Design, synthesis and biological evaluation of hetaryl-nucleoside derivatives as inhibitors of chitin synthase

Article abstract of DOI:10.1016/S0960-894X(03)00239-7

We report here the design, synthesis and biological evaluation of new models of sugar analogues for chitin synthase. These UDP-GlcNAc mimetics associate a sugar-mimicking hetaryl group and uridine, linked with different pyrophosphate bioisosteres. The compounds displayed weak inhibition activity on chitin synthase and their antifungal potencies have been assayed against a large variety of pathogenic fungi.

Full text of DOI:10.1016/S0960-894X(03)00239-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1713–1716
Design, Synthesis and Biological Evaluation of Hetaryl-Nucleoside
Derivatives as Inhibitors of Chitin Synthase
Jean-Bernard Behr, Thierry Gourlain, Abdellatif Helimi and Georges Guillerm*
´ ´
Laboratoire Reactions Selectives et Applications UMR 6519, UFR Sciences, CNRS BP 1039, 51687 Reims Cedex 2, France
Received 3 January 2003; revised 4 February 2003; accepted 3 March 2003
Abstract—We report here the design, synthesis and biological evaluation of new models of sugar analogues for chitin synthase.
These UDP-GlcNAc mimetics associate a sugar-mimicking hetaryl group and uridine, linked with different pyrophosphate bioi-
sosteres. The compounds displayed weak inhibition activity on chitin synthase and their antifungal potencies have been assayed
against a large variety of pathogenic fungi.
# 2003 Elsevier Science Ltd. All rights reserved.
In the 25 past years, a steep increase in life-threatening
fungal infections has occurred and resistance develop-
ment to the currently administered treatments has urged
the development of new antifungal agents.1 In this con-
text, chitin synthase (CS) a specific enzyme present in
fungi and essential for cell viability was envisaged as an
attractive target.2 The first isolated CS inhibitors were
the peptidyl nucleoside Polyoxins and Nikkomycins,
among which Nikkomycin Z 1 (Fig. 1) was the most
promising candidate as antifungal drug.3 Some recent
studies described new nucleoside analogues as potent
competitive inhibitors of CS,4 as well as non-competi-
tive inhibitors, lacking any structural resemblance with
the natural substrate UDP-GlcNAc, but displaying Ki’s
in the sub-nanomolar range.5 However, the modest
antifungal properties displayed by these new CS inhibi-
tors, implied further research in this field.
In this study, malonic as well as tartaric acid or carbo-
hydrate-based linkages were chosen as non-hydrolizable
analogues of the pyrophosphate portion of UDP-
GlcNAc, mimicking the probable six-membered ring
pyrophosphate–M2+ complex formed in the active site
(Fig. 1).8 In this paper, we report the synthesis of these
In view of our interest in new antifungal agents, we
designed hetaryl nucleoside derivatives 2–4 (Fig. 1) as
new models of CS inhibitors, in the prospect of
improving their antifungal properties. The easily syn-
thesized heteroaromatic ring might serve as suitable
glycopyranosyl surrogate6 and might favourably inter-
act with the hydrophobic binding area in the active site
of CS.4b It is worth noting that Nikkomycin Z utilizes
an analogous hydroxypyridine, providing conforma-
tional mimicry of the transition-state in the presumed
glycosyl transfer reaction catalyzed by chitin synthase.7
*Corresponding author. Tel.:+33-326-913-308; fax:+33-326-913-166;
e-mail: georges.guillerm@univ-reims.fr
Figure 1. Design of new chitin synthase inhibitors 2–4.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00239-7

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J.-B. Behr et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1713–1716
new sugar-nucleoside analogues, their interaction with
CS at the active site and their antifungal activity.
assayed to evaluate the influence of hydrophobic charac-
ter of the pyridinyl moiety on enzyme’s recognition.
