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(19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0203693 A1
CHAPARIAN et al.
(54) 4,6-SUBSTITUTED
Aug. 8, 2013
Publication Classi?cation
(51) Int- Cl C07H 15/234
2,5-DIDEOXYSTREPTAMINE
AMINOGLYCOSIDE ANTIBIOTICS
(2006.01)
(72) Inventors: Michael G. CHAPARIAN, Washington Township, MI (US); Michael Brady, Haverhill, MA (US); Scott Moe, Sudbury, MA (US); Babu Rao
(57)
ABSTRACT
R7
A
/ \N
R HN
O
R,
OH
(22) Filed:
(60)
US 2013/0203693 A1
Aug. 8,2013
4,6-SUBSTITUTED 2,5-DIDEOXYSTREPTAMINE
AMINOGLYCOSIDE ANTIBIOTICS CROSS-REFERENCE TO RELATED APPLICATIONS
infections such as Pae and Klebsiella pneumoniae (Kpn), seem Well suited to address this problem if compounds can be created that effectively overcome the mo st clinically relevant mechanisms of AG resistance. In addition to overcoming resistance and increasing potency and spectrum, it is desir
[0001] This application claims priority from Us. provi sional application 61/594,663, ?led Feb. 3, 2012, the entire
contents of Which are incorporated herein by reference.
FIELD OF THE INVENTION
able to improve the therapeutic index, particularly by decreasing the nephrotoxicity and/or ototoxicity.
SUMMARY OF THE INVENTION
[0002]
HZN
OH
[0003]
R3
R2.
R7
R8
A\ N/
HO
RlHN
O
o
OH
R,
HZN
[0005]
4C(:O)Rlo, Wherein
[0018] R10 is chosen from i(Cl-C2O)alkyl, i(C3
easily by plasmids in clinical isolates and inactivate AGs by chemical modi?cation resulting in greatly reduced ribosomal binding. Three general classes of AGME
exist:
4CHi may be replaced With iNi, tWo 4CHi may be replaced by 4C:Ci, and one or
tWo 4CH2i may be replaced by Oi, iSi, iSOi, isozi, 4CECi, a (C3-Clo)car
bocycle or a (C3-C6)heterocycle and
ing in broad, intermediate level resistance to all the AGs. [0010] 3. Ef?ux. Drugs are pumped out of the cell before they can cause cell death. This generally results in broad resistance to all AGs. AGs are affected by both general antibiotic ef?ux pumps and also by AG speci?c pumps.
(C3-C9)heterocycle,
(Cl-C8)alkyl(C3-ClO)car
[0021] R5 is chosen from H, halogen, N3, i(Cl-C4) alkynyl and iNHRSO, wherein R50 is chosen from H,
plasmid mediated.
[0012] Coincident With the emergence of AG resistance is the rapid emergence of a variety of serious gram-negative
noacid;
[0022] R7 is chosen from H, i(Cl-C6)alkyl and hydroxy- (C l -C6)alkyl; [0023] R8 is chosen from i(Cl-C2O)alkyl, i(C3-Clo)
rently marketed and once effective antibiotics (aminoglyco sides and beta-lactams) and thus pose a signi?cant and urgent need for neW or improved antibiotics. Aminoglycosides, hav
US 2013/0203693 A1
Aug. 8,2013
[0024]
[0036]
and (C l-C6)alkyl;
[0025] in said (Cl-C2O)alkyl or in the (Cl-C8)alkyl portion of said (C1-C8)alkyl(C3-Clo)carbocycle or
(C1-C8)all<yl(C3 -C9)heterocycle, one or tWo iCHi
embodiments, R1 is iC(:O)R1O.
[0037] In some embodiments of the invention, R10 is (C1 C2O)alkyl. In other embodiments of the invention, R10 is (C3
C 1O)carbocycle. In some embodiments of the invention, R10 is
a (C3-C9)heterocycle. In some embodiments of the invention, R10 is a i(C1-C8)alkyl(C3-C1O)carbocycle. In still other
may be replaced With iNi, tWo 4CHi may be replaced by 4C:Ci, and one or tWo iCHZi may
cycle; and
embodiments of the invention, R10 is a i(Cl-C8)alkyl(C3 C9)heterocycle. In some embodiments of the invention, in the
(C3-C9)heterocycle,
(Cl-Cs)alkyl(C3-Clo)car
i(C3-C9)heterocycle, i(Cl-C8)alkyl(C3-Clo)carbocycle,
or i(Cl-C8)alkyl(C3-C9)heterocycle additionally substi
tuted With one, tWo or three substituents. In some embodi
%(:O)Rlo. In some ofthese embodiments, R10 is (Cl-C15) alkyl. In other embodiments of the invention, R10 is (C3-C6)
carbocycle. In some embodiments of the invention, R10 is a (C3-C5)heterocycle. In some embodiments of the invention, R10 is a i(Cl-C3)alkyl(C3-C6)carbocycle. In still other embodiments of the invention, R10 is a i(Cl-C3)alkyl(C3 C5)heterocycle. As above, in some embodiments of the inven
pound described above. [0031] In another aspect, the invention relates to pharma ceutical compositions comprising a pharmaceutically accept
able carrier and a compound described above.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In one embodiment, the invention relates to a com
tion, in the alkyl portion ofR1O [that is, when R10 is (Cl-C15) alkyl, i(Cl-C3)alkyl(C3-C6)carbocycle, or i(Cl-C3)alkyl
(C3-C5)heterocycle], one or tWo 4CHi may be replaced With iNi. In other embodiments of the invention in which
R10 contains an alkyl portion, tWo 4CHi may be replaced by 4C:Ci. In still other embodiments in which R10 con tains an alkyl portion, one or tWo 4CH2i may be replaced
C6)carbocycle,
or
i(Cl-C3)alkyl(C3-C5)heterocycle
HZN
/
additionally substituted With one, tWo or three sub stituents. In some embodiments, these substituents are chosen indepen
Rs
HO
R HN
0 OH
R5
CONH2, iNHC(:NH)NH2, iCN or halogen. In yet other embodiments of the invention, R10 is selected from optionally
substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyrrole, imidaZole, furan, tetrahydrofuran, piperi
dine, imidaZolylmethyl and (Cl-C1O)alkyl. In other embodi
ments, R10 is (Cl-C1O)alkyl in Which one or tWo 4CHi may
be replaced With iNi. In still other embodiments, tWo 4CHi may be replaced by 4C:Ci, and one or tWo
[0033]
[0034]
4C(:O)Rlo is 4-amino-2-hydroxybutyryl.
