Pyranmycins, a Novel Class of Aminoglycosides with Improved Acid Stability: The SAR of D-Pyranoses on Ring III of Pyranmycin

Article abstract of DOI:10.1021/ol0269042

(Matrix Presented) The synthesis of a novel class of aminoglycosides, pyranmycins, is reported along with the structure activity relationship (SAR) of their antibacterial activity against Escherichia coli. Two pyranmycins show prominent activity (9 μM). P

Full text of DOI:10.1021/ol0269042

ORGANIC
LETTERS
2002
Vol. 4, No. 26
4603-4606
Pyranmycins, a Novel Class of
Aminoglycosides with Improved Acid
Stability: The SAR of D-Pyranoses on
Ring III of Pyranmycin
Cheng-Wei Tom Chang,* Yu Hui, Bryan Elchert, Jinhua Wang, Jie Li, and
Ravi Rai
Department of Chemistry and Biochemistry, Utah State UniVersity,
0300 Old Main Hill, Logan, Utah 84322-0300
chang@cc.usu.edu
Received September 14, 2002
ABSTRACT
The synthesis of a novel class of aminoglycosides, pyranmycins, is reported along with the structure activity relationship (SAR) of their
antibacterial activity against Escherichia coli. Two pyranmycins show prominent activity (9 µM). Pyranmycins also manifest superior stability
in acidic media. The SAR information will lead to the future designs of pyranmycin against drug resistant bacteria.
Neomycin belongs to a group of aminoglycoside antibiotics
containing a 4,5-disubstituted 2-deoxystreptamine core and
have been used against both gram-positive and gram-negative
bacteria for more than fifty years (Figure 1).1 Neomycin
exerts its antibacterial activity by binding selectively to the
A-site of 16S ribosomal RNA of bacteria, and thereby
inhibits the protein synthesis of these microorganisms.
Although neomycin is still widely used for the treatment of
serious infections, there are two problems associated with
its use. The first is the rapid emergence of drug resistance
in infectious microorganisms,2 due to the prolonged misuse
and overuse of aminoglycoside antibiotics. The second is
its relatively high cytotoxicity.1
III and IV).1a Because the glycosidic bond of a furanose is
more acid sensitive than that of a pyranose,2 it is conceivable
that by replacing the ring III furanose with a pyranose,
superior acid stability will result, possibly leading to a
reduction in the required dose for oral administration and,
hopefully, a lowering of the associated cytotoxicity. To this
end, we have modified neomycin by substituting the neo-
Neomycin degrades readily in acidic media into less active
neamine (rings I and II) and inactive neobiosamine (rings
(1) (a) Hooper, I. R. Aminoglycoside Antibiotics; Springer-Verlag: New
York, 1982. (b) Haddad, J.; Kotra, L. P.; Mobashery, S. In Glycochemistry
Principles, Synthesis, and Applications; Wang, P. G., Bertozzi, C. R., Eds.;
Marcel Dekker: New York, 2001; p 307.
(2) Cohen, M. L. Science 2002, 257, 1050-1055. (b) Neu, H. C. Science
2002, 257, 1064-1072.
Figure 1. Structures of Neomycin and Pyranmycin.
10.1021/ol0269042 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/21/2002

