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US 20130203693A1

(19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0203693 A1
CHAPARIAN et al.
(54) 4,6-SUBSTITUTED

(43) Pub. Date:

Aug. 8, 2013

Publication Classi?cation
(51) Int- Cl C07H 15/234

2,5-DIDEOXYSTREPTAMINE
AMINOGLYCOSIDE ANTIBIOTICS

(2006.01)

(71) Applicant: SelectX Pharmaceuticals, Inc., Boston,


MA (Us)

(52) US, Cl,


CPC .................................. .. C07H 15/234 (2013.01)
USPC .......................................... .. 514/40; 536/168

(72) Inventors: Michael G. CHAPARIAN, Washington Township, MI (US); Michael Brady, Haverhill, MA (US); Scott Moe, Sudbury, MA (US); Babu Rao

(57)

ABSTRACT

Aminoglycoside antibiotics of the formula


HZN OH

Renikuntla, ShreWsbury, MA (US);


Srinivas Gadthula, Dublin, OH (US); Srinivasarao Meneni, ShreWsbury, MA
(US); Venkata Sai Prakash
R3
00 R2,

Chaturvedula, Alpharetta, GA (US)


HZN

(73) Assignee: SelectX Pharmaceuticals, Inc., Boston, MA (US)


R8

R7
A
/ \N

R HN
O

R,
OH

(21) Appl.No.: 13/755,275


HO

(22) Filed:

Jan. 31, 2013


Related US. Application Data
are disclosed. The compounds are useful for treating bacterial

(60)

Provisional application No. 61/594,663, ?led on Feb. 3, 2012.

infections, particularly infections resistant to known antibi


otics.

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

[0013] In one aspect the invention relates to compounds of formula I:

[0002]

The invention relates to aminoglycoside antibiotics.


BACKGROUND OF THE INVENTION
I

HZN

OH

[0003]

Since their ?rst clinical use With the introduction of

streptomycin in 1947, the aminoglycosides (AG) have been


one of the most important and Widely used classes of antibi
0
HZN 0

R3
R2.

otics against most gram-negative and serious gram-positive


infections. Aminoglycosides bind the A-site of the 30S ribo

some, blocking bacterial protein synthesis through disruption


of initiation and translation. AGs are actively transported into the bacterial cell by an energy-requiring process. Defective membrane proteins resulting from translational errors further

R7

R8

A\ N/
HO

RlHN
O

o
OH

R,

enhance the activity of AGs by alloWing passive entry of the


antibiotic into the cell. [0004] Over the past several decades of use, clinical resis tance to the AGs has emerged. Aminoglycoside resistance generally occurs by one of several mechanisms, described here in order of clinical relevance:

HZN

[0005]

1. EnZyme-mediated chemical modi?cation of

the drug by aminoglycoside modifying enZymes


(AGME). These enZymes are carried and transferred

[0014] [0015] [0016] [0017]

Wherein R2 is chosen from iOH and iNHZ;


R3 is chosen from H and OH; R1 is chosen from H, 4C(:NH)NH2, and

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:

Clo)carbocycle, i(C3-C9)heterocycle, i(C1-C8)


alkyl(C3-Clo)carbocycle, and i(Cl-C8)alkyl(C3-C9)
heterocycle Wherein
[0019] in said (C1-C2O)alkyl or in the (Cl-C8)alkyl portion of said (C l-C8)alkyl(C3 -C 1O)carbocycle or
(Cl-C8)alkyl(C3-C9)heterocycle, one or tWo

[0006] a. N-Acetyltransferases (AAC)4catalyZes acetyl CoA-dependent acetylation of an amino group

[0007] b. O-Adenyltransferases (ANT)4catalyZes ATP-dependent adenylation of hydroxyl group


[0008] c. O-Phosphotransferases (APH)4catalyZes ATP-dependent phosphorylation of a hydroxyl group
[0009] 2. Reduced uptake or decreased cell permeability. Most typically seen in Pseudomonas aeruginosa (Pae),
this form of resistance is due to a transport defect result

