Design and synthesis of a structurally constrained aminoglycoside

Article abstract of DOI:10.1021/jo0707636

(Chemical Equation Presented) The synthesis of a constrained tricyclic aminoglycoside derivative is described. This constrained compound fixes the spatial orientation of two critical rings for the minimal motif for binding to biological macromolecules such as RNA and proteins. Methanolysis of neomycin B under acidic conditions produced the bicyclic neamine. Transient protection by the Cu²⁺ ion and regioselective introduction of protective groups led to intermediate 7, which was used for a key annulation reaction that introduced the tricyclic nucleus into the structural framework. A final hydrogenolysis step to remove the protective groups produced the desired target molecule. The efficient eight-step synthesis was accomplished in 8% overall yield.

Full text of DOI:10.1021/jo0707636

Design and Synthesis of a Structurally
Constrained Aminoglycoside
Dale Kling, Dusan Hesek, Qicun Shi, and
Shahriar Mobashery*
Department of Chemistry and Biochemistry, UniVersity of
otre Dame, Notre Dame, Indiana 46556
mobashery@nd.edu
ReceiVed April 11, 2007
FIGURE 1. Stereoview of a capped stick representation for 15
superimposed conformers of (A) compound 3 and (B) compound 9.
Atoms are colored according to atom types (C, N, and O shown in
yellow, blue, and red, respectively).
The synthesis of a constrained tricyclic aminoglycoside
derivative is described. This constrained compound fixes the
spatial orientation of two critical rings for the minimal motif
for binding to biological macromolecules such as RNA and
proteins. Methanolysis of neomycin B under acidic condi-
tions produced the bicyclic neamine. Transient protection
by the Cu2+ ion and regioselective introduction of protective
groups led to intermediate 7, which was used for a key
annulation reaction that introduced the tricyclic nucleus into
the structural framework. A final hydrogenolysis step to
remove the protective groups produced the desired target
molecule. The efficient eight-step synthesis was accom-
plished in 8% overall yield.
for resistance to these agents have emerged, the most common
are acquisition of resistance enzymes.9,12,13 These enzymes
modify the structures of the antibiotics, whereby binding to the
ribosomal site either is eliminated or becomes less effective.
The typical aminoglycoside antibiotic may have up to five
rings in its structure. Though each ring has its own preferred
conformation, where these molecules exhibit considerable
conformational flexibility is at the glycosidic bonds. Indeed,
part of the effectiveness of aminoglycosides from a therapeutic
standpoint comes from their ability to adopt multiple conforma-
tions. A manifestation of this structural flexibility is that
aminoglycosides tend to lack high RNA target selectivity,
binding not only to bacterial ribosomal RNA but also to
biologically relevant ribozymes and the HIV-1 RNAs known
as RRE and TAR.14 In addition, bacterial resistance enzymes
that target these compounds take advantage of their conforma-
tional adaptability, binding aminoglycosides in conformationally
distinct manners.15,16 There exist now opportunities for the
generation of conformationally constrained aminoglycosides that
could be used in studies of both RNA targets (ribosomal and
otherwise) and resistance enzymes. These analogues might
discriminate in binding to one or the other sites and thus prove
to be valuable tools in studies of the functions of these
biologically important targets.17
Aminoglycoside antibiotics have been in clinical use since
the 1940s.1-3 These antibiotics have been effective in light of
their predictable pharmacokinetics and reliability in treatment
of difficult bacterial infections. Although examples of resistance
to these antibiotics have emerged over the years,4-9 they
continue to be used clinically to the present day.
These antibiotics bind to the 30S bacterial ribosomal subunit,
interfering with the protein biosynthetic machinery, which results
in a bactericidal outcome.10,11 Whereas different mechanisms
(1) Davies, J. E. J. Antibiot. 2006, 59, 529-532.
(2) Sutcliffe, J. A. Curr. Opin. Microbiol. 2005, 8, 534-542.