Scheme 1. Synthesis of Glycosyl Donors and Protected Pyranmycins
biosamine component with a glycopyranose (Figure 1). We
only one example uses D-glucopyranose as the ring III
component via R and â linkages.15 However, the resulting
adducts were less active than neamine. With the envisioned
advantages of our pyranmycin design and our recently
synthesized library of glycopyranosyl donors,16 we report a
general procedure for the synthesis of pyranmycins together
with the results of structure activity relationship studies
targeting the ring III pyranose of pyranmycin.
Glycosyl trichloroacetimidates (glycosyl donors) were
obtained from the corresponding glycosyl acetate via selec-
tive hydrolysis of anomeric acetate with hydrazine acetate
in DMF, followed by treatment with trichloroacetonitrile in
the presence of catalytic amount of DBU. Acid-catalyzed
(BF3-OEt2) glycosylation of these glycosyl donors with
neamine derivative9 afforded the designed protected pyran-
mycins (Scheme 1). The â-glycosidic bond on ring III was
have named this novel aminoglycoside pyranmycin.
Another advantage of the pyranmycin design is that it can
serve as a template for further chemical modifications. With
recent advances in the structural analysis of the binding of
aminoglycosides to rRNA,4-7 these modifications can be
designed to increase the binding affinity toward the targeted
RNA molecules and disrupt enzyme-catalyzed inactivations,8
which are the most prevalent mechanisms of aminoglycoside
resistance from resistant bacteria. Hence, the resulting
aminoglycoside entities may regain the antibacterial activity
against resistant strains.
While many neomycin or ribostamycin analogues contain-
ing a ring III furanose with â linkage have been reported,9-14
(3) (a) Bochkov, A. F.; Zaikov, G. E. Chemistry of the O-Glycosidic
Bond: Formation and CleaVage; Pergamon Press: Elmsford, NY, 1979.
(b) Shallenberger, R. S.; Birch G., G. Sugar Chemistry; Avi Publishing
Co., Inc.: 1975.
(4) Fourmy, D.; Recht, M. I.; Puglisi, J. D. J. Mol. Biol. 1998, 277, 347-
362.
(10) Wong, C.-H.; Hendrix, M.; Manning, D. D.; Rosenbohm, C.;
Greenberg, W. A. J. Am. Chem. Soc. 1998, 120, 8319-8327.
(11) Yoshikawa, M.; Ikeda, Y.; Takenaka, K. Chem. Lett. 1984, 2097-
2100.
(5) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science
1996, 274, 1367.
(12) Girodeau, J.-M.; Pineau, R.; Masson, M.; Le Goffic, F. J. Antibiotics
1984, 37, 150-158.
(6) Fourmy, D.; Yoshizawa, S.; Puglisi, J. D. J. Mol. Biol. 1998, 277,
333-345.
(13) Woo, P. W. K.; Haskell, T. H. J. Antibiotics 1982, 35, 692-702.
(14) (a) Kumar, V.; Remers, W. A. J. Org. Chem. 1978, 43, 3327-
3331. (b) Kumar, V.; Remers, W. A. J. Med. Chem. 1979, 22, 432-436.
(c) Kumar, V.; Jones, G. S., Jr.; Blacksberg, I.; Remers, W. A. J. Med.
Chem. 1980, 23, 42-49. (d) Kumar, V.; Remers, W. A. J. Org. Chem.
1981, 46, 4298-4300.
(7) Ma, C.; Baker, N. A.; Joseph, S.; McCammon, J. A. J. Am. Chem.
Soc. 2002, 124, 1438-1442.
(8) For example: Haddad, J.; Kotra, L. P.; Liano-Sotel, B.; Kim, C.;
Azucena, E. F., Jr.; Liu, M.; Vakulenko, S. B.; Chow, C. S.; Mobashery,
S. J. Am. Chem. Soc. 2002, 124, 3229-3237.
(15) (a) Endo, T.; Perlman, D. J. Antibiot. 1972, 25, 681-682. (b) Suami,
T.; Nishiyama, S.; Ishikawa, Y.; Katsura, S. Carbohydr. Res. 1977, 56,
414-418.
(9) Greenberg, W. A,; Priestley, E. S.; Sears, P. S.; Alper, P. B.;
Rosenbohm, C.; Hendrix, M.; Hung, S.-C.; Wong, C.-H. J. Am. Chem. Soc.
1999, 121, 6527-6541.
(16) Elchert, B.; Hui, Yu.; Chang, C.-W. T. Unpublished results.
4604
Org. Lett., Vol. 4, No. 26, 2002