The 2-hydroxymethylquinoline 5a9 and the correspond-
ing bromide 6a10 were prepared in one step according to
known procedures. The hydroxypyridine derivatives 5b
and 6b were obtained starting from 2-methyl-5-hydroxy-
pyridine 7 after protection of the free hydroxyl group
with MOM-chloride (Scheme 1).11
Synthesis of non-symmetrical l-tartaric acid derivatives
3a,b (Scheme 3) implied the differentiation of both link-
ages. For this reason, we synthesized the known 50-
amino-50-deoxy uridine derivative 1612 by direct azida-
tion of uridine,13 protection with TBDMS-Cl and azide
reduction with a catalytic amount of 1,3-propanedithiol
and NaBH4 as a co-reagent. Tartaric monomethyl ester
18, readily obtained by mono-saponification of com-
mercial 17, was reacted with amine 16 under the condi-
tions stated above, to give tartaric nucleoside 19. As
expected, saponification with KOH/MeOH occurred
specifically on the ester function without affecting the
amide bond and yielded acid 20, after neutralization
with Dowex (H+) resin. Esterification of 20 with alco-
hols 5a,b, purification of the resulting esters 21a,b (FC,
ethyl acetate/petroleum ether, v/v, 7/3) and subsequent
deprotection (90% aqueous TFA) gave pure crystalline
UDP-GlcNAc analogues 3a,b after subsequent wash-
ings in CHCl3.
Malonic models 2a,b were obtained by coupling alco-
hols 5a,b with the known uridinyl malonate 11 (Scheme
2).7 This esterification was best achieved with DCC and
HOBT as activators in the presence of a catalytic
amount of pyrrolidinopyridine (PPY). Conversion was
somewhat low and could be improved by adding an
excess of DCC and acid 11 during the course of the
reaction. After filtration to remove DCU, purification
of the crude material by FC (ethyl acetate/diethyl ether,
v/v, 1/1) gave malonic diesters 12a,b. Deprotection of
the acid-sensitive protective groups and purification by
FC (ethyl acetate/CH3CN, v/v, 9/1) gave pure 2a,b. The
partially deprotected intermediate 13b was isolated and
The structural similarity of a monosaccharide moiety
and a pyrophosphate–M2+ complex was first pointed
out to explain the high potency of tunicamycin to bind
to the catalytic site of GlcNAc phosphotransferase.14 In
search for new galactosyltransferase inhibitors, Wong
and co-workers demonstrated that (1,4)-linked glucose
could also serve as a pyrophosphate mimic.8 In this
study, we investigated (1,3)-, (1,4)- or (1,6)-linked glu-
cose units as pyrophosphate substitutes. In each pro-
posed model, the chair-like carbohydrate could mimic
the six-membered diphosphate–metal complex as
Scheme 1. (i) MOMCl, iPr2NEt, CH2Cl2 (94%); (ii) m-CPBA, CHCl3
(94%); (iii) Ac2O, 120 ꢀC (75%); (iv) KOH, MeOH (85%); (v) PPh3,
CBr4, CH2Cl2 (100%).
Scheme 3. (i) PPh3, CBr4, NaN3, DMF (80%); (ii) TBDMSCl, imi-
dazole, pyr. (89%); (iii) 1,3-propanedithiol, NaBH4, NEt3, iPrOH
Scheme 2. (i) DCC, HOBT, PPY, CH2Cl2/DMF (46%); (ii) 80% aq
HCO2H, CH3CN (73%); (iii) 90% TFA (52%).
(90%); (iv) KOH (1.2 equiv), MeOH, then Dowex 50WX-8 (H+
(80%); (v) DCC, HOBT, PPY, CH2Cl2/DMF; (vi) 90% TFA.
)

J.-B. Behr et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1713–1716
1715
discussed above. In addition, comparison of the binding
affinity of the corresponding 4a–c regioisomers should
give an evaluation of the distance fitting the O-P-O-P-O
five-atom bridge. The (1,3)-substituted glucosyl residue
4a was prepared starting from diacetone-d-glucose after
alkylation with bromide 6a (Scheme 4). Deprotection of
acetonides (80% aqueous TFA) and subsequent acetyl-
ation gave 23, which was glycosylated to 24 with 20,30-
O-isopropylideneuridine 25, using standard procedures.
Pure 24 (silica gel FC, CH2Cl2/MeOH, v/v, 95/5) was
treated with MeONa/MeOH and formic acid (80%
aqueous solution) to remove protecting groups. Com-
pound 4a was purified by FC on a HP20hydrophobic
support, eluted with a gradient of water/methanol (from
v/v, 50/50 to 20/80) before biological evaluation.
either the 6-OH or the 4-OH was kept free to yield
respectively 27 or 28 as major product. Subsequent
alkylation with 6a gave hetaryl-glucose compounds 29,
30 which were deprotected (80% aqueous TFA) and
further acetylated (Ac2O, pyridine) to give 31, 32 as
suitable substrates for the glycosylation step with 25.
Deprotection and purification in the conditions descri-
bed above, led to pure UDP-GlcNAc analogues 4b,c.