US 2013/0203693 A1
Aug. 8,2013
[0039] In some embodiments, n is Zero. In other embodi ments, n is one. In other embodiments, n is tWo. In other
embodiments, n is three. In other embodiments, n is four. In other embodiments, n is ?ve. In other embodiments, n is six.
[0040]
[0041]
i(C:O)i. In other embodiments of the invention, A is 4C(:O)Oi. In still other embodiments of the invention, A is iNH(C:O)i. In yet other embodiments of the inven tion, A is i(C:O)NHi. In some embodiments of the invention, A is iNH(C:O)NHi. In some embodiments of the invention, A is i(C:S)NHi. In other embodiments of the invention, A is iNH(C:S)i. In some embodiments of the invention, A is iNH(C:S)NHi. [0050] In some embodiments of the invention, R7 and RSA, taken together With the nitrogen to Which they are attached,
form a (C3-C9)heterocycle. In some of these embodiments,
embodiments, R13 is optionally substituted phenyl. In some embodiments, R13 is optionally substituted 5- or 6-membered
the (C3-C9)heterocycle is optionally substituted With one, tWo or three substituents chosen independently from CH3,
ring heterocycle.
[0043] In some embodiments, R5 is H. In some embodi
bocycle. In some embodiments of the invention, R50 is (C3 C9)heterocycle. In some embodiments of the invention, R50 is i(C l-C8)alkyl(C3-C 1O)carbocycle. In some embodiments of the invention, R50 is i(C1-C8)alkyl(C3-C9)heterocycle. In some embodiments of the invention, R50 is the deshydroxy
residue of an aminoacid. In some embodiments, R50 is
ents chosen independently from CH3, iCH2CH3, iOH, %H2OH, iNHz, %H2NH2, iCOOH, :O, iNH
CONH2, iNHC(:NH)NH2, 4CN and halogen. In other embodiments, R7 and RSA, taken together With the nitrogen
to Which they are attached, form a piperidine, piperaZine, tetrahydropyrimidine or pyrrolidine, any of Which are option
ally substituted With CH3, 4CH2CH3, iOH, 4CH2OH, iNHz, %H2NH2, %OOH, :O, iNHCONHz, iNHC
(:NH)NH2, 4CN or halogen.
[0052] In some embodiments, A is a direct bond and R7 and
(Cl-C6)alkyl.
[0053] In some embodiments of the invention, R7 is H; A is chosen from a direct bond, i(C:O)i, iC(:O)Oi, and
alkyl.
[0046] In some embodiments of the invention, R8 is (C1 C2O)alkyl. In some embodiments of the invention, R8 is (C3 C 1O)carbocycle. In some embodiments of the invention, R8 is (C3-C9)heterocycle. In some embodiments of the invention, R8 is i(Cl-C8)alkyl(C3-Clo)carbocycle. In some embodi ments of the invention, R8 is i(Cl-C8)alkyl(C3-C9)hetero cycle. In some embodiments of the invention, R8 is C(:NH) NH2. In some embodiments of the invention, R8 is NRSORSI. In some embodiments of the invention, in the alkyl portion of
embodiments of the invention in Which R8 contains an alkyl portion, tWo 4CHi may be replaced by 4C:Ci. In still
other embodiments in Which R8 contains an alkyl portion, one
chosen independently from CH3, iCH2CH3, iOH, %H2OH, iNHZ, %OOH, :O, iNHCONH2, iNHC (:NH)NH2, and halogen.
[0054] In some embodiments, R7 is H; A is a direct bond;
chosen independently from CH3, iCH2CH3, iOH, iCHZOH, iNH2, 4CH2NH2, iCOOH, :O, iNH
CONH2, iNHC(:NH)NH2, iCN or halogen.
[0047] [0048] In some embodiments of the invention, R80 is H. In In some embodiments of the invention, R81 is H. In
NH2.
[0055] In some embodiments of the invention, R7 is H; A is chosen from a direct bond and i(C:O)i; and R8 is chosen
US 2013/0203693 A1
Aug. 8,2013
iCH2CH3, ADH, %H2OH, iNHz, %OOH, :O, iNHCONHZ, iNHC(:NH)NH2, and halogen.
[0056] In some embodiments of the invention, R7 is H; A is chosen from a direct bond, i(C:O)i, iC(:O)Oi, and iNH(C:O)i; and R8 is (C1-Cl5)alkyl. In some of these
embodiments, one or tWo iCHi of the (Cl-C15)alkyl may
through sulfur.