(3) Weizman, H.; Tor, Y. Carbohydr.-Based Drug DiscoVery 2003, 2,
661-683.
We describe herein the design and synthesis of a conforma-
tionally restricted aminoglycoside poised for mechanistic studies
(4) Afnsa, J. A.; Martin, C. Recent Res. DeV. Antimicrob. Agents
Chemother. 2000, 4, 1-10.
(5) Hobson, R. P.; Gould, I. M. Antiinfect. Drugs Chemother. 1995, 13,
143-147.
(6) Shannon, K.; Phillips, I. J. Antimicrob. Chemother. 1982, 9, 91-
102.
(12) Vakulenko, S. B.; Mobashery, S. Clin. Microbiol. ReV. 2003, 16,
430-450.
(7) Smith, C. A.; Baker, E. N. Curr. Drug Targets 2002, 2, 143-160.
(8) Trieu-Cuot, P.; Courvalin, P. J. Antimicrob. Chemother. 1986, 18,
93-102.
(9) Wright, G. D.; Berghuis, A. M.; Mobashery, S. AdV. Exp. Med. Biol.
1998, 456, 27-69.
(10) Carter, A.; Clemons, W.; Brodersen, D.; Morgan-Warren, R.;
Wimberly, B.; Ramakrishnan, V. Nature 2000, 407, 340-348.
(11) Cate, J.; Yusupov, M.; Yusupova, G.; Earnest, T.; Noller, H. Science
1999, 285, 2095-2104.
(13) Wright, G. D. Chem. Biol. 2000, 7, R127-R132.
(14) Chow, C.; Bogdan, F. Chem. ReV. 1997, 97, 1489-1513.
(15) Bastida, A.; Hidalgo, A.; Chiara, J. L.; Torrado, M.; Corzana, F.;
Perez-Canadillas, J. M.; Groves, P.; Garcia-Junceda, E.; Gonzalez, C.;
Jimenez-Barbero, J.; Asensio, J. L. J. Am. Chem. Soc. 2006, 128, 100-
116.
(16) Sucheck, S.; Wong, C. Curr. Opin. Chem. Biol. 2000, 4, 678-686).
(17) Blount, K. F.; Zhao, F.; Hermann, T.; Tor, Y. J. Am. Chem. Soc.
2005, 127, 9818-9829.
10.1021/jo0707636 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/19/2007
5450
J. Org. Chem. 2007, 72, 5450-5453

SCHEME 1. Synthesis of a Structurally Constrained Aminoglycoside 3
with biological targets. The structural template is that of neamine
investigated earlier by our group by detailed measurements of
(1). A series of computational analyses revealed that tethering
the paramagnetic contributions of the cupric ion to T1 relaxation
times by H NMR spectroscopy.21 This transient metal-ion
protection strategy efficiently results in just two products: the
desired di-Boc derivative 5 with protection at positions 3 and
6′ as the major component and a tri-Boc neamine species (Boc
at positions 1, 3, and 6′), which was easily separated by column
chromatography. Subjecting 5 to benzyl chloroformate and
aqueous sodium carbonate gave 6 in good yield.
Deprotection of the Boc groups in 6 proceeded smoothly
using 25% triflouroacetic acid in dichloromethane. Concentra-
tion of the reaction in vacuo and subsequent dilution with
anhydrous ether led to precipitation of crude 7, which was used
in the next step without further purification. This material was
dissolved in pyridine in high dilution before allowing it to react
with 1 equiv of succinic anhydride. We presume that intermedi-
ate 8 should form preferentially over the corresponding 3-N
regioisomer. Analysis by LC/MS at this point revealed a single
new species with the correct mass, which was not isolated.
