Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside for the Treatment of Drug-Resistant Enterobacteriaceae and Other Gram-Negative Pathogens

Article abstract of DOI:10.1021/jacs.9b01693

Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4′-deoxy-4′-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or rRNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy.

Full text of DOI:10.1021/jacs.9b01693

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Article
Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation
of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine
Class Aminoglycoside for the Treatment of Drug-Resistant
Enterobacteriaceae and Other Gram-Negative Pathogens
Takahiko Matsushita, Girish Sati, Nuwan Kondasinghe, Michael Pirrone, Takayuki Kato,
Prabuddha Waduge, Harshitha Kumar, Adrian Sanchon, Malgorzata Dobosz-Bartoszek,
Dimitri Shcherbakov, Mario Juhas, Sven N. Hobbie, Thomas Schrepfer, Christine S
Chow, Yury Polikanov, Jochen Schacht, Andrea Vasella, Erik C. Böttger, and David Crich
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b01693 • Publication Date (Web): 22 Feb 2019
Downloaded from http://pubs.acs.org on February 22, 2019
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Design, Multigram Synthesis, and in Vitro and in Vivo
Evaluation of Propylamycin: A Semisynthetic 4,5-
Deoxystreptamine Class Aminoglycoside for the Treatment of
Drug-Resistant Enterobacteriaceae and Other Gram-Negative
Pathogens
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Takahiko Matsushita,a Girish C. Sati,a Nuwan Kondasinghe,a Michael G. Pirrone,a Takayuki Kato,a
Prabuddha Waduge,a Harshitha Santhosh Kumar,b Adrian Cortes Sanchon,b Malgorzata Dobosz-
Bartoszek,c Dimitri Shcherbakov,b Mario Juhas,b Sven N. Hobbie,b Thomas Schrepfer,d Christine
S. Chow,a Yury S. Polikanov,†,c,e Jochen Schacht,d Andrea Vasella,*,f Erik C. Böttger,*,b and David
Crich*,a
† To whom enquiries about the X-ray crystal structure should be addressed: yuryp@uic.edu
* Corresponding authors: vasella@org.chem.ethz.ch; boettger@imm.uzh.ch;
dcrich@chem.wayne.edu
a: Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA
b: Institut für Medizinische Mikrobiologie, Universität Zürich, 28 Gloriastrasse, 8006 Zürich,
Switzerland
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c: Department of Biological Sciences, University of Illinois at Chicago, 900 South Ashland
Avenue, Chicago, IL 60607, USA
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d: Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan,
1150 West Medical Center Drive, Ann Arbor, MI 48109, USA
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e: Department of Medicinal Chemistry and Pharmacognosy, 900 South Ashland Avenue,
Chicago, IL 60607, USA
f: Laboratorium für Organische Chemie, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zürich,
Switzerland
Abstract: Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant
Enterobacteriaceae (CREs) present a major and growing threat to human health and society, providing
an urgent need for the development of improved potent antibiotics for their treatment. We describe
the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of
propylamycin. Propylamycin is a 4’-deoxy-4’-alkyl paromomycin whose alkyl substituent conveys
excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections,
including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside
modifying enzymes or ribosomal RNA methyl transferases. Importantly, propylamycin is demonstrated
not to be susceptible to the action of the ArmA resistance determinant whose presence severely
compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine
aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid
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as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance
when used in combination with carbapenems. Cell-free translation assays, quantitative ribosomal
footprinting, and X-ray crystallography support a model in which propylamycin functions by interference
with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were
used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In
mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, that is
better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available
aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple
mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of
aminoglycoside therapy.
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Introduction
Seventy years after their introduction, the aminoglycoside antibiotics (AGAs) remain some of the most
efficacious and cost-effective treatments against life-threatening Gram-negative bacterial infections.1
Decades of clinical use, however, have led to the development of widespread resistance, which has
resulted in diminished efficacy. The most common mechanism of resistance arises from AGA
modification by aminoglycoside modifying enzymes (AMEs),2,3 which reduce AGA affinity for their target
– the decoding A site in helix 44 of the bacterial ribosome.4,5 A second mechanism of resistance,
modification of the target by the ribosomal RNA methyltransferases (RMTs), especially the A1405 N7
methyltransferases, is an increasing threat, particularly as the RMT genes are frequently encoded on the
same mobile genetic elements as the metallocarbapenemases.6-11 In addition, potentially serious side
effects to the kidney and inner ear have contributed to the decline in use of AGAs. The potential for
toxic side effects of AGAs is minimized in the clinic by limiting treatment to short duration regimens with
once daily dosing.12 A subgroup of patients is rendered hypersusceptible to AGAs by mutations in the
decoding A site of the mitochondrial ribosome.13-18
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Astute chemical modification of AGAs enables circumvention of the action of the more problematic
AMEs,3,19-24 as exemplified by the semisynthesis of the drugs amikacin, arbekacin, and most recently of
plazomicin, a doubly modified version of the natural AGA sisomicin, that is active in the presence of
most resistance determinants.25-27 However, recent analyses of the AGA resistome indicate that a much
broader range of AMEs act on the 4,6-series of AGAs, typified by the clinical comparators gentamicin,
tobramycin, and amikacin,19,28 suggesting that further development of the 4,6-AGAs will meet greater
challenges than that of the 4,5-series that we favor. Guided by cell-free translation assays with bacterial
and hybrid bacterial ribosomes carrying the decoding A sites of the wild-type and mutant human
mitochondrial ribosomes,29 we have demonstrated that chemical modification of AGAs can also lead to
reduced ototoxicity in a guinea pig model.30 An alternative approach to moderation of ototoxicity
employs chemical modification of AGAs in such a way as to impair their uptake into inner ear hair cells
by the mechanotransducer channels.31 AGA ototoxicity-reducing modifications, when installed at or
proximal to the sites targeted by AMEs, can also suppress resistance due to the presence of AMEs.