Scheme 2. Synthesis of the Designed Pyranmycins
a
MIC: minimum inhibitory concentration
formed exclusively due to the expected neighboring group
assistance.17 The protected pyranmycins were subjected to
the final synthetic steps, including hydrolysis of acetyl groups
with K2CO3 in MeOH, reduction of azido group using the
Staudinger reaction, and deprotection of benzyl groups with
hydrogenation (H2, Pd(OH)2/C, Degussa-type in HOAc/H2O
solution). Purification was accomplished using flash chro-
matography, followed by an ion-exchange (Dowex 1 ×
8-200, Cl- form) (Scheme 2).
The constructed pyranmycins were assayed against E. coli
(ATCC 25922), and the minimum inhibitory concentrations
(MIC) were determined18 using neomycin, ribostamycin, and
neamine as positive controls (MIC ) 2, 5, 36 µM, respec-
tively). From the MIC values, a structure activity relationship
can be elucidated. TC001 has a MIC slightly higher than
that of the neamine, which is consistent with literature
results.15 Three pyranmycins were constructed to probe the
effect of monoamino substitution at different positions on
the ring III pyranose, including TC002 (6′′-NH2), TC007
(3′′-NH2), and TC012 (4′′-NH2). Among them, TC002 (6′′-
NH2) is the most potent. Attempts to incorporate the
glucosamine donor bearing a C′′-2 amino group were,
however, unsuccessful.
The diamino analogue, TC003, which has a novel gluco-
1,3-diamine binding motif, is less potent than TC002. This
result implies that simply increasing the number of amino
groups may not lead to an increase in the antibacterial
activity, even though the binding affinity between the
constructed compounds and the RNA molecules is likely to
increase in vitro.19 We also incorporated pyranoses with
galacto- and allo-configurations (TC004, TC008, and TC018),
and found no significant differences in the activity among
these scaffolds.
The most promising results came from TC005 and TC006,
which have an amino group at the equatorial and axial
positions of C-4′′, respectively, and a C-6′′ CH3 group. The
potencies of TC005 and TC006 are similar to those of
neomycin and ribostamycin, despite having weaker trans-
and cis-1,2-hydroxyamine binding motifs, respectively.20
(19) Wong and colleagues have shown in their studies (ref 9) that binding
affinity and antibacterial activity cannot be correlated.
(20) Hendrix, M.; Alper, P. B.; Priestley, S.; Wong, C.-H, Angew. Chem.,
Int. Ed. Engl. 1997, 36, 95-98.
(21) Glucose pentaacetate was purchased from Acros Chemical Co.
(22) The glycosyl acetate was obtained from acid-catalyzed hydrolysis
and peracetylation of 1:2,3:4-di-O-isopropylidene-D-galactopyranose (Sza-
rek, W. A.; Jones, J. K. N. Can. J. Chem. 1965, 43, 2345).
(23) Glycosyl acetate was obtained from acid-catalyzed hydrolysis and
peracetylation of 3-azido-3-deoxy-1:2,5:6-di-O-isopropylidene-D-galacto-
pyranose (Knapp, S.; Gore, V. K. Org. Lett. 2000, 2, 1391-1393).
(24) Weinges, K.; Haremsa, S.; Mauer, W. Carbohydr. Res. 1987, 155,
453-458.
(25) Glycosyl acetate was obtained from selective acid-catalyzed hy-
drolysis (TsOH, MeOH), tosylation, azide substitution, hydrolysis, and
peracetylation of 3-azido-3-deoxy-1:2,5:6-di-O-isopropylidene-D-galacto-
pyranose (ref 22).
(17) The â-glycosidic bond is essential for the formation of intramolecular
hydrogen bonding between 2′-NH2 and O-5′′ of the constructed pyranmy-
cins. Please refer to the NMR study for the binding of aminoglycoside
toward RNA in ref 5.
(18) Methods for Dilution Antimicrobial Susceptibility Testing for
Bacteria that Grow Aerobically. Approved standard M7-A5; Performance
Standards for Antimicrobial Disk Susceptibility Tests. Approved standard
M2-A7; National Committee for Clinical Laboratory Standards; Wayne,
PA.
(26) (a) Banaszek, Z. Rocz. Chem. 1971, 45, 391-397. (b) Paulsen, H.;
Sumfleth, B.; Redlich, H. Chem. Ber. 1976, 109, 1362-1368.
Org. Lett., Vol. 4, No. 26, 2002
4605

