Heterocycl. Chem. 1995, 32, 299; (f) Kotovskaya, S. K.;
Baskakova, Z. M.; Charushin, V. N.; Chupakhin, O. N.; Belanov, E. F.; Bormotov, N.
I.; Balakhnin, S. M.; Serova, O. A. Khim.-Farm. Zh. 2005, 39, 12.
7. Analytical data of compound 15e: Yellow solid. Mp 211–214 °C; IR (ATR) 2222,
1628, 1589, 1466, 1344, 1298, 1263, 1186, 1134 cmꢀ1
;
1H NMR (CDCl3) d 1.98–
2.09 (3H, m), 2.12 (6H, s), 2.31 (3H, s), 2.72–3.69 (4H, m), 7.20–7.38 (3H, m),
7.48–7.60 (4H, m), 7.89–8.14 (1H, m), 8.02 (1H, d, J = 8.1 Hz); 13C NMR (DMSO-
d6) d 150.22, 147.39, 145.63, 144.59, 134.73, 130.63, 130.60, 129.35, 128.84,
128.79, 128.38, 125.64, 121.31, 121.27, 118.89, 115.92, 115.51, 95.43, 65.10,
>128 lg/ml in the conditions used, the morphological change on
the surface of the cell wall of C. albicans was observed under a micro-
scope.8 Therefore it was considered that the compounds had a cer-
tain effect on the synthesis of the cell wall. We are currently
studying the evaluation of the growth inhibition of C. albicans. By
contrast, neither antifungal activity nor a morphological change
was observed against A. fumigatus. Kre6p is our primary target and
its homologues have been found in Aspergillus species,5 but it was
reported that Aspergillus species contain no b-1,6-glucan polymer.9
The functions of these homologues in Aspergillus species are not well
54.21, 49.84, 43.41, 29.17, 19.95; ½a D25:0
ꢀ56.4° (c 1.030, CHCl3); MS (ESI), m/z
ꢁ
396 [M+H]+; Anal. Calcd for C25H25N5: C, 75.92; H, 6.37; N, 17.71. Found: C,
75.79; H, 6.32; N, 17.78.
8. (a) Kitamura, A.; Higuchi, S.; Hata, M.; Kawakami, K.; Yoshida, K.; Namba, K.;
Nakajima, R. Antimicrob. Agents Chemother. 2009, 53, 3963; (b) Kitamura, A.;
Someya, K.; Okumura, R.; Hata, M.; Takeshita, H.; Nakajima, R. Biol. Pharm. Bull.
2010, 33, 192.
9. Fontaine, T.; Simenel, C.; Dubreucq, G.; Adam, O.; Delepierre, M.; Lemoine, J.;
Vorgias, C. E.; Diaquin, M.; Latge, J. P. J. Biol. Chem. 2000, 275, 27594.