Unfortunately, while this duality of action has been amply demonstrated, it is typically accompanied by
a loss of antibacterial activity.30-33
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We report here on a simple minimal modification of the 4,5-disubstituted 2-deoxystreptamine (DOS)
AGA paromomycin 1 that blocks the action of multiple AMEs, and results in increased ribosomal
selectivity and reduced ototoxicity in the guinea pig model, with no loss of and even increased
antibacterial activity against Enterobacteriaceae and other Gram-negative pathogens for which the
AGAs are often the preferred treatment. It is noteworthy that this simple modification does not cause
any reduction in antibacterial activity in the presence of the G1405 RMTs, whose presence compromises
the activity of all 4,6-DOS AGAs currently employed in the clinic including plazomicin.6-8,26,27 Based on
structural studies of an AGA-ribosome complex, simple physical organic considerations, and exploration
of structure-activity parameters, we further develop a structure-based model for increased AGA activity.
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This new modification and the model it stimulates open the way for the rational design of further AGAs
displaying improved antibacterial activity in the presence and absence of AMEs and RMTs with
minimization of hearing impairment, such as are increasingly recognized as necessary to combat the
growing epidemic of multidrug resistant infectious diseases.
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Design and Discovery of Propylamycin
Earlier work from our laboratories revealed that 4’-O-ethylation 3 of the 4,5-DOS paromomycin
1 provokes a significant increase in ribosomal selectivity and a corresponding reduction in
ototoxicity in guinea pigs, but is accompanied by a loss of antibacterial activity.30 4’-
Deoxygenation 2 of paromomycin on the other hand causes only a minor loss in activity but no
increase in selectivity.30 Combining these results we hypothesized that replacement of the 4’-
ethoxy group of 3 by a similar and ideally isosteric substituent lacking the electron-withdrawing
C-O bond would yield compounds with reduced ototoxicity and little or no loss of antibacterial
activity. Accordingly, we targeted 4’-deoxy-4’-ethylthioparomomycin 4 and ultimately 4’-
deoxy-4’-propylparomomycin 5 for synthesis (Figure 1).
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4
6'
OH
4'
O
X
I
HO
H2N
H2N
5
6
O
II
NH2
OH
O
HO
O
III
7
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H2N
OH
IV O
OH
O
9
OH NH2
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5
paromomycin
, X = OH,
, X = H
, X = OEt
, X = SEt
propylamycin
, X = CH2CH2CH3,
R2
H
NHR1
O
N
O
OH
NH2
NH2
H2N
O
HO
H2N
OH
NH2
H
O
N
HO
O
O
O
O
HO
NH2
O
HO
HN
OH
HN
OH
Gentamicin C1A, C1 and C2
Plazomicin
(R1R2 = H = C1A, R1R2 = Me = C1,
R1 = H, R2 = Me = C2)
NH2
O
NH2
O
HO
HO
HO
NH2
NH2
OH
H2N
HO
H
O
HO
O
NH2
N
HO
O
O
O
NH2
OH
OH
OH
OH
O
O
HO
Tobramycin
HO
H2N
H2N
Amikacin
Figure 1. Paromomycin and selected derivatives at the 4’-position, and the current clinical drugs
plazomicin, gentamicin, tobramycin and amikacin.
The synthesis of the 4’-deoxy-4’-ethylthio derivative 4 began with the previously described
galacto-configured triflate 6,34 which was converted to the thioethers 7-10 and the thioester 11
by standard nucleophilic substitution reactions. Peroxide-mediated oxidation of 7 gave the
sulfoxide 12 and the sulfone 13 in a temperature-dependent manner, while the thioester 11
served as precursor to the thioether 14. A two-step deprotection sequence of Staudinger
reduction34 followed by hydrogenolysis then afforded 4′-deoxy-4’-ethyl paromomycin 4 and the
analogs 15-20 to which we return below (Scheme 1).
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OBn
O
TfO
BnO
BnO
OBn
O
R
BnO
N3
N3
N3
N3
O
N3
i)
O
O
N3
O
OBn
O
OBn
7
8
9
: R = SEt, 83%
: R = SCH2CH2Me, 70%
: R = SCHMe2, 75%
: R = SCH2CHMe2, 78%
: R = SAc, 59%
N3
OBn
O
OBn
O
ii)
10
11
OBn N3
iii)
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iv), v)
12
13
14
: R = SOEt, 83%
: R = SO2Et, 81%
: R = SCH2CH2F, 37%
OH
O
RS
HO
H2N
H2N
vi), vii)
O
NH2
OH
O
HO
O
H2N
OH
O
OH
O
OH NH2
: R = SEt, 20%
18
19
20
: R = SCH2CH2F, 27%
: R = SOEt, 6%
: R = SO2Et, 41%
4
15
16
17
: R = SCH2CH2Me, 7%
: R = SCHMe2, 36%
: R = SCH2CHMe2, 11%
Scheme 1. Synthesis of the sulfur-based paromomycin derivatives 4 and 15-20. i) RSNa or AcSK; ii)
mCPBA, -78 oC; iii) mCPBA, room temp; iv) NH2NH2.HOAc; v) NaH, FCH2CH2OTf; vi) PMe3, NaOH; vii) H2,
Pd/C.
