6-Hydroxy to 6?-amino tethered ring-to-ring macrocyclic aminoglycosides as probes for APH(3′)-IIIa kinase

Article abstract of DOI:10.1016/j.bmcl.2007.03.014

Based on molecular modeling and available X-ray structure data on aminoglycosides complexed with a bacterial ribosomal surrogate or with a kinase, two analogues of paromomycin were prepared by tethering the 6-OH and the 6?-NH₂ group with a five

Full text of DOI:10.1016/j.bmcl.2007.03.014

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3221–3225
6-Hydroxy to 6000-amino tethered ring-to-ring macrocyclic
aminoglycosides as probes for APH(30)-IIIa kinase
Stephen Hanessian,* Janek Szychowski, Natalhie B. Campos-Reales Pineda,
Alexandra Furtos and Jeffrey W. Keillor*
Department of Chemistry, Universite´ de Montre´al, C.P. 6128, Succ. Centre-Ville, Montre´al, P.Q., Canada H3C 3J7
Received 7 February 2007; revised 5 March 2007; accepted 5 March 2007
Available online 12 March 2007
Abstract—Based on molecular modeling and available X-ray structure data on aminoglycosides complexed with a bacterial ribo-
somal surrogate or with a kinase, two analogues of paromomycin were prepared by tethering the 6-OH and the 6000-NH2 group with
a five-carbon bridge. Only one of two possible hydroxyl groups was phosphorylated by the kinase. The application of ring closure
metathesis is presented for the first time to construct bridged macrocyclic analogues in the aminoglycoside series.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Antibiotics have been used as effective drugs to combat
infection for nearly 75 years.1 Among these, the amino-
glycoside family holds a historically and clinically prom-
inent position as potent therapeutic drugs against Gram
positive and Gram negative infections.2 Aminoglyco-
sides are administered parenterally under a hospital
regimen. However, their promiscuous use as broad spec-
trum antibiotics has caused the emergence of bacterial
resistance. Resistance can develop by enzymatic deacti-
vation of the drugs, by target modification, or by de-
creased intracellular concentration of the active entity
by efflux.3 Oto- and nephrotoxicity are also associated
with dose-related practices of aminoglycoside therapy
in the clinic.4
AAC6' (when R = NH2)
AAC1
H2N
HO
NH2
O
R
O
OH
O
HO
O
AAC2'''
O
H2N
OH
ANT4'
AAC2'
APH3'
OH
H2N
HO
O
OH
NH2
1, R = OH, Paromomycin
2, R = NH2, Neomycin
Figure 1. Susceptibility to enzymatic deactivation in the neomycin–
paromomycin group of aminoglycosides.
The cellular target for bactericidal aminoglycosides is
the A-site of the 16S ribosomal RNA.5 In recent years,
elegant X-ray crystallographic6 and NMR7 studies of
A-site—aminoglycoside complexes have shown highly
conserved interactions, especially in the spatially com-
mon rings I and II of the pseudosaccharide portion of
4,5- and 4,6-disubstituted aminoglycosides (Fig. 1). In
general, aminoglycosides such as paromomycin6b (1)
and neomycin (2) bind to the A-site in their lowest
energy conformation. Other conformations may also pre-
vail for different inactivating enzymes.8 Coincidentally,
aminoglycoside-modifying enzymes such as the kinase
APH(30)-IIIa, that catalyzes the phosphorylation of
the 30OH and 500OH groups of ring I in paromomycin
and related congeners, also recognize these lowest
energy conformers of their substrates.3,9 In fact, X-ray
crystallographic studies have shown a virtual superposi-
tion of the conformations observed in the ternary com-
plex APH(30)-IIIa with neomycin B, and that observed
in the crystal structure of the 30S ribosomal subunit with
paromomycin (Fig. 2).6b,9 Apart from specific van
der Waals interactions, several functional groups utilized
in binding to the bacterial ribosome are also identical
to those required to act as a substrate of APH(30)-IIIa.
Keywords: Aminoglycosides; Phosphotransferase; APH(30)-IIIa kinase;
Paromomycin.
*
Corresponding authors. E-mail addresses: stephen.hanessian@
umontreal.ca; jw.keillor@umontreal.ca
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.014

