Application of the Synthetic Aminosugars for Glycodiversification: Synthesis and Antimicrobial Studies of Pyranmycin

Article abstract of DOI:10.1021/jo035290r

A divergent approach was employed for the synthesis of aminosugars, from which a novel library of aminoglycoside antibiotics (pyranmycins) was synthesized. Pyranmycins have comparable antibacterial activity as neomycin, a clinically used aminoglycoside antibiotic, against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Mycobacterium smegmatis. In addition, pyranmycins, like streptomycin, are bacteriocidal while isoniazid (INH) is bacteriostatic. Therefore, pyranmycins may provide new therapeutic options in the treatment against tuberculosis. Several members of pyranmycins also manifest modest anti-Tat and anti-Rev activities, which may aid in the development of new anti-HIV agents. Although the antibacterial activity of pyranmycins against aminoglycoside resistant bacteria is less than expected, the synthetic methodologies of utilizing a library of aminosugars can be a model for future studies of glycodiversification or glycorandomization.

Full text of DOI:10.1021/jo035290r

Ap p lica tion of th e Syn th etic Am in osu ga r s for
Glycod iver sifica tion : Syn th esis a n d An tim icr obia l Stu d ies of
P yr a n m ycin
Bryan Elchert,† J ie Li,† J inhua Wang,† Yu Hui,† Ravi Rai,† Roger Ptak,‡ Priscilla Ward,‡
J on Y. Takemoto,§ Mekki Bensaci,§ and Cheng-Wei Tom Chang*,†
Department of Chemistry and Biochemistry, and Department of Biology, Utah State University,
0300 Old Main Hill, Logan, Utah 84322-0300, and Infectious Disease Research Department,
Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701
chang@cc.usu.edu
Received September 2, 2003
A divergent approach was employed for the synthesis of aminosugars, from which a novel library
of aminoglycoside antibiotics (pyranmycins) was synthesized. Pyranmycins have comparable
antibacterial activity as neomycin, a clinically used aminoglycoside antibiotic, against Escherichia
coli, Staphylococcus aureus, Bacillus subtilis, and Mycobacterium smegmatis. In addition, pyran-
mycins, like streptomycin, are bacteriocidal while isoniazid (INH) is bacteriostatic. Therefore,
pyranmycins may provide new therapeutic options in the treatment against tuberculosis. Several
members of pyranmycins also manifest modest anti-Tat and anti-Rev activities, which may aid in
the development of new anti-HIV agents. Although the antibacterial activity of pyranmycins against
aminoglycoside resistant bacteria is less than expected, the synthetic methodologies of utilizing a
library of aminosugars can be a model for future studies of glycodiversification or glycorandom-
ization.
In tr od u ction
ever, there are two problems often associated with these
results. The first one is the synthetic challenge involved
in the preparation of a library of aminosugars. The second
problem is the lack of methodologies for converting the
synthetic aminosugars into glycosyl donors. Most of the
glycosyl donors, such as glycosyl halides, glycosyl ace-
tates, and glycosyl trichloroacetimidates, are not suitable
for the procedures of aminosugar synthesis. Therefore,
phenylthio or ethylthio groups, which are stable enough
to endure the conditions for amino group incorporation
and can be activated for glycosylation directly, are often
used for the synthesis of aminosugars and corresponding
derivatives.12-17 Nevertheless, the starting materials,
ethyl thioglucopyranoside or phenyl thioglucopyranoside,
are inconvenient to prepare in large quantities for the
synthesis of an aminosugar library. Commercially avail-
able thioglucopyranose is too expensive to be a practical
option.
Aminosugars have attracted a growing interest due to
their broad spectrum of application in chemistry, bio-
chemistry, medicine, and pharmaceutical fields.1-6 Since
aminosugars are often found in the naturally occurring
antibiotics, one of the applications of aminosugars will
be the library construction of naturally occurring anti-
biotic analogues with the original sugar replaced by a
synthetic one leading to the concept of glycodiversification
or glycorandomization. Extensive studies have been done
toward the synthesis of various aminosugars.7-11 How-
* To whom correspondence should be addressed. Fax: 435-797-3390.
† Department of Chemistry and Biochemistry, Utah State Univer-
sity.
‡ Southern Research Institute.
§ Department of Biology, Utah State University.
(1) Kirschning, A.; Bechthold, A. F.-W.; Rohr, J . Top. Curr. Chem.
1997, 188, 1-84.
(2) J ohnson, D. A.; Liu, H.-w. Curr. Opin. Chem. Biol. 1998, 2, 642-
649.
(3) Weymouth-Wilson, A. C. Nat. Prod. Rep. 1997, 99-110.
(4) Hooper, I. R. Aminoglycoside Antibiotics; Springer-Verlag: New
York, 1982.
To provide solutions for these two problems, we de-
signed a divergent approach for the expedient synthesis
of an azidosugar (aminosugar) library with designed
(5) Priebe, W.; Van, N. T.; Burke, T. G.; Perez-Soler, R. Anticancer
Drugs 1993, 4, 37-48.
(11) For examples see: (a) Knapp, S.; Kukkola, P. J .; Sharma, S.;
Dhar, T. G. M.; Naughton, A. B. J . J . Org. Chem. 1990, 55, 5700-
5710. (b) Bundle, D. R. J . Chem. Soc., Perkin Trans. 1 1979, 2751-
2755. (c) Dent, B. R.; Furneaux, R. H.; Gainsford, G. J .; Lynch, G. P.
Tetrahedron 1999, 55, 6977-6996.
(6) Lothstein, L.; Sweatman, T, W.; Priebe, W. Bioorg. Med. Chem.
Lett. 1995, 5, 1807-1812.
(7) For reviewing: (a) Brimacombe, J . S. Angew. Chem., Int. Ed.
1971, 10, 236-248. (b) Umezawa, S.; Tsuchiya, T. Aminoglycoside
Antibiotics; Springer-Verlag: New York, 1982; pp 37-110.
(8) Zunszain, P. A.; Varela, O. Tetrahedron: Asymmetry 1998, 9,
1269-1276.
(9) Saotome, C.; Ono, M.; Akita, H. Tetrahedron: Asymmetry 2000,
11, 4137-4151.
(10) For the synthesis of 1,3-diaminopentose see: Yuasa, H.; Hash-
imoto, H. J . Am. Chem. Soc. 1999, 121, 5089-5090.
(12) Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C.-H. J . Am. Chem.
Soc. 1998, 120, 1965-1978.
(13) Crich, D.; Sun, S. J . Am. Chem. Soc. 1998, 120, 435-436.
(14) Crich, D.; Sun, S. J . Org. Chem. 1997, 62, 1198-1199.
(15) Crich, D.; Sun, S. J . Org. Chem. 1997, 62, 4506-4507.
(16) Crich, D.; Cai, W. J . Org. Chem. 1999, 64, 4926-4930.
(17) Garegg, P. J .; Olsson, L.; Oscarson, S. J . Org. Chem. 1995, 60,
2200-2204.
10.1021/jo035290r CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/28/2004
J . Org. Chem. 2004, 69, 1513-1523
1513

Elchert et al.
F IGURE 1. Prospective applications of glycodiversification.
binding motifs from commercially available and inex-
pensive methyl glucoside. The anomeric methoxyl group
of methyl glucoside is stable enough in the conditions for
amino-substitution or deoxygenation. However, it is too
stable to be activated for glycosylation. Hence, we have
also developed a general procedure for converting syn-
thetic sugar derivatives into glycosyl donors ready for
glycosylation. The amino groups are installed as azido
groups. After completion of the aminosugar synthesis, we
further utilize this library for the synthesis of a novel
class of aminoglycoside antibiotics, which we have termed
pyranmycins. Through our research, a model for the
study of carbohydrate-containing compounds has been
established. This includes the divergent synthesis of
unusual sugars with designed binding motifs, the library
construction of unusual sugar-containing compounds, and
the glycodiversification approach for the structure-
activity relationship studies of these compounds (Figure
1).
Given the complexity of these aminosugars and deoxy-
sugars found in the naturally occurring aminoglycoside
antibiotics, it is arduous to outline or predict the conclu-
sions regarding the structure-activity relationship of
unusual sugars among the vast numbers of unusual
sugar-containing antibiotics. Therefore, Wong and co-
workers have proposed several binding motifs based on
the commonly observed structural features of unusual
sugars on aminoglycosides (Figure 2).18,19 These include
gluco-/galacto-1,3-hydroxylamino and cis-/trans-1,2-hy-
droxylamino substructures. Following these binding mo-
tifs, we propose four new natural and nonnatural binding
motifs, including gluco-/galacto-1,3-diamino and variants
of gluco-/galacto-1,3-hydroxylamino substructures (Fig-
ure 2).20 These scaffolds will be the targets for our
synthesis of unusual sugars (Figure 1).