In line with expectation, introduction of the ethylsulfanyl group 4 resulted in only a minor loss
in activity against the bacterial ribosome compared to the parent, but reduced activity for the
eukaryotic ribosomes and so excellent selectivity (Figure 2 and Table 1). Extrapolating further,
we designed the 4’-deoxy-4’-propyl derivative of paromomycin 5, that retains the beneficial
three atom chain at the 4’-position but replaces the 4’-oxygen by a methylene group, and which
we name propylamycin.
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Figure 2. Decoding A sites of the human mitochondrial, A1555G mutant mitochondrial and cytoplasmic
ribosomes, and of the bacterial ribosome. The bacterial AGA binding pocket is boxed. The bacterial
numbering scheme is illustrated for the AGA binding pocket. Changes from the bacterial ribosome
binding pocket are coloured green. The A1555G mutant conferring hypersusceptibility to AGA
ototoxicity is coloured red.
Propylamycin 5 was obtained by sequential conversion of paromomycin 1 to the
pentatrifluoroacetamide 21, its benzylidene acetal 22, and the subsequent hexabenzoate 23 in
essentially quantitative yield on a 100 g scale. Cleavage of the acetal gave the diol 24, which
was selectively benzoylated at the primary position with benzoyl cyanide35 to afford 25.
Triflation followed by displacement with sodium iodide then gave the iodide 26 in 39% yield. In
the key C-C bond forming step, reaction of 80 g of 26 with allyl phenyl sulfone36 in α,α,α-
trifluorotoluene37 at 0 oC with initiation by triethylborane and air38-40 afforded 92% of crude 27
as a single diastereoisomer. The purification of 27 on a large scale was complicated by the
formation of significant amounts of the byproduct 29. Accordingly, the mixture of 27 and 29
was taken forward to the next step when saturation of the double bonds in the two substances
gave a still difficult-to-separate mixture of 28 and 30. Finally, removal of the benzoate esters
from 28 with magnesium methoxide in methanol and subsequent cleavage of the
trifluoroacetamides with barium hydroxide gave 5 in 64% yield for the three steps after
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purification by Sephadex chromatography (Scheme 2). Noteworthy features of this synthesis
include the use of the trifluoroacetamide amine protecting group, which was selected because
it: i) rendered all intermediates solid and easy to handle on a large scale; ii) afforded sharp and
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readily interpreted H-NMR spectra at all stages; and iii) could be cleaved under relatively mild
basic conditions, yet was resistant to the conditions for the removal of the benzoate esters with
magnesium methoxide,41 thereby avoiding potential problems of O→N benzoate migration
during deprotection. The excellent equatorial selectivity of the radical C-C bond forming step is
consistent with the precedent for such reactions at the 4-position of glucopyranosides.42,43
OH
Ph
O
O
O
HO
HO
O
XO
XHN
NHTFA
OX
XHN
TFAHN
O
O
O
NHX
NHTFA
O
O
HO
XO
NHTFA
O
OH
XHN
OH
O
OX
O
OH
OX
O
O
ii)
OH NHX
OX NHTFA
1
22
: X = H
: X = H
iii)
i)
21
23
1
)
: X = TFA
: X = Bz, ~100% (from
I
OX
O
OBz
O
HO
BzO
BzO
NHTFA
NHTFA
TFAHN
O
TFAHN
O
O
iv)
NHTFA
vi), vii)
NHTFA
O
O
BzO
BzO
NHTFA
O
OBz
OBz
NHTFA
OBz
OBz
O
OBz
O
O
OBz
O
BzO
v)
NHTFA
BzO
NHTFA
24
: X = H, 94%
26
, 39%
25
: X = Bz, 95%
OH
O
OBz
O
viii)
X
HO
BzO
NH2
NHTFA
H2N
TFAHN
O
O
NH2
NHTFA
O
O
HO
BzO
NHTFA
O
O
OH
OBz
NH2
OH
O
OBz
O
OH
OBz
O
O
x), xi)
OH NH2
BzO
NHTFA
27
28
: X = CH2CH=CH2, 92%
: X = CH2CH2CH3, 53%
5
28
)
: 64% (from
ix)
PhO2S
SO2Ph
PhO2S
SO2Ph
30
29
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Scheme 2. Synthesis of the 4′-C-alkyl paromomycin derivative 5 and structures of the byproducts 29 and
30. i) (CF3CO)2O; ii) PhCH(OMe)2, CSA; iii) Bz2O, py; iv) AcOH, H2O; v) BzCN, Et3N; vi) Tf2O, py; vii) NaI; viii)
PhSO2CH2CH=CH2, Et3B, air, 0 oC; ix) Pd(OH)2, H2; x) Mg(OMe)2; xi) Ba(OH)2.
7
8
9
Gratifyingly, 5 displayed excellent selectivity for bacterial over the humanized ribosomes
(Figure 2) in a series of cell-free translation assays (Table 1) surpassing that of the earlier lead 3
and of the ethylsulfanyl derivative 4, without any significant loss of activity against the bacterial
ribosome. The selectivity of 5 for the bacterial over the mitrochondrial and mutant
mitochondrial ribosomes also exceeds that of the clinical comparators gentamicin, tobramycin,
amikacin and plazomicin (Table 1). Screening against a panel of Gram-positive and Gram-
negative reference strains (Table 2) revealed excellent levels of antibacterial activity for 5,
comparable to and in some cases better than those of the clinical comparators gentamicin,
tobramycin, amikacin, and plazomicin, and even across-the-board improved levels of activity
when compared to the parent paromomycin.