3222
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3221–3225
the L-shaped topology of paromomycin and its bioac-
tive conformation within the 16S ribosomal A-site,6b,7
we considered the spatial proximity of 6-OH and
6000-NH2 to connect them within energetically favorable
macrocyclic motifs (Fig. 3). Access to functionally dis-
tinct alkene appendages would provide the opportunity
to explore ring closing metathesis reactions16 in the ami-
noglycoside series for the first time. We were particularly
interested to see if the macrocyclic variants of paromo-
mycin would protect the ring I and/or ring III
landscapes normally exposed to the kinase for phos-
phorylation. Recently, Tor,17 Hermann,18 Asencio,19
and their respective coworkers have independently pre-
pared conformationally constrained derivatives of neo-
mycin B, and consisting of bridging the C20 amino
group with extended 500-methano and ethano tethers,
respectively. The specificity of these analogues toward
different RNA targets was studied by NMR spectros-
copy and molecular dynamics simulations.
Figure 2. Superposition of X-ray structures of neomycin in the
APH(30)-IIIa ternary complex (magenta) and paromomycin in the
A-site (blue). APH(30)-IIIa can phosphorylate the 30-OH of ring I or
the 500-OH of ring III.
The readily available 40,60-O-benzylidene penta-N-Cbz
paromomycin 320 was selectively O-benzoylated, then
O-allylated at 6-OH which remained free to give 4
(Scheme 1). Treatment with NaOH in aqueous dioxane
cleaved both the ester groups and the 6000N-Cbz group,
presumably via cyclic carbamate 5, to give the corre-
sponding 6000 amine 6. Amide formation with 3-butenoic
acid followed by O-acetylation gave the diene 7. Ring
closing metathesis in the presence of Grubbs’ second
generation catalyst21 followed by deacetylation gave
macrocyclic amide 8 as a mixture of cis/trans isomers.
Cleavage of the benzylidene group under acidic condi-
tions and hydrogenation gave macrocyclic amide 9 in
excellent yield (Scheme 1).
In spite of the promiscuity of this enzyme in detoxifying
several aminoglycosides containing a 30-OH group,
there is a fundamental topological feature that distin-
guishes it from its ribosomal RNA-based counterpart
`
vis-a-vis the substrate. Thus, the face of the aminoglyco-
side that is exposed to APH(30)-IIIa is opposite to that
which interacts with the prokaryotic ribosomal site.9,10
This is also observed in all available X-ray structures
for aminoglycoside-deactivating enzyme complexes
such as AAC(20)-Ic,11 AAC(60)-Iy,12 ANT(40),13 and
APH(30)-IIa.14
This fundamental difference based on van der Waals
interactive forces constitutes an important observation
that can be exploited in the design and synthesis of
chemically modified aminoglycosides.15 Considering
Treatment of penta-N-Cbz paromomycin 1020 with
aqueous base effected a selective cleavage of the 6000-
N-Cbz group to give 11 (Scheme 2). N-3-Butenylation
a
Tether
H2N
H2N
b
NH2
NH2
OH
OH
OH
OH
O
O
O
O
O
O
HO
HO
O
O
O
O
O
O
H2N
OH
H2N
OH
HO
O
HO
O
H2N
HO
H2N
HO
O
NH
NH
9
15
OH
OH
not phosphorylated
by APH(3')-IIIa
not phosphorylated
by APH(3')-IIIa
Figure 3. (a) Proposed ligation of selected functional groups within paromomycin to study the influence of the topology on the
APH(30)-IIIa-mediated phosphorylation; (b) structures of macrocycles 9 and 15.