[0064] Aryl and heteroaryl mean a 5- or 6-membered aro
matic or heteroaromatic ring containing 0-3 heteroatoms selected from O, N, or S; a bicyclic 9- or l0-membered aromatic or heteroaromatic ring system containing 0-3 het
eroatoms selected from O, N, or S; or a tricyclic 13- or l4-membered aromatic or heteroaromatic ring system con
iCH3, iCH2CH3, ADH, iCHzOH, iNHZ, iCOOH, :O, iNHCONHZ, iNHC(:NH)NH2, and halogen.
[0057] In some embodiments of the invention, R7 is H; A is
matic 6- to l4-membered carbocyclic rings include, e.g., benZene, naphthalene, indane, tetralin, and ?uorene and the 5- to l0-membered aromatic heterocyclic rings include, e. g.,
iOH, iNH2, and iNHC(:NH)NH2. In some of these embodiments, R8 is aminopropyl. In some of these embodi ments, R5 is ?uorine. In some of these embodiments,
imidaZole, pyridine, indole, thiophene, benZopyranone, thia Zole, furan, benZimidaZole, quinoline, isoquinoline, quinoxa
line, pyrimidine, pyraZine, tetraZole and pyraZole. As used
herein aryl and heteroaryl refer to residues in Which one or more rings are aromatic, but not all need be. [0065] Arylalkyl as a substituent means an aryl ring attached to the parent structure via an alkyl residue. Examples are benZyl, phenethyl and the like. Heteroarylalkyl means a
i(C:O)RlO is 4-amino-2-hydroxybutyryl.
[0058] In some embodiments of the invention, R10 is
elemental constituents and includes alkyl, cycloalkyl, poly cycloalkyl, alkenyl, alkynyl, aryl and combinations thereof.
ments, R5 is chosen from H, ?uorine, N3 and iNHRSO, and R50 is chosen from H, cyclopropyl, cyclopropylmethyl, pyr
rolidinyl, and the deshydroxy residue of citrulline or serine.
of heterocycles. Examples of heterocycles that fall Within the scope of the invention include pyrrolidine, pyraZole, pyrrole,
[0061] Alkyl is intended to include linear and branched hydrocarbon structures. A combination of alkyl With
cycloalkyl such as i(Cl -C8)alkyl(C3-C 1O)carbocycle Would be, for example, cyclopropylmethyl. LoWer alkyl refers to
alkyl groups of from 1 to 6 carbon atoms. Examples of loWer
[0070]
may be used interchangeably With unsubstituted or substi tuted. The term substituted refers to the replacement of
one or more hydrogen atoms in a speci?ed group With a
replaced by ?uorine; indeed, all available hydrogen atoms could be replaced by ?uorine.
US 2013/0203693 A1
Aug. 8,2013
acid fragments.
[0074] The term amino acid as used herein refers to the
metric asymmetry, and unless speci?ed otherWise, it is intended that the compounds include both E and Z geometric
isomers. Likewise, all tautomeric forms are also intended to be included. [0072] Substituents R are generally de?ned When intro
arginine, citrulline, cysteine, glutamic acid, glutamine, gly cine, histidine, isoleucine, leucine, lysine, methionine, phe nylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, sarcosine, norvaline, norleucine, homoserine, allo threonine, hydroxynorvaline, statine, hydroxyproline, orni thine, 2-aminoadipic acid, penicillamine, homocysteine,
S-methylcysteine, ethionine and phenylglycine. For the pur
pose of this invention, the term amino acid also includes a
duced and retain that de?nition throughout the speci?cation and in all independent claims.
[0073] The term residue of an amino acid as used herein refers to an amino acid (as de?ned beloW) minus one
hydroxyl or carboxyl that is considered part of the linkage to the parent aminoglycoside scaffold. When the residue of the amino acid is deshydroxy, it Will be minus the hydroxyl of the acid function. If the amino acid has tWo carboxyls (e.g. glutamic acid) the hydroxy can be from either carboxylic acid. When the residue of the amino acid is descarboxy,
it Will be minus 4COOH; if the amino acid has tWo car
unless expressly further limitediis intended to include salts of that compound. Thus, for example, the recitation a com pound of formula I as depicted above, in which R1 is 4C(:NH)NH2, Would include salts in which R1 is 4C(:NH)NH3+X_, Wherein X is any counterion. In a par ticular embodiment, the term compound of formula I refers to the compound or a pharrnaceutically acceptable salt thereof. [0076] The compounds of the invention may be present as
late), benZoate, bicarbonate, bisulfate, carbonate, camphor sulfonate, citrate, ethanesulfonate, fumarate, gluconate, glutamate, glycolate, bromide, chloride, isethionate, lactate, maleate, malate, mandelate, methanesulfonate, mucate, nitrate, pamoate, pantothenate, phosphate, succinate, sulfate,
tartrate, tri?uoroacetate, p-toluenesulfonate, acetamidoben
NH NH 0 0
HO
enecitrate, ascorbate, aspartate, calcium edetate, camphorate, camsylate, caprate, caproate, caprylate, cinnamate, cycla mate, dichloroacetate, edetate (EDTA), edisylate, embonate,
HZN
the circled residue R10 is the descarboxy residue of the natural amino acid serine. Similarly in the molecule:
estolate, esylate, ?uoride, formate, gentisate, gluceptate, glu curonate, glycerophosphate, glycolate, glycollylarsanilate, hexylresorcinate, hippurate, hydroxynaphthoate, iodide, lac tobionate, malonate, mesylate, napadisylate, napsylate, nico tinate, oleate, orotate, oxalate, oxoglutarate, palmitate, pecti nate, pectinate polymer, phenylethylbarbiturate, picrate, pidolate, propionate, rhodanide, salicylate, sebacate, stearate,
tannate, theoclate, tosylate and the like. Although pharma
ceutically acceptable counter ions Will be preferred for pre paring pharmaceutical formulations, other anions are quite acceptable as synthetic intermediates. That is, pharmaceuti cally undesirable anions, such as iodide, oxalate, tri?uo
romethanesulfonate and the like, may be present When such
salts are chemical intermediates.
