Design, synthesis, and antibacterial activities of conformationally constrained kanamycin A derivatives

Article abstract of DOI:10.1021/jo302247x

A series of conformationally constrained kanamycin A derivatives with a 2′-hydroxyl group in ring I and a 5-hydroxyl group in ring II tethered by carbon chains were designed and synthesized. Pivotal 5,2′-hydroxyl groups were exposed, and the kanamycin A intermediate was synthesized from 5, 2′, 4″, 6″-di-O-benzylidene-protected tetraazidokanamycin A. Cyclic kanamycin A derivatives with intramolecular 8-, 9-, 10-, and 11-membered ethers were then prepared by cesium carbonate mediated Williamson ether synthesis or a ring-closing metathesis reaction. The kanamycin A derivatives were assayed against both susceptible and resistant bacterial strains. Although no derivative showed better antibacterial activities than kanamycin A, the antibacterial activities of these cyclic kanamycin A derivatives indeed varied with the length of the bridge. Moreover, different variations of activities were observed between the susceptible and resistant bacterial strains. More tightly constrained derivative 2 with a one-carbon bridge showed better activity than the others against susceptible strains, but it was much less effective for resistant bacterial strains than derivative 3 with a two-carbon bridge and derivative 6 with an unsaturated four-carbon bridge.

Full text of DOI:10.1021/jo302247x

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Article
Design, Synthesis and Antibacterial Activities of
Conformationally Constrained Kanamycin A Derivatives
Wenxuan Zhang, Ying Chen, Qingzhao Liang, Hui Li, Hongwei
Jin, Liangren Zhang, Xiangbao Meng, and Zhongjun Li
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo302247x • Publication Date (Web): 10 Dec 2012
Downloaded from http://pubs.acs.org on December 11, 2012
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Design, Synthesis and Antibacterial Activities of Conformationally
Constrained Kanamycin A Derivatives
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Wenxuan Zhang, † Ying Chen, † Qingzhao Liang, † Hui Li, † Hongwei Jin,‡ Liangren Zhang, ‡
Xiangbao Meng,*,† and Zhongjun Li*,†
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†The State Key Laboratory of Natural and Biomimetic drugs; Department of Chemical Biology,
School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
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‡The State Key Laboratory of Natural and Biomimetic drugs; Department of Medicinal Chemistry,
School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
*Corresponding author. Tel.: 86 10 82801714; eꢀmail: xbmeng@bjmu.edu.cn; zjli@bjmu.edu.cn
Abstract: A series of conformationally constrained kanamycin A derivatives with 2′ꢀhydroxyl group in ring I
and 5ꢀhydroxyl group in ring II tethered by carbon chains were designed and synthesized. Pivotal 5, 2′ꢀhydroxyl
groups exposed kanamycin A intermediate was synthesized from 5, 2′, 4′′, 6′′ꢀdiꢀOꢀbenzylideneꢀprotected
tetraazidoꢀkanamycin A. Cyclic kanamycin A derivatives with intramolecular eight, nine, ten and
elevenꢀmembered ether were then prepared by cesium carbonate mediated Williamson ether synthesis or
ringꢀclosing metathesis reaction. The kanamycin A derivatives were assayed against both susceptible and
resistant bacterial strains. Although no derivative showed better antiꢀbacterial activities than kanamycin A, the
antiꢀbacterial activities of these cyclic kanamycin A derivatives indeed varied with the length of the bridge.
Moreover, different variations of activities were observed between the susceptible and resistant bacterial strains.
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More tightly constrained derivative 2 with oneꢀcarbon bridge showed better activity than the others against
susceptible strains, but it was much less effective for resistant bacterial strains than derivative 3 with a
twoꢀcarbon bridge and derivative 6 with an unsaturated fourꢀcarbon bridge.
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Introduction
Aminoglycosides are frequently prescribed broad spectrum antibiotics, which have been used clinically for
70 years. However, the emergence of resistant bacteria is a threat for their use. One of the major causes of
bacterial resistance is the enzymatic modification of the antibiotics.1 The aminoglycosideꢀmodifying enzymes
can be broadly classified as Nꢀacetyltransferases (AACs), Oꢀadenyl transferases (ANTs), and
Oꢀphosphotransferases (APHs). Various strategies have been investigated to overcome the resistance.2 Over the
past decade many structural analogues of natural aminoglycosides were designed and synthesized, and some of
the structures showed considerable antibacterial activities.3
Neamine (1a) is a conserved pseudo disaccharide that constitutes the core structures of many
aminoglycosides such as neomycin B (1b), paromomycin (1c), kanamycin A (1d) and kanamycin B (1e)
(Figure. 1). Among the natural aminoglycosides containing neamine motif, neomycin B and paromomycin have
a 4, 5ꢀdisubstitution on 2ꢀdeoxystreptamine (2ꢀDOS, ring II), while kanamycin has a 4, 6ꢀdisubstitution on
2ꢀdeoxystreptamine. Herein we report the design, synthesis and biological evaluation of conformationally
constrained kanamycin A analogues through the linkage of 5ꢀ and 2′ꢀhydroxyl groups.
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R
6'
HO
HO
O
I
NH2
4
6'
2'
H2N
H2N
5''
O
II
HO
HO
I
O
O
H2N
O
NH2
HO
3
H2N
OH
5
O
III
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OH
HO
NH2
O
OH
6'''
OH
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IV
H2N
NH2
O
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HO
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Neamine 1a
R=NH2 Neomycin B 1b
R=OH paromomycin1c
NH2
I
O
HO
HO
NH2
2'
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R
O
NH2
HO
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5
O
6''
OH
III
O
HO
OH
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H2N
R=OH kanamycin A 1d
R=NH2 kanamycin B 1e
Figure 1. Structures of representative aminoglycosides. The neamine core is composed of ring I and ring II.
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Results and Discussion
Design
The 3D structures of kanamycin A in complex with 16S RNA Aꢀsite and aminoglycosideꢀmodifying
enzymes show that the conformation of kanamycin A recognized by the 16S RNA Aꢀsite and the enzymes are
different.4 Indeed, these detailed structural information has already provided insights for the rational design of
new aminoglycoside analogues.
Asensio and coꢀworkers have developed a strategy to overcome bacterial resistance with conformationally
locked neomycin B (1b).5a NeomycinꢀB derivative with a methylene bridge between the 2′ꢀNH2 of ring I and
5′′ꢀOH of ring III represents an improved activity against bacteria expressing Staphylococcus aureus ANT(4′).
Hanessian’s group also synthesized paromomycin (1c) analogues by tethering the 6ꢀOH of ring II and the
6′′′ꢀNH2 of ring IV with a fiveꢀcarbon bridge.5b However, the synthesized compounds were inactive at the
concentrations lower than 40 ꢀg/mL. Mobashery and van Delft also reported the syntheses of structurally
constrained Neamine (1a) derivatives, but no biological activity was reported.5c,d
The 3D structure of kanamycin A (1d) bound to the target ribosomal Aꢀsite RNA has been determined.4l In
addition, several 3D structures of kanamycin Aꢀenzyme complexes have been determined recently.4 The 3D
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structures of the antibiotic in complex with the enzymes significantly differ from that observed in kanamycin A
/Aꢀsite RNA complex (Figure. 2). For instance, kanamycin A exists in higher energy conformation when bound
with ANT(4′)4a and AAC(2′)4g, but it adopts the minimum energy conformation in the RNAꢀbound state.
Therefore, it is possible to design analogues that can retain the ribosomal Aꢀsite RNA binding property but
resist enzyme modifications.
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Figure 2. Xꢀray structures of kanamycin A bound to Aꢀsite (a), ANT4′ (b) or AAC2′ (c). Kanamycin A is shown as gray backbone.
Atoms are colored by CPK. The 2′Oꢀtoꢀ5O distances are indicated.