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3221–3225
3223
CbzHN
HO
CbzHN
O
NHCbz
O
NHCbz
O
O
O
O
O
O
Ph
O
O
Ph
HO
BzO
O
O
O
a, b
O
O
OH
CbzHN
OH
OBz
CbzHN
OBz
CbzHN
HO
CbzHN
O
O
BzO
OH
NHCbz
OBz
NHCbz
3
4
CbzHN
O
NHCbz
O
O
c, d, e
O
O
Ph
AcO
O
f, g
O
OR
O
CbzHN
HO
OAc
CbzHN
OAc
CbzHN
AcO
O
O
6, R = H
7, R =
O
NH
AcO
NHR
O
5
O
H2N
CbzHN
NHCbz
O
NH2
OH
OH
OH
O
O
O
O
O
Ph
O
O
HO
HO
O
O
O
O
O
h, i
O
H2N
CbzHN
OH
HO
HO
H2N
HO
CbzHN
O
O
O
O
NH
NH
HO
OH
OH
9
8
Scheme 1. Reagents and conditions: (a) BzCl, pyridine, 75%; (b) CH2@CHCH2I, KHMDS, THF, 43%; (c) 0.5 M NaOH, dioxane/H2O (2:1);
(d) EDCl, 3-butenoic acid, Et3N, CH2Cl2/DMF (9:1); (e) Ac2O, pyridine, 23% for three steps; (f) Grubbs’ 2nd generation catalyst, CH2Cl2, reflux;
(g) NaOMe/MeOH, 48% for two steps; (h) HOAc/H2O (4:1), 60 ꢁC, 4 h; (i) Pd(OH)2/C, H2, HOAc/H2O (4:1), 85% for two steps.
followed by restoration of the N-Cbz group gave 12
which was selectively acetylated, then O-allylated at
the remaining 6-OH group to give diene 13. Ring closing
metathesis as described above gave the macrocyclic ole-
fin 14 as a mixture of cis/trans isomers. Deacetylation
followed by hydrogenolysis gave the macrocyclic amine
15.22
correspond to two different modes of monophosphory-
lation of paromomycin. This behavior has been
observed previously24 and has been attributed to the
phosphorylation of either the 30-OH or the 500-OH
groups from two different Michaelis complexes formed
from the same substrate that is bound in two different
ways, with different affinities. Separate fitting of the
low- and high-concentration data provided the kinetic
parameters for the high-affinity phosphorylation of
The main objective of this study was to explore the abil-
ity of the kinase APH(30)-IIIa to phosphorylate 30-OH
and 500-OH in the macrocyclic analogues 9 and 15 com-
pared to the parent paromomycin.23 Both compounds
were found to be substrates for phosphorylation,
albeit poorer than paromomycin, as shown in Figure
4a for compound 9 (kcat = 0.073 sꢀ1, Km = 21.8 lM,
kcat/Km = 3.35 · 103 Mꢀ1 sꢀ1).22 By way of comparison,
we also measured the kinetic parameters for phosphory-
lation of paromomycin at 4 ꢁC. As can be seen from
Figure 4b, the initial rates of paromomycin phosphory-
lation showed biphasic hyperbolic dependence on
substrate concentration. The consumption of one
equivalent of ATP over the duration of this phosphory-
lation reaction demonstrates that these results
paromomycin (kcat(1) = 0.190 sꢀ1
kcat(1)/Km(1) = 1.12 · 104 Mꢀ1 sꢀ1) and the low-affinity
,
Km(1) = 16.9 lM,
phosphorylation, respectively (kcat(2) = 0.304 sꢀ1, Km(2)
=
51.1 lM, kcat(2)/Km(2) = 5.95 · 103 Mꢀ1 sꢀ1). Our high-
affinity kinetic parameters compare favorably to those
measured at 37 ꢁC and published previously (i.e., k37
¼
3:62 sꢀ1, Km37 ¼ 19:5 lM, kc3a7t=K3m7 ¼ 1:86 ꢁ 105 Mꢀ1 sꢀc1a)t.23
Unlike paromomycin, analogues 9 and 15 were phos-
phorylated only at 500-OH which was determined by
LC–MS with in-source fragmentation.22 Mono- and
bis-phosphorylated paromomycins were used as test
samples to validate our experimental approach to these
product studies.24,25 The single phosphorylation at