[0077] It Will be recogniZed that the compounds of this invention can exist in radiolabeled form, i.e., the compounds
may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number
usually found in nature. Radioisotopes of hydrogen, carbon, phosphorous, ?uorine, and chlorine include 2H, 3 H, 13C, 14C,
15 N, 3 5 S, 1 8F, and 3 6Cl, respectively. Compounds that contain
the circled residue R50 is the deshydroxy residue of the natu ral amino acid citrulline. These and similar structures of amino acids that lack a functional group at the point of attach
those radioisotopes and/or other radioisotopes of other atoms are Within the scope of this invention. Among the isotopically altered compounds of the invention, deuterated, i.e. 2H, com pounds are of particular interest. Selective incorporation of
US 2013/0203693 A1
Aug. 8,2013
deuterium in place of hydrogen (deuteration) has the unique effect of retaining the biochemical potency and selectivity of
7,514,068; 7,608,737; 7,678,914 and others.] Tritiated, ie 3H, and carbon-14, i.e., l4C, radioisotopes are in certain cir
cumstances preferred for their ease in preparation and detect
Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled
reagent for a non-radiolabeled reagent.
NH2 ~57iN\"/NH2
0H AHB H G NH
[0078] Although this invention is susceptible to embodi ment in many different forms, preferred embodiments of the
invention are shoWn. It should be understood, hoWever, that the present disclosure is to be considered as an exempli?ca tion of the principles of this invention and is not intended to limit the invention to the embodiments illustrated. It may be found upon examination that certain members of the claimed genus are not patentable to the inventors in this application. In
~iiN\/\/NHZ ii A
N H PDA oH H CPA H N
iiiNQVNHZ jgi L}
N H 2-OH PDA O 3_Ap
2i \/\ NHZ
EDA OH ISO
NHZ.
[0084]
Reagent?ert-butoxy-bis-(dimethylamino)
est require protection and deprotection during the synthesis procedure. Terminology related to protecting, deprotect
ing and protected functionalities occurs throughout this application. Such terminology is Well understood by persons
of skill in the art and is used in the context of processes Which involve sequential treatment With a series of reagents. In that
context, a protecting group refers to a group Which is used to
Bu:butyl
DCC:dicyclohexyl carbodiimide
mask a functionality during a process step in Which it Would otherWise react, but in Which reaction is undesirable. The protecting group prevents reaction at that step, but may be
HOBt:hydroxybenZotriaZole HOSU:N-hydroxysuccinimide
[0081] LiHMDSIIithium hexamethyldisilaZide MCPBAImeta-ChloroperoxybenZoic Acid
is beloW, the person of ordinary skill can readily envision those groups that Would be suitable as protecting groups.
Suitable groups for that purpose are discussed in standard
Me:methyl
MICIminimum inhibitory concentration MMP:matrix metalloproteinase NaH:sodium hydride
in Organic Synthesis by T. W. Greene [John Wiley & Sons, NeW York, 1991], Which is incorporated herein by reference. [0085] While it may be possible for the compounds of
formula (I) to be administered as the raW chemical, it is
preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of
US 2013/0203693 A1
Aug. 8,2013
formula (I) or a pharmaceutically acceptable salt or solvate thereof, together With one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredi ents. The carrier(s) must be acceptable in the sense of being
[0086]
cant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the poWdered compound moistened With an inert liquid diluent. The tablets may optionally be
coated or scored and may be formulated so as to provide
[0089]
positions, oral dosage forms and topical pharmaceutical com positions are preferred. Tablets, capsules, intraocular topical
formulations and parenteral solutions are common among
aqueous and non-aqueous sterile injection solutions Which may contain anti-oxidants, buffers, bacteriostats and solutes Which render the formulation isotonic With the blood of the
aminoglycosides. The formulations may conveniently be pre sented in unit dosage form and may be prepared by any of the
methods Well knoWn in the art of pharmacy. All methods include the step of bringing into association a compound of formula (I) or a pharmaceutically acceptable salt or solvate
example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use. Extemporaneous injection solu tions and suspensions may be prepared from sterile poWders, granules and tablets of the kind previously described.
[0090] Preferred unit dosage formulations are those con taining an effective dose, as hereinbeloW recited, or an appro
shaping the product into the desired formulation. [0087] Formulations of the present invention suitable for
oral administration may be presented as discrete units such as
?avoring agents.