F IGURE 2. Proposed binding motifs of aminosugars.
Resu lts a n d Discu ssion
Diver gen t Syn th esis of Un u su a l Su ga r s. The syn-
thesis of our aminosugar library began with the com-
mercially available methyl glucopyranoside. Benzylation
of compound 121 yielded compound 221 (Scheme 1).
Compound 2 was treated with NaBH3CN/HCl in THF to
selectively deprotect the C-4 hydroxyl group providing
compound 3.21 Triflation followed by azide substitution
of compound 3 generated azidosugar 4 with intrinsic 1,3-
hydroxyamine binding motif in the galacto configuration.
Epimerization of the 4-OH of 3 was achieved with Swern
oxidation and NaBH4 reduction offering compound 5,
which allowed an SN2 azide substitution that furnished
(18) Wong, C.-H.; Hendrix, M.; Manning, D. D.; Rosenbohm, C.;
Greenberg, W. A. J . Am. Chem. Soc. 1998, 120, 8319-8327.
(19) Hendrix, M.; Alper, P. B.; Priestley, S.; Wong, C.-H. Angew.
Chem., Int. Ed. Engl. 1997, 36, 95-98.
(21) Hanessian, S. Preparative Carbohydrate Chemistry; Marcel
Dekker: New York, 1996; pp 53-67.
(20) Hui, Y.; Chang, C.-W. T. Org. Lett. 2002, 4, 2245-2248.
1514 J . Org. Chem., Vol. 69, No. 5, 2004

Synthesis and Antimicrobial Studies of Pyranmycin
SCHEME 1a
SCHEME 3a
a
Reagents: (a) Ac2O, concentrated H2SO4; (b) (1) H2NNH2-
a
Reagents: (a) PhCH(OMe)2, PPTS, DMF; (b) BnBr, NaH,
HOAc, DMF, (2) CCl3CN, DBU, CH2Cl2.
TBAI, THF; (c) NaBH3CN, HCl, THF; (d) (1) Tf2O, py, CH2Cl2, (2)
NaN3, DMF; (e) (1) (COCl)2, DMSO, DIPEA, (2) NaBH4, MeOH.
substitution generating azidosugar 10 with the intrinsic
cis-1,2-hydroxylamino motif in the galacto configuration.
Alternatively, compound 9 underwent the Swern oxida-
tion/NaBH4 reduction protocol to invert the C-4 hydroxyl
group that allowed the synthesis of azidosugar 12 with
the intrinsic trans-1,2-hydroxylamino motif in the gluco
configuration. In the last path, compound 7 was treated
with TsCl, followed by azide substitution to selectively
place an azido group on the C-6 position. The free C-4
hydroxyl group in the equatorial position of compound
13 was converted to the axial position yielding compound
14, which enabled the synthesis of azidosugar 15 with a
novel intrinsic gluco-1,3-diamino binding motif.
SCHEME 2a
We tried various methods22-26 to selectively hydrolyze
the anomeric methoxyl group into the hydroxyl group.
In every attempt, low yield and complex mixtures were
often encountered due to the concomitant deprotection
of the benzyl groups. Therefore, we decided to convert
the anomeric methoxyl group and all the benzyl groups
into acetyl groups using Ac2O with a catalytic amount of
H2SO4 (Scheme 3). The resulting acetyl glycosides, how-
ever, failed to undergo direct glycosylation with various
Lewis acids as catalysts or stoichiometric agents. As a
result, we transformed the acetyl glucosides to the
glycosyl trichloroacetimidate as the glycosyl donor. Treat-
ment of the acetyl glucosides with hydrazine acetate
resulted in the unprotected hydroxyl group at the ano-
meric position. Further treatment with trichloroacetoni-
trile and DBU as a base yielded the glycosyl trichloro-
acetimidate donor,28 which could then be coupled to the
acceptor of our choice. The constructed aminosugar
a
Reagents: (a) TsOH-H2O, MeOH; (b) (1) Tf2O, py, CH2Cl2,
(2) NaN3, DMF; (c) (1) TsCl, py, (2) LiAlH4, THF; (d) (1) (COCl)2,
DMSO, DIPEA, (2) NaBH4, MeOH; (e) (1) TsCl, py, (2) NaN3, DMF.
azidosugar 6 with intrinsic 1,3-hydroxylamino binding
motif in the desired gluco configuration.
In another route (Scheme 2), compound 2 was treated
with TsOH to give compound 7 with free hydroxyl groups
at the C-4 and C-6 positions, which branched into three
distinct routes. In the first route, triflation followed by
azide substitution of compound 7 provided azidosugar 8
with a novel intrinsic galacto-1,3-diamino binding motif.
In the second route, compound 7 was selectively deoxy-
genated at the C-6 position by sequential tosylation and
LiAlH4 reduction. Compound 9 was subjected to azide
(22) Demuynck, M.; De Clercq, P.; Vandewalle, M. J . Org. Chem.
1979, 44, 4863.
(23) Hanessian, S.; Guindon, Y. Tetrahedron Lett. 1980, 21, 2305.
(24) Bhatt, M. V.; El-Morey, S. S. Synthesis 1982, 1048.
(25) Ganem, B.; Small, V. R., J r. J . Org. Chem. 1974, 39, 3728.
(26) Tsunoda, T.; Amaike, M.; Tambunan, U. S. F.; Fujise, T.; Ito,
S.; Kodama, M. Tetrahedron Lett. 1987, 28, 2537.
(27) Wang, J .; Li, J .; Tuttle, D.; Takemoto, J .; Chang, C.-W. T. Org.
Lett. 2002, 4, 3997-4000.
(28) Chang, C.-W. T.; Hui, Y.; Elchert, B.; Wang, J .; Li, J .; Rai, R.
Org. Lett. 2002, 4, 4603-4606.
J . Org. Chem, Vol. 69, No. 5, 2004 1515

Elchert et al.
SCHEME 4a
The construction of the new pyranmycins begins with
the synthesis of a common intermediate, compound 39.
Compound 39 was synthesized by the glycosylation of
neamine derivative (Schemes 5 and 6) 35 and 3-O-allyl
glycosyl donor 38, which can be synthesized from 36 via
a similar procedure as in our previous report.29 The
glycosylated precursor, 39, was then directed into differ-
ent synthetic routes yielding various final products
(Scheme 7). Treatment of 39 with our final synthesis
protocol provides TC040. Hydroboration of 39 gave 40,
which was converted to TC041 with the final synthesis
protocol. Alternatively, fluorination of the 3-hydroxyl-
propyl group with DAST offers TC044 after completed
deprotection. Glycosylation of 40 with the glycosyl donors
4129 generated 42, which led to the synthesis of TC045.
The 6′′-fluronated analogue was synthesized from
methyl 6-fluoroglucopyranoside30 with the same protocols
(Scheme 8).
An tiba cter ia l Activity of P yr a n m ycin s. The previ-
ous synthesized and newly constructed pyranmycins were
assayed against Escherichia coli (ATCC 25922), Staphy-
lococcus aureus (ATCC 25923), Bacillus. subtilis, and
Mycobacterium smegmatis following the standard proce-
dure.31 The minimum inhibitory concentrations (MIC)
were determined with use of neomycin, ribostamycin,
neamine, or isoniazid (INH) as the controls. Previously,
we have reported the SAR of pyranmycins against E.
coli.27-29 We observed a similar trend of activity among
pyranmycins against S. aureus, B. subtilis, and M.
smegmatis, which implies that the ribosomal target of
pyranmycins is highly conserved and, likely, to be the
16S rRNA (Table 1 and Figure 4). For example, TC005,
TC006, and TC010 (entries 9, 10, and 14, respectively)
are typically the most active members against all four
strains of bacteria. Compounds from the model studies,
32 and 34, are found to be inactive although they
resemble the scaffold of rings II and III of pyranmycins.
This result indicates the indispensable role of ring I in
the neamine core-containing antibiotics. From our previ-
ous SAR studies, we learned that 6′′-CH3 group is crucial
for the increased antibacterial activity. We speculated
that the 6′′-CH3 group may contribute to an unusual
hydrophobic interaction. Therefore, a 6′′-fluornated ana-
logue, TC033, was produced and evaluated with the
expectation that fluoride may form better hydrophobic
interaction than the hydroxyl group. However, we dis-
covered that TC033 (entry 31) is completely inactive
against E. coli, and less active than TC001, the nonflu-
orinated counterpart, against S. aureus. We also noticed
that, in the disk assay against M. smegmatis, pyranmy-
cins, like streptomycin, are bacteriocidal in which the
inhibition remained clear throughout the testing period
while INH is bacteriostatic where significant bacterial
grow back was observed after 2 days. Therefore, pyran-
a
Reagents: (a) (1) MsCl, pyr, (2) NaN3, DMF; (b) BzOH, PPh3,
DEAD, THF; (c) NaOMe, MeOH; (d) TMSOTf, CH2Cl2, 4 Å MS;
(e) (1) K2CO3, MeOH, (2) H2, Pd/C.
library will undergo glycosylation in favor of the forma-
tion of the â-glycosidic bond due to the presence of an
acetyl group at the O-2 position.