3224
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3221–3225
CbzHN
HO
CbzHN
HO
NHCbz
O
OH
OH
NHCbz
O
OH
OH
O
HO
O
O
O
O
HO
O
a
O
b, c
O
OH
CbzHN
OH
CbzHN
HO
OH
CbzHN
OH
O
CbzHN
O
HO
OH
NHCbz
OH
NH2
10
11
CbzHN
HO
CbzHN
O
NHCbz
NHCbz
OH
OH
OAc
OAc
O
O
O
HO
O
O
O
AcO
O
O
d, e
O
O
f
OH
CbzHN
OH
OAc
CbzHN
OAc
CbzHN
HO
CbzHN
AcO
O
O
HO
CbzN
12
AcO
CbzN
13
CbzHN
H2N
NHCbz
O
OAc
OAc
NH2
O
O
OH
OH
OH
O
O
AcO
O
HO
O
O
O
O
g, h
O
O
CbzHN
OAc
AcO
H2N
CbzHN
AcO
HO
H2N
HO
O
O
NCbz
NH
OAc
OH
14
15
Scheme 2. Reagents and condition: (a) 0.5 M NaOH, dioxane/H2O (2:1); (b) CH2@CHCH2CH2Br, NaHCO3, DMF; (c) Cbz-Cl, NaHCO3, MeOH/
H2O (20:1), 40% for three steps; (d) Ac2O, pyridine, 65%; (e) CH2@CHCH2I, KHMDS, THF, 35%; (f) Grubbs’ 2nd generation catalyst, CH2Cl2,
reflux, 45%; (g) NaOMe/MeOH, 85%; (h) Pd(OH)2/C, H2, HOAc/H2O (4:1), 80%.
Figure 4. APH(30)-IIIa-mediated phosphorylation of paromomycin and 9. (a) Phosphorylation of 9 at 500-OH; (b) phosphorylation of paromomycin
at 30-OH or 500-OH.
500-OH may be due to a shielding of the 30-OH of ring I
in 9 and 15 by the bridged tether. Ideally, one would
wish to have an analogue that is modified in such a
way that it is no longer recognized as a substrate by
the kinase, while maintaining its ability to bind to the
ribosomal A-site. Such an analogue would have the
attributes for a potentially important antibacterial.
Alternatively, one could design an analogue that is
strongly bound by the kinase but not phosphorylated.
Such an analogue would act as an inhibitor of the kinase

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3221–3225
3225
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Unfortunately, neither 9 nor 15 was found to inhibit
growth of Staphylococcus aureus or Escherichia coli at
concentrations lower than 40 lg/mL (MIC > 40 lg/mL
in all cases). The incorporation of the five-carbon tether
between the 6-OH and 6000-NH2 groups may alter the
fidelity of binding of the crucial rings I and II in the A-
site, possibly due to the exclusion of bound water mole-
cules by the hydrophobic aliphatic motif. The reasons for
the monophosphorylation at the 500-OH, but not at the
30-OH, are less evident, and may also involve subtle con-
formational changes. Nevertheless, this selectivity
augurs well for a continued exploration of specific mod-
ifications toward effective aminoglycoside congeners.
The macrocyclic analogues 9 and 15 represent the first at-
tempts to chemically and topologically discriminate be-
tween A-site accommodation and kinase recognition9.
Further studies in this area are in progress in our labora-
tories and will be reported in due course.
16. (a) For an authoritative collection of relevant work in this
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Wiley-VCH: Weinhem, Germany, 2003; Vols. 1, 2 & 3, see
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Org. Chem. 2006, 10, 185; (c) Martin, S. F. Pure Appl.
Chem. 2005, 77, 1207; (d) Grubbs, R. H. Tetrahedron
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Organomet. Chem. 2004, 7, 223; (f) Connon, S. J.;
Blechert, S. Top. Organomet. Chem. 2004, 7, 93; (g)
Connon, S. J.; Blechert, S. Angew. Chem., Int. Ed. 2003,
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Acknowledgments
We thank NSERC and CIHR for financial assistance and
Boehringer-Ingelheim Canada for a fellowship to J.S.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.03.014.
2001, 34, 18; (i) Furstner, A. Angew. Chem., Int. Ed. 2000,
39, 3012.
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F.; Zhao, Q.; Blount, K. F.; Han, Q.; Tor, Y.; Hermann,
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