[0092] Table 1 (below) presents representative members of
the genus of the invention:
[0088]
Substituents
Example #
l
ID Code
SXPl2l2-56"7
R1
R5
F
R2
OH
1B
OH
H
R8iAiN(R7)i
lllHZ E
NH2
ENWNTNHZ
O O
lllHz
E H
N\H/ NH2
0 NH
US 2013/0203693 A1
Aug. 8, 2013
-continued
Substituents
Example #
23
ID Code
SXP1212-56"-41 O
R1
R5
F
R2
OH
16'
OH
R8iAiN(R7)i
NHZ
NH; NH
24
SXP1212-56"-42
OH
OH
NH;
25 SXP1212-56"-43 O F OH OH
N/\/\NH2 H
NH;
N C /\ N
H
26
2554
O
NH
NH2 H
LNH
2
F NH2 H
>{N
H
27
2523
NH;
28 2524 0 F NH2 H
>N/\/\NH2
NMNHZ
H OH
NH;
29 2525 O F NH2 H
NH
H
NH2
[0093] For evaluation of ef?cacy, We assembled panels of
%N
TABLE 2
MIC values of reference AGs against indicator strains.
representative
gram-negative
pathogens,
including
MIC (ug/mL)
Standard AG PAWT KPWT ABWT ECWT
Pseudomonas aeruginosa (Pae, PAWT, ATCC #27853), Klebsiella pneumoniae (Kpn, KPWT, ATCC #700603), Acinelobacler baumannii (Aba, ABWT, ATCC #BAA-747)
and Escherichia coli (Eco, ECWT, ATCC #25922). These
reference strains are useful indicator organisms to character
tobralnycin sisomycin
dibekacin arbekacin
0.25
0.25-
8 1-2
16 0.25 0.5
0.25
0.125-
l
0.25
0.5
0.25 0.5
0.25
0.25 0.5
0.5
l-2 l
gentalnycin
alnikacin
l
l-2
8
l
0.25-0.5
1
0.5
2-4
l6 l6 32
l-2 l6 4-8
4 2 l
US 2013/0203693 A1
11
Aug. 8,2013
TABLE 2-continued
MIC values of reference AGs against indicator strains.
ug/mL. This re?ects the dif?culty in treating clinical Pseudomonas aeruginosa infections. Aminoglycoside and
[3-lactam pro?ling shoWed the presence of most all represen
ECWT
MIC (ug/mL)
Standard AG PAWT KPWT ABWT
tative aminoglycoside modifying enzymes, e?llux and [3-lac tamase mechanisms of resistance. Using this stringent panel,
We tested a subset of compounds of the invention (Table 4).
TABLE 4
kanalnycin A spectinomycin
>64 >64
32 64
1 16
4 8-16
MIC (ug/mL)
Pseudomonas aeruginosa
SXP #
SXP1212-56"-7 SXP1212-56"-8 SXP1212-56"-16 2523 2524 2554 2525
PR1
16 8 2-8 1-2 4 2 8
PR2
16 8 <0.25-4 0.5 0.5 0.5 1
P16
128 64 32 4 8 8 8
MIC90
4 4 4 4
third clinical isolate (PR3) possessing high-level, e?llux-me diated, pan-aminoglycoside resistance (impermeable). For
each strain and antibiotic, the mechanism(s) of resistance Was inferred from the resistance pro?le. The presence of speci?c AGMEs Was con?rmed by colony PCR.
TABLE 3
Potency of reference and test compounds against Pseudomonas aeruginosa
Wild type and resistant strains:
[0095] In general, MIC9O values Were Within a single dilu tion of MIC values against PR3, suggesting that PR3 is an
appropriate indicator strain. [0096] Klebsiella pneumoniae (Kpn) and Escherichia coli (Eco) are gram-negative (like Pae), enteric pathogens With increasing clinical relevance. Many clinical isolates of Kpn
and Eco harbor extended-spectrum beta-lactamases and are no longer sensitive to most beta-lactam antibiotics. We
MIC (ug/mL)
PAWT TOB DIB SISO ARB GEN AMK STREP KAN-B NEO KAN-A SPE SXP1212-56"-7 SXP1212-56"-8 SXP1212-56"-16 2523 2554 2524 2525 0.25 0.25-0 5 0.25-0.5 0.5 1 1-2 16 16 32 >64 >64 4 4 1 0.25 0.25 0.25 0.25-0.5 PR1 2 4 4 8 8 8 64 64 32 >64 >64 16 8 2-8 1-2 2 4 8 PR2 >64 >64 >64 8-16 >64 8-16 16 >64 8-16 >64 >64 16 8 <0.25-4 0.5 0.5 05 1 PR3 >64 >64 >64 16 >64 64 64 >64 >64 >64 >64 128 64 32 4 8 8 8
activity against the test panel and only ARB and AMK
shoWed potency against 3 of 4 strains tested. Strain KR3 Was
largely recalcitrant to aminoglycoside exposure (except GEN). Recognizing strain KR3 is largely recalcitrant to AGs,
[0094] Next, We considered Whether the SAR observed against select Pae strains 1, 2 or 3 Was indicative of general potency against Pseudomonas aeruginosa. To assess this, a panel of more than ?fty resistant contemporary clinical iso lates of Pseudomonas aeruginosa Were obtained from mul
sidered Whether the SAR observed against select Klebsiella pneumoniae strains 1, 2 or 3 Was indicative of general
antibiotics including aminoglycosides, [3-lactams and ?uoro quinolones. The MIC9O values for ?fteen antibiotics (amika
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TABLE 5
MIC values against Enterobacteriaceae
KZebsieZZa pneumonia E. coli
of the rats Were aseptically removed, Weighed, homogenized, serially diluted, and plated on MacConkey medium. The
plates Were incubated overnight at 370 C. in 5% CO2. CFU
SXP #
SXP1212-56"-16 2554 2524 2525 2523
KPWT
<0.25 0.125 0.5 0.5 0.5-1
KRl
0.5-1 0.25 0.5-1 1 0.5-1
KR2
1 0.125 0.5 0.5 0.5-1
KM
16 0.25-0.5 0.5-1 1 1
MIC90
0.5 1 1 1
MICg0
2 2-4 2 2
per gram of thigh Was calculated by enumerating the plated colonies then adjusting for serial dilutions and the Weight of the thigh. The folloWing table 7 summarizes the results Table
6: TABLE 7
Rat neutropenic thigh model results
Change
Acinelobacler baumannii is another clinically important and very challenging gram negative pathogen. Members of the
Acinelobacler genus, including baumannii, have a remark
Total Drug
Concentration
Log
CFU/
Change
from 24 hr.
from
T = RX
test article
T = Rx
(mgkg)
i
n
5
thigh
4.18
St. Dev.