Instead, EDCI was added directly to the reaction mixture, and
stirring was continued for 2 days. This reaction lead to
compound 9 and according to LC/MS analysis conversion was
in excess of 95%. Removal of the two Cbz groups at positions
1 and 2′ over palladium under an atmosphere of hydrogen
produced the desired 3 in 92% yield. The overall yield for the
eight-step procedure was 8% (from neomycin trisulfate, 4). A
combination of homonuclear decoupling experiments, two-
dimensional NMR, and IR spectroscopy were used to establish
the connectivities of the linker element uniting the two rings.
Annulation of species 8 was key in the success of this
synthesis. In designing this synthesis we were keenly aware that
the spatial predisposition of the amines at positions 6′ and 3
would be critical in the success of the reaction. Computation
revealed that this should be likely, and the product of the
annulation, compound 9, turned out to be stable. The results of
of the 6′-amine group to that at position 3 would be favorable
in locking the conformations of the two rings of neamine
roughly orthogonal to each other (Figure 1A), as will be
described below.
Quantum mechanics methods were applied in computing the
suitable number of methylenes within the tether while keeping
the energy to a minimum (2, n ) 0-4 were investigated). These
analyses revealed that with both amide bonds in a trans
configuration the minimal preferred number for methylenes is
two. Molecular dynamics simulations were performed for the
species with two methylene groups (3) to document the stability
of the molecule. A 50 000-step energy minimization followed
by 500 ps nonperiodic molecular dynamics ensued. We selected
15 conformations from the course of the simulation, which are
overlapped in Figure 1A.
The synthesis of 3 (Scheme 1) started with methanolysis of
commercially available neomycin B trisulfate (4) to afford
neamine 1 (as an HCl salt), which was converted to the free
base form in the presence of ammonia in methanol.18-20
Protection of the 3-NH2 and 6′-NH2 groups in 1 by di-tert-
butyldicarbonate was carried out in the presence of Cu2+. This
protection strategy, which relies upon preferential chelation of
Cu2+ ions to the amines at positions 1 and 2′ of neamine, was
(18) Dutcher, J.; Donin, M. J. Am. Chem. Soc. 1952, 74, 3420-3422.
(19) Ford, J.; Bergy, M.; Brooks, A.; Garrett, E.; Alberti, J.; Dyer, J.;
Carter, H. J. Am. Chem. Soc. 1955, 77, 5311-5314.
(20) Grapsas, I.; Cho, Y. J.; Mobashery, S. J. Org. Chem. 1994, 59,
1918-1922.
(21) Grapsas, I.; Massova, I.; Mobashery, S. Tetrahedron 1998, 54,
7705-7720.
J. Org. Chem, Vol. 72, No. 14, 2007 5451

SCHEME 2. Synthesis of Constrained Aminoglycoside 11
(m, 4H, Cbz CH2Ph), 3.40 (s, 1H, H-5′), 3.28 (overlapping multiplet,
3H, H-3, H-3′, H-4 or H-5), 3.07-2.89 (overlapping muliplets, 7H,
H-1, H-2′, H-3 H-4 or H-5, H-4′, H-6, H-6′), 1.98 (ddd, ω ) 21
the dynamics simulations of 9 are depicted in Figure 1B. These
analyses revealed that the two Cbz groups segregate to one side
of the molecule, leaving the 6′ and 3 amines available in close
proximity for the annulation reaction to proceed.
Hz, assigned as a 1:2:1:1:2:1 six line m, J2eq-2ax ) 13 Hz, J2eq-1
)
J2eq-3 ) 4 Hz, 1H, H-2eq), 1.29 (ddd, ω ) 37, assigned as a 1:3:
3:1 four line m, J2ax-2eq ) 13 Hz, J2ax-1 ) J2ax-3 ) 12 Hz, 1H,
H-2ax). 13C NMR (CD3CN, 125 MHz) δ 162.6, 162.3, 158.4, and
157.7 (CdO), 138.1, 138.0, 129.6,129.0, 128.8, (C6H5), 118.4, 97.6
(C-1′), 77.8 (C-4), 75.8 (C-6), 73.6 (C-5), 71.7 (C-3′), 70.0 (C-4′),
67.7 (C-5′), 67.3 (C-2′), 56.4 (C-1), 52.0 (C-3), 51.2 (C-6′), 42.7
(C-2). LC/MS using a Pro C18 YMC reverse-phase column (Scan
ES+) provided a single peak at Rt ) 4.30 min corresponding to
m/z 591 [M + H]+. HRMS (FAB+) for C28H38N4O10 [M + H]+:
calcd 591.2666, found 591.2648. Melting point: 79-81 °C.