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Table 1. In Vitro Translation Inhibitory Activity (μg.mL-1) and Selectivity.
OH
OH
OH
O
O
O
X
X
X
HO
HO
HO
H2
N
H2
N
H2N
H2
N
H2
N
H2N
O
O
O
NH2
NH2
NH2
O
O
O
HO
HO
HO
O
O
O
OH
OH
OH
H2
N
H2
N
H2N
OH
O
OH
O
OH
O
OH
OH
OH
O
O
O
OH NH2
OH NH2
OH NH2
6, X = SCH2CH2CH3
7, X = SCH(CH3
1, X = OH,
35, X = Et
36, X = CH2CH2OH
37, X = CH2CHOHCH2OH
paromomycin
)
2
2, X = H,
4'-deoxy
8, X = SCH2CH(CH3
)
2
3, X = OEt
4, X = SEt
9, X = SCH2CH2
F
10, X = SOEt (sulfoxide)
11, X = SO2Et (sulfone)
5, X = CH2CH2CH3
IC50 (μg.mL-1)
Mit13 A1555G Cyt 14
50 5.4 9.4
Selectivity
Cmpd 4’-Subs
OH
wt
Mit 13 A1555G Cyt14
1
0.02
2500
270
470
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2
H
0.05
0.08
0.04
0.03
0.07
0.10
0.10
0.06
0.50
8.5
74
24
28
1480
1075
2775
5567
1788
1210
1800
1700
236
480
1188
1150
1733
1571
1250
1500
867
278
21
560
3
OEt
86
95
88
1100
1500
2133
1857
1110
1610
1150
246
4
SEt
111
167
125
121
180
102
118
207
182
31
46
60
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5
CH2CH2CH3
SCH2CH2CH3
SCH(CH3)2
SCH2CH(CH3)2
SCH2CH2F
S(O)Et
52
64
15
16
17
18
19
20
35
36
37
110
125
150
52
130
111
161
69
139
175
129
25
123
136
112
51
SO2Et
24
16
CH2CH3
CH2CH2OH
0.06
0.03
3033
1033
320
2150
833
330
33
1867
1700
590
CH2CHOHCH2OH 0.10
32
33
59
Gentamicin
Tobramycin
Amikacin
0.015
0.02
0.02
0.06
9
0.5
0.5
0.42
2.8
33
600
2200
1350
4500
6983
15
27
750
25
12
90
600
21
Plazomicin
50
419
833
47
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Table 2. Antibacterial Activity Against MRSA and ESKAPE Pathogens (μg.mL-1)
5
6
7
8
9
MRSA
E. coli
K. pneumoniae E. cloacae A. baumannii
P. aeruginosa
AG031c AG220c
>64 >64
Cmpd 4’-Subs
AG038
4
AG042a AG001 AG003b
AG215
AG290
AG225
2-4
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1
OH
>256
8-16
8-16
2-4
2
2-4
nd
8-16
32
1
4
2
4-8
8
2
H
16
nd
>64
32
nd
>64
nd
2-4
32
3
OEt
8-16
4
16
32
8
16-32
4
4
SEt
8
8
2
2-4
0.5-1
2
5
CH2CH2CH3
SCH2CH2CH3
SCH(CH3)2
SCH2CH(CH3)2
SCH2CH2F
S(O)Et
1-2
1-2
4-8
2
1-2
8
1-2
4
0.5
2-4
8
2
8
15
16
17
18
19
20
2
8
32
2-4
2
16-32
4-8
16-32
>64
>64
16-32
4-8
16-32
>128
>64
8
16-32
8
>128
64
nd
64
4
2
8
4-8
64-128
>64
4
4
8-16
>128
>128
128
nd
nd
nd
nd
64-128
>64
64
>128
64
>128
SO2Et
nd
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35
36
37
CH2CH3
8
1
4-8
2
8
2-4
16
1-2
1-2
2
8
4
4
2-4
4-8
1-2
4-8
0.25
0.5-1
1
16-32
64-128
nd
32
16
1
CH2CH2OH
2-4
16
CH2CHOHCH2OH
Gentamicin
Tobramycin
Amikacin
4
8
8
8
8
0.5-1
1
16-32
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0.25
0.5-1
4
64
32-64
4
0.25
0.5-1
1
1
1
4
4
≥256
16-32
2
1
2
2
2
Plazomicin
2
2
2-4
0.25-0.5
0.5
2
2-4
a) strain AG042 carries the AAC(6)’, ANT(4’,4’’) and APH(3’) genes. b) strain AG003 carries the AAC(3) gene. c) strains AG031 and AG220
carry the APH(3’) gene.
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Quantitative dimethyl sulfate footprinting44 of the E coli 70S ribosome in the presence of 5,
paromomycin, and the early hit 3 afforded apparent Kd values of 0.34±0.05, 0.59±0.05, and
1.03±0.20 μM, respectively (Figures 3 and 4), consistent with the trend in IC50 values and
confirming the tight association of the compounds with the decoding A site of helix 44.
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Figure 3. Autoradiogram for DMS footprinting of 5, 1, and 3 binding to the helix 44 region of E. coli 70S
ribosomes. Autoradiogram of reacted rRNA followed by primer extension using a radiolabeled primer is
shown (G, U, C, and A dideoxy sequencing; DMS, dimethyl sulfate; ND, no DMS; 0, 0.1, 0.5, 1, 2.5, 5, 10,
and 20 correspond to the compound concentrations (μM)).