0.25
control
controls
able ability to upregulate and acquire resistance determinants. Coupled With its ability to survive for prolonged periods in a hospital environment, A. baumannii is an emerging threat for healthcare institutions globally. We acquired clinical isolates
of Acinelobacler baumannii and used this panel to test com
24 hr
7.26
0.37
3.08
controls
SXP-2523 Amikacin 10.0 60.0 5 5 6.55 5.77 0.44 0.37 0.71 1.49 2.37 1.59
(ABWT). Of the 4,6-di sub stituted-2 -deoxystreptamine refer ence compounds, only arbekacin demonstrated activity (28
ug/mL) against 3 of the 4 indicator strains.
[0098] As in the case of Pseudomonas aeruginosa and Klebsiella pneumoniae, We considered Whether the SAR observed against select Acinelobacler baumannii strains 1, 2 or 3 Was indicative of general potency. The MIC9O values for
It can be seen that SXP-2523 is effective against P aerugi nosa in an appropriate animal model. The relative potency vis-a-vis amikacin cannot be determined from this experi ment because the dose of SXP-2523 Was one-sixth the dose of amikacin. [0100] The compound identi?ed as SXP 2523 Was further tested in vivo against a resistant strain of P aeruginosa.
TWenty male Sprague DaWley rats Were rendered neutropenic by treatment With cyclophosphamide on day 4 and day 1 With 100 mg/kg and 75 mg/kg, respectively. Rats Were infected With R aeruginosa 6294 MLP-3, via injection into
the right thigh muscle of 0.1 mL per rat. TWo hours post
infection rats Were treated intravenously With either SXP 2523 or amikacin in a total dose of 10 or 60 mg/kg, respec
(TIRX). TWenty-four hours post infection; rats Were eutha nized by C02 inhalation. The right thigh muscles of the rats
MIC (ug/rnL)
Acinelobacler baumannii
SXP #
2554 2524 2525 2523
ABWT
0.25 0.5 0.5 0.5
AB1
0.5 2 2-4 2-4
AB2
0.25-0.5 1 1 1
MICg0
2 2 2 4
diluted, and plated on BHI medium. The plates Were incu bated overnight at 370 C. in 5% CO2. CFU per gram ofthigh Was calculated by enumerating the plated colonies then adjusting for serial dilutions and the Weight of the thigh. The folloWing table 8 summarizes the results:
TABLE 8
Rat neutropenic thigh model results against resistant P aeruginosa
[0099]
Change
Log
CFU/
Change
from 24 hr.
from
T = RX
test article
T = Rx
(mgkg)
i
n
5
thigh
3.49
St. Dev.
0.46
control
i
controls
i
MLP-3, via injection into the right thigh muscle of 0.1 mL per
rat. TWo hours post infection rats Were treated intravenously
With either SXP-2523 or amikacin in a total dose of 10 or 60
24 hr
6.65
0.37
3.16
controls
SXP-2523 Amikacin 10.0 60.0 5 5 1.71 3.92 1.13 0.72 4.94 2.73 1.78 0.43
mg/kg, respectively. The test article and control agent Were delivered at 2, 4, and 6 hours post infection. Five rats Were treated With each drug concentration. One group of ?ve rats
Was euthanized at initiation of treatment and thigh CFUs Were
processed (TIRX). TWenty-four hours post infection rats Were euthanized by C02 inhalation. The right thigh muscles
[0101]
US 2013/0203693 A1
Aug. 8,2013
protected With Boc and acetyl groups, then the 5 -position Was converted to 5-F (or to other substituents R5) as described below. The acetyl protecting groups Were then removed With base (eg sodium methoxide) and the 6" hydroxyl Was tosy
[0102] The general procedure for the protection of amines and acetylation of the hydroxyls in the kanamycin amino gly cosides is reported by Shitara et al [Shitara T, Umemura E, Tsuchiya T, Matsuno T Carbohydrate Research 276, 75-89
(1 995)].
[0103] Modi?cation of the R5 hydroxyl group is generally
accomplished as folloWs: For R5 -?uoro compounds, the pro tected aminoglycoside is dissolved in dichloromethane and
logs:
SCHEMEl HN
2 PIN OH
00
HN
BocHN
OAc
OH
0A0
Mon
NH2
o
OH
0 l.Boc2O
,
unuOAc
BocHN
o
0A0
O
2.AC2O
HN
OH HO 0
g
OH A00 0
HO
OH
AcO
0A0
HZN
as.
BocHN
bb
R5 modi?cations
(see text)
B
0
HN
BocHN
OAc
HN
BocHN
OH
OAc
OH
UnOAc
BocHN o 0
0A0
1. NaOMe/MeOH o
Mon
BocHN 0
OH
R5
A00 0
R5
TsO O
A00 BocHN
0A0
HO BocHN
OH
cc
dd
HN 2
HZN 0
\\\\OH
OH OH
OH
NHRI O
1. R
s_
HNR
o HN 2. dlOXin?/Hcl
R5
R8ANR7 o
0
HO
OH
66
US 2013/0203693 A1
Aug. 8,2013
trated. The residue Was thoroughly Washed With Water (10 ml)
and dried in vacuum at 400 C. to give compounds dd
(~60%).