After completion of the synthesis of aminosugars with
designed binding motifs, we carried out a model study of
glycosylation (Scheme 4). One glycosyl acceptor, cis,cis-
3,5-diazidocyclohexanol (30) with an intrinsic naturally
occurring cis-1,3-diamino binding motif that can be found
in the 2-deoxystreptamine of clinically used aminoglyco-
side antibiotics, such as neomycin, was selected for the
model glycosylation and final syntheses. cis,cis-3,5-di-
azidocyclohexanol was synthesized from cis,cis-cyclohex-
anetriol via selective azido incorporation and Mitsunobu
reaction. The glycosylations were carried out with the
catalysis of TMSOTf or BF3-OEt2 (Scheme 4). The acetyl
groups were hydrolyzed with K2CO3. Final hydrogenation
with Pd/C and H2 at atmospheric pressure reduced the
azido groups into amino groups.
Libr a r y Con str u ction of P yr a n m ycin s. Previously,
we have reported the synthesis of 26 pyranmycins
(Scheme 5).27-29 Pyranmycins contain a neamine core
(rings I and II) and a variable ring III pyranose compo-
nent attached at the O-5 of neamine via a â linkage.
Herein, we wish to report the synthesis of five additional
members (Figure 3) based on our structure-activity
relationship (SAR) results, which reveals O-3′′ could be
the optimal position for more structural modifications.
We wish to explore the effect of fluoro substitution at
O-3′′ and O-6′′ positions on the activity of pyranmycins
since fluorination is one of the common strategies of
derivatizing aminoglycosides.32
(31) Methods for Dilution Antimicrobial Susceptibility Testing for
Bacteria that Grow Aerobically; Approved standard M7-A5, and
Performance Standards for Antimicrobial Disk Susceptibility Tests.
Approved standard M2-A7, National Committee for Clinical Laboratory
Standards: Wayne, PA.
(32) For reviewing: (a) Mingeot-Leclercq, M.-P.; Glupczynski, Y.;
Tulkens, P. M. Antimicrob. Agents Chemother. 1997, 43, 727-737. (b)
Kotra, L. P.; Haddad, J .; Mobashery, S. Antimicrob. Agents Chemother.
2000, 44, 3249-3256. (c) Wright, G. D. Curr. Opin. Microbiol. 1999,
2, 499-503.
(29) Li, J .; Wang, J .; Hui, Y.; Chang, C.-W. T. Org. Lett. 2003, 5,
431-434.
(30) Card, P. J . J . Org. Chem. 1983, 48, 393-395.
1516 J . Org. Chem., Vol. 69, No. 5, 2004

Synthesis and Antimicrobial Studies of Pyranmycin
SCHEME 5a
a
Reagents: (a) BF3-OEt2, CH2Cl2, 4 Å MS; (b) (1) K2CO3, MeOH, (2) PMe3, THF, H2O, (3) H2, Pd(OH)2/C.
F IGURE 3. Newly synthesized pyranmycins.
SCHEME 6a
a
Reagents: (a) (1) HOAc, TFA, H2O; (2) Ac2O, cat. H2SO4; (b) (1) H2NNH2-HOAc, (2) CCl3CN, DBU, CH2Cl2; (c) neamine acceptor,
BF3-OEt2, CH2Cl2, 4 Å MS.
mycins may provide new therapeutic options in the
treatment against tuberculosis.
Escherichia coli (TG1) containing pTZ19-3 plasmid coded
with APH-3′ and Escherichia coli (TG1) containing
pSF815 plasmid coded with AAC6′/APH2′′. Pyranmycins
showed no significant antibacterial activity as compared
to amikacin and streptomycin. Therefore, only the dif-
fusion assays were carried out (Table 2). However,
pyranmycins (TC005, TC006, and TC010) manifest
similar activity against E. coli (TG1) (AAC6′/APH2′′) as
neomycin (entries 5, 6, 7, and 8), and are also slightly
more active than gentamicin and kanamycin (entries 2
and 3). The results suggest that the aminoglycoside with
4,5-disubstituted 2-deoxystreptamine (ring II) could be
a better template for the modifications aiming to regain
activity against resistant bacteria equipped with AAC6′
and APH2′′ aminoglycoside-modifying enzymes than the
aminoglycoside with 4,6-disubstituted 2-deoxystreptamine.
In general, pyranmycins are prominent in serving as the
lead structures for additional modification, which may
pave the way to the development of a new library of
antibiotics against drug-resistant bacteria.
We have discovered that the O-3′′ position is the only
place that can tolerate chemical modification without
losing significant antibacterial activity.29 Therefore, we
anticipated that the attachment of complex functional-
ities at the O-3′′ position would result in new antibiotics
that would be poor substrates for the aminoglycoside-
modifying enzymes, and thereby avoid enzyme-catalyzed
inactivation. However, the newly synthesized members
with diverse modification at the O-3′′ position are less
active or inactive compared to the lead compound, 5. It
is clear to us that the O-3′′ position can tolerate only
small structural modifications. For example, even a small
extension of a CH2 unit (TC026 vs TC041) results in the
loss of activity.
The most prevalent mode of aminoglycoside resistance
is enzyme-catalyzed modification. Over 50 different types
of aminoglycoside-modifying enzymes, found in resistant
bacteria, are known to be responsible for the inactivation
of aminoglycosides (Figure 5).32 These aminoglycoside-
modifying enzymes, which are grouped into three classes
including aminoglycoside phosphotransferases (APHs),
aminoglycoside acetyltransferases (AACs), and aminogly-
coside nucleotidyltransferases (ANTs), introduce chemi-
cal groups almost exclusively on the neamine core (rings
I and II) of the aminoglycosides including neomycin and
kanamycin.
An ti-HIV-1 Ta t a n d Rev Activity of P yr a n m ycin s.
Two HIV-1 regulatory proteins, Tat and Rev, mediate
their functions in HIV-1 replication through binding to
the viral TAR and RRE RNA stem-loop structures,
respectively. Tat transactivates the 5′-long terminal
repeat (LTR) promoter of the viral RNA genome, thereby
increasing the level of viral mRNA transcription and
We also examined the antibacterial activity of pyran-
mycins against resistant strains of bacteria, including
J . Org. Chem, Vol. 69, No. 5, 2004 1517

Elchert et al.
SCHEME 7a
a
Reagents: (a) H3B, THF then NaOH, H2O2; (b) DAST, CH2Cl2; (c) (1) K2CO3, MeOH, (2) PMe3, THF, NaOH, (3) H2, Pd(OH)2/C; (d)
K2CO3, MeOH; (e) BF3-Et2O, CH2Cl2.
SCHEME 8a
function via cell-based assay. We observed several can-
didates that showed modest activity against HIV-1 Tat
and/or Rev (Table 3). For example, TC009 showed the
highest activity against Rev among the pyranmycin
members. Interestingly, TC009 is almost inactive against
both gram-positive and gram-negative bacteria. TC016,
TC018, and TC021 manifested the highest activities
against Tat among the pyranmycin members. It should
also be noted that with the exception of TC045, which
appeared to be highly toxic at 100 µM in both assays,
the pyranmycin members had minimal to no cytotoxicity
in these tissue culture systems. Although we did not
observe prominent results since the activities demon-
strated required relatively high test concentrations of 50
to 100 µM (significant inhibitors would be expected to
reduce the Tat or Rev function by >90% at concentrations
of <1.0 µM), such a novel aminoglycoside library does
present a unique model for screening of possible lead
structures for future modifications.
a
Reagents: (a) Ac2O, cat. H2SO4; (b) (1) H2NNH2-HOAc, (2)
CCl3CN, DBU, CH2Cl2, (c) 35, BF3-OEt2, CH2Cl2, 4 Å MS; (d) (1)
K2CO3, MeOH, (2) PMe3, THF, NaOH, (3) H2, Pd(OH)2/C.
protein synthesis.33 Rev protein prevents the splicing of
viral mRNA and promotes the nuclear export of unspliced
and incompletely spliced forms of the mRNA to the
cytoplasm. Both proteins play essential roles in the
proliferation of HIV-1, and the bindings of Tat and Rev
proteins to viral RNA’s are, therefore, targeted for the
development of drugs against HIV-1. Several reports
have explored or suggested the potential of using ami-
noglycosides as RNA binding surrogates for the inhibition
of HIV-1 replication and potential therapeutic applica-
tions.34,35 We therefore evaluated the library of pyran-
mycins as potential inhibitors of HIV-1 Tat and/or Rev
Con clu sion
We have prepared a library of novel aminosugars with
natural and nonnatural binding motifs employing an
efficient divergent synthetic strategy starting from a
common starting material. We have demonstrated the
feasibility of using such an aminosugar library for
glycorandomization or glycodiversification research, lead-
ing to one of the prospective applications, the library
(33) For reviewing: Ptak, R. G. Expert Opin. Invest. Drugs, 2002,
11, 1099-1115.