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

[0020] said (C1-C2O)alkyl, (C3-Clo)carbocycle,

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

bocycle, (Cl-C8)alkyl(C3-C9)heterocycle may be


additionally substituted With from one to three sub

stituents chosen independently from CH3,

ADH, iNHZ, %OOH, :O, iNHCONH2,


iNHC(:NH)NH2, 4CN or halogen;

[0011] 4. Altered ribosome binding sites, typically by


methylation, facilitated by 1 6S RNA methylases. Modi
?cation at the site of aminoglycoside interaction inter feres With ribosomal binding. These enZymes are also

[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

i(C3-Clo)carbocycle, i(C3-C9)heterocycle, i(Cl C8)alkyl(C3-C1O)carbocycle, i(Cl-C8)alkyl(C3-C9)


heterocycle and the deshydroxy residue of an ami

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)

infections, most notably hospital based (nosocomial) infec


tions. Many of these infections are not susceptible to cur

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

carbocycle, i(C3-C9)heterocycle, i(Cl-C8)alkyl(C3 Clo)carbocycle, i(Cl-C8)alkyl(C3-C9)heterocycle,


iNRSORSI, and iC(:NH)NH2, Wherein

ing a long history of effective use against gram-negative

US 2013/0203693 A1

Aug. 8,2013

[0024]

R80 and R81 are chosen independently from H

[0036]

In some embodiments of the invention, R1 is H. In

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

other embodiments, R1 is 4C(:NH)NH2. In still other

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

be replaced by 40*, iSi, iSOi, isozi,


iCECi, a (C3 -C 1O)carbocycle or a (C3 -C6)hetero

cycle; and

[0026] said (C1-C2O)alkyl, (C3-Clo)carbocycle,

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

alkyl portion of R10 [that is, when R10 is (Cl-C2O)alkyl, (C1


C8)alkyl(C3-C1O)carbocycle, or (Cl-C8)alkyl(C3-C9)hetero
cycle], one or tWo 4CHi may be replaced With iNi. In other embodiments of the invention in which R10 contains an

bocycle, (Cl-C8)alkyl(C3-C9)heterocycle may be


additionally substituted With from one to three sub

stituents chosen independently from CH3,

%H2CH3, iOH, %H2OH, iNHz, %H2NH2, iCOOH, :O, iNHCONH2,


iNHC(:NH)NH2, 4CN and halogen;
[0027] or

alkyl portion, tWo 4CHi may be replaced by 4C:Ci. In


still other embodiments in which R10 contains an alkyl por tion, one or tWo 4CH2i may be replaced by 40*, iSi,
iSOi, isozi, 4CECi, a (C3-Clo)carbocycle or a

[0028] R7 and RSA, taken together With the nitrogen to


Which they are attached, form a (C3 -C9)heterocycle, said

(C3 -C6)heterocycle. In yet other embodiments of the inven

tion, R1O may be i(Cl-C2O)alkyl, i(C3-Clo)carbocycle,

(C3-C9)heterocycle optionally substituted With from


one to three substituents chosen independently from

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

ments, these substituents are chosen independently from

(:NH)NH2, 4CN and halogen; and


[0029] A is chosen from a direct bond, i(C:O)i,

%H3, iOH, iNHZ, %OOH, :O, iNHCONH2,


iNHC(:NH)NH2, 4CN or halogen.
[0038] In some embodiments of the invention, R1 is

%(:O)Oi, iNH(C:O)i, i(C:O)NHi, iNH(C:O)NHi, i(C:S)NHi, iNH(C:S)i,


and iNH(C:S)NHi.
[0030] In another aspect, the invention relates to method of treating a mammal suffering from a bacterial infection, by
administering a therapeutically effective amount of a com

%(: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

pound of the formula (I) shoWn beloW:

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

by 40*, iSi, iSOi, iSOZi, 4CECi, a (C3-Clo)


carbocycle or a (C3-C6)heterocycle. In yet other embodi
HZN OH

ments of the invention, R1O may be i(Cl-C15)alkyl, i(C3

C6)carbocycle, i(C3-C5)heterocycle, i(Cl-C3)alkyl(C3


R3
00 R2,

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

dently from CH3, iOH, iNHZ, 4COOH, :O, iNH


R7
1 0 / A\ N

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]

In some embodiments of the invention, R2 is iOH.