All the new compounds 2–4 gave satisfactory analytical
data.17
For the inhibition studies, enzymatic assays were per-
formed using Saccharomyces cerevisiae cells (X2180
strain), which were permeabilized according to a
recently reported procedure.18 Activity of Chitin Syn-
thase I was assayed specifically by measuring the rate of
formation of [14C]-chitin from UDP-N-acetyl-[14C]-glu-
cosamine, according to the standard method reported
by Choi and Cabib.19 The reaction rate of chitin syn-
thase showed normal Michaelis–Menten kinetics with
UDP-GlcNAc as the variable substrate and GlcNAc as
the fixed substrate. A Km value of 0.5 mM was deter-
mined for UDP-GlcNAc from Lineweaver–Burk plot,
which was in good agreement with earlier reported
values. The standard Nikkomycin Z 1 strongly inhibited
our chitin synthase preparation (IC50=0.50 mM,
Ki=0.34 mM, competitive). The CS inhibitory activity
of 2–4 were examined under the same conditions and
the corresponding IC50 values were determined by the
Dixon method (Table 1). As shown on Table 1, com-
pounds 2–4 displayed weak activity on CS, when com-
pared to 1 used as reference.
The (1,4)- and (1,6)-substituted monosaccharide link-
ages were prepared by regioselective ring opening of the
readily prepared methoxybenzylidene-glucose derivative
26 (Scheme 5).15 According to the reaction conditions,16
Antifungal activity was also evaluated against Candida
albicans, Saccharomyces cerevisiae, Aspergillus fumiga-
tus, Trichophyton mentagrophytes, Trichophyton rubrum
or Cryptococcus neoformans, according to the standard
protocol.20 No significant activity of compounds 2–4
was noted on this panel of strains. Growth inhibition
was only detected with 4b, at 256 mg/mL (80% inhibi-
tion for C. neoformans, 30% inhibition for C. albicans,
Scheme 4. (i) 6a, NaH, DMF (80%); (ii) 80% aq TFA, then
Ac2O, pyr. (63%); (iii) TMSOTf, 3 A MS, CH2Cl2 (36%); (iv) MeONa/
MeOH, then 80% HCOOH (63%).
Scheme 5. (i) p-CH3OPhCH(OCH3)2, TsOH, DMF, 70 ꢀC, 150torr (70%); (ii) MOMCl, iPr2NEt, CH2Cl2, 45 ꢀC (90%); (iii) NaBH3CN, TMS, 3 A
MS, CH3CN (75%); (iv) NaBH3CN, TFA, 3 A MS, DMF (75%); (v) NaH, DMF, 6a; (vi) 80% aq TFA, then Ac2O, pyr.; (vii) 25, TMSOTf, 3 A
MS, CH2Cl2; (viii) MeONa/MeOH, then 80% HCO2H.

1716
J.-B. Behr et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1713–1716
Table 1. Inhibition of chitin synthase with compounds 1–4
9. Chiu, Y.-H.; Dos Santos, O.; Canary, J. W. Tetrahedron
1999, 55, 12069.
10. Rieger, B.; Abu-Surrah, A. S.; Fawzi, R.; Steinman, M. J.
Organomet. Chem. 1995, 497, 73.
11. Linderman, R. J.; Jaber, M.; Griedel, B. D. J. Org. Chem.
1994, 59, 6499.
12. Peterson, M. A.; Nilsson, B. L.; Sarker, S.; Doboszewski,
B.; Zhang, W.; Robins, M. J. J. Org. Chem. 1999, 64, 8183.
13. Yamamoto, I.; Sekine, M.; Hata, T. J. Chem. Soc., Perkin
Trans. 1 1980, 306.
14. Camarasa, M.-J.; Fernandez-Resa, P.; Garcia-Lopez,
M.-T.; Heras, F.; Mendez-Castrillon, P. P.; Alarcon, B.; Car-
rasco, L. J. Med. Chem. 1985, 28, 40 .
15. Furstner, A.; Radkowski, K.; Grabowski, J.; Wirtz, C.;
¨
Mynott, R. J. Org. Chem. 2000, 65, 8758.
Compd
CSI inhibition (IC50, mM)
1
0.00034 (ꢂ0.00002)
2.9 (ꢂ0.4)
2a
13b
2b
3a
3b
4a
4b
4c
10.8 (ꢂ2)
2.8 (ꢂ0.4)
2.0( ꢂ0.2) (Ki=0.8 mM)a
3.2 (ꢂ0.4)
0.8 (ꢂ0.1) (Ki=0.25 mM)a
3.2 (ꢂ0.4)
2.0( ꢂ0.2)
aCompetitive.