(34) Baker, T. J .; Luedtke, N. W.; Tor, Y.; Goodman, M. J . Org.
Chem. 2000, 65, 9054-9058.
(35) Luedtke, N. W.; Baker, T. J .; Goodman, M.; Tor, Y. J . Am.
Chem. Soc. 2000, 122, 12035-12036.
1518 J . Org. Chem., Vol. 69, No. 5, 2004

Synthesis and Antimicrobial Studies of Pyranmycin
TABLE 1. Min im u m In h ibitor y Con cen tr a tion (µM) of
4.80 (d, J ) 11.9 Hz, 1H), 4.72 (d, J ) 11.5 Hz, 1H), 4.62 (d,
J ) 11.9 Hz, 1H), 4.564 (d, J ) 3.6 Hz, 1H), 4.562 (d, J ) 11.5
Hz, 1H), 4.49 (d, J ) 11.9 Hz, 1H), 4.00 (dd, J ) 9.0 Hz, J )
3.6 Hz, 1H), 3.99 (s, 1H), 3.92 (dd, J ) 6.3 Hz, J ) 6.6 Hz,
1H), 3.81 (dd, J ) 9.0 Hz, J ) 3.6 Hz, 1H), 3.57 (dd, J ) 9.1
Hz, J ) 6.6 Hz, 1H, H-6), 3.51 (dd, J ) 9.1 Hz, J ) 6.3 Hz,
1H), 3.33 (s, 3H); 13C NMR (68 MHz, CDCl3) δ 138.3 (s), 138.2
(s), 137.8 (s), 128.54 (s), 128.48 (s), 128.2 (s), 127.96 (s), 127.89
(s), 127.8 (s), 98.8 (s), 77.8 (s), 76.2 (s), 73.9 (s), 73.7 (s), 73.3
(s), 69.0 (s), 67.1 (s), 61.5 (s), 55.5 (s); LRFAB m/e 507 [M +
NH4]+; HRFAB calcd for C28H35N4O5 [M + NH4]+ m/e 507.2607,
measured m/e 507.2611.
P yr a n m ycin s a ga in st Va r iou s Str a in s of Ba cter ia
E. coli S. aureus
(ATCC
25922)
(ATCC Bacillus
25923) subtilis smegmatis
M.
entry
compd
1
2
3
4
5
6
7
8
9
Neomycin B
Ribostamycin
Neamine
Isoniazid (INH)
TC001
TC002
TC003
TC004
TC005
2
5
36
0.3
2
7
51
3
6
12
6
42
16
19
25
9
12
4
8
>32
3
3
Meth yl 2,3,6-Tr i-O-ben zyl-r-D-ga la ctop yr a n osid e (5).
To a solution of (COCl)2 (1.1 mL, 13.0 mmol) in 30 mL of
anhydrous CH2Cl2 at -78 °C was added anhydrous DMSO (1.8
mL, 26.0 mmol) and the resulting solution was stirred for 15
min allowing the mixture to warm to -65 °C. To the reaction
flask was added a solution of 3 (3.0 g, 6.5 mmol) in 20 mL of
CH2Cl2. The reaction mixture was stirred for 0.5 h allowing
the solution to warm to -45 °C. To this solution was added
anhydrous DIPEA (9.1 mL, 52.0 mmol), and the reaction was
allowed to warm to -10 to 0 °C in 0.5 h. After completion of
the reaction, the reaction mixture was quenched with 1 N HCl
and diluted with CH2Cl2. The organic layer was washed with
pH 7 buffer (three times) and dried over Na2SO4. After removal
of solvent, the crude product was obtained as a viscous oil.
The crude product was dissolved in methanol (30 mL) and
NaBH4 (0.74 g, 19.5 mmol) was slowly added at 0 °C. The
reaction was allowed to stir overnight. After the completion
of the reaction, the reaction mixture was quenched with 1 N
HCl(aq) then concentrated. The slurry crude product was
dissolved in EtOAc and washed with water, saturated
NaHCO3(aq), and brine, then dried over Na2SO4(s). Removal of
the solvent followed by gradient column chromatography
(hexane:EtOAc ) 90:10 to 40:60) afforded the product as a
yellowish oil (2.4 g, 5.17 mmol, 80%). 1H NMR (270 MHz,
CDCl3) δ 7.2-7.4 (m, 15H), 4.81 (d, J ) 11.9 Hz, 1H), 4.79 (d,
J ) 11.6 Hz, 1H), 4.69 (d, J ) 11.6 Hz, 1H), 4.66 (d, J ) 2.0
Hz, 1H), 4.64 (d, J ) 11.9 Hz, 1H), 4.59 (d, J ) 11.9 Hz, 1H),
4.52 (d, J ) 11.9 Hz, 1H), 4.04 (s, 1H), 3.88 (dd, J ) 5.3 Hz, J
) 6.3 Hz, 1H), 3.85 (d, J ) 2.0 Hz, 1H), 3.85 (s, 1H), 3.72 (dd,
J ) 9.9 Hz, J ) 5.3 Hz, 1H), 3.65 (dd, J ) 9.9 Hz, J ) 6.3 Hz,
1H), 3.37 (s, 3H); 13C NMR (68 MHz, CDCl3) δ 138.4 (s), 138.2
(s), 138.1 (s), 128.6 (s), 128.5 (s), 128.1 (s), 127.92 (s), 127.90
(s), 127.8 (s), 127.7 (s), 98.7 (s), 77.7 (s), 75.8 (s), 73.7 (s), 73.6
(s), 72.8 (s), 69.7 (s), 68.4 (s), 68.2 (s), 55.4 (s); LRFAB m/e 482
[M + NH4]+; HRFAB calcd for C28H36N1O6 [M + NH4]+ m/e
482.2543, measured m/e 482.2523.
2
10 TC006
9
3
2
11 TC007
12 TC008
13 TC009
14 TC010
15 TC012
16 TC015
17 TC016
18 TC017
19 TC018
20 TC019
21 TC020
22 TC021
23 TC022
24 TC023
25 TC024
26 TC025
27 TC026
28 TC028
29 TC031
30 TC032
31 TC033
32 TC040
33 TC041
34 TC044
35 TC045
36 32
26
29
16
inactive
3
12
inactive >50
9
20
inactive
28
45
12
inactive
19
inactive
inactive 26
38
40
inactive inactive
27
13
inactive inactive
39
inactive 25
>50
inactive
inactive
2
3
8
6
24
51
6
11
18
8
3
13
52
inactive
inactive
inactive
4
190
58
8
inactive
inactive
inactive inactive
inactive inactive
37 34
synthesis of pyranmycins. Pyranmycins have comparable
antibacterial activity to neomycin. We believe this new
addition of aminoglycoside can also lead to the develop-
ment of antibiotics against drug-resistant bacteria. We
are currently constructing another library of aminosugars
that will favor the formation of the R-glycosidic bond.
When combined with our current library of aminosugars,
the newly constructed library will offer the opportunity
for complete evaluation of the roles of carbohydrate
components on molecules of biological importance.