Previous investigations demonstrated that neamine is the optimal pharmacophore to manifest the antibiotic
activity.6 The 3D structures of RNA–aminoglycoside complexes revealed that aminoglycosides bind to RNA in
a similar fashion. The neamine sugar rings of different aminoglycosides adopt the same orientation, bind to
relatively the same binding site, and have almost the same conserved contacts .4i, 4l, 4m In contrast, the other
sugar segments of aminoglycosides looks disorderly. These observations suggest that neamine moiety might be
a better target for modifications to gain unique antibacterial properties than other segments. In fact,
modifications of ring III of neomycin B or ring IV of paromomycin lead to significant decreases in affinity to
16S RNA in previous reports.5a,b We hope a direct restriction of the pseudoꢀdisaccharide of the neamine moiety
can reduce the potentially negative impact on the affinity to 16S RNA, while maintaining the conformational
restriction. Moreover, electrostatic interaction is considered to be the main mode of aminoglycoside and RNA
binding.7 The ammonium positive ions play a major role in electrostatic interactions. Therefore, the
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modifications of the hydroxyl groups may be more likely than the modifications of the amino groups to
generate active analogues. The crystal structure of kanamycin A in complex with ribosomal Aꢀsite RNA shows
that both the 5ꢀhydroxyl group of ring II and the 2′ꢀhydroxyl group of ring I have no critical interaction with the
RNA and these two hydroxyl groups are proximate to each other (Figure 3). In addition, the distances between
Oꢀ5 and Oꢀ2′ of kanamycin A are different in the complexes with Aꢀsite, ANT(4′) or AAC(2′) (Figure 2). Based
on these analyses, kanamycin A derivatives with their structures constrained by linking the 5ꢀhydroxyl group of
ring II and the 2′ꢀhydroxyl group of ring I with carbon bridges were designed (compounds 2ꢀ5, Figure 4).
Different lengths (1ꢀ4 carbons) of the bridges were used to restrict the conformation of the kanamycin A at
different degrees. Moreover, a double bond was introduced to enhance the conformational restriction
(compound 6). Because hydrogen bonding interactions also play a key role in the binding of aminoglycosides
with RNA, derivative 7 with protected hydroxyl groups was also prepared to evaluate the influence of the
modifications of these two hydroxyl groups.
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Figure 3. The crystal structure of kanamycin A–A site complex. Kanamycin A is shown as green backbone. The RNA is shown as line.
Atoms are colored by CPK. Hydrogen bonds between kanamycin A and RNA are shown with green dash lines.
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Figure 4. Structures of designed kanamycin A analogues (2ꢀ7)
Synthesis
We prefer to use azido groups as the precursors of amines on kanamycin since the azido derivatives have
excellent solubility in organic solvents. Tetraazidoꢀkanamycin A (8) was synthesized via a threeꢀstep procedure
in 80% yield (Scheme 1). The commercially available kanamycin A (1d) was firstly converted into perazido
species by the modified diazo transfer procedure.8 In order to facilitate the purification, the reaction residues
were treated with acetic anhydride in pyridine to give peracetylated product. After purification by column
chromatography, the peracetylated product was then deacetylated with NaOMe–MeOH to provide compound 8.
Our synthesis required the selective protection of the hydroxyl groups of kanamycin A except the 5ꢀhydroxyl
and 2′ꢀhydroxyl groups. We previously isolated a byꢀproduct 5, 2′, 4′′, 6′′ꢀdiꢀOꢀbenzylideneꢀprotected
tetraazidoꢀkanamycin A (10) during the synthesis of 4′′, 6′′ꢀOꢀbenzylidene protected tetraazidoꢀkanamycin A (9).
1
Its structure was determined by the Hꢀ1H COSY, HMBC, HMQC, and DEPT of its acetylated derivative 10a
(Scheme 1).9 The unique 5, 2′ꢀbenzylidene group is an eightꢀmembered structure, which should be relatively
unstable and could be selectively hydrolyzed. Based on these results, a threeꢀstep protection strategy was
designed. Compound 8 was firstly treated with 2.4 equiv of benzaldehyde dimethyl acetal and catalytic amount
of pꢀtoluenesulfonic acid under reduced pressure in DMF at 68ºC on a rotary evaporator for 12h to furnish the
desired product 10 in 46% yield. Further increase of the amount of benzaldehyde dimethyl acetal and the
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prolongation of reaction time did not improve the yield, but accompanied by the formation of byproducts. When
acetonitrile was used as the solvent, compound 10 was afforded in 60% yield with the same proportion of the
reactants as above reacted on a rotary evaporator at 40ºC for 5 min (Scheme 2). Compound 10 was then treated
with NaH and benzyl bromide in DMF to afford the fully protected product 11. We subsequently tested HCl
(aq), acetic acid, or H2SO4 at different concentrations to selectively remove the 5, 2′ꢀOꢀbenzylidene group, but
they all failed to hydrolyze the 5, 2′ꢀOꢀbenzylidene group selectively. It seems that this ringꢀcross benzylidene
group was not as unstable as we previously assumed. Therefore, a twoꢀstep reaction was carried out. Complete
hydrolysis of the two benzylidene groups with 0.004M HCl (aq) at 50ºC for 3 hours produced compound 12 in
93% yield, which was treated with 1.2 equiv of benzaldehyde dimethyl acetal in acetonitrile at room
temperature for 3 hours to afford the 4′′, 6′′ꢀOꢀbenzylidened product 13 in 75% yield.
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Scheme 1. Synthesis of compound 10 and 10a
Conditions: (a) (i) Tf2O, NaN3, CH3CN, 0oC ; (ii) CuSO4, H2O, CH3CN, NEt3, r.t; (b) Ac2O, Pyridine, r.t, yield 82% for two steps; (c)
MeOH, MeONa, r.t, 98%;(d) PhCH(OCH3)2, DMF, pꢀTsOH, 68oC, 20% for 9, 46% for 10; (e) Ac2O, Pyridine, r.t, 98%.
The synthesis of compounds 2ꢀ5 involved the formation of eight, nine, ten and elevenꢀmembered
intramolecular ether rings similar to crown ethers. Generally speaking, the preparations of medium sized crown
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ethers bearing eight to twelve ring atoms often face considerable difficulties.10,11 However, a dramatic increase
of the yields could be obtained with the use of cesium carbonate as base in the synthesis of crown
compounds.10a Treatment of compound 13 with dibromomethane in the presence of 8 equiv Cs2CO3 in DMF at
90ºC for 24h afforded compound 14 with a methylene bridge in 67% yield (Scheme 2). However, when 1,
2ꢀdibromoethane was used to synthesize compound 15 with Cs2CO3 as the base, no product was observed even
at higher temperature or longer reaction time. Similarly, 1, 3ꢀdibromo propane could not yield compound 16.
Stronger bases such as NaOH or NaH failed to promote these reactions. When compound 13 was treated with 1
equiv 1, 2ꢀdiꢀmethyl sulfonyloxyethane and 8 equiv Cs2CO3 in DMF at 80ºC for 2 days, cyclic product 15 was
prepared in 10% yield with the formation of 2′ or 5ꢀOꢀsulfonate byproducts. When 3 equiv 1,
2ꢀdiꢀmethylsulfonyloxyethane was used, the yield increased to 25% but it was still not satisfactory. However,
further increase of the amount of 1, 2ꢀdiꢀmethylsulfonyloxyethane did not improve the yield. 1, 2ꢀdiꢀpꢀtoluene
sulfonyloxyethane (3 equiv) was then used and the yield increased to 45%. As before, further increase of the
amount of 1, 2ꢀdiꢀpꢀtoluene sulfonyloxyethane, reaction temperature and reaction time did not improve the
yield. Compared with previous reports of the similar reactions, the yield here would be satisfactory.10ꢀ13
Similarly, compound 13 treated with 3 equiv 1, 3ꢀdiꢀpꢀtoluene sulfonyloxy propane, 8 equiv Cs2CO3 in DMF at
80 ºC for 2 days afforded cyclic ether product 16 in 45% yield and 2′ or 5ꢀOꢀallylated byproduct in 30% yield,
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while 1, 3ꢀdiꢀmethyl sulfonyloxy propane under the same condition gave 16 only in 20% yield and 2′ or
5ꢀOꢀallylated byproduct in 40% yield. We attempted to suppress this elimination reaction by using weaker base
Na2CO3 or K2CO3, but no reaction was observed.