In some embodiments of the invention, R3 is H. In

4CH2i may be replaced by iOi, iSi, iSOi,


iSOZi or CEO In some embodiments, R10 is the des carboxy residue of a natural ot-amino acid. In other embodi

In other embodiments, R2 is iNH2.


other embodiments, R3 is iOH. [0035] In some embodiments of the invention, R2 is iNHZ
and R3 v is H.

ments, R10 is iCH(Rll)i(CH2)niNHRl2. In still other


embodiments, R10 is i(CH2)niRl3 . In some embodiments,

4C(:O)Rlo is 4-amino-2-hydroxybutyryl.

US 2013/0203693 A1

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[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]

In some embodiments, R11 is 40H. In other


In some embodiments, R12 is H. In other embodi

embodiments, R11 is iNHZ.


ments, R12 is (Cl-C6)haloalkyl. In still other embodiments, R12 is iC(:NH)NH2. In yet other embodiments, R12 is the
deshydroxy residue of a natural ot-amino acid. [0042] In some embodiments, R13 is iOH. In some

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

%H2CH3, ADH, %H2OH, iNHz, iCHzNHz,


%OOH, :O, iNHCONH2, iNHC(:NH)NH2, %N and halogen.
[0051] In some embodiments, R7 and RSA, taken together With the nitrogen to Which they are attached, form a (C3 -C6) heterocycle. In some embodiments, this (C3-C6)heterocycle
may be optionally substituted With from one to three substitu

ments, R5 is halogen. In other embodiments, R5 is iN3. In


still other embodiments, R5 is (Cl-C4)alkynyl. In some embodiments, R5 is iNHRSO. In some embodiments, R5 is
?uorine. [0044] In some embodiments of the invention, R50 is H. In some embodiments of the invention, R50 is (C3-Clo)car

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

selected from H, cyclopropyl, cyclopropylmethyl, pyrrolidi


nyl, and the deshydroxy residue of citrulline or serine. [0045] In some embodiments of the invention, R7 is H. In some embodiments of the invention, R7 is (Cl-C6)alkyl. In some embodiments of the invention, R7 is hydroXy(C1-C6)

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

R8 are chosen independently from (C l-C6)alkyl and hydroxy

(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

iNH(C:O)i; and R8 is chosen from (C1-Cl5)alkyl, (C3

Clo)carbocycle, (C3-C6)heterocycle, i(Cl-C3)alkyl(C3-C6) carbocycle, i(Cl-C3)alkyl(C3-C6)heterocycle, iN(CH3)


2iNH2, and 4C(:NH)NH2. In some of these
embodiments, one or tWo of the iCHi residues of the

(C1-Cl5)alkyl or the (Cl-C3)alkyl portion of the i(Cl-C3) alkyl(C3-C6)carbocycle or i(Cl-C3)alkyl(C3-C6)hetero


cycle may be may be replaced With iNi, tWo 4CHi may
be replaced by i-C:Ci, or one or tWo 4CH2i may be

R8 [that is, When R8 is (C1-C2O)alkyl, i(Cl-C8)alkyl(C3


Clo)carbocycle, or i(Cl-C8)alkyl(C3-C9)heterocycle], one or tWo iCHi may be replaced With iNi. In other

replaced by iOi, isozi, 4CECi, a (C5-C6)car


bocycle or a (C3-C4)heterocycle. Additionally or altema

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

tively, in some of these embodiments, the (Cl-C15)alkyl, (C3

Clo)carbocycle, (C3-C6)heterocycle, i(Cl-C3)alkyl(C3-C6)


carbocycle, or i(Cl-C3)alkyl(C3-C6)heterocycle may be
additionally substituted With from one to three substituents

or tWo 4CH2i may be replaced by iOi, iSi, iSOi,


isozi, 4CECi, a (C3-Clo)carbocycle or a (C3-C6)het

erocycle. In yet other embodiments of the invention, R8 may

be (C1-C2O)alkyl, (C3-C1O)carbocycle, (C3-C9)heterocycle,


i(Cl-C8)alkyl(C3-Clo)carbocycle, or i(Cl-C8)alkyl(C3
C9)heterocycle additionally substituted With one, tWo or three
substituents. In some embodiments, these substituents are