Derivative 9. To a flask containing 250 mg (0.3 mmol) of 7
was added 60 mL of pyridine. This mixture was stirred for 15 min
at room temperature before addition of succinic anhydride (31 mg,
0.3 mmol). The reaction was aged for 12 h. Analysis by LC/MS
(Scan ES+) revealed a single peak at Rt ) 4.5 min representing
m/z 691 [M + H]+, which corresponds to intermediate 8. No
purification was performed at this point. To the crude product 8
was added EDCI (150 mg, 75 mmol, 2.5 equiv). After 16 h LCMS
revealed a 50:50 mixture of starting material and the desired product
9. The reaction was stirred for an additional 32 h (48 h total) to
bring the reaction to completion. Pyridine was removed in vacuo
to obtain a brown residue. Addition of 50 mL of water resulted in
precipitation of a tan-colored solid, which was filtered. Product 9
was obtained (32 mg) in 34% yield. 1H (DMSO, 500 MHz) δ 7.87,
7.64, 7.16, and 7.08 (4H, NH′s), 7.37 (m, 10H, C6H5), 5.68 (1H,
H-1′), 5.04 (m, 4H, CH2Ph), 3.77 (m, 1H, H-3), 3.57-3.13
(overlapping multiplets, 12H, H-2′ H-3′ H-4, H-4′, H-5, H-5′, H-6,
H-6′ and 4 OH′s), 2.90 and 2.71 (m, 2H, H-1, H-6′), 2.34 (m, 4H,
bridge CH2's), 1.80 (1H, H-2eq), 1.25 (1H, H-2ax). 13C (DMSO,
125 MHz) δ 172.4, 171.1, 156.8, and 156.2 (C)O), 147.7, 138.0,
137.7, 128.8, 128.0, 127.8, 127.6 (C6H5), 101.4 (C-1′), 85.6 (C-4),
76.6 (C-6), 74.6 and 73.9 (C-3′ and C-5), 72.8 (C-4′), 71.6 (C-5′),
65.6 and 65.5 (CH2Ph), 57.3 (C-2′), 51.6 (C-1), 49.4 (C-3), 42.7
(C-6′), 35.2 and 33.6 (bridge CH2 ‘s), 32.6 (C-2). LC/MS using a
Pro C18 YMC reverse phase column (Scan ES+) provided a single
peak at Rt ) 4.93 min corresponding to m/z 673 [M + H]+. HRMS
(FAB+) for C32H40N4O12 [M + H]+: calcd 673.2721, found
673.2722. Melting point: > 310 °C.
We hasten to add that two other groups have also prepared
a certain constrained aminoglycoside 11 previously.15,17,22 This
synthesis (Scheme 2) proceeded through protection of the four
amino groups of the neomycin B (4) with benzyl chloroformate
followed by protection of the primary alcohol function of the
five-membered ring with TIBS chloride to give derivative 10.
Deprotection of the amines by hydrogenolysis was followed by
heating the aqueous solution for 7 days to produce 11.
Experimental Section
Free-base neamine (1)18,19 and the di-Boc derivative 521 were
prepared by literature methods.