Scheme 2. Synthesis of the cyclic kanamycin A derivatives 2ꢀ4
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Conditions: (a) 2.4eq PhCH(OCH3)2, pꢀTsOH, CH3CN, 40oC, 60%; (b) BnBr, DMF, NaH, r.t, 95%; (c) THF, MeOH, HCl(aq), 50oC,
93%; (d) 1.2eq PhCH(OCH3)2, CH3CN, pꢀTsOH, r.t, 75%; (e) for 14, Cs2CO3, DMF, CH2Br2, 90oC, 24h, 67%; for 15,
TsOCH2CH2OTs, Cs2CO3, DMF, 80oC, 2 days, 45% ; for 16, TsOCH2CH2CH2OTs, Cs2CO3, DMF, 80oC, 2 days, 45%; (f) H2S, Py,
Et3N, H2O, 6h; (g) HCl(aq), Pd(OH)2/C, H2, H2O, 50ꢀ60% for two steps.
To construct the fourꢀcarbon bridge, ringꢀclosing metathesis reaction was employed. Allylic etherification
of 5ꢀhydroxyl and 2′ꢀhydroxyl groups with NaH and ally bromide gave compound 17 in 90% yield (Scheme 3).
Phosphineꢀfree secondꢀgeneration Hoveyda–Grubbs catalyst was applied for ring closure metathesis reaction
because of the incompatibility of azido groups with the phosphine ligand of the secondꢀgeneration Grubbs’
catalyst.5d Treatment of compound 17 with 0.1 equiv catalyst in toluene for 24h gave only cisꢀcyclic product 18
in 60% yield (J=10.8 Hz). Increase of the amount of catalyst to 0.15 equiv and 0.2 equiv or elevation of
temperature did not improve the yield significantly.
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The deprotections of 14ꢀ16, 18 were achieved by a twoꢀstep process, because direct hydrogenation
catalyzed by palladium carbon could not give a clean reaction.8,9 At first, reduction of azido groups with
hydrogen sulfide gas afforded the corresponding amines. After purification with column chromatography,
debenzylation was then carried out by palladium carbon catalyzed hydrogenation under acidic conditions
(pH=3ꢀ4) to give final products 2ꢀ5 as hydrogen chloride salt (Scheme 2 and 3). These final products were
purified by HPLC for biological activity test. To synthesize compound 6 with an unsaturated fourꢀcarbon bridge,
boron trichloride14 was tried to deprotect benzyl groups of compound 18 selectively. However, it did not give a
clean reaction. Birch reduction15 was tried subsequently, but the intramolecular allyl ether bond was
decomposed (scheme 3)16.
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Scheme 3. Synthesis of the cyclic kanamycin A derivative 5
N3
N3
N3
O
O
BnO
BnO
BnO
BnO
O
BnO
BnO
N3
N3
N3
O
O
b
HO
O
O
a
O
HO
O
N3
O
N3
N3
O
O
O
O
O
O
O
O
O
BnO
O
BnO
O
BnO
O
N3
13
N3
17
18
N3
Ph
Ph
Ph
c,d
NH2
O
NH3
HO
HO
4Cl
O
HO
HO
H2N
O
H3N
O
O
O
O
NH2
NH3
O
O
O
OH
O
OH
HO
OH
O
HO
OH
H2N
6
H3N
5
MesN
Cl
NMes
MesN
Cl
NMes
Ru
O
Ru
Cl
Cl
Ph
PCy3
iPr
Second-generation Hoveyda-Grubbs Catalyst
Second-generation Grubbs catalyst
Conditions: (a) NaH, DMF, Allyl bromide, r.t, 3h, 90%; (b) Hoveyda catalyst, toluene, r.t., 24h, 60%; (c) H2S, Py, Et3N,
H2O; (d) HCl(aq), Pd(OH)2/C, H2, H2O, twoꢀstep yields: 60%.
To synthesize compound 6, different protecting strategy has to be used. The pꢀmethoxy benzyl protecting
group was used because it could be deprotected by trifluoroacetic acid without affecting the allyl ether.17
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Treatment of 5, 2′, 4′′, 6′′ꢀOꢀbenzylidene tetraazidoꢀkanamycin A (10) with pꢀmethoxy benzyl chloride and
NaH in DMF gave compound 19 in 93% yield, which was converted to 23 as shown in Scheme 4 as the
synthesis of 18 from 11 in Scheme 3. After reduction of the azides to the corresponding amines with hydrogen
sulfide gas, trifluoroacetic acid was then employed to remove PMB protective groups and benzylidene group to
give compound 6 as the trifluoroacetate salt. Similarly, 5, 2′ꢀdiallylꢀkanamycin A (7) was also prepared from
compound 22. These two final products were purified by HPLC for biological activity test.
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Scheme 4. Synthesis of the cyclic kanamycin A derivative 6 and 5, 2′ꢀdiallylꢀkanamycin A (7)
Conditions: (a) PMBCl, NaH, DMF, r.t., 6h, 93%; (b) MeOH, THF, HCl, 60oC, 90%; (c) PhCH(OCH3)2, CH3CN, pꢀTsOH, r.t., 70%;
(d) NaH, DMF, Allyl bromide, 90%; (e)Hoveyda catalyst, toluene, r.t., 24h, 50%; (f) H2S, Et3N, Py, H2O, 6h; (g) TFA, H2O, r.t., 2h.
Antibacterial activity
The synthesized aminoglycosides 2ꢀ7 were assayed against susceptible bacterial strains, using kanamycin
A as the control. Aminoglycoside susceptible Staphylococcus aureus (ATCC 25923), Staphylococcus
epidermidis (ATCC 12228) and Escherichia coli (ATCC 25922) were used. The minimum inhibitory
concentrations (MICs) are summarized in Table 1. All tested compounds showed decreased activity compared
with kanamycin A. These results indicated that modifications of 5, 2′ ꢀhydroxyl groups led to the decrease of
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activities against susceptible bacterial strains, as previosusly reported.5a,b,18 However, conformational
restrictions lead to higher activity than simple alkylation of hydroxyl groups. In addition, antiꢀbacterial
activities of these cyclic kanamycin A derivatives indeed varied with the length of the bridge. When the number
of the carbon atoms in the bridges is less than or equal to three (derivatives 2ꢀ4), the antibacterial activities of
kanamycin A derivatives are getting weaker along with the extension of the carbon chain length. When the
number of the carbon atoms in the bridges is four, derivative 6 with a double bond in the bridge showed better
activity than derivative 5 with a saturated carbon chain, which showed similar activity as derivaive 7. These
results indicate the conformational recognition is important for Aꢀsite as a target.19
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The activities of these synthesized aminoglycosides 2ꢀ7 against drugꢀresistant strains were assayed then.
Clinically significant pathogens methicillinꢀresistant S. aureus (MRSA) (Gram positive), Klebsiella
pneumoniae (Gram negative) and Escherichia coli (09ꢀ1) were used. Methicillinꢀresistant S. aureus (ATCC
33591) is the leading cause of bacterial infections, which express APH(3′), ANT(4′), and AAC(6′)/APH(2′′) and
render the bacteria resistant to various aminoglycosides.20 K. pneumoniae (ATCC 700603) is resistant to several
aminoglycosides (caused by ANT(2′′).21 Escherichia coli (09ꢀ1) is another clinical isolate obtained from patients
in China in 2009.
The results are summarized in Table 1. As expected, the MIC of kanamycin A for the resistant
Methicillinꢀresistant S. aureus (MRSA) is significantly increased compared with the susceptible S. aureus
(ATCC 25923). Surprisingly, compound 2 showed 8ꢀfold weaker activity against the methicillinꢀresistant S.
aureus (MRSA) than the susceptible S. aureus (ATCC 25923). In contrast, the cyclic derivative 3 and 6 showed
only 2ꢀfold less activity against S. aureus (MRSA) than against the nonresistant bacteria. In fact, compound 3
and 6 showed better activity than compound 2 in all the tested resistant bacterial strains. However, these two
compounds did not show better activity than kanamycin A. These results indicated that conformational
restrictions do not enhance the antibacterial activity of kanamycin A against the resistant bacterial strains. In
fact, similar result has been reported by Asensio and coꢀworkers.22
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Table 1 MIC of the synthesized aminoglycosides against susceptible bacterial strains and resistant bacterial strains a
3
4
5
6
7
8
9
S. aureusb
S. epidermidisc E. colid
S. aureuse
16
K. pneumoniaeb f
E. colig
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4
4
4
32
1d
2
8
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>64
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>64
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>64
>64
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>64
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>64
>64
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>64
>64
64
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aUnit: ꢁg/mL. bStaphylococcus aureus (ATCC 25923). c Staphylococcus epidermidis (ATCC 12228). dEscherichia coli (ATCC 25922).
eStaphylococcus aureus (ATCC 33591). fKlebsiella pneumoniae (ATCC 700603). gEscherichia coli (09ꢀ1).