Figure 4. Quantification for DMS footprinting of 5 (DC79), 1 (paromomycin), and 3 (AC3) binding to the
helix 44 region of E. coli 70S ribosomes. Average calculated % protection of N1 of A1408 from three
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trials for 5 and two trials for 1 and 3 was plotted as a function of aminoglycoside concentration and
fitted to a simple binding equation to obtain apparent Kd values of 0.34 ± 0.05 μM (R2=0.99), 0.59 ± 0.05
μM (R2=0.99), and 1.03 ± 0.20 μM (R2=0.99) for 5 (DC79), 1 (paromomycin), and 3 (AC3), respectively.
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Confirmation of the Bacterial Decoding A Site as Drug Target
To support the hypothesis that the antibacterial activity of propylamycin 5 arises from its
interaction with the bacterial decoding A site we tested for activity against the wild type Gram
positive eubacterium Mycobacterium smegmatis and a single allelic derivative with the A1408G
mutation obtained by site-directed mutagenis and RecA-mediated gene conversion (Table
3).34,45-48 The A1408G mutation disrupts the canonical pseudobase interaction of ring I of the 2-
deoxystreptamine class of AGAs with A1408 in the drug binding pocket and results in a
significant loss of activity.47 Accordingly, the parent 1 and propylamycin 5 both displayed an
approximately 100-200-fold loss of activity against M. smegmatis in the presence of the A1408
mutation. This loss of activity reveals the activity of 5 to be the result of binding to the
decoding A site, not of an off-target effect, and analogous to that of the parent paromomycin.
As expected mutation A1408G decreases the antibacterial activity of the comparators
gentamicin, tobramycin, amikacin, and plazomicin, which were used as positive controls, to a
greater extent than that of paromomycin or propylamycin. This is because gentamicin,
tobramycin, amikacin and plazomicin all carry an amino group at the 6’-position, whereas
paromomycin and propylamycin are 6’-hydroxy AGAs. In the 6’-amino series the A1408G
mutation results in a repulsive interaction between the base and the AGA with a
correspondingly high loss of activity, whereas in the 6’-hydroxy series the loss of activity is
smaller consistent with the simple loss of the canonical pseudobase interaction.47,49
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Table 3. Antibacterial Activity (μg.mL-1) for Wild-type and A1408G Mutant M. smegmatis.
5
6
7
Wild-type A1408G Activity Lossa
8
9
Paromomycin 1
Propylamycin 5
Gentamicin
Tobramycin
Amikacin
0.12
0.16
0.12
0.12
0.06
0.03
16
133
100-200
>1067
>1067
>2133
>4267
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48
49
50
51
52
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54
55
56
57
58
59
60
16-32
>128
>128
>128
>128
Plazomicin
a) Activity Loss = MIC Mutant/MIC wild-type
X-Ray Crystal Structure of Propylamycin 5 in Complex with the Bacterial Ribosome
To unambiguously identify the mode of binding of the 4’-deoxy-4’-propyl derivative of
paromomycin, 5, to its target, we solved the crystal structure of the Thermus thermophilus 70S
ribosome associated with mRNA, A-, P- and E-site tRNAs and 5 at 2.75 Å resolution (Supporting
Information Table S1). In this study we used deacylated valine-specific tRNA as the A-site
substrate and initiator methionine-specific tRNA as the P-site substrate. The E site of the
ribosome contained tRNAVal. The difference electron density maps (Fobs – Fcalc) were used to
localize the antibiotic on the ribosome. A strong peak of positive electron density (Figure 5)
resembling distinct features of 5 was observed in the decoding site at the top of the helix 44 of
the 16S rRNA in both copies of the ribosome in the asymmetric unit. Atomic models of the
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ribosome-bound 5, generated from its chemical structure and the restraints based on idealized
5
6
7
3D geometry were used to fit the drug into the observed electron density (Figure 5).
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Figure 5. Unbiased Fo-Fc (A) and 2Fo-Fc (B) electron density maps of 5 in complex with the T.
thermophilus 70S ribosome. The refined model of 5 is displayed in its electron density maps before and
after refinement, respectively. Carbon atoms are colored yellow, nitrogen atoms are blue, and oxygen
atoms are red. Key chemical moieties of the drug are indicated.
The structure reveals that 5 binds to the 70S ribosome in the canonical AGA binding site located
4,50
in the decoding region of the small ribosomal subunit
(Figure 6A and B) with bases A1492
and A1493 of the 16S rRNA in the flipped out conformation. The hydroxyl group at position 3’
of ring I forms a hydrogen bond with the phosphate groups of A1492 and A1493, thereby
further stabilizing the location of ring I. Ring II of 5 forms hydrogen bonds with G1494 and
U1495 as well as with the phosphate groups of A1493 and G1494. Rings III and IV of 5 are
oriented towards base pairs 1409–1491 and 1410–1490, enabling the hydroxyl group at
position 5’’ of ring III to contact N7 of G1491. The newly appended propyl group of 5 makes no
direct contact with the ribosome but, like the alkyl group of the 4’-O-alkyl series of AGAs,49
extends into a highly hydrated area of the ribosome close to the backbone phosphates of
G1491 and A1492. As with all other 4,5-DOS AGAs, ring I of 5 stacks upon G1491 and forms a
pseudo base pair with A1408 (Figure 6 C,D).4,49,51 In addition to the primary site of action of 5,
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electron density peaks corresponding to this compound were observed in two additional
locations on the large ribosomal subunit. Unlike the primary binding site in the decoding
region, these two secondary sites are far from any known ribosome functional centers and are
likely functionally irrelevant, because there are no known mutations around those secondary
sites that can confer resistance to AGA antibiotics.