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;

and R8 is chosen from iN(CH3)2, iNHZ, and iC(:NH)

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

other embodiments of the invention, R80 is (C l-C6)alkyl.


other embodiments of the invention, R81 is (Cl-C6)alkyl.
[0049] In some embodiments of the invention, A is a direct bond. In some embodiments of the invention, A is

from (C1-Cl5)alkyl, (C3-Clo)carbocycle, (C3-C6)hetero cycle, i(Cl-C3)alkyl(C3-C6)carbocycle, and i(Cl-C3)


alkyl(C3-C6)heterocycle. In some of these embodiments, the

(C1-Cl5)alkyl, (C3-C1O)carbocycle, (C3-C6)heterocycle,


i(Cl-C3)alkyl(C3-C6)carbocycle, or i(Cl-C3)alkyl(C3
C6)heterocycle may be additionally substituted With from one to three substituents chosen independently from CH3,

US 2013/0203693 A1

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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

poxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the


like. LoWer-alkoxy refers to groups containing one to four carbons. [0063] Similarly, alkylthio refers to groups of from 1 to 6 carbon atoms of a straight, branched, or cyclic con?guration and combinations thereof attached to the parent structure

be replaced With iNi, tWo 4CHi may be replaced by


iC:Ci, or one or tWo 4CH2i may be replaced by

through sulfur.
[0064] Aryl and heteroaryl mean a 5- or 6-membered aro

iOi, iSOzi, iCECi, cyclopentyl, cyclohexyl, furan


or dioxole. In still other embodiments, the (C1-Cl5)alkyl or

replaced (C1-Cl5)alkyl may additionally be substituted With


from one to three substituents chosen independently from

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

taining 0-3 heteroatoms selected from O, N, or S. The aro

a direct bond; and R8 is (C l-C6)alkyl additionally substituted


With one to or tWo substituents chosen independently from

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

selected from optionally substituted cyclopropyl, cyclobutyl,

cyclopentyl, cyclohexyl, phenyl, pyrrole, imidaZole, furan,


tetrahydrofuran, piperidine, imidaZolylmethyl, (C 1 -C l0)alkyl
[in Which one or tWo 4CHi may be replaced With iNi, tWo iCHi may be replaced by 4C:Ci, and one or tWo

heteroaryl ring attached to the parent structure via an alkyl

residue. Examples include, e.g., pyridinylmethyl, pyrimidi


nylethyl and the like. [0066] Hydrocarbon means a linear, branched, or cyclic residue comprised of hydrogen and carbon as the only

iCHzi may be replaced by 40*, iSi, iSOi,


iSOZi or iCEC], the descarboxy residue of a natural

ot-amino acid, 4CH(Rll)i(CH2)niNHRl2, and i(CH2)


niRl3 . In these embodiments, A is chosen from a direct bond

elemental constituents and includes alkyl, cycloalkyl, poly cycloalkyl, alkenyl, alkynyl, aryl and combinations thereof.

and i(C:O)i, and R8 is chosen from (Cl-C15)alkyl, (C3

Clo)carbocycle, (C3-C6)heterocycle, i(Cl-C3)alkyl(C3-C6)


carbocycle, and i(Cl-C3)alkyl(C3 -C6)heterocycle. The (C 1

Examples include benZyl, phenethyl, cyclohexylmethyl, camphoryl and naphthylethyl.


[0067] Unless otherWise speci?ed, the term carbocycle is
intended to include ring systems in Which the ring atoms are all carbon but of any oxidation state. Thus (C3-C1O) car bocycle refers to both non-aromatic and aromatic systems,

Cl5)alkyl, (C3-C1O)carbocycle, (C3-C6)heterocycle, (Cl-C3) alkyl(C3-C6)carbocycle, (Cl-C3)alkyl(C3-C6)heterocycle


may be additionally substituted With from one to three sub

stituents chosen independently from CH3, iCH2CH3,

including such systems as cyclopropane, benZene and cyclo


hexene. Carbocycle, if not otherWise limited, refers to mono

iOH, iCHzOH, iNHz, %OOH, :O, iNHCONH2,


iNHC(:NH)NH2, and halogen. In some of these embodi

cycles, bicycles and polycycles.