16. Johansson, R.; Samuelsson, B. J. Chem. Soc., Perkin
Trans. 1 1984, 2371.
S. cerevisiae and A. fumigatus). Analogue 4c showed
some weak activity against C. albicans at the same high
concentration.
17. Selected data, 2a: 1H NMR (DMSO-d6, 500 MHz, d ppm,
J=Hz) 8.25 (d, 1H, J=8.6 Hz, Ar–H), 7.90(d, 1H, J=8.6 Hz,
Ar–H), 7.85 (d, 1H, J=8.6 Hz, Ar–H), 7.66 (t, 1H, J=2ꢁ8.6,
Ar-H), 7.50(m, 3H, Ar–H, H-6), 5.80(d, 1H, J=3.7 Hz, H-
10), 5.60(d, 1H, J=8.0Hz, H-5), 5.41 (s, 2H, Ar–CH 2O), 4.40
In conclusion, the new hetaryl nucleosides 2–4, designed
to mimic the sugar-nucleoside donor at the transition-
state during the glycosyl transfer process, did not
improve the binding affinity as expected. From these
results and others,21 it appeared until now that attempts
to inhibit the catalytic activity of CS with a single UDP-
GlcNAc analogue, were mostly inefficient. Targeting,
with a multicomponent adduct, the multi-binding sites
suggested to be present in this processive glycosyl-
transferase,22 could help the discovery of potent inhibi-
tors of this enzyme. Work is in progress to evaluate this
new model.
0
(m, 2H, H-50), 4.10(m, 2H, H-3 , H-40), 4.06 (m, 1H, H-20),
3.71 (s, 2H, C(O)CH2C(O)). –13C NMR (DMSO-d6,
62.5 MHz) 175.5, 166.1, 165.7, 163.2, 150.5, 146.9, 140.2,
136.9, 129.6, 128.5, 127.6, 127.1, 126.5, 119.1, 102.0, 89.2, 80.9,
73.2, 69.5, 67.6, 64.5, 41.0. – CI-MS (NH3): m/z=472 (MH+,
1
10%), 160 (100%). 3a: H NMR (CD3OD, 500 MHz) 8.47 (d,
1H, J=8.5 Hz, Ar-H), 8.07 (d, 1H, J=8.5 Hz, Ar-H), 7.99 (d,
1H, J=8.5 Hz, Ar-H), 7.82 (t, 1H, J=2ꢁ8.5 Hz, Ar-H), 7.73
(d, 1H, J=8.5 Hz, Ar-H), 7.68 (d, 1H, J=8.1 Hz, H-6), 7.65
(t, 1H, Ar-H), 5.82 (d, 1H, J=4.7 Hz, H-10), 5.72 (d, 1H,
J=8.1 Hz, H-5), 5.60(d, 1H, JAB=14.1 Hz, ArCH2O), 5.53
(d, 1H, JAB=14.1 Hz, ArCH2O), 4.79 (d, 1H, J=2.2 Hz,
CHOH), 4.64 (d, 1H, J=2.2 Hz, CHOH), 4.201 (t, 1H,
J=2ꢁ4.9 Hz, H-20), 4.10(t, 1H, J=2ꢁ5.0Hz, H-3 ), 4.02 (q,
1H, J=3ꢁ4.9 Hz, H-40), 3.75 (dd, 1H, J=4.9 Hz 10.5, H-50a),
3.51 (dd, 1H, J=4.9 Hz 10.5, H-50b). ). –13C NMR (CD3OD,
125 MHz) 169.7, 169.6, 166.3, 159.4, 152.0, 147.5, 143.1, 139.9,
131.8, 129.0, 128.2, 127.8, 127.6, 120.5, 103.0, 91.1, 83.7, 74.3,
73.6, 73.5, 71.5, 67.3, 40.8. – CI-MS (NH3): m/z=517 (MH+,
5%), 303 (60%), 160 (100%). 4a: 1H NMR (CD3OD,
500 MHz) 8.48 (bs, 1H, NH), 8.34 (d, 1H, J=8.5 Hz, Ar-H),
8.04 (d, 1H, J=8.1 Hz, H-6), 8.00 (d, 1H, J=8.5 Hz, Ar-H),
7.80(d, 1H, J=8.5 Hz, Ar-H), 7.77 (t, 1H, J=2ꢁ8.5 Hz, Ar–