Conclusion
We have designed and synthesized five conformationally constrained kanamycin A derivatives with
modifications at 5, 2′ꢀhydroxyl groups of the neamine core. After 5, 2′ꢀhydroxyl groups exposed kanamycin A
derivative was synthesized as the pivotal intermediate, cyclic kanamycin A derivatives with intramolecular 8, 9,
10, 11ꢀmembered ether were prepared by cesium carbonate mediated Williamson ether synthesis or ringꢀclosing
metathesis reaction. The antiꢀbacterial activities of these cyclic kanamycin A derivatives indeed varied with the
lengths of the bridges. However, they did not showed better antiꢀbacterial activities against tested resistant
bacterial strains.
Experimental Section
1.Chemistry
1H and 13C NMR spectra were recorded on 300M or 400M instruments using TMS as the internal standard. High resolution mass
spectra (HRMS) were obtained in positive ion electrospray ionization (ESI) mode using TOF (timeꢀofꢀflight) analyzer. Optical
rotations were measured at 25oC using polarimeter. Column chromatography was performed on Silica Gel H60. Solvents were
purified by standard procedures. The purification of the final products is carried out by HPLC (Refractive index detector).
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1, 3, 6′, 3′′ꢀTetraazidoꢀkanamycin A (8)
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To an iceꢀcooled suspension of NaN3 (5.4 g, 83.08 mmol) in anhydrous CH3CN (50 mL), trifluoromethanesulfonic anhydride
(11.56 mL, 69.22 mmol) was added dropꢀwise over 30 min and the mixture was stirred for 2 h at 0 ºC. The mixture was filtered
quickly at 0 ºC and the filtrate was added slowly into a mixture of kanamycin A (5.82 g, 10 mmol), CuSO4 (0.1 g, 0.6 mmol),
triethylamine (9.08 mL) and H2O (25mL). The reaction was stirred for 24 h at room temperature. After completion of the reaction
(monitored by TLC, acetone–EtOAc 1:1, v/v), the mixture was concentrated under reduced pressure to a green oil. To this green oil,
100 mL dried pyridine was added, followed by 20 mL acetic anhydride. The mixture was stirred for 4 h at room temperature and
MeOH was added to quench the extra Ac2O in ice bath. The solvent was removed at reduced pressure, and the remained mixture was
precipitated by the addition of water to give a yellow solid. This crude product was purified with column chromatography (PE–EtOAc
5:1, v/v) to provide the product as colorless syrup (6.89 g, 8.2 mmol, 82%), Rf=0.43 (PE–EtOAc 3:1, v/v). To a solution of the above
product (840 mg, 1 mmol) in 50 mL methanol, sodium methoxide (about 1 M solution in methanol) was added dropꢀwise to give a pH
of 11. After completion of the reaction (in about 4 h, monitored by TLC, CH2Cl2–MeOH 3:1, v/v), the reaction was neutralized with
cationic resin. Filtered the mixture and concentrated the filtrate under reduced pressure to give 8 (580 mg, 0.98 mmol, 98%) as orange
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oil: Rf=0. 26 (CHCl3–MeOH 3:1, v/v). [α]D25+80 (c 1.0, CHCl3); H NMR (300 MHz, DMSOꢀd6): δ 5.32 (br s, 4H), 5.08 (d, 1H,
J=3.6 Hz), 5.06 (d, 1H, J=3.6 Hz), 3.93 –3.82 (m, 2H), 3.69 (m, 1H), 3.56 – 3.06 (m, 14H), 2.28 – 2.24 (m, 1H), 1.59 –1.47 (m, 1H);
13C NMR (75 MHz, DMSOꢀd6): δ 100.9, 97.7, 83.3, 79.2, 73.9, 72.7, 72.0, 72.0, 71.8, 70.5, 70.3, 67.5, 67.1, 60.3, 59.7, 58.9, 51.4,
31.9; HR ESI MS: calcd for C18H28N12NaO11 ([M+Na]+) 611.1889, found 611.1893.
1, 3, 6′, 3′′ꢀTetraazidoꢀ5, 2′, 4′′, 6′′ꢀdiꢀOꢀbenzylideneꢀkanamycin A (10)
To a solution of compound 8 (5.88 g, 10 mmol) in dry CH3CN (100 mL) was added pꢀtoluenesulfonic acid monohydrate (50 mg),
followed by benzaldehyde dimethyl acetal (3.6 mL, 24 mmol). The mixture was rotated under reduced pressure in CH3CN at 40ºC on
the rotary evaporator for 5min. After completion of the reaction (monitored by TLC, CH2Cl2–MeOH 12:1, v/v), the reaction was
quenched by addition of Et3N (5 mL) and evaporated to dryness. The resulting crude product was purified with column
chromatography (CH2Cl2–MeOH 50:1, v/v) to provide colorless foam 10 (4.58 g, 6.0 mmol, 60%). Rf=0.56 (CH2Cl2–MeOH 12:1,
1
v/v). [α]D25+92.8 (c 0.99, CHCl3); H NMR (300 MHz, CDCl3): δ 7.46–7.26 (m,10H), 5.99 (s, 1H), 5.49 (d, 1H, J= 3.9 Hz), 5.34(s,
1H), 5.17 (d, 1H, J= 3.6 Hz), 4.00 (t, 1H, J=8.4 Hz), 3.87 – 3.23 (m, 15H), 2.90 (s, 1H), 2.29 (m, 1H),1.53 (m, 1H,); 13C NMR (75
MHz, CDCl3): δ 136.5, 134.6, 129.3, 129.1, 128.8, 128.1, 126.4, 126.0, 101.3, 101.0, 100.5, 99.0, 85.4, 81.1, 79.8,79.3, 73.1, 72.0,
71.9, 71.1, 69.2, 68.2, 63.9, 62.9, 59.6, 50.8, 31.5; HR ESI MS: calcd for C32H37N12O11 ([M+H]+) 765.2705, found 765.2708.
1, 3, 6′, 3′′ꢀTetraazidoꢀ5, 2′, 4′′, 6′′ꢀdiꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀ benzylꢀ kanamycin A (11)
To a solution of 10 (764 mg, 1 mmol) in anhydrous DMF (30 mL) was added NaH (3 eq.) at 0 ºC. After stirring for 5 min, BnBr
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(4.5 eq.) was added. Then the reaction mixture was warmed slowly to room temperature. After completion of the reaction (in about 6
h, monitored by TLC, PE–EtOAc 5:1, v/v), the reaction was quenched by addition of methanol. The reaction was concentrated under
reduced pressure. The residue was dissolved in EtOAc, washed with brine, dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified by column chromatography (PE–EtOAc 10:1, v/v) to give compound 11 as yellow foam in 95%
yield. Rf=0.34 (PE–EtOAc 5:1, v/v); 1H NMR (300 MHz, CDCl3): δ 7.43–7.10 (m, 25H), 6.02 (s, 1H), 5.45 (d, 1H, J =4.2 Hz), 5.43
(d, 1H, J= 3.6 Hz), 5.25 (s, 1H), 5.00 (s, 1H), 5.00 (m, 1H), 4.83 (m, 4H), 4.50 (m, 1H), 4.10 (m, 4H), 3.87 (m, 4H), 3.66 – 3.57 (m,
5H), 3.46 – 3.22(m, 6H), 3.19 (m, 1H), 2.25 (m, 1H), 1.50 (m, 1H); 13C NMR (75 MHz, CDCl3): δ 138.6, 137.9, 137.1, 136.5, 134.8,
129.2, 128.9, 128.7, 128.4, 128.3, 128.2, 127.9, 127.8, 127.6, 126.4, 126.2, 101.3, 100.8, 99.1, 96.9, 85.5, 81.8, 79.3, 79.2, 78.2, 76.6,
75.6, 75.1, 73.5, 73.1, 70.5, 68.4, 63.1, 61.2, 60.3, 57.7, 50.8, 32.0; HR ESI MS: calcd for C53H58N13O11 ([M+NH4]+)1052.4367, found
1052.4373.