Compound 6. 3,6′-Di-N-(tert-butoxycarbonyl)neamine (5) (3.5
g, 6.7 mmol) was added to 300 mL of p-dioxane, and the mixture
was stirred at room temperature for 15 min to dissolve most of the
solid. Benzyl chloroformate (2.40 g, 14.1 mmol) and Na2CO3 (7.6
g, 20 mmol) were then added along with 100 mL of deionized
water. The reaction proceeded for 3 h, after which time TLC
(CHCl3/MeOH/concentrated ammonia 4:1:0.1) revealed a single
spot with Rf ) 0.35. The solvent was removed in vacuo, and the
remaining solid was washed with 150 mL portions of water (4×).
Product 6 was obtained (3.85 g) in 72% yield. 1H NMR (CD3OD,
500 MHz) δ 7.30 (overlapping multiplet, 10H, Cbz), 5.30 (s, 1H,
H-1′), 5.10 (m, 4H, CH2Ph), 3.76 (unresolved multiplet, 1H, H-5′),
3.40-3.64 (overlapping multiplets, 8H, H-1, H-2′, H-3, H-3′, H-4,
H-5, H-6′), 3.24 (t, J ) 8.5 Hz, 1H, H-4′), 3.20 (t, J ) 10 Hz, 1H,
H-6), 2.02 (m, 1H, H-2eq), 1.34 (s, 18H, OC(CH3)3), 1.25 (m, 1H.
H-2ax). 13C NMR (CD3OD, 125 MHz) δ 157.9, 157.7, 157.2, 156.5
(CdO), 136.9, 128.1, 127.9, 127.6, 127.5 (C6H5), 98.8 (C-1′), 80.6,
79.3, 77.4, 75.2 (C-3′, C-4, C-4, C-6), 71.2 (C-5′) 70.9 (C-4′) 66.3
and 66.0 (CH2C6H5), 56.0 (C-2′), 51.4 (C-1), 49.6 (C-3), 40.5 (C-
6′), 35.5 (C-2), 27.5 and 27.4 (OC(CH3)3). LC/MS using a Pro C18
YMC reverse-phase column (Scan ES+) provided a single peak at
Rt ) 7.68 min, corresponding to m/z 791 [M + H]+. HRMS (FAB+)
for C38H54N4O14Na [M + Na+]+: calcd 813.3534, found 813.3535.
Melting point: 245 °C.
Compound 3. To a flask containing of 9 (82 mg 0.12 mmol)
was added 30 mL of a DMF/MeOH/AcOH solution (5:4:1). The
flask was placed under an atmosphere of nitrogen before addition
of 50 mg of palladium (10%) on activated carbon. Evacuation was
then carried out with hydrogen atmosphere replacements (3×). The
mixture was stirred overnight at room temperature under an
atmosphere of hydrogen. The mixture was filtered through celite,
and the residue was washed with MeOH (10 mL, 3×) and water
(10 mL). Upon evaporation of the solvent, compound 3 was isolated
Compound 7. To 1.47 g (1.9 mmol) of 6 was added 100 mL of
25% triflouroacetic acid in CH2Cl2. This solution was stirred for 1
h at room temperature under an atmosphere of nitrogen, after which
time the solvent was evaporated. p-Dioxane (200 mL) was then
added, stirred for 5 min, and decanted to help remove residual TFA.
To the remaining golden brown residue was added 150 mL of
diethyl ether, which resulted in precipitation of a white solid. This
material corresponded to the desired 7, which was filtered and dried
1
under vacuum. H NMR (D2O, 500 MHz) δ 8.25, 8.10, and 7.56
(4H, NH′s), 6.90 (10H, Cbz), 5.18 (d, J ) 4 Hz, 1-H, H-1′), 4.61
1
as a beige solid (44 mg, a 92%). H (D2O, 500 MHz) δ 5.1 (d, J
) 3.5 Hz, 1H, H-1′), 3.75 (m, 1H, H-3), 3.61 (overlapping
multiplets, 3H, H-4, H-5′, H-6′), 3.46 (m, 2H, H-6, H-3′ or H-5),
3.38 (t, J ) 10.5 Hz, 1-H, H-3′ or H-5), 3.19 (overlapping
multiplets, 2H, H-2′, H-4′), 3.09 (overlapping multiplets, 2H, H-1,
(22) Asensio, J. L.; Hidalgo, A.; Bastida, A.; Torrado, M.; Corzana, F.;
Chiara, J. L.; Garcia-Junceda, E.; Canada, J.; Jimenez-Barbero, J. J. Am.