Meth yl 2,3-Di-O-ben zyl-r-D-glu cop yr a n osid e (7). A so-
lution of 2 (46 g, 0.10 mol) and TsOH-H2O (ca. 2 g) in 500 mL
of MeOH was stirred overnight. After completion of the
reaction, the reaction was quenched by the addition of Et3N
(20 mL). The solvents were evaporated. The slurry crude
product was dissolved in EtOAc and washed with 1 N HCl(aq)
,
water, saturated NaHCO3(aq), and brine, then dried over Na2-
SO4(s). Removal of the solvent followed by gradient column
chromatography (hexane:EtOAc ) 65:35 to EtOAc:MeOH )
90:10) afforded the product as a yellowish oil, which solidify
Exp er im en ta l Section
Meth yl 4-Azid o-2,3,6-tr i-O-ben zyl-4-d eoxy-r-D-ga la cto-
p yr a n osid e (4). To a solution of 3 (3.0 g, 6.4 mmol) and
pyridine (0.83 mL, 10.3 mmol) in anhydrous CH2Cl2 at 0 °C
was added Tf2O (1.5 mL, 9.0 mmol) slowly. After being stirred
for 30 min, the reaction mixture was diluted with CH2Cl2,
washed with water, saturated NaHCO3(aq), and brine, and dried
over Na2SO4(s). The solution was filtered through Celite and
transferred into a solution of excess NaN3 and DMF. The
reaction mixture was stirred overnight while the solvents were
slowly evaporated with an aspirator. After completion of the
reaction, the reaction mixture was filtered through Celite. The
residue was washed with EtOAc. The combined solutions were
concentrated and purified with gradient column chromatog-
raphy (hexane:EtOAc ) 90:10 to 40:60) providing the product
1
slowly at room temperature (37 g, 0.099 mol, 99%). H NMR
(270 MHz, CDCl3) δ 7.2-7.4 (m, 10H), 5.04 (d, J ) 11.7 Hz,
1H), 4.78 (d, J ) 12.1 Hz, 1H), 4.70 (d, J ) 12.1 Hz, 1H), 4.66
(d, J ) 11.7 Hz, 1H), 4.60 (d, J ) 3.7 Hz, 1H, 1-H), 3.79 (dd,
J ) 9.5 Hz, J ) 8.8 Hz, 1H, 3-H), 3.7-3.8 (m, 2H), 3.62 (ddd,
J ) 4.0 Hz, J ) 4.0 Hz, J ) 9.9 Hz, 1H), 3.50 (dd, J ) 9.5 Hz,
J ) 3.7 Hz, 1H, 2-H), 3.5-3.6 (m, 1H), 3.38 (s, 3H); 13C NMR
(68 MHz, CDCl3) δ 138.6 (s), 137.9 (s), 128.7 (s), 128.5 (s), 128.1
(s), 128.0 (s), 127.9 (s), 98.2 (s), 81.2 (s), 79.8 (s), 75.4 (s), 73.1
(s), 70.6 (s), 70.4 (s), 62.5 (s), 55.3 (s); LRCI m/e 392.4 [M +
NH4]+; HRCI calcd for C21H30NO6 [M + NH4]+ m/e 392.2073,
measured m/e 392.2068.
1
as a light yellowish oil (2.70 g, 5.5 mmol, 86%). H NMR (270
MHz, CDCl3) δ 7.2-7.4 (m, 15H), 4.82 (d, J ) 11.9 Hz, 1H),
Meth yl 2,3-Di-O-ben zyl-6-d eoxy-r-D-glu cop yr a n osid e
(9). To a solution of 7 (12.0 g, 32.0 mmol) in anhydrous
J . Org. Chem, Vol. 69, No. 5, 2004 1519

Elchert et al.
F IGURE 4. Structures of previously reported pyranmycins.
F IGURE 5. Modifications from resistance bacteria against aminoglycoside antibiotics.
TABLE 2. Zon e of In h ibition (m m ) a ga in st
Dr u g-Resista n t Str a in s
product was dissolved in THF (30 mL) and LiAlH4 (3.60 g, 96.0
mmol) was added. The reaction was stirred at room temper-
ature overnight then refluxed for 2 h. After completion of the
reaction, the reaction mixture was quenched by slow addition
to ice then filtered through Celite. The filtered cake was
washed with EtOAc, and the resulting cloudy yellowish
solution was filtered through Celite. The combined organic
solutions were washed with 1 N HCl(aq), water, saturated
NaHCO3(aq), and brine and dried over Na2SO4(s). After removal
of the solvent followed by purification with gradient column
chromatography (hexane:EtOAc ) 90:10 to 40:60), the product
E. coli E. coli (TG1)
E. coli (TG1)
entry
compd
(TG1)
(APH(3′)-I)
(AAC6′/APH2′′)
1
2
3
4
5
6
7
8
9
Amikacin
Gentamicin
Kanamycin
Streptomycin
Neomycin B
TC005
TC006
TC010
TC028
20
20
19
22
15
18
16
14
17
21
18
inactive
18
inactive
inactive
inactive
inactive
inactive
20
12
9
24
16
15
14
14
12
1
was obtained as a white powder (7.89 g, 22.0 mmol, 69%). H
NMR (270 MHz, CDCl3) δ 7.2-7.4 (m, 10H), 5.02 (d, J ) 11.6
Hz, 1H), 4.75 (d, J ) 11.9 Hz, 1H), 4.67 (d, J ) 11.6 Hz, 1H),
4.65 (d, J ) 11.9 Hz, 1H), 4.54 (d, J ) 3.3 Hz, 1H), 3.71 (dd,
J ) 9.6 Hz, J ) 9.0 Hz, 1H), 3.64 (dq, J ) 9.2 Hz, J ) 6.3 Hz,
1H), 3.50 (dd, J ) 9.6 Hz, J ) 3.3 Hz, 1H), 3.36 (s, 3H), 3.14
(dd, J ) 9.2 Hz, J ) 9.0 Hz, 1H), 2.12 (broad, 1H, 4-OH), 1.22
(d, J ) 6.3 Hz, 3H); 13C NMR (68 MHz, CDCl3) δ 138.8 (s),
138.1 (s), 128.6 (s), 128.2 (s), 128.1 (s), 128.04 (s), 128.00 (s),
98.0 (s), 81.3 (s), 80.1 (s), 75.4 (s, 2 carbons), 73.1 (s), 66.9 (s),
pyridine (30 mL) was added TsCl (7.34 g, 38.5 mmol) slowly
at 0 °C. The reaction mixture was stirred overnight allowing
the reaction to warm to room temperature. After the comple-
tion of the reaction, the reaction mixture was diluted with
EtOAc. The combined organic layers were washed with 1 N
HCl(aq) 3 times, saturated NaHCO3(aq), and brine, then dried
over Na2SO4(s). After removal of solvent, the tosylated crude
1520 J . Org. Chem., Vol. 69, No. 5, 2004

Synthesis and Antimicrobial Studies of Pyranmycin
TABLE 3. An ti-Rev a n d An ti-Ta t Activities of P yr a n m ycin s
HIV-1 Rev assay
HIV-1 Tat assay
% inhibition of
Rev functiona
% reduction in
cell viabilityb
% inhibition of
% reduction in
cell viabilityb
entry
compd
TC001
TC002
TC003
TC004
TC005
TC006
TC007
TC009
TC010
TC012
TC015
TC016
TC017
TC018
TC019
test concn
Tat functiona
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
50 µM
100 µM
100 µM
100 µM
100 µM
100 µM
100 nM
1 µM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10.3
36.6
20.4
15.2
46.5
17.1
28.7
14.1
42.4
4.9
25.4
71.1
49.9
70.4
46.3
65.2
7.7
20.8
12.7
20.4
50.9
100.0
98.3
100.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
4.6
11.6
9.9
13.2
11.0
13.6
5.4
0.0
8.9
0.0
0.0
49.6
15.8
21.9
3.0
16.6
24.3
38.4
38.1
14.7
0.0
29.4
30.9
21.9
28.6
100.0
100.0
53.4
12.3
10.9
16.1
6.8
13.2
11.6
18.4
11.6
0.0
TC021
TC022
TC038
TC040
TC041
TC044
TC045
0.0
0.0
6.0
22.5
20.5
14.4
1.8
99.9
60.6
35.5
21.0
11.6
21.4
20.6
100.0
73.8
42.8
Lep tom ycin Bc
Ro24-7429d
a
b
d
Renilla Luciferase analysis. Firefly Luciferase analysis. c Reported inhibitor of the Rev function. Reported inhibitor of the Tat
function.
55.2 (s), 17.7 (s); LRFAB m/e 376 [M + NH4]+; HRFAB calcd
40:60), the product was obtained as a yellowish oil containing
R-anomer as the major product.
for C21H30N1O5 [M + NH4]+ m/e 376.2124, measured m/e
376.2119.
Gen er a l P r oced u r e for th e P r ep a r a tion of Glycosyl
Tr ich lor oa cetim id a te. To a solution of starting material in
DMF was added hydrazine acetate (1.2 equiv). The solution
was stirred at room temperature until the complete consump-
tion of the starting material (4-5 h). The reaction mixture was
filtered through a short column packed with a layer of silica
gel on top of a layer of Celite. The column was eluted
thoroughly with EtOAc. After removal of the solvents, the
crude product can be used directly for the next step, or can be
further purified with column chromatography providing a light
yellowish oil. We do, however, recommend a purification of the
crude product with flash column chromatography. To a solu-
tion of glycosyl hydroxide and trichloroacetonitrile (12 equiv)
in anhydrous CH2Cl2 was added a catalytic amount of DBU
dropwise. The solution was stirred at room temperature until
the complete consumption of the starting material. The reac-
tion mixture was added with charcoal and filtered through a
short column packed with a layer of silica gel on top of a layer
of Celite. The column was eluted thoroughly with ether. After
removal of the solvents, the crude product was purified with
column chromatography providing glycosyl trichloroacetimi-
date as a light yellowish oil. The glycosyl trichloroacetimidate
may undergo slow decomposition at room temperature. There-
fore, these compounds have been characterized only by 1H and
13C NMR.
tr a n s,tr a n s-3,5-Dia zid ocycloh exa n ol (30a ). To a solution
of cis,cis-1,3,5-cyclohexanetriol (3.50 g, 0.0265 mol) in pyridine
(15 mL) at 0 °C was added MsCl (6.38 g, 4.3 mL, 0.0557 mol).