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1, 3, 6′, 3′′ꢀTetraazidoꢀ3′, 4′, 2′′ꢀtriꢀOꢀ benzylꢀkanamycin A (12)
To a solution of 11 (1.25g, 1.21mmol) in THF–MeOH (2:1) 15mL, 60ꢀL 1M HCl (aq) was added. The reaction mixture was
kept at 50 ºC. After completion of the reaction (in about 3 h, monitored by TLC, CH2Cl2: MeOH =10:1, v/v), the reaction was
neutralized with anion resin. Filtered the mixture and concentrated the filtrate under reduced pressure, followed by chromatography
(CH2Cl2: MeOH =30:1) gave 12 (968mg, 1.13mmol, 93%) as colorless oil. [α]D25+130 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ
7.42ꢀ7.26 (m, 17H), 5.18ꢀ5.16(m, 2H), 5.05(d, J = 3.6 Hz, 1H), 4.92ꢀ4.79 (m, 4H), 4.67(d, J = 11.6 Hz, 1H), 4.61(d, J = 10.4 Hz, 1H),
4.17ꢀ4.95 (m, 3H), 3.84ꢀ3.46 (m, 8H), 3.39ꢀ3.21(m, 6H), 2.32ꢀ2.29(m, 1H), 155ꢀ1.46 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 138.4,
137.7, 137.1, 128.6, 128.6, 128.5, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.7, 101.6, 96.9, 85.1, 82.1, 81.9, 78.1, 77.6, 77.2, 75.3,
75.2, 74.6, 72.9, 72.7, 71.5, 71.4, 69.4, 65.2, 62.2, 60.0, 51.1, 32.1. HR ESI MS: Calcd for C39H46N12NaO11 ([M+Na]+) 881.3301,
found 881.3289.
1, 3, 6′, 3′′ꢀTetraazidoꢀ 4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀbenzylꢀkanamycin A (13)
To a solution of compound 12 (968mg, 1.13mmol) in dry CH3CN (10 mL) was added pꢀtoluenesulfonic acid monohydrate
(10mg, 0.05mmol), followed by benzaldehyde dimethyl acetal (0.2ml, 1.35mmol, 1.2eq). The mixture was stirred at room temperature.
After completion of the reaction (monitored by TLC, PE–EtOAc 2:1, v/v), the reaction was quenched by addition of Et3N and
evaporated to dryness. The resulting crude product was purified with column chromatography (PE–EtOAc 10:1, v/v) to provide
colorless oil 13 (810mg, 0.86mmol, 75%). [α]D25+80 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.52ꢀ7.25 (m, 20H), 5.53 (s, 1H),
5.17 (d, J = 3.6 Hz, 1H), 5.11 (d, J =3.6 Hz, 1H), 4.94ꢀ4.77 (m, 5H), 4.65 (m, 1H), 4.25 (m, 2H), 4.04 (m, 2H), 3.82 (m, 1H),
3.65ꢀ3.23 (m, 12H), 2.19 (m, 1H), 1.63 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 138.1, 137.7, 137.1, 136.8, 129.0, 128.6, 128.5, 128.3,
128.2, 128.1, 128.0, 127.9, 126.0, 101.3, 97.6, 85.6, 81.8, 81.3, 79.7, 78.0, 77.4, 75.4, 75.1, 74.1, 73.3, 72.7, 71.4, 68.7, 63.0, 61.8,
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60.0, 59.0, 51.1, 32.2. HR ESI MS: calcd for C46H50N12O11Na ([M+Na]+) 969.3614, found 969.3612.
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1, 3, 6′, 3′′ꢀTetraazidoꢀ 4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀbenzylꢀ5, 2′ꢀO ꢀmethyleneꢀkanamycin A (14)
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A mixture of 13 (190mg, 0.2 mmol), dibromomethane(0.3ml) and Cs2CO3 (0.5 g, 1.54mmol) in anhydrous DMF (20 mL) was
stirred at 90 °C for 24h under argon. After cooling down to r.t., the mixture was filtered, and concentrated to dryness under reduced
pressure. The residue was poured into H2O (20 mL), and extracted with CH2Cl2 (3×10mL). The combined organic phases were dried
(Na2SO4), and the solvent was evaporated. The residue was purified with column chromatography (PE–EtOAc 10:1, v/v) to provide
colorless oil 14 (128mg, 0.13 mmol, 67%). 1H NMR (300 MHz, CDCl3) δ 7.41ꢀ7.14 (m, 20H), 5.49 (d, J = 4.2 Hz, 1H), 5.43 (s, 1H),
5.34 (d, J = 3.9 Hz, 1H), 4.90ꢀ4.66 (m, 7H), 4.57ꢀ4.52 (m, 1H), 4.17ꢀ4.12 (m, 1H), 4.04ꢀ3.73 (m, 4H), 3.69ꢀ3.29 (m, 12H), 2.15 (m,
1H), 1.40 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 138.5, 137.9, 136.9, 136.7, 129.1, 128.5, 128.4, 128.3, 128.1, 127.8, 127.7, 127.6,
127.1, 126.9, 126.0, 101.5, 99.2, 96.7, 94.6, 84.8,81.9, 80.4, 79.3, 78.6, 77.7, 77.2, 76.6, 75.6, 75.1, 73.2, 70.8, 68.6, 63.0, 61.6, 60.3,
57.4, 50.7, 32.0. HR ESI MS: calcd for C47H50N12O11Na ([M+Na]+) 981.3614, found 981.3614.
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1, 3, 6′, 3′′ꢀTetraazidoꢀ 4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀbenzylꢀ5, 2′ꢀO ꢀ1, 2 ethylꢀ kanamycin A (15)
A mixture of 13 (100mg, 0.11 mmol), 1, 2ꢀdiꢀpꢀtoluene sulfonyloxyetane (122.1 mg, 3eq) and Cs2CO3 (275mg, 0.845mmol) in
anhydrous DMF (20 mL) was stirred at 80 °C for 48h under argon. After cooling down to r.t., the mixture was filtered, and
concentrated to dryness under reduced pressure. The residue was poured into H2O (20 mL), and extracted with CH2Cl2 (3×10mL).
The combined organic phases were dried (Na2SO4), and the solvent was evaporated. The residue was purified with column
chromatography (PE–EtOAc 10:1, v/v) to provide colorless oil 15 (45mg, 0.047 mmol, 45%).1H NMR (400 MHz, CDCl3) δ 7.45ꢀ7.25
(m, 21H), 5.59 (d, J = 3.2 Hz, 1H), 5.51(s, 1H), 5.37(d, J =4.0 Hz, 1H), 4.93(t, 2H), 4.82ꢀ4.76(m, 3H), 4.62ꢀ4.59(m, 1H), 4.14ꢀ4.02(m,
2H), 4.02ꢀ3.95(m, 2H), 3.86(t, 1H), 3.71ꢀ3.64(m, 2H), 3.56ꢀ3.31(m, 12H), 2.38ꢀ2.34(m, 1H), 1.60ꢀ1.50 (m, 1H); 13C NMR (100 MHz,
CDCl3) δ 138.0, 137.4, 137.0, 136.8, 129.7, 128.5, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.5, 126.0, 101.6, 99.3, 96.0,
87.8, 83.0, 79.6, 77.9, 77.5, 77.3, 77.2, 75.3, 75.1, 73.2, 70.6, 68.8, 62.8, 61.5, 60.2, 58.6, 51.3, 32.2. HR ESI MS: Calcd for
C48H52N12NaO11 ([M+Na]+) 995.3771, found 995.3759.
1, 3, 6′, 3′′ꢀTetraazidoꢀ 4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀbenzylꢀ5, 2′ꢀO ꢀ1, 3 propylꢀ kanamycin A (16)
A mixture of 13 (100mg, 0.11 mmol), 1, 3ꢀdiꢀpꢀtoluene sulfonyloxy propane (126.7 mg, 3eq) and Cs2CO3 (275mg, 0.845mmol)
in anhydrous DMF (20 mL) was stirred at 80 °C for 48h under argon. After cooling down to r.t., the mixture was filtered, and
concentrated to dryness under reduced pressure. The residue was poured into H2O (20 mL), and extracted with CH2Cl2 (3×10mL).