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Figure 6. Overview of the propylamycin 5 (CMP5) binding sites (yellow) on the T. thermophilus 30S
subunit (A) and 70S ribosome (B). The 30S and 50S subunits are, respectively, light and dark grey.
mRNA is shown in blue and tRNAs are displayed in green for the A site, in magenta for the P site, and in
orange for the E site. In (A), the 30S subunit is viewed from the subunit interface, as indicated by the
inset; the 50S subunit and parts of tRNAs are removed for clarity. (C, D) Close-up views of the AGA
canonical binding site showing the interactions of 5 with the 16S rRNA.
Rationale for Activity and Selectivity of Propylamycin and Structure-Activity Relation at the
Target Level
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We hypothesize that the increased activity of the 4’-deoxy-4’-C-propyl paromomycin derivative
5 as compared to the 4’-O-ethyl derivative 3 and to paromomycin arises from the influence of
the modification on the strength of the pseudobase pair interaction with A1408. Thus, by
analogy with the influence of β-bonds on the pKa values of amines,52 replacement of the 4’-C-O
bond in 3 by a C-C bond results in enhanced basicity of the ring oxygen (O5’) in ring I rendering
it a better acceptor for the hydrogen bond from N6 of A1408. This also accounts for the greater
activity of 4’-deoxy paromomycin 2 over the 4’-O-ethyl derivative 3. The increased activity of 5
over 2 is necessarily due to the presence of the hydrophobic propyl group at the 4’-position,
possibly retarding dissociation of the complex and/or interfering with the hydration shell.
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In comparison to paromomycin, propylamycin 5 affords a significant increase in selectivity for
the bacterial over the cytoplasmic ribosome. The same modification affords an increase in
selectivity for the bacterial over the mutant mitochondrial ribosome for which the structural
basis has yet to be determined.
We synthesized a small series of cognate derivatives to further explore the space available to
the 4’-substituent and challenge our hypotheses. Specifically, compounds were designed to
test the influence of length and branching on the alkyl group as well as the effects of electron-
withdrawing groups and increased hydrophilicity. Working first in the alkylsulfanyl series − for
ease of synthesis − we prepared three analogs of 4 with variation in the length and branching of
the substituent (15-17, Scheme 1). Cell-free translation assays of these compounds with the
wild-type bacterial ribosome revealed that each of these modifications is accompanied by a
two- to three-fold reduction in inhibition of luciferase production (Table 1). Similar reductions
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in the inhibition of the humanized ribosomes were also observed, resulting in an overall
5
6
7
comparable selectivity to that seen with the ethylthio derivative 4.
8
9
The availability of 27, a key intermediate in the synthesis of 5, in multigram amounts enabled
the formation for the further 4′-deoxy-4′-alkyl derivatives by standard manipulations of the
alkene (Scheme 3). Thus, ozonolysis of the alkene 27 followed by reduction with sodium
borohydride gave the alcohol 31, which, on treatment with iodine, triphenylphosphine and
imidazole afforded the iodide 32. Hydrogenolysis of 32 over palladium on charcoal in the
presence of triethylamine then gave the derivative 33. Treatment of 27 with N-
methylmorpholine N-oxide and catalytic osmium tetroxide afforded the diol 34 as an
inseparable mixture of diastereomers. Exposure of each of 33, 31, and 34 to magnesium
methoxide and then barium hydroxide followed by Sephadex chromatography afforded the
paromomycin derivatives 35-36, respectively.
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OH
OBz
OBz
O
O
HO
X
BzO
BzO
NHTFA
NHTFA
TFAHN
TFAHN
O
O
O
NHTFA
NHTFA
O
BzO
NHTFA
BzO
NHTFA
O
O
OBz
OBz
OBz
O
OBz
O
vii)
OBz
OBz
O
O
8
9
BzO
NHTFA
BzO
NHTFA
34
: 49%
27
31
32
33
: X = CH2CH=CH2, 92%
: X = CH2CH2OH, 26%
: X = CH2CH2I, 51%
: X = CH2CH3, 86%
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i), ii)
iii)
iv)
v), vi)
v), vi)
OH
O
OH
O
HO
HO
HO
HO
NH2
NH2
H2N
O
H2N
O
O
O
O
O
NH2
NH2
HO
OH
OH
NH2
NH2
OH
O
OH
O
OH
OH
O
O
OH NH2
35
OH NH2
: 58%
36
: 66%
OH
OH
O
HO
HO
NH2
v), vi)
H2N
O
O
NH2
HO
O
OH
NH2
OH
O
OH
O
OH NH2
37
: 74%
Scheme 3. Synthesis of the 4′-C-alkyl paromomycin derivatives 35-37. i) O3; ii) NaBH4; iii) PPh3,
imidazole, I2; iv) Pd/C, Et3N, H2; v) Mg(OMe)2; vi) Ba(OH)2; vii) OsO4, NMNO.
The 4’-deoxy-4’-ethyl derivative 35 displayed a minor reduction in antibacterioribosomal
activity compared to its higher homolog 5, which was accompanied by a small loss of selectivity
(Table 1). Overall, it is clear that the optimal chain length is three linear non-hydrogen atoms
from the 4’-position.