The reaction mixture was stirred overnight allowing the
solution to warm to room temperature. After completion of the
reaction, EtOAc was added, and the organic layer was washed
with 1 N HCl, water, saturated Na2CO3, and brine then dried
over Na2SO4. After removal of solvent, the crude mesylated
compound was added with DMF (25 mL) and NaN3 (7 g, 0.108
mol). The reaction mixture was stirred at 70 °C overnight.
After removal of most of the DMF, the reaction mixture was
diluted with EtOAc and filtered through Celite. After purifica-
tion with gradient column chromatography (hexane:EtOAc )
90:10 to 40:60), the product was obtained as a volatile pale
Met h yl 6-Azid o-2,3-d i-O-b en zyl-6-d eoxy-r-D-glu cop y-
r a n osid e (13). To a solution of 7 (6.01 g, 16.1 mmol) in
anhydrous pyridine (30 mL) was added TsCl (4.06 g, 21.3
mmol) slowly at 0 °C. The reaction mixture was stirred
overnight allowing the reaction to warm to room temperature.
After the completion of the reaction, the reaction mixture was
diluted with EtOAc. The combined organic layers were washed
with 1 N HCl(aq) (3 times), water, saturated NaHCO3(aq), and
brine, then dried over Na2SO4(s). After removal of solvent, the
tosylated crude product was dissolved in DMF (200 mL) and
excess NaN3 (3-5 equiv) was added. The reaction solution was
stirred at 80 °C overnight. After removal of DMF, EtOAc was
added, and the resulting cloudy yellowish solution was filtered
through Celite. Removal of the solvent followed by gradient
column chromatography (hexane:EtOAc ) 90:10 to 50:50)
afforded the product as a pale yellowish oil (6.41 g, 16.1 mmol,
99%). 1H NMR (270 MHz, CDCl3) δ 7.2-7.4 (m, 10H), 5.30 (d,
J ) 11.6 Hz, 1H), 4.76 (d, J ) 11.9 Hz, 1H), 4.66 (d, J ) 11.6
Hz, 1H), 4.65 (d, J ) 11.9 Hz, 1H), 4.63 (d, J ) 3.3 Hz, 1H),
3.74 (dd, J ) 9.2 Hz, J ) 9.0 Hz, 1H), 3.71 (m, 1H), 3.52 (dd,
J ) 9.6 Hz, J ) 3.6 Hz, 1H), 3.4-3.5 (m, 2H), 3.40 (s, 3H),
3.3-3.4 (m, 1H), 2.18 (broad, 1H); 13C NMR (68 MHz, CDCl3)
δ 138.6 (s), 137.9 (s), 128.8 (s), 128.6 (s), 128.2 (s), 128.1 (s),
98.1 (s), 81.1 (s), 79.8 (s), 75.5 (s), 73.2 (s), 70.7 (s), 70.3 (s),
55.5 (s), 51.6 (s); LRFAB m/e 417 [M + NH4]+; HRFAB calcd
for C21H29N4O5 [M + NH4]+ m/e 417.2138, measured m/e
417.2140.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Acet yl
Glycosid es. To a solution of methyl glycoside in Ac2O at 0 °C
was added concentrated H2SO4 (catalytic amount) slowly. More
concentrated H2SO4 can be added if the reaction does not go
to completion in 5-6 h. After completion of the reaction, the
reaction mixture was poured into a solution of saturated
NaHCO3(aq) and EtOAc. After the mixture was stirred over-
night, the organic layer was separated, washed with water,
saturated NaHCO3(aq), and brine, and dried over Na2SO4(s)
.
After removal of the solvent followed by purification with
gradient column chromatography (hexane:EtOAc ) 90:10 to
J . Org. Chem, Vol. 69, No. 5, 2004 1521

Elchert et al.
yellowish oil (3 g, 0.0165 mol, 62%). 1H NMR (270 MHz, CDCl3)
δ 4.30 (t, J ) 3.0 Hz, 1H), 3.74 (tt, J ) 11.6 Hz, J ) 3.9 Hz,
2H), 2.29 (dm, J ) 14.2 Hz, 1Heq), 2.01 (dm, J ) 14.2 Hz),
1.46 (td, J ) 11.6 Hz, J ) 3.0 Hz), 1.32 (q, J ) 11.9 Hz); 13C
NMR (68 MHz, CDCl3) δ 65.6 (s), 54.2 (s), 37.5 (s), 36.9 (s);
LREI (compound degraded quickly in mass spec) m/e 155 ([MH
°C in a water bath for 1 h. The acids were removed by addition
of 100 mL of H2O, followed by coevaporation at 100 °C with a
rotovap twice. After being further pump-dried under vacuum,
the crude product was added to Ac2O (40 mL) and 2 drops of
concentrated H2SO4 at 0 °C. Upon completion of the reaction
(monitored by TLC, hexane:EtOAc ) 1:1), the reaction mixture
was poured into a solution of saturated NaHCO3(aq) and EtOAc.
After the solution was stirred for 1 D, the organic layer was
washed with 1 N HCl(aq), water, saturated NaHCO3(aq), and
brine and dried over Na2SO4(s). After removal of the solvent
followed by purification with gradient column chromatography
(hexane:EtOAc ) 90:10 to 30:70), the product was obtained
as a white powder (3.22 g, 9.75 mmol, 96%). 1H NMR (270
MHz, CDCl3) (mixture of R and â anomers) δ 6.22 (d, J ) 4.0
Hz, 1H), 5.7-5.9 (m, 2H), 5.60 (d, J ) 8.3 Hz, 1H), 5.1-5.2
(m, 4H), 5.07 (dd, J ) 9.6 Hz, J ) 8.3 Hz, 1H), 4.98 (dd, J )
9.9 Hz, J ) 4.0 Hz, 1H), 4.83 (dd, J ) 9.9 Hz, J ) 9.6 Hz, 1H),
4.82 (dd, J ) 9.9 Hz, J ) 9.6 Hz, 1H), 4.0-4.1 (m, 3H), 3.89
(m, 1H), 3.77 (dd, J ) 9.9 Hz, J ) 9.6 Hz, 1H), 3.57 (dd, J )
9.6 Hz, J ) 9.6 Hz, 1H), 3.5-3.6 (m, 2H), 2.13 (s, 3H), 2.09 (s,
3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.1-
1.3 (m, 6H); 13C NMR (68 MHz, CDCl3) (mixture of R and â
anomers) δ 169.7 (s), 169.6 (s), 169.5 (s), 169.2 (s), 169.1 (s),
134.6 (s), 134.3 (s), 117.1 (s), 116.7 (s), 92.1 (s), 89.6 (s), 79.8
(s), 77.1 (s), 74.4 (s), 74.1 (s), 73.5 (s), 73.0 (s), 71.9 (s), 71.8
(s), 71.3 (s), 68.3 (s), 21.0 (s), 20.9 (s), 20.8 (s), 17.44 (s), 17.38
(s); LRFAB m/e 353 [M + Na]+; HRFAB calcd for C16H26O8Na
[M + Na]+ m/e 353.1212, measured m/e 353.1198.
Gen er a l P r oced u r e for Glycosyla tion . A solution of
glycosyl trichloroacetimidate, neamine acceptor, 35 (1.2 equiv)
or cis,cis-3,5-diazidocyclohexanol (1.2 equiv), and activated
powder 4Å molecular sieve was stirred in anhydrous CH2Cl2
(ca. 5 mL) at room temperature for 1 h then cooled to -50 °C.
To this cloudy solution was added BF3-OEt2 or TMSOTf (0.3
equiv). The solution was stirred at low temperature until the
complete consumption of the glycosyl trichloroacetimidate (ca.
40 min). The reaction mixture was quenched by the addition
of NaHCO3 powder. After being stirred for 15 min, the reaction
mixture was filtered through Celite. The residue was washed
thoroughly with CH2Cl2 and ethyl acetate. After removal of
the solvents, the crude product was purified with column
chromatography.