The combined organic phases were dried (Na2SO4), and the solvent was evaporated. The residue was purified with column
1
chromatography (PE–EtOAc 10:1, v/v) to provide colorless oil 15 (45mg, 0.047 mmol, 45%). H NMR (400 MHz, CDCl3) δ
7.44ꢀ7.26 (m, 24H), 5.52ꢀ5.50 (s, 2H), 5.20(d, J=3.3 Hz, 1H), 4.96ꢀ4.75 (m, 5H), 4.93(t, 2H), 4.62ꢀ4.59(m, 1H), 4.23ꢀ4.09(m, 4H),
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4.00ꢀ3.83 (m, 4H), 3.73ꢀ3.38(m, 12H), 3.26ꢀ3.16(m, 2H), 2.49ꢀ2.44(m, 1H), 1.92ꢀ1.87(m, 1H), 1.74ꢀ1.62(m, 2H), 1.60ꢀ1.50 (m, 1H);
13C NMR (100 MHz, CDCl3) δ 138.9, 136.9, 129.2, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 127.5, 126.1, 101.9, 99.6, 96.3, 85.4,
82.7, 80.7, 79.6, 77.9, 77.3, 77.2, 75.2, 75.1, 73.2, 70.6, 62.8, 61.8, 61.5, 60.6, 60.4, 58.4, 51.3, 31.8, 28.8. HR ESI MS: Calcd for
C49H54N12NaO11 ([M+Na]+) 1009.3927, found 1009.3931.
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1, 3, 6′, 3′′ꢀTetraazidoꢀ 4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀbenzylꢀ5, 2′ꢀdiꢀO ꢀallylꢀkanamycin A (17)
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To a solution of 13 (190mg, 0.2 mmol) in anhydrous DMF (10 mL) was added NaH (3 eq) at 0ºC. After stirring for 5 min, allyl
bromide (2 eq) was added. Then reaction mixture was warmed slowly to room temperature. After completion of the reaction
(monitored by TLC, PE–EtOAc 5:1, v/v), the reaction was quenched by addition of methanol. The mixture was poured into H2O (20
mL), and extracted with CH2Cl2 (3×10mL). The combined organic phases were dried (Na2SO4), and the solvent was evaporated. The
residue was purified by column chromatography (PE–EtOAc 10:1, v/v) to afford 17 (185mg, 0.18mmol, 90%). [α]D25+60 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.48ꢀ7.24 (m, 20H), 5.96ꢀ5.81 (m, 2H), 5.60 (d, J=3.6Hz, 1H), 5.52 (d, J=3.6Hz, 1H), 5.47 (s,
1H), 5.34ꢀ5.08 (m, 2H), 4.93ꢀ4.74 (m, 6H), 4.63ꢀ4.50 (m, 2H), 3.70ꢀ3.33 (m, 11H), 2.41ꢀ2.36 (m, 1H), 1.67ꢀ1.55 (m, 1H); 13C NMR
(75 MHz, CDCl3) δ 138.4, 138.0, 137.1, 137.0, 134.2, 133.4, 129.0, 128.5, 128.4, 128.2, 127.8, 127.7, 126.2, 117.8, 116.5, 101.6, 99.1,
96.0, 82.3, 81.8, 79.7, 79.1, 78.3, 77.9, 77.4, 76.6, 75.6, 74.9, 73.9, 73.1, 70.6, 68.7, 62.5, 61.5, 60.2, 59.0, 51.2, 32.1. HR ESI MS:
Calcd for C52H62N13O11 ([M+NH4]+) 1044.4685, found 1044.4686.
1, 3, 6′, 3′′ꢀTetraazidoꢀ 4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀbenzylꢀ5, 2′ꢀOꢀ(1, 4 butylꢀ2ꢀene)ꢀkanamycin A (18)
Compound 17 (51mg, 0.05mmol) was coꢀevaporated three times with toluene and dissolved in toluene (10mL) again. Argon was
bubbled through the solution for 10 min. To the reaction mixture was added Hoveyda–Grubbs catalyst (0.1eq). The mixture was
stirred at room temperature. After the reaction was complete (approximately after 24 h, monitored by TLC), the mixture was treated
with DMSO (50 equiv, relative to catalyst) overnight. The solution was concentrated in vacuo and purified by column
chromatography (PE–EtOAc 8:1, v/v) to provide the RCM product 18 (29.9mg, 0.03mmol, 60%) as colorless oil. [α]D25+84 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.46ꢀ7.25 (m, 2H), 5.99ꢀ5.92 (m, 1H), 5.72ꢀ5.67(m, 1H), 5.58(d, J =4.4 Hz, 1H), 5.51(s, 1H),
5.49(d, J =3.6 Hz, 1H), 5.11(dd, J1=9.6Hz, J2=11.2Hz, 1H), 4.95ꢀ4.75(m, 5H), 4.65ꢀ4.60(m, 2H), 4.32ꢀ4.18(m, 4H), 4.13ꢀ4.08 (m,
1H), 3.98 (t, 1H), 3.87(t, 1H), 3.69ꢀ3.57(m, 2H), 3.51ꢀ3.36(m, 10H), 2.46ꢀ2.40(m, 1H), 1.64(dd, J1=12.4Hz, J2=24.8Hz); 13C NMR
(100 MHz, CDCl3) δ 138.6, 138.0, 137.0, 136.9, 130.6, 130.0, 129.1, 128.6, 128.5, 128.4, 128.4, 128.2, 128.0, 127.9, 127.8, 127.7,
126.2, 101.8, 99.2, 96.5, 82.7, 82.3, 82.0, 78.1, 77.7, 77.3, 77.2, 76.0, 75.8, 75.1, 73.2, 70.5, 70.3, 68.7, 62.7, 61.8, 60.8, 60.4, 58.3,
51.2, 32.0. HR ESI MS: Calcd for C50H58N13O11 ([M+NH4]+) 1016.4413, found 1016.4373.
5, 2′ꢀOꢀMethyleneꢀkanamycin A (2)
Compound 14 (96mg, 0.1mmol) was dissolved in Et3N–pyridine–H2O (3:2:1), H2S gas was bubbled into the solution for 1 h. The
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mixture was stirred for another 5 h. The solvent was removed under reduced pressure, followed by column chromatography (CHCl3:
MeOH: NH4OH =20: 1: 0.5) to provide benzylated kanamycin A as a colorless oil. The benzylated kanamycin A was added with
catalytic amount of Pd(OH)2/C and 5 mL CH3OH (pH=3ꢀ4). The reaction mixture was stirred at room temperature under hydrogen of
5 atm for 2 day. Then the mixture was filtered through Celite. The residue was washed with water, and the combined solutions were
concentrated affording crude product as a HCl salt in 54% yield. The product was purified by HPLC (0.025M CF3COOH in H2O and
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CH3OH (19:1)) to afford compound 2 as a salt of trifluoroacetic acid. H NMR (300 MHz, D2O) δ 5.56 (s, 1H), 4.95 (s, 1H), 4.84 (s,
2H), 3.85ꢀ2.93 (m, 16H), 2.38 (m, 1H), 1.83ꢀ1.53 (m, 1H); 13C NMR (75 MHz, D2O) δ 101.9, 99.1, 95.5, 82.3, 80.5, 99.6, 78.8, 73.7,
72.5, 70.7, 68.9, 65.7, 60.2, 55.3, 50.5, 48.3, 40.9, 28.4. HR ESI MS: calcd for C19H37N4O11 ([M+H]+) 497.2453, found 497.2440.
5, 2′ꢀOꢀ1, 2ꢀEthylꢀkanamycin A (3)
Compound 3 was prepared in the similar way as 2. Yield: 52% for two steps. 1H NMR (400 MHz, D2O) δ 5.50 (d, J = 3.6 Hz,
1H), 5.08(d, J =3.2 Hz, 1H), 4.31ꢀ4.29(m, 1H), 4.02ꢀ3.95(m, 2H), 3.99ꢀ3.89(m, 3H), 3.85ꢀ3.32(m, 17H), 3.25(t, J =10.0 Hz, 1H),
3.04(dd, J1=8.4Hz, J2=13.6Hz, 1H), 2.46ꢀ2.43(m, 1H), 1.88 (dd, J1=12.4Hz, J2=25.2Hz, 1H); 13C NMR (100 MHz, D2O) δ 101.7, 98.5,
88.3, 83.5, 80.5, 80.0, 71.2, 70.5, 68.5, 68.4, 65.0, 59.4, 54.6, 49.7, 47.8, 27.9. HR ESI MS: Calcd for C20H39N4O11 ([M+H]+)
511.2610, found 511.2610.