The replacement of the optimal ethylthio group in 4 by a 2-fluoroethylthio group with the
intention of increasing hydrophilicity53,54 while maintaining the optimal chain length and shape
provoked a minor decrease in inhibition of the bacterial ribosome and a small decrease in
selectivity. This is consistent with a decrease in basicity of the ring I oxygen on incorporation of
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the strongly electron-withdrawing fluorine atom and so with the model. Oxidation of the
thioether in 4 to either the corresponding sulfoxides 19 or the sulfone 20 resulted in a dramatic
drop in antiribosomal activity suggesting that the combination of steric bulk with a strongly
electron withdrawing group directly attached to ring I is not tolerated (Table 1). The 4’-deoxy-
4’-(2-hydroxyethyl) derivative 36 had comparable activity to 5 against the bacterial ribosome,
but significantly lower selectivity, whereas the 4’-deoxy-4’-(2,3-dihydroxypropyl) derivative 37
displayed reduced activity and lower selectivity (Table 1), indicating that increased
hydrophilicity of the 4’-substituent is detrimental.
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Overall, the optimum combination of high activity for the bacterial ribosome and high
selectivity over the humanized ribosomes is found in the 4’-deoxy-4’-propyl derivative 5. The
inhibitory effect on translation is affected by either lengthening or shortening of the three-
atom backbone of the 4’-substituent, by the incorporation of branching, and by hydroxylation
of the substituent as it results in a significant loss of selectivity.
Antibacterial Activity in the Presence and Absence of Specific Resistance Determinants
All compounds were screened for activity against the same panel of reference strains employed
for 5 (Table 2), with results largely consistent with the levels of inhibition of the wild type
bacterial ribosomes.
Finally, 4’-deoxy-4’-C-propylparomomycin 5 was tested for antibacterial activity against a panel
of clinical wild-type E. coli, K. pneumoniae, and E. cloacae clinical isolates resulting in the
distributions shown in Figure 7, and suggesting MIC90s of 2, 1, and 1 μg.mL-1, respectively.
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Figure 7. MIC distribution of 5 for clinical wild-type Enterobacteriaceae isolates
To determine the effectiveness of the novel 4’-modifications in thwarting the action of
resistance determinants, selected compounds were screened for activity against a panel of
wild-type and engineered E. coli carrying specific AMEs or RMTs (Table 4). While it is not
surprising that the replacement of the 4’-hydroxy group in the parent paromomycin restores
activity in the presence of ANT(4’,4’’) enzymes, it is of note that the 4’-C-propyl modification
affords continued high levels of activity in the presence of the APH(3’). Indeed, the ability to
circumvent the action of the APH(3’) obviates the need for modification at the 3’-position. The
activity of 4’-deoxy-4’-C-propyl paromomycin 5 is also not affected by the presence of the AMEs
AAC(3)-I, AAC(2’) and AAC(6’), consistent with the known resistome of the 4,5-AGAs19 It is also
of note that the structure of propylamycin 5 is such that it is not susceptible to the ANT(2’’)
class of AMEs, which are the primary cause of resistance to the 4,6-AGAs in clinical use in North
America.28 Importantly, the 4’-deoxy-4’-C-propyl modification does not interfere with the
inherent resilience of the 4,5-AGAs to the ArmA ribosomal methyltransferase resistance
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mechanism that severely limits the action of all 4,6-DOS AGAs including the newly introduced
plazomicin.6,7,26,27 Therefore, propylamycin will not suffer from problems of cross-resistance
when used in combination therapy with carbapenems, unlike the 4,6-AGA plazomicin. This
latter conclusion is borne out by a final set of screens in which a series of clinical isolates
carrying two or more resistance determinants (AMEs and/or Rmts) were challenged with
propylamycin 5 and the clinical comparators (Table 5).
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The ability of the 4’-C-propyl modification to overcome the influence of the AAC(2’) resistance
mechanism was further demonstrated in Mycobacterium abscessus. Thus, the MIC of 5 was
either unchanged (4 μg.mL-1) or exhibited only a two-fold reduction (from 4 to 2 μg.mL-1) when
the AAC(2’) gene was deleted from this increasingly problematic mycobacterium that presents
a rapidly increasing threat in hospitalized patients with cystic fibrosis or chronic pulmonary
disease.55 In contrast, the MIC of the parent paromomycin was reduced from 64 to 16 μg.mL-1.
The antimycobacterial activity of 5 was further explored with clinical isolates of Mycobacterium
tuberculosis which showed MICs of 1 μg.mL-1. As the parent paromomycin had MICs of 2-4
μg.mL-1 the beneficial effects of the 4’-deoxy-4’-C-propyl modification potentially extend to the
treatment of Mtb.