has less of an effect on activity compared to substitution at
C-4′′ and C-6′′.
We also carried out acid degradation experiments for
TC002 and TC005. The compounds were dissolved in D2O,
purged with anhydrous HCl (pH ca. 1), sealed in NMR tubes
1
and incubated at 37 °C, and monitored by H NMR. Under
identical conditions, neomycin underwent a time-dependent
acid degradation (20, 40, 60, and 80% degradation after 2,
6, 10, and 14 days, respectively), rendering a significant
decrease in antibacterial activity (the MIC of acid-treated
neomycin increases from 2 to 50 µM). On the contrary, both
TC002 and TC005 showed no sign of degradation and
maintained the same level of antibacterial activity.
Figure 2. Summary of the SAR of Ring III of Pyranmycins.
In conclusion, we have reported the structure activity
relationships of pyranmycins ring III D-pyranose. Pyranmy-
cins have comparable antibacterial activity to that of neo-
mycin, but exhibit much-improVed acid stability. Although
the effectiveness of alleviating cytotoxicity by improving acid
stability will require more detailed studies, we have dem-
onstrated that pyranmycins have great potential for develop-
ment into clinically useful antibiotics. We are currently
assaying pyranmycins for activity against resistant strains
of bacteria. We are also studying the in vitro binding affinity
of the pyranmycins with RNA constructs to validate the
postulated van der Waals interaction.
Two possibilities may account for the unexpected high
potency of TC005 and TC006: the steric hindrance from
C-6′′ and a van der Waals interaction between the rRNA
and the C-6′′ methyl group. Similar trends of potency are
observed between TC001 and TC020 and between TC018
and TC006. To further study the role of the C-6′′ methyl
group, we prepared analogue TC022, with a D-xylopyranose
as the ring III sugar. TC022 was completely inactiVe,
favoring the presence of a possible van der Waals interaction
between the RNA and C-6′′ methyl group.
The dideoxygenated analogue TC017 showed a dramatic
decrease in activity. The result manifests the importance of
having either NH2 or OH at the C-4′′ position. Further
modifications such as glycosylations on the 4′′-OH with
either glucopyranose (TC021) or galactopyranose (TC019)
destroy the activity, presumably due to the steric hindrance
of an overexpanded substituent on C-4′′. Finally, the structure
activity relationship (SAR) at the ring III D-pyranose of
pyranmycin is summarized as follows (Figure 2): (1) no
significant differences in the antibacterial activity are ob-
served among the pyranoses with allo-, gluco-, and galacto-
configurations; (2) deoxygenation at C-6′′ (6′′-CH3) sub-
stantially increases the activity; (3) an NH2 or OH group at
C-4′′ position is essential for activity, and deoxygenation of
4′′-OH or glycosylation on 4′′-OH results in a dramatic
decrease in activity; and (4) amino group substitution at C-3′′
Acknowledgment. We are thankful for the financial
support from the Utah State University (CURI grant) and
the support from Department of Chemistry and Biochemistry,
Utah State University. We also thank Prof. Bradley Davidson
from Utah State University for assistance on composing the
manuscript, Prof. Paul Savage from Brigham Young Uni-
versity for reexamining the MIC value of TC005, and Prof.
Hung-wen (Ben) Liu from University of Texas, Austin, for
providing valuable discussion and suggesting the name,
pyranmycin. Mass spectrometry was provided by the Wash-
ington University Mass Spectrometry Resource and the Mass
Spectrometry Facilities, University of California, Riverside.
Supporting Information Available: General procedures
for the preparation of compounds 2b-p and 3a-p and
pyranmycins and the corresponding 1H and 13C NMR spectra
and mass spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
(27) These glycosyl acetates were prepared from peracetylation of lactose,
maltose, and xylose purchased from Acros Chemical Co.
(28) Glycosyl acetate was obtained from methyl glucoside via tosylation,
deoxygenation (LiAlH4), and acetylation (Ac2O, concentrated H2SO4).
(29) Schmidt, R. R.; Jung K.-H. In PreparatiVe Carbohydrate Chemistry;
Hannessian, S., Ed.; Marcel Dekker: New York, 1997; p 297.
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