5, 2′ꢀOꢀ1, 3ꢀPropylꢀkanamycin A (4)
Compound 4 was prepared in the similar way as 2. Yield: 59% for two steps. 1H NMR (400 MHz, D2O) δ 5.50 (d, J = 2.4 Hz,
1H), 5.14(d, J =2.4 Hz, 1H), 4.18ꢀ4.16(m, 1H),4.05ꢀ4.03(m, 1H), 3.96ꢀ3.29(m, 22H), 3.18ꢀ3.15(m, 1H), 2.58ꢀ2.55(m, 1H), 1.98ꢀ1.74
(m, 1H); 13C NMR (100 MHz, D2O) δ 101.6, 99.8, 83.4, 83.0, 82.5, 77.2, 73.2, 71.1, 70.5, 70.2, 68.4, 68.3, 59.5, 54.6, 49.9, 48.2, 46.7,
39.8, 28.2, 27.8. HR ESI MS: Calcd for C21H41N4O11 ([M+H]+) 525.2766, found 525.2760.
5, 2′ꢀOꢀ1, 4ꢀButylꢀkanamycin A (5)
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Compound 5 was prepared in the similar way as 2. Yield: 55% for two steps. H NMR (400 MHz, D2O) δ 5.50 (d, J = 3.2 Hz,
1H), 5.07(d, J =2.0 Hz, 1H), 3.93ꢀ3.63(m, 16H), 3.55ꢀ3.33(m, 8H), 3.21ꢀ3.17(m, 1H), 2.47(d, J = 12.0 Hz, 1H), 1.92ꢀ1.75 (m, 4H),
1.56ꢀ1.44(m, 1H); 13C NMR (100 MHz, D2O) δ101.9, 101.2, 83.2, 82.8, 79.6, 78.2, 75.5, 73.8, 73.3, 70.9, 70.8, 68.6, 68.5, 64.8, 59.1,
54.6, 49.9, 48.2, 40.2, 28.4, 28.1, 24.1. HR ESI MS: Calcd for C22H43N4O11 ([M+H]+) 539.2923, found 539.2922.
1, 3, 6′, 3′′ꢀTetraazidoꢀ5, 2′, 4′′, 6′′ꢀdiꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀpꢀmethoxyꢀ benzylꢀ kanamycin A (19)
To a solution of 10 (2.5g, 3.27 mmol) in anhydrous DMF (30 mL) was added NaH (3 eq) at 0 ºC. After stirring for 5 min,
pꢀmethoxyꢀbenzyl chloride (4.5 eq) was added. Then reaction mixture was warmed slowly to room temperature. After completion of
the reaction (monitored by TLC, PE–EtOAc 3:1, v/v), the reaction was quenched by addition of methanol. The mixture was poured
into H2O (20 mL), and extracted with CH2Cl2 (3×10mL). The combined organic phases were dried (Na2SO4), and the solvent was
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evaporated. The residue was purified by column chromatography (PE–EtOAc 10:1, v/v) to afford 19 (3.42g, 3.04 mmol, 93%). H
NMR (400 MHz, CDCl3) δ 7.44ꢀ7.25 (m, 15H), 7.19ꢀ7.13(m, 4H), 6.93ꢀ6.86(m, 7H), 6.06 (s, 1H), 5.47 (d, J = 4.8 Hz, 1H), 5.45 (d, J
= 3.2Hz, 1H), 5.27 (s, 1H), 4.94(d, J =10.8 Hz, 1H), 4.78ꢀ4.70 (m, 4H), 4.46(d, J =10.8 Hz, 1H), 4.18ꢀ4.11 (m, 2H), 3.91ꢀ3.78 (m,
14H), 3.74ꢀ3.60 (m, 6H), 3.51ꢀ3.46 (m, 1H), 3.41ꢀ3.31(m, 5H), 3.21(t, J =10.0 Hz, 1H), 2.32ꢀ2.29 (m, 1H), 1.61 (m, 1H); 13C NMR
(100 MHz, CDCl3) δ 159.7, 159.3, 136.6, 135.5, 134.9, 130.9, 130.2, 130.0, 129.6, 129.5, 129.2, 129.1, 128.9, 128.7, 127.9, 126.5,
126.3, 113.9, 113.8, 113.7, 101.3, 100.8, 99.1, 96.9, 85.5, 81.8, 79.3, 79.2, 78.2, 77.8, 77.2, 76.5, 75.6, 75.3, 74.8, 73.2, 70.5, 68.4,
63.1, 61.2, 60.3, 57.7, 55.3(2), 50.8, 32.1. HR ESI MS: Calcd for C56H60N12NaO14 ([M+Na]+) 1147.4244, found 1147.4227.
1, 3, 6′, 3′′ꢀTetraazidoꢀ 4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀ pꢀmethoxyꢀbenzylꢀ kanamycin A (21)
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To a solution of 19 (0.625g, 0.56mmol) in THF–MeOH (2:1) 7.5mL, 30ꢀL 1M HCl (aq) was added. The reaction mixture was
kept at 50 ºC. After completion of the reaction (in about 3 h, monitored by TLC, CH2Cl2: MeOH =10:1, v/v), the reaction was
neutralized with cationic resin. Filtered the mixture and concentrated the filtrate under reduced pressure, followed by chromatography
(CH2Cl2: MeOH =30:1) gave 20 (477mg, 90%) as colorless oil. To a solution of compound 20 (477mg, 0.5mmol) in dry CH3CN (10
mL) was added pꢀtoluenesulfonic acid monohydrate (10mg, 0.05mmol), followed by benzaldehyde dimethyl acetal (0.09ml, 0.6 mmol,
1.2eq). The mixture was stirred at room temperature. After completion of the reaction (monitored by TLC, PE–EtOAc 2:1, v/v), the
reaction was quenched by addition of Et3N and evaporated to dryness. The resulting crude product was purified with column
chromatography (PE–EtOAc 10:1, v/v) to provide colorless oil 21 (362mg, 0.35mmol, 70%).Yield for two steps: 63%.1H NMR (400
MHz, CDCl3) δ 7.51ꢀ7.49 (m, 2H), 7.40ꢀ7.34(m, 5H), 7.29ꢀ7.22(m, 7H), 6.92ꢀ6.86(m, 6H), 5.53(s, 1H), 5.13 (m, 2H), 4.88ꢀ4.79(m,
2H), 4.76ꢀ4.70 (m, 2H), 4.66(d, J =1.6 Hz, 1H), 4.56(d, J =10.8 Hz, 1H), 4.26ꢀ4.23(m, 1H), 4.10ꢀ3.99(m, 2H), 3.81ꢀ3.79 (m, 7H),
3.77(s, 1H), 3.72ꢀ3.63 (m, 2H), 3.59ꢀ3.48 (m, 5H), 3.44ꢀ3.38(m, 3H), 3.33(t, J =10.4 Hz, 1H), 3.29ꢀ3.26(m, 2H), 2.94(d, J =4.4 Hz,
1H), 2.41ꢀ2.38 (m, 1H), 1.61 (t, J =5.6Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 159.6, 159.5, 159.4, 136.7, 135.5, 134.9, 130.4, 130.0,
129.6, 129.5, 129.2, 129.1, 128.3, 126.0, 114.0, 113.9, 113.8, 101.4, 101.3, 97.6, 85.5, 81.8, 81.3, 79.7, 77.8, 77.6, 77.2, 75.1, 74.8,
74.2, 73.0, 72.8, 71.4, 68.8, 63.1, 61.8, 60.0, 59.0, 55.3, 51.1, 32.3. HR ESI MS: Calcd for C49H56N12NaO14 ([M+Na]+) 1059.3931,
found 1059.3930.