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Table 4. Activity in the Presence of Specific Resistance Determinants (μg.mL-1)
Strain
AG006
WT
1-2
AG007 AG105
AG009
AG008
ANT(2’’)
2-4
AG036
AG166
AG103
armA
4
Resistance Mechanism
AAC(3) AAC(2′) AAC(6’)
ANT(4′,4′′) APH(3′)
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1
OH
4
2
4
32-64
8-16
1-2
1
>128
64
4
3
OEt
8-16
1-2
16-32
8-16
2-4
1
16
4
8
≥64
8
4
SEt
4
1
5
Pr
0.5-1
1-2
2
1-2
2-4
4
0.5-1
1
1-2
8
2
15
17
SPr
4
2
2-4
1-2
0.5
4-8
SCH2CH(CH3)2
2
4
2-4
32
1
1-2
8
8
Gentamicin
0.5
32
4
1-2
64-128
32
2
8-16
8
0.5
0.5-1
2
>128
>128
>128
>128
Tobramycin
Amikacin
0.5
16-32
4-8
1
1-2
2
2
1-2
Plazomicin
0.5-1
2
1-2
0.5-1
1
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Table 5. Activity of Propylamycin and Clinical Comparators in the Presence of Multiple Resistance Determinants (μg.mL-1)
5
6
7
8
Isolate
Resistance Mechanism
Propylamycin Gentamicin Tobramycin Amikacin
9
E. coli
2014405344 AAC(3), APH(3’), RmtB
201430298 AAC(3), AAC(6’)
2014251408 AAC(3), AAC(6’)
201441114 AAC(6’), ANT(2’’)
2014310108 AAC(3), AAC(6’), RmtB
201440494 AAC(3), AAC(6’)
2014310114 AAC(3), AAC(6’)
201385168 AAC(3), AAC(6’), RmtC
201333207 AAC(3), AAC(6’), APH(3’)
201435487 AAC(3), AAC(6’)
201430601 AAC(6’), ANT(2’’)
4
>256
128
256
16
>256
256
128
32
>256
8
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45
46
47
48
1
2
64
64
>256
2
1
K. pneumoniae
E. cloacae
1
>256
32
>256
16
0.5
0.25
1
256
>256
64
256
>256
32
8
>256
16
4
0.5
0.5
0.5
64
16
16
32
8
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Efficacy in vivo
5
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In a mouse thigh infection model for E coli 5 displayed a 1.5 log reduction in CFUs compared to
vehicle with a dose of 3 mg/kg similar to that observed with 6 mg of the parent paromomycin,
and with 3 mg/kg of plazomicin (Figure 8a). In a neutropenic mouse E. coli septicemia model a
4 mg/kg dose of 5 reduced the bacterial burden in the blood by approximately 1.5 log units, 1
log more than the parent paromomycin at the same dose level (Figure 8b).
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a)
b)
Figure 8. In-vivo efficacy of 5. a) CFU reduction in a neutropenic mouse thigh infection model. b) CFU
reduction in blood in a mouse septicemia model (mpk = milligrams per kilogram)
Toxicity
Compounds 4 and 5 were screened for cytotoxicity against NIH 3T3 mouse embryonic fibroblast
cells, with both showing only minimal loss of cell viability after 48 and 72 h at up to 2 M
concentrations (Supporting Information, Figure S1).
To assess ototoxicity, 4’-deoxy-4’-C-propyl paromomycin 5 was administered once daily SC to
guinea pigs for 14 days at dose levels of 80 and 100 mg/kg with comparisons to controls (saline)
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and the clinical 4,6-AGA gentamicin (Figure 9, left column). Thresholds of auditory brain stem
responses (ABR) were obtained before and 14 days after the end of treatment at 8, 16 and 32
kHz and the shift in thresholds was calculated as a measure of ototoxicity. At all three
frequencies the threshold shifts for 5 are indistinguishable from the control with a dose of 80
mg/kg and only marginally increased over the control at 100 mg/kg. In contrast, gentamicin
already shows significant threshold shifts at 80 mg/kg, comparable to those with 5 at the more
elevated dose, and displays even higher shifts at the 100 mg/kg dose level. This trend of the
ABR thresholds is consistent with the pattern at the target level with 5 displaying very
significantly greater selectivity for the bacterial ribosome over the mitochondrial, mutant
mitochondrial and cytoplasmic ribosomes.
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The reduced ototoxicity of 5 compared to gentamicin is confirmed by microscopic examination
of the cochlear hair cells (shown here at the higher 100 mg/kg dose level; Figure 9, center
column) which are essentially unchanged from the control even in the basal turn, the most
sensitive target of aminoglycosides, while gentamicin treatment results in extensive damage.
Quantitative assessment of hair cell loss along the entire length of the cochlea confirms this
pattern. Both saline and 100 mg/kg of 5 show no effective loss of inner or outer hair cells,
whereas with gentamicin very high levels of outer hair cell loss are seen already beginning at 6
mm from the apex (Figure 9, right column). We have previously demonstrated by means of an
ex-vivo mouse cochlear ex-plant study that both tobramycin and plazomicin display similar ex-
vivo ototoxicity to gentamicin, consistent with their ribosomal selectivity patterns.27
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Figure 9. Comparative ototoxicity of gentamicin and propylamycin (5). Threshold shifts induced by
treatment as determined by ABR (left column). Representative surface preparations outlining one row
of inner hair cells (IHC) and three rows of outer hair cells (OHC) in sections from the base of the cochlea
(Center column). Quantification of hair cell loss along the entire length of the cochlea (Right column). A:
Control animals injected with saline for 14 days. B: Threshold shifts after treatment with gentamicin at
80 and 100 mg/kg body weight, respectively, for 14 days; surface preparation and hair cell counts are
from the same animal after treatment with 100 mg/kg gentamicin. C: Threshold shifts after treatment
with propylamycin (5) at 80 and 100 mg/kg body weight, respectively, for 14 days; surface preparation
and hair cell counts are from the same animal after treatment with 100 mg/kg propylamycin. N = 3 for
each drug treatment, n = 2 for controls. * significantly different from saline control and from
propylamycin (p < 0.05).
Conclusion
A single modification to the 4,5-DOS-AGA paromomycin, replacing an hydroxyl group at the 4’-
position by an ethylthio group, or optimally by a propyl group, has multiple beneficial effects
including increased ribosomal selectivity, enhanced activity against bacterial pathogens, and
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