Chem. Soc. 2005, 127, 8278-8279.
5452 J. Org. Chem., Vol. 72, No. 14, 2007

H-6′), 2.43 (m, 4H, bridge CH2′s), 2.1 (ddd, ω ) 21 Hz, assigned
as a 1:2:1:1:2:1 6 line m, J2eq-2ax ) 13 Hz, J2eq-1 ) J2eq-3 ) 4 Hz,
1H, H-2eq), 1.30 (ddd, ω ) 38 Hz, assigned as a 1:3:3:1 4 line m,
J2ax-2eq ) 13 Hz, J2ax-1 ) J2ax-3 ) 12.5 Hz, 1H, H-2ax). 13C (D2O,
125 MHz) δ 174.8, 174.2 (CdO), 98.8 (C-1′), 84.4 (C-4), 75.4
(C-6), 72.8 and 72.4 (C-3′ and C-5), 71.5 (C-4′), 69.4 (C-5′), 55.0
(C-2′), 49.8 (C-1), 49.1 (C-3), 39.9 (C-6′), 33.4 and 31.8 (bridge
CH2 ‘s), 30.3 (C-2). FT-IR (KBr pellet), 3395, 1654, 1560, 1412,
1050 cm-1. LC/MS using a Pro C18 YMC reverse phase column
(Scan ES+) provided a single peak at Rt ) 0.85 min corresponding
to m/z 405 [M + H]+. HRMS (FAB+) for C16H28N4O8 [M + H]+:
calcd 405.1985, found 405.2003. Melting point: 170-172 °C.
Computational Methods. The structures of compounds 3 and
9 were prepared by Sybyl 7.0 (Tripos) (SYBYL 7.0, Tripos Inc.,
1699 South Hanley Rd., St. Louis, MO, 63144), starting with the
crystal coordinates for neamine.23 The geometry of the structure
was energetically optimized at the Hartree-Fock (HF) level with
the 6-31G(d) basis set.24 The structures were heated from 0 to 300
K in the gas phase using the Amber Molecular Dynamics Package
programs (version 8).25 The generated electrostatic potentials from
the HF optimization were fit using the two-step RESP procedure.26
The Antechamber program in the Amber package was used for
assigning atom charge for the non-amino-acid molecules and
documented in the format readable to Sybyl. Subsequently, Amber
xleap was applied to generate a geometry documented in the PDB
format, and all atom information was packed into a topological
document. The structures were energy-minimized in 50 000 steps
with the 30 steps steepest descent method followed by the conjugate
gradient method for the remaining steps. The resultant structure
was then used for molecular dynamic simulation. A total of 25
iterative simulation runs were carried out, each for 20 ps. The
fluctuation of temperature about the preset value of 300 K was
less than 0.6 K. The coordinate but not velocity parameters of the
structures from each simulation were used as starting points in the
next simulation. For each simulation run 10 000 snapshots were
taken. In more than 200 000 conformations from the molecular
dynamics sampling, 15 representative examples were selected for
superimposition shown in Figure 1.
(23) Murray, J. B.; Meroueh, S. O.; Russell, R. J. M.; Lentzen, G.;
Haddad, J.; Mobashery, S. Chem. Biol. 2006, 13, 129-138.
(24) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K.
N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li,
X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels,
A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.;
Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz,
P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson,
B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03;
Gaussian Inc.: Wallingford, CT, 2004.
Supporting Information Available: NMR spectra for the
synthetic molecules 3, 5, 6, 7, 8, and 9. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO0707636
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Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman, D. A.;
Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.; Hornak, V.;
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