1, 3, 6′, 3′′ꢀTetraazidoꢀ4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀpꢀmethoxyꢀbenzylꢀ5, 2′ꢀdiꢀOꢀallylꢀkanamycin A (22)
Compound 22 was prepared in the similar way as 17. Yield: 90%.1H NMR (400 MHz, CDCl3) δ 7.47ꢀ7.44 (m, 2H), 7.38ꢀ7.33(m,
5H), 7.26(t, J =4.8 Hz, 1H), 7.21(d, J =8.4 Hz, 1H), 6.91(d, J =8.4 Hz, 1H), 6.88ꢀ6.84(m, 4H), 5.93ꢀ5.85(m, 2H), 5.55(d, J =3.6Hz,
1H), 5.50(d, J =3.6Hz, 1H), 5.47(s, 1H), 5.32ꢀ5.22(m, 2H), 5.16(dd, J1=1.2Hz, J2=10.4Hz, 1H), 5.09(dd, J1=1.2Hz, J2=10.8Hz, 1H),
4.85ꢀ4.75(m, 4H), 4.69(d, J =11.2 Hz, 1H), 4.55ꢀ4.49 (m, 2H), 4.34ꢀ4.30(m, 2H), 4.26ꢀ4.05(m, 5H), 3.82ꢀ3.79(m, 9H), 3.66ꢀ3.31(m,
13H), 2.41ꢀ2.37(m, 1H), 1.66ꢀ1.58(m, 1H); 13C NMR (100 MHz, CDCl3) δ 159.6, 159.3, 159.2, 137.1, 134.4, 133.5, 130.7, 130.2,
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129.9, 129.5, 129.5, 129.2, 129.0, 128.0, 126.2, 117.7, 116.5, 114.0, 113.8, 101.7, 97.2, 96.2, 82.3, 81.7, 79.8, 79.2, 78.0, 77.5, 77.2,
76.9, 76.6, 75.3, 74.6, 73.9, 72.8, 70.7, 68.8, 62.5, 61.5, 60.3, 59.1, 55.3, 51.3, 32.2. HR ESI MS: Calcd for C55H64N12NaO14
([M+Na]+) 1139.4557, found 1139.4561.
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1, 3, 6′, 3′′ꢀTetraazidoꢀ4′′, 6′′ꢀOꢀbenzylideneꢀ3′, 4′, 2′′ꢀtriꢀOꢀpꢀmethoxyꢀbenzylꢀ5, 2′ꢀOꢀ(1, 4 butylꢀ2ꢀene)ꢀkanamycin A (23)
Compound 23 was prepared in the similar way as 18. Yield: 50%, only cisꢀproduct was detected. 1H NMR (400 MHz, CDCl3) δ
7.47ꢀ7.44 (m, 4H), 7.36ꢀ7.34(m, 5H), 7.30ꢀ7.26(m, 8H), 7.22(d, J =8.8 Hz, 2H), 6.92ꢀ6.86(m, 6H), 5.98ꢀ5.93 (m, 1H), 5.72ꢀ5.68(m,
1H), 5.56(d, J =4.4 Hz, 1H), 5.51(s, 1H), 5.45(d, J =3.6 Hz, 1H), 5.11(t, J=10.2Hz, 1H), 4.87ꢀ4.76(m,4H), 4.69(d, J =11.2 Hz, 1H),
4.54(d, J =10.8 Hz, 1H), 4.33ꢀ4.08 (m, 5H), 3.96(t, J =9.6 Hz, 1H), 3.85ꢀ3.78(m, 10H), 3.69ꢀ3.33(m, 11H), 2.46ꢀ2.41(m, 1H), 1.64(dd,
J1=12.4 Hz, J2=25.6Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 159.7, 159.4, 159.3, 137.0, 130.8, 130.6, 130.2, 130.1, 130.0, 129.7,
129.6, 129.5, 129.1, 128.9, 128.3, 126.2, 114.0, 113.9, 113.8, 101.8, 99.2, 96.6, 82.7, 82.3, 82.0, 80.1, 79.6, 77.8, 75.5, 74.7, 72.8,
70.5, 70.3, 70.2, 68.7, 62.7, 61.8, 60.6, 58.3, 55.3, 51.2, 32.0. HR ESI MS: Calcd for C53H60N12NaO14 ([M+Na]+) 1111.4244, found
1111.4246.
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5, 2′ꢀOꢀ(1, 4ꢀButylꢀ2ꢀene)ꢀkanamycin A (6)
Compound 23 (100mg, 0.09mmol) was dissolved in NEt3–pyridine–H2O (3:2:1), H2S gas was bubbled into the solution for 1 h.
The mixture was stirred for another 5 h. The solvent was removed under reduced pressure, followed by column chromatography
(CHCl3: MeOH: NH4OH =20: 1: 0.5) provided pꢀmethoxy benzylated kanamycin A as a colorless oil. To the solution of pꢀmethoxy
benzylated kanamycin A in H2O (1mL), 0.5 ml trifluoroacetice acid was added. The mixture was stirred for 2h. The solvent was
removed under reduced pressure, followed by column chromatography (ODS, water) to give the crude product in 53% yield. The
product was purified by HPLC (0.025M CF3COOH in H2O and CH3OH 19:1) to afford compound 6 as a salt of trifluoroacetic acid.
1H NMR (400 MHz, D2O) δ 5.87ꢀ5.80(m, 2H), 5.64(d, J =4.0 Hz, 1H), 5.04(d, J =2.4 Hz, 1H), 4.47(d, J =15.2Hz, 1H), 4.30(dd,
J1=4.4Hz, J2=9.2Hz, 1H), 4.02 (dd, J1=5.2Hz, J2=15.2Hz, 1H), 3.50ꢀ3.25(m, 7H), 2.93(dd, J1=7.2Hz, J2=14.4Hz, 1H), 2.41(dd,
J1=4.4Hz, J2=8.0Hz, 1H), 1.91(dd, J1=12.4Hz, J2=25.2Hz, 1H); 13C NMR (100 MHz, D2O) δ 131.6, 129.6, 101.7, 99.0, 83.5, 82.8,
79.6, 77.4, 73.2, 71.2, 71.1, 70.6, 68.8, 68.5, 65.0, 59.5, 54.6, 50.0, 48.1, 40.3, 28.2. HR ESI MS: Calcd for C22H41N4O11 ([M+H]+)
537.2766, found 537.2766.
5, 2′ꢀDiꢀOꢀallylꢀkanamycin A (7)
Compound 7 was prepared in the similar way as 6. Yield: 68%. 1H NMR (400 MHz, D2O) δ 5.94ꢀ5.79(m, 2H), 5.56 (d, J = 2.4
Hz, 1H), 5.28ꢀ5.14(m, 4H), 5.07(d, J =2.4 Hz, 1H), 4.30ꢀ4.20(m, 4H), 4.11(dd, J1=6.4Hz, J2=12.0Hz, 1H), 4.03(t, J=9.6Hz, 1H),
3.90(t, J=9.6Hz, 1H), 3.84ꢀ3.63(m, 9H), 3.49ꢀ3.26(m, 6H), 3.09(dd, J1=8.0Hz, J2=13.6Hz, 1H), 2.41ꢀ2.38(m, 1H), 1.93(dd, J1=12.0Hz,
J2=25.2Hz, 1H); 13C NMR (100 MHz, D2O) δ 133.5, 133.5, 119.4, 118.2, 101.1, 95.2, 81.5, 81.4, 77.9, 73.8, 73.6, 73.0, 71.4, 71.3,
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71.2, 68.8, 68.5, 64.9, 59.2, 54.6, 49.9, 48.4, 40.4, 27.7. HR ESI MS: Calcd for C24H45N4O11 ([M+H]+)565.3079, found 565.3084.
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2. Antibacterial assay
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Minimum inhibitory concentrations (MIC) in ꢀg/mL were determined by the microdilution broth method according to Clinical
and Laboratory Standards Institute (CLSI) guidelines. Inoculum was prepared by diluting actively growing broth cultures to a
McFarland value of 0.5 (1–108 cfu/mL). Antibiotic dilutions were made by dispensing 0.1 mL into each of the 96 wells of a standard
microtitre tray in 2ꢀfold dilutions. The inoculum was added to give a final concentration of 0.03ꢀ64 ꢀg/mL. The trays were covered
and incubated at 35ºC for 16–20 h. The MIC was defined as the lowest concentration of drug inhibiting visible growth. The
microdilution broth susceptibility testing was performed in triplicate.
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Acknowledgements
This work was financially supported by the National Basic Research Program of China (Grant No. 2012CB822100) and the Ministry
of Science and Technology of China (2012ZX09502001ꢀ001).
Supporting Information
Copies of 1H and 13C NMR spectra of compounds 2ꢀ7, 11ꢀ19 and 21ꢀ23. This material is available free of charge via the Internet at
http:// pubs.acs.org.
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