[0108] General procedure for deprotection of Boc groups: Compound dd (200 mg) Was dissolved in dioxane/HCl (10
mL) and stirred at 100 C. for 5-6 hrs. Dioxane Was removed under vacuum and the solid obtained Was Washed With iso
NaHCO3. The reaction is Worked up by Washing the organic layer With aq. NaHCO3, Water, sodium hypochlorite solution, and then Water. The organic layer is dried and then evaporated to provide the R5-epi-?uoro-5R5-desoxy analogs.
[0104] For R5-chloro, bromo and iodo compounds, the pro tected aminoglycoside is dissolved in dichloromethane (20
completion of the reaction, the product is concentrated under vacuum and suspended in Water (20 mL) and extracted With
Antibiotics 1990, 858-872]. Selective guanidinylation of amines Was achieved by reacting protected aminoglycosides With the corresponding equivalents of TfN:C(NHBoc)2. Thus, for example, if a guanidine is desired at R1, compound
h of Scheme 2 or compound n of Scheme 3 beloW can be reacted With TfN:C(NHBoc)2 in the presence of a base such as triethylamine in aqueous solution. [0110] When analogs are desired in which R1 is not AHB,
bromides, the intermediate is dissolved in DMF (10 mL) and NaBr (4 moles) is added. The reaction is re?uxed at 1000 C.
for 18 hours. Work-up of the reaction mixture as above fur
nishes R5-deoxy-R5 -bromo compounds. For iodides, the intermediate is dissolved in DMF (10 mL) and Nal (6 moles)
is added. The reaction is re?uxed at 1000 C. for 18 hours. Work-up of the reaction mixture as above furnishes
[0111] Synthesis of Examples 26-29 is shoWn in schematic form in Schemes 2 and 3.As shoWn in Scheme 2, tobramycin
Was ?rst protected With Boc and acetyl groups, then the
5-position Was converted to 5-F. The protecting groups Were then removed. The 3" and 1 amines Were then chelated With Zn(OAc)2 and the non-chelated amines at 3, 2' and 6' Were protected With Boc groups. The 3"-amine Was protected as its
R5-deoxy-R5-iodo compounds.
[0105] For R5 aZides and amines, the mesitylene sulfonate
intermediate obtained as above is dissolved in DMF (10 mL) and NaN3 (8 moles) is added. The reaction is re?uxed at 1000 C. for 18 hours. Work-up of the reaction mixture as above
tri?uoroacetamide (TFA amide) and the remaining R1 -amine Was coupled With various carboxylic acids, including for
furnishes 5-deoxy-R5 -aZide compounds. The R5 -deoxy-R5 aZide (500 mg) can be dissolved in dry THF (10 mL) and reduced by adding LiAlH4 in THF (5 mL) at 100 C. The
reaction is stirred for 3 hours at room temperature. After the
centrated under vacuum and diethyl ether (20 mL) plus dil.
HCl (20%, 10 mL) are added With stirring at 100 C. The
organic layer is separated and the aqueous layer is Washed With excess diethyl ether (2><25 mL). The combined ether
layers are Washed With brine and died over anhydrous Na2SO4 folloWed by concentration under vacuum to furnish
substitution reaction With primary or secondary amines to form an amide. The term includes esters activated by neigh
R5 -deoxy-R5 -amine compounds. The amine canbe alkylated With the halide of the appropriate (C3-C1O)carbocycle, (C3 C9)heterocycle, (Cl-C8)alkyl(C3-C1O)carbocycle or (Cl-C8)
alkyl(C3-C9)heterocycle. Or the amine can be reacted With an activated ester of an N-Boc-protected aminoacid to provide
boring electron WithdraWing substituents. Examples include esters of phenols, particularly electronegatively substituted
phenol esters such as penta?uorophenol esters; O-esters of isourea, such as arise from interaction With carbodiimides;
O-esters of N-hydroxyimides and N-hydroxy heterocycles; speci?c examples include S-t-butyl esters, S-phenyl esters,
[0106]
conditions
to
provide
5F-tobramycin-NH-AHB-R6"
RSANR7 analogs:
US 2013/0203693 A1
16
Aug. 8,2013
-continued
OH BocHN TFA BocHN OH
OO
BocHN
NHBoc W
BocHN
O O
NHBoc
HZN
HO HO 0 O F OH
HZN
HO O
HZN
g
BocHN BocHN
OH
1) NH3 MeOH
BocHN NHBoc
O O OH BOCHN O
2) E0020
E
HO
HO TFAHN i 0 O
F
OH
BOCHN BocHN
OH
O
O
OH BOCHN o
NHBoc &>
2) OH-resin
g
R8A(R7)N
HO BocHN k 0
F
O
OH H0
[0113]
tobramycin-Zinc complex Was reacted With Boc-anhydride and then ethyl tri?uoroacetate. The remaining Rl position
Was then acylated With AHB-Boc. The 3"-TFA Was cleaved With base, then protected as a Boc group. The R6"-OH Was
The free amines Were protected With Boc, folloWed by selec tive acylation of the hydroxyl groups, leaving the 5-OH
unprotected. The 5-OH Was ?uorodeoxygenated With DAST [(diethylamino)sulfur tri?uoride] or DEOXO-FLUOR to give the epi-F-desoxy intermediate. This material Was then
treated With mesitylene sulfonyl chloride folloWed by dis placement of the mesitylenesulfonate With various amines.