5-O-(2,4-Di-O-a cetyl-3-O-(3-h yd r oxyp r op yl)-6-d eoxy-â-
D-glu cop yr a n osyl)-6,3′4′-tr i-O-ben zyl-1,3,2′6′-tetr a a zid o-
n ea m in e (40). To a solution of 39 (0.10 g, 0.10 mmol) in
anhydrous THF (5 mL) was added borane/THF complex (1.0
M in THF; 0.10 mL, 0.10 mmol) at room temperature. The
reaction bottle was stirred for 3 h and monitored with TLC
(eluted with EtOAc:hexane ) 50:50). After completion of the
reaction, several drops of H2O were added to destroy excessive
borane followed by the addition of 3 N NaOH (5 mL) and H2O2
(30%) solution (5 mL). The solution was stirred for several
minutes until no bubble was produced. The resulting solution
was diluted with EtOAc and the organic layer was washed by
brine solution and dried over Na2SO4. After removal of the
solvents and purification by column chromatography, the
product was afforded as a colorless oil (0.073 g, 0.071 mmol,
69%). 1H NMR (270 MHz, CDCl3) δ 7.2-7.4 (m, 15H), 5.69 (d,
J ) 3.6 Hz, 1H), 3.8-5.0 (m, 7H), 4.70 (d, J ) 9.9 Hz, 1H),
4.59 (d, J ) 11.2 Hz, 1H), 4.2-4.3 (m, 1H), 4.0-4.1 (m, 3H),
3.64 (dd, J ) 5.6 Hz, J ) 5.6 Hz, 4H), 3.2-3.6 (m, 9H), 2.28
(m, 1H), 2.15 (s, 3H), 2.08 (s, 3H), 1.4-1.7 (m, 3H), 1.20 (d, J
) 6.3 Hz, 3H); 13C NMR (68 MHz, CDCl3) δ 169.9 (s), 169.2
(s), 137.9 (s, 2 carbons), 137.2 (s), 128.9 (s), 128.5 (s), 128.4
(s), 128.2 (s), 127.9 (s), 99.0 (s), 97.6 (s), 85.3 (s), 80.5 (s), 79.5
(s), 78.7 (s), 77.3 (s), 75.6 (s, 2C), 75.3 (s), 75.0 (s), 73.9 (s),
72.5 (s), 71.1 (s), 70.5 (s), 68.5 (s), 63.0 (s), 60.7(s), 60.4 (s),
59.5 (s), 51.2 (s), 32.6 (s), 29.8 (s), 21.2 (s), 21.0 (s), 16.9 (s);
MALDI calcd for C46H56N12O13K [M + K]+ m/e 1023.3721,
measured m/e 1023.3777.
- N2]+, 10), 137 ([M - OH - N2]+, 10), 127 ([M - H - N2
-
N2]+, 15), 112 ([M - N3 - N2]+, 80).
cis,cis-3,5-Dia zid ocycloh exyl Ben zoa te (30b). To a solu-
tion of 30a (1 g, 0.0055 mol) in anhydrous THF (8 mL) at -50
°C were added PhCO2H (1.1 g 0.0088 mol), Ph3P (2.30 g, 0.0088
mol), and DEAD (1.40 mL, 1.531 g, 0.0088 mol). The reaction
mixture was allowed to warm to room temperature. After
completion of the reaction, Et2O was added, and the organic
layer was washed with 1 N HCl, water, saturated Na2CO3,
and brine then dried over Na2SO4. After purification with
gradient column chromatography (hexane:EtOAc ) 95:5 to 60:
40), the product was obtained as a pale yellowish solid (0.72
g, 0.00252 mol, 46%). 1H NMR (270 MHz, CDCl3) δ 7.4-8.1
(m, 5H), 5.01 (tt, J ) 11.6 Hz, J ) 4.4 Hz), 3.48 (tt, J ) 12 Hz,
J ) 4.2 Hz), 2.49 (dm, J ) 10.4 Hz), 2.37 (dm, J ) 12 Hz),
1.56 (q, J ) 12.4 Hz), 1.43 (q, J ) 12.4 Hz); 13C NMR (68 MHz,
CDCl3) δ 165.1 (s), 132.8 (s), 129.7 (s), 127.9 (s), 67.8 (s), 54.4
(s), 36.1 (s), 35.9 (s); LREI (compound degraded quickly in mass
spec) m/e 137 ([M - OBz - N2]+, 5), 109 ([M - OBz - N3
-
N2]+, 55).
cis,cis-3,5-Dia zid ocycloh exa n ol (30). To a solution of 30b
(0.1 g, 0.35 mmol) in anhydrous methanol (2 mL) was added
NaOMe/MeOH (0.1 mL, 0.5 M) slowly. After the solution was
stirred for 4 h, Amberlite IR-120 (plus) was added, and the
reaction mixture was filtered through glass wool. After re-
moval of solvent and purification by gradient column chroma-
tography (hexane:EtOAc ) 90:10 to 40:60), the product was
obtained as a volatile pale yellowish oil (0.06 g, 0.33 mmol,
1
94%). H NMR (270 MHz, CDCl3) δ 3.72 (tt, J ) 11.2 Hz, J )
4.0 Hz, 1H), 3.35 (tt, J ) 12.0 Hz, J ) 4.0 Hz, 2H), 2.30 (m),
1.32 (m); 13C NMR (68 MHz, CDCl3) δ 66.2 (s), 55.2 (s), 39.8
(s), 36.4 (s); LREI (compound degraded quickly in mass spec)
m/e 155 ([MH - N2]+, 10), 137 ([M - OH - N2]+, 10), 127 ([M
- H - N2 - N2]+, 75), 112 ([M - N3 - N2]+, 50).
cis,cis-3,5-Dia m in ocycloh exyl â-D-6-Am in o-6-d eoxyglu -
cop yr a n osid e (32). A solution of 31 and K2CO3 (4-5 equiv)
was stirred in MeOH (5 mL) at room temperature until the
complete consumption of starting material (ca. 5 h). The
solvent was removed, and the reaction mixture was diluted
with EtOAc and filtered through a short column packed with
TLC silica gel and Celite. After removal of solvents, the crude
azido compound was added with a catalytic amount of Pd/C
(10%) and 5 mL of degassed HOAc/H2O (1/1). After being
further degassed, the reaction mixture was stirred at room
temperature under atmospheric H2 pressure. After being
stirred for 1 day, the reaction mixture was filtered through
Celite. The residue was washed with water, and the combined
solutions were concentrated affording product as an acetate
salt. The final product with Cl- salt can be prepared with an
ion-exchange column packed with Dowex 1 × 8-200 (Cl- form)
and eluting with water. After the desired fractions were
collected and the solven removed, the final products were
subjected to bioassay directly. 1H NMR (270 MHz, D2O)
(acetate salt) δ 4.63 (d, J ) 8.0 Hz, 1H, H-1′), 4.00 (tt, J )12.0
Hz, J ) 4.4 Hz, 1H), 3.68 (dd, J ) 10.5 Hz, J ) 3.2 Hz, 1H),
3.64 (dd, J ) 10.5 Hz, J ) 3.6 Hz, 1H), 3.50 (t, J ) 9.5 Hz,
1H), 3.37 (t, J ) 9.5 Hz, 1H), 3.25 (dd, J ) 9.5 Hz, J ) 8.0 Hz,
1H), 3.10-3.50 (m, 3H), 2.00-2.60 (m, 3H), 1.90 (s, 9H). 1.50
(q, J ) 12.0 Hz, 2H), 1.40-1.60 (m, 1H); 13C NMR (68 MHz,
D2O) δ 100.4 (s), 74.7 (s), 72.4 (s), 72.2 (s), 71.3 (s), 70.6 (s),
44.8 (s), 44.7 (s), 39.9 (s), 35.3 (s), 34.0 (s), 32.5 (s); LRFAB
m/e 292 ([M3+ - 2H+]+); HRFAB calcd for C12H26N3O5 ([M3+
2H+]+) m/e 292.1872, measured m/e 292.1882.