H), 7.60(m, 2H, Ar–H), 5.94 (d, 1H, J=4.9 Hz, H-10), 5.80(d,
1H, J=8.1 Hz, H-5), 5.22 (s, 2H, ArCH2O), 4.49 (d, 1H,
J=7.8 Hz, H-100), 4.35 (t, 1H, J=2ꢁ4.9 Hz, H-30), 4.30(t, 1H,
J=4.9 Hz, H-20), 4.24 (m, 2H, H-40, H-50a), 3.93 (dd, 1H,
J=2.0Hz 12.2, H-60000a), 3.85 (m, 1H, H-50b), 3.74 (dd, 1H,
J=5.9 Hz 12.2, H-6 b), 3.59 (m, 2H, H-400, H-300), 3.49 (t, 1H,
J=2ꢁ7.8 Hz, H-200), 3.42 (m, 1H, H-500). –13C NMR
(CD3OD, 125 MHz) 166.5, 160.4, 152.6, 147.5, 142.6, 138.7,
131.0, 128.9, 128.7, 127.8, 127.6, 120.5, 103.5, 102.6, 89.9, 87.1,
84.5, 77.3, 75.2, 74.9, 74.6, 70.9, 70.8, 69.1, 61.9. – CI-MS
(NH3): m/z=549 (MH+, 15%), 220 (100%).
Acknowledgements
The authors thank Dr. N. S. Ryder (Novartis Research
Institute, A-1235 Vienna) for the antifungal evaluation.
This work was supported by a grant (T.G.) from the
´ ´
Conseil general de la Marne and Europol’Agro.
References and Notes
1. (a) Turner, W. W.; Rodriguez, M. J. Curr. Pharm. Des.
1996, 2, 209. (b) Fostel, J. M.; Lartey, P. A. Drug Discov.
Today 2000, 5, 25. (c) Hossain, M. A.; Ghannoum, M. A. Exp.
Opin. Invest. Drugs 2000, 9, 1797.
2. Maertens, J. A.; Boogaerts, M. A. Curr. Pharm. Des. 2000,
6, 225.
3. Zhang, D.; Miller, M. J. Curr. Pharm. Des. 1999, 5, 73.
4. (a) Suda, A.; Ohta, A.; Sudoh, M.; Tsukuda, T.; Shimma,
N. Heterocycles 2001, 55, 1023. (b) Obi, K.; Uda, J.-I.; Iwase,
K.; Sugimoto, O.; Ebisu, H.; Matsuda, A. Bioorg. Chem. Med.
Lett. 2000, 10, 1451.
5. Sudoh, M.; Yamazaki, T.; Masubuchi, K.; Taniguchi, M.;
Shimma, N.; Arisawa, M.; Yamada-Okabe, H. J. Biol. Chem.
2000, 275, 32901.
18. Crotti, L. B.; Drgon, T.; Cabib, E. Anal. Biochem. 2001,
292, 8.
19. Choi, W.-J.; Cabib, E. Anal. Biochem. 1994, 219, 368.
20. Ryder, N. S.; Wagner, S.; Leitner, I. Antimicrob. Agents
Chemother. 1998, 42, 1057.
21. (a) Xie, J.; Thellend, A.; Becker, H.; Vidal-Cros, A. Carbo-
hydrate Res. 2001, 334, 177. (b) Gautier-Lefevre, I.; Behr, J.-
B.; Guillerm, G.; Ryder, N. S. Bioorg. Med. Chem. Lett. 2000,
10, 1483.
6. (a) Knapp, S.; Dong, Y.; Withers, S. G. Bioorg. Med.
Chem. Lett. 1995, 5, 763. (b) Bhattacharya, A. K.; Stolz, F.;
Schmidt, R. R. Tetrahedron Lett. 2001, 42, 5393.
7. Behr, J.-B.; Gautier-Lefebvre, I.; Mvondo-Evina, C.; Guil-
lerm, G. J. Enz. Inhib. 2001, 16, 107.
8. Wang, R.; Steensma, D. H.; Takaoka, Y.; Joanne, W. Y.;
Kajimoto, T.; Wong, C. H. Bioorg. Med. Chem. 1997, 5, 661.
`
22. Saxena, I. M.; Brown, R. M.; Fevre, M.; Geremia, R. A.;
Henrissat, B. J. Bacteriol. 1995, 177, 1419.