US 2013/0203693 A1
Aug. 8, 2013
-continued
BOCNH BocNH
8 7
OH
BOCNH BocNH
OH
O
-\\OH BOCHN
o o
NHBoc %
MOH BOCHN
o
O
0
NHBoc
g
Mesity1SO2O O
OH
g
R8ANR7 O
OH
BocHN
I
BocHN
S
B0020
BOCNH BocNH
OH
BOCNH BocNH
0A0
NHBOC &
o
NHBOC
0
-\\OH BOCHN
o
-\OACBOCHN
o
g
R8ANR7 O
OH
g
RsANRv o
OH
Howoir
BocHN
t
AcOwOAc
BocHN
u
DEOXOFLUOR
BOCNH
0A0
HZN
OH
BocNH
HZN
o
0
. nOAC BOCHN
NHBOC 1) NH3M6OH
2)HC1
. nOH HZN
o
O
NH2
g
R8ANR7 0
g
RsANRv 0
A00
BocHN
0A0
Howoir
HZN
W
[0114] Tetrahydrofuran (250 mL) Was added to Zn(OAc)2. 2H2O (29.3 g. 133.8 mmol) in a 500 mL Erlenmeyer ?ask,
sWirled for 5 min, and the solid Was collected on a fritted glass Buchner funnel and Washed With another 250 mL of THF. The solid Was completely dried under vacuum for 1 hour at 400 C.
hours (after 20-25 min clear solution Was observed). Et3N (22.5 mL, 160.5 mmol) Was added With stirring, and the initial precipitate that formed dissolved Within 30 min to give a clear solution. Boc-anhydride (46.6 g, 214.0 mmol) Was added to this solution, and the stirring Was continued for 16 h. The progress of the reaction Was monitored by LC-MS, Which shoWed about 10% of Tob-Boc5 as the only impurity. The excess Boc-anhydride Was quenched by the addition of 28% NH4OH (3.5 mL) and stirred for 1 hour. Volatiles Were
US 2013/0203693 A1
Aug. 8,2013
by agitating it With 50 mL of Water for 15 minutes folloWed by collection of the solid by ?ltration. The solid is dried under vacuum overnight to give 6.2 g of 3"-carbamate (q) as free
layers Were evaporated using a Genevacg Rocket Evaporator (45 C., 3 h). The resulting solid Was suspended in methanol (60 mL) and then Water (30 mL) Was added. Again the combined Water and methanol layers Were removed using
temperature for 20 h, until LC/MS-ELSD shoWed that it contained at least 90% of the desired product. The major contaminants Were 5% remaining starting material and 5%
TLC (Ninhydrin) CH2Cl2:MeOH:28% NH4OH, 8.5: 1 .0:0.5) Rf 0.25. The impurity Was removed from the product by
selectively dissolving the product in Water. This Was achieved by stirring the solid With Water (350 mL) for at least 1 hour, folloWed by ?ltration through a fritted glass Buchner funnel. This procedure Was repeated another three times (total of four
Washes) until LC-MS shoWed no product in the residual solid. The combined aqueous layers Were concentrated using Gene
crude yield) Was split into tWo portions. The ?rst portion (27.0
g) Was dry-packed With silica gel, loaded onto a pre-packed 330 g silica gel column and eluted using a lsco Combi?ash Rf200 system With a dichloromethane/methanol solvent sys
to Tob-Boc3-TFA (n) With approximately 2-3% Tob-Boc3 TFA2 side product according HPLC/ELSD. (Small-scale
reactions at various concentrations of Tob-Boc3 suggest that the amount of Tob-Boc3-TFA2 by-product can be substan tially eliminated by performing the reaction at 0.1 M Tob Boc3 in DMF.) To the reaction mixture Were added 31 mL of
of Tob-Boc4-N1-AHB-Boc-6"-O-mesitylene sulfonate (33%) Rt 11.7 min (Phenomenex C18, 50><3 mm, 4 um, gra dient elution With 95% Water (0.1% formic acid) gradient 100% methanol); LC/MS: m/Z 1251.44 (M') observed, 1251. 44 calcd. TLC Rf0.25 (DCM:MeOH:9.5:0.5).
[0118] To a stirred solution ofTob-Boc4-N1-AHB-Boc-6"
O-mesitylene sulfonate (r) (17.26 g, 13.8 mmol) in acetoni trile Was added 1,3-propanediamine (11.5 mL, 74.12 mmol)
at room temperature. The resulting solution Was stirred in an
76.6 mmol), 1-ethyl-3-[3-dimethylaminopropyl]carbodiim ide hydrochloride (EDC.HCl)(5.8 g, 76.6 mmol) and triethy
lamine (4.2 mL, 76.6 mmol). The mixture Was stirred for 16 hours. LC-MS shoWed completion of the reaction. Solvents
Were removed under reduced pressure and to the residue
oil bath at 70 C. for 18 h, until LC/MS indicated that the reaction Was complete. The reaction Was Worked up by removing the volatiles under reduced pressure and residual
Water (400 mL) Was added and stirred for 30 minutes. The solid Was ?ltered, placed in a beaker (1 L) and stirred With Water (400 mL) for 1 hour and ?ltered. This Was repeated one more time and Washed With excess Water (400 mL). The ?nal
solid Was sucked dry on the ?lter and then dried under vacuum
1).
[0119] A round-bottomed ?ask (oven-dried) equipped With
a magnetic stir bar Was charged With a solution of Tob-N1