-
Acetyl 2,4-Di-O-a cetyl-3-O-a llyl-6-d eoxy-D-glu cop yr a -
n osid e (37). A solution of 36 (2.48 g, 10.2 mmol) in a 100 mL
aqueous mixture of 1% TFA and 80% HOAc was stirred at 70
5-O-(2,4-Di-O-a cetyl-3-O-(3-flu or op r op yl)-6-d eoxy-â-D-
glu cop yr a n osyl)-6,3′4′-t r i-O-b en zyl-1,3,2′6′-t et r a a zid o-
1522 J . Org. Chem., Vol. 69, No. 5, 2004

Synthesis and Antimicrobial Studies of Pyranmycin
n ea m in e (43). To a solution of 40 (0.34 g, 0.35 mmol) in
anhydrous CH2Cl2 in a PE vial was added DAST (0.10 g, 0.62
mmol) at -30 °C. The reaction mixture was stirred for 3 h
allowing the temperature to rise to -10 °C. After completion
of the reaction (monitored by TLC, eluted with EtOAc:hexane
) 50:50), the reaction was quenched by addition of 5 mL of
MeOH. After removal of solvents and purification with column
chromatography, the product was afforded as a light yellowish
oil (0.23 g, 0.23 mmol, 68%). 1H NMR (270 M Hz, CDCl3) δ
7.2-7.6 ( m, 15H), 5.70 (d, J ) 4.0 Hz, 1H), 5.03 (d, J ) 7.9
Hz, 1H), 4.97(d, J ) 9.2 Hz, 1H), 4.8-4.9 (m, 5H), 4.70 (d, J )
9.9 Hz, 1H), 4.59 (d, J ) 11.6 Hz, 1H), 4.53 (t, J ) 5.6 Hz,
1H), 4.35 (t, J ) 5.6 Hz, 1H), 4.1-4.2 (m, 1H), 4.07 (dd, J )
9.2 Hz, J ) 8.9 Hz, 1H), 4.02 (dd, J ) 10.2 Hz, J ) 8.9 Hz,
1H), 3.1-3.6 (m, 12H), 2.30 (ddd, J ) 13.2 Hz, J ) 4.5 Hz, J
) 4.5 Hz, 1H), 2.13 (s, 3H), 2.06 (s, 3H), 1.7-1.9 (m, 2H), 1.46
(ddd, J ) 13.2 Hz, J ) 12.5 Hz, J ) 12.5 Hz, 1H), 1.19 (d, J )
6.3 Hz, 3H); 13C NMR (68 MHz, CDCl3) δ 169.6 (s), 169.0 (s),
137.9 (s, 2C), 137.3 (s), 128.7 (s), 128.5 (s), 128.4 (s), 128.2 (s),
127.9 (s), 99.0 (s), 97.6 (s), 85.3 (s), 80.5 (d, J CF ) 163.5 Hz),
80.7 (s), 79.5 (s), 78.7 (s), 77.5 (s), 75.6 (s, 2 carbons), 75.3 (s),
75.0 (s), 74.6 (s), 73.1 (s), 71.1 (s), 70.6 (s), 67.4 (d, J CF ) 4.7
results in a suspension containing approximately 1 × 108 to 2
× 108 CFU/mL for E coli ATCC 25922, which was transferred
into the Mueller-Hinton agar plate by swab. A disk containing
20 µL (1 mg/mL) of the pyranomycin solution was placed onto
the plate. The plate was incubated at 37 °C for 16 h. The
diameter of the zone of inhibition was measured, to the nearest
whole millimeter.
P r oced u r e for MIC Deter m in a tion . E. coli ATCC 25922
was inoculated into Trypticase Soy Broth, and the resulting
solution was incubated at 37 °C until it achieved or exceeded
the turbidity of the 0.5 McFarland standard. The turbidity of
inoculated medium was adjusted to an absorbance of 0.08 to
0.10 at 625 nm. The adjusted inoculated medium was diluted
by 10-fold, then 5 µL of the inoculated medium was mixed with
45 µL of broth in the designated wells of a 96-well plate. A
series of varying concentrations (2-fold dilution) of the tested
compounds (50 µL) were added to the designated wells. The
growth control was prepared by loading 50 µL of 0.9% brine
onto the well inoculated with bacteria. The blank control was
prepared by loading 0.1 mL of 0.9% brine. The plate was
incubated at 37 °C for 16 h. The MIC is determined by the
compound concentration that renders at least 90% inhibition
of the growth of bacteria. The MIC results are repeated in
duplicate.
Hz), 63.0 (s), 60.7 (s), 59.5 (s), 51.2 (s), 32.6 (s), 31.1 (d, J CF
)
19.7 Hz), 21.1 (s, 2 carbons), 16.9 (s); MALDI calcd for C46H55
-
FN12O12 [M + Na]+ m/e 1009.3939,; measured m/e 1009.3882.
Gen er a l P r oced u r e for th e F in a l Syn th esis. A solution
of starting material and K2CO3 (4-5 equiv) was stirred in
MeOH (5 mL) at room temperature until the complete con-
sumption of starting material (ca. 5 h). The solvent was
removed, and the reaction mixture was diluted with EtOAc
and filtered through a short column packed with a layer of
silica gel on top of a layer of Celite. The column was eluted
with EtOAc/MeOH (1/1 solution). Removal of solvents afforded
crude product mixed with some K2CO3 solid. The crude product
was added with THF (6 mL) and filtered through glasswool
into the reaction flask for the next step. To the perbenzylated
azidoaminoglycoside/THF solution in a reaction flask (or vial)
equipped with a reflux condenser were added 0.1 M NaOH(aq)
(0.5 mL) and PMe3 (1 M in THF, 4-5 equiv). The reaction
mixture was stirred at 50 °C for 2 h. The product has a Rf of
0 when eluted with EtOAc/MeOH (9/1) solution and a Rf of
0.9 when eluted with iPrOH/1 M NH4OAc (2/1) solution. After
completion of the reaction, the reaction mixture was cooled to
room temperature and loaded to a short column (5 cm in
height) packed with a layer of silica gel on top of a layer of
Celite. The column was eluted with a series of solutions as
follows: THF, THF/MeOH, MeOH, and MeOH/concentrated
NH4OH (from 0 to 28% of concentrated NH4OH). The fractions
containing the desired product were analyzed by TLC and
collected. After removal of solvents, the crude perbenzylated
aminoglycoside was added with a catalytic amount of Pd-
(OH)2/C (20% Degussa type) and 5 mL of degassed HOAc/H2O
(1/1). After being further degassed, the reaction mixture was
stirred at room temperature under atmospheric H2 pressure.
After being stirred for 1 day, the reaction mixture was filtered
through Celite. The residue was washed with water, and the
combined solutions were concentrated affording pure final
product as an acetate salt. Most of the reported final products
are characterized by 1H and 13C NMR at this stage. The
product has a Rf of 0 when eluted with iPrOH/1 M NH4OAc
(2/1) solution and a Rf of 0.1-0.2 when eluted with concen-
trated NH4OH/MeOH (2/5) solution. The final product with
Cl- salt can be prepared with an ion-exchange column packed
with Dowex 1 × 8-200 (Cl- form) and eluting with water. After
the desired fractions were collected and the solvent removed,
the final products were subjected to bioassay directly.
Cell-Ba sed Assa ys for HIV-1 Ta t a n d Rev. Inhibition of
the HIV-1 Rev function was determined by using the pDM128
Rev reporter plasmid previously described by Hope et al.36 The
pDM128 plasmid was modified by replacing the chlorampheni-
col acetyltransferase coding sequence with that of Renilla
Luciferase. HeLa cells engineered to express HIV-1 Rev and
Firefly Luciferase were subsequently transfected with the
modified pDM128 plasmid to generate a cell line in which
Renilla Luciferase expression is dependent upon Rev function.
In contrast, Firefly Luciferase expression in this cell line is
independent of Rev function and is used to assay for nonspe-
cific or toxic compounds. Using this system, compounds that
inhibit the Rev function are identified by their ability to reduce
the expression of Renilla Luciferase with no effect on the
expression of Firefly Luciferase. A similar cell line expressing
HIV-1 Tat in place of Rev and that was transfected with a
standard Tat/LTR-reporter gene system37 (using Renilla Lu-
ciferase) instead of pDM128 was generated for testing inhibi-
tors of HIV-1 Tat. Assays were performed in 96-well format
by plating cells (2 × 104/well) in the presence of test compound
(triplicate wells) and incubating for 24-48 h. Luciferase
expression levels were subsequently determined with use of
Dual-Luciferase assay reagents (Promega) following the manu-
facturer’s instructions.
Ack n ow led gm en t. We acknowledge the Utah State
University (CURI Grant), DARPA (DAAD 19-03-1-
0050), and National Foundation for Infectious Diseases
(New Investigator Matching Grant) for generous finan-
cial support. We also acknowledge the support from the
Department of Chemistry and Biochemistry, Utah State
University. We also thank Prof. Mobashery, Wayne
State University, for the generous gift of the pTZ19-3
and pSF815 plasmids and Dr. Thomas J . Hope, Uni-
versity of Illinois at Chicago, for the generous gift of the
pDM128 plasmid.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C spectra
of the synthesized compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
P r oced u r e for Disk Assa y of An tim icr obia l Activity.
E. coli ATCC 25922 was inoculated into Trypticase Soy Broth,
and the resulting solution was incubated at 37 °C until it
achieved or exceeded the turbidity of the 0.5 McFarland
standard. The turbidity of inoculated medium was adjusted
to be the same as that of the 0.5 McFarland standard. This
J O035290R
(36) Hope, T. J .; Huang, X. J .; McDonald, D.; Parslow, T. G. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 7787-7791.
(37) J eeninga, R. E.; Hoogenkamp, M.; Armand-Ugon, M.; de Baar,
M.; Verhoef, K.; Berkhout, B. J . Virol. 2000, 74, 3740-3751.
J . Org. Chem, Vol. 69, No. 5, 2004 1523