[0068] Heterocycle means a cycloalkyl or aryl residue in Which one to four of the carbons is replaced by a heteroatom such as oxygen, nitrogen or sulfur. Heteroaryls form a subset

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.

[0059] In some embodiments, R2 is iNHZ and R3 is H.


[0060] Throughout this speci?cation the terms and sub
stituents retain their de?nitions.

of heterocycles. Examples of heterocycles that fall Within the scope of the invention include pyrrolidine, pyraZole, pyrrole,

indole, quinoline, isoquinoline, tetrahydroisoquinoline, ben


Zofuran, benZodioxan, benZodioxole (commonly referred to
as methylenedioxyphenyl, When occurring as a substituent),

[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

tetraZole, morpholine, thiaZole, pyridine, pyridaZine, pyrimi


dine, thiophene, furan, oxaZole, oxaZoline, isoxaZole, diox
ane, tetrahydrofuran and the like. [0069] The term halogen means ?uorine, chlorine, bro
mine or iodine. In one embodiment, halogen may be ?uorine
or chlorine.

alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl,


s-butyl and t-butyl, and the like. Preferred alkyl groups are those of C20 or beloW. Cycloalkyl or carbocycle includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms.

[0070]

As used herein, the term optionally substituted

Examples of cycloalkyl groups include c-propyl, c-butyl,


c-pentyl, norbomyl and the like. Alkylene is a divalent alkyl

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

residue, for example propylene is iCH2CH2CH2i.


[0062] Alkoxy or alkoxyl refers to groups of from 1 to 6 carbon atoms of a straight, branched, or cyclic con?guration and combinations thereof attached to the parent structure

speci?ed radical. In one embodiment, l, 2 or 3 hydrogen


atoms are replaced With a speci?ed radical. In the case of alkyl and cycloalkyl, more than three hydrogen atoms can be

through an oxygen. Examples include methoxy, ethoxy, pro

replaced by ?uorine; indeed, all available hydrogen atoms could be replaced by ?uorine.

US 2013/0203693 A1

Aug. 8,2013

[0071] Virtually all of the compounds described herein


contain one or more asymmetric centers and thus give rise to

ment to the glycoside scaffold are referred to herein as resi

dues of amino acids. One might also refer to them as amino

enantiomers, diastereomers, and other stereoisomeric forms


that may be de?ned, in terms of absolute stereochemistry, as (R)- or (S)-. The present invention is meant to include all such possible isomers, as Well as their racemic and optically pure

acid fragments.
[0074] The term amino acid as used herein refers to the

racemates and all optical isomers of the folloWing naturally

forms. Optically active (R)- and (S)-isomers may be prepared


using chiral synthons or chiral reagents, or resolved using

occurring ot-amino acids: alanine, asparagine, aspartic acid,

conventional techniques. When the compounds described


herein contain ole?nic double bonds or other centers of geo

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

single naturally occurring [3-amino acid, [3-alanine.


[0075] As used herein, and as Would be understood by the
person of skill in the art, the recitation of a compoun i

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

boxyls, either carboxylic acid can be removed. For example,


in the molecule illustrated beloW:

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

salts, i.e. cationic species. The term pharmaceutically


acceptable salt refers to salts Whose counter ion (anion)

derives from pharrnaceutically acceptable non-toxic acids


HZN OH

including inorganic acids and organic acids. Suitable phar


maceutically acceptable anions for the compounds of the present invention include acetate, benZenesulfonate (besy

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

Zoate, adipate, alginate, aminosalicylate, anhydromethyl


on

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

physiologically active compounds While, in certain instances,


modifying metabolic fate to substantially alter their overall therapeutic pro?le. In favourable cases, this modi?cation has the potential to have a positive impact effect on safety, e?i cacy and/or tolerability. [See The Development of Deute

NHS:N-hydroxysuccinimide [0082] Phrphenyl PhOH:phenol


rt:room temperature satdIsaturated

TBDMS?-butyldimethylsilyl THF?etrahydrofuran TIPS?riisopropylsilyl TMS?rimethylsilyl


[0083] The folloWing abbreviations are also used in the description of the substituents in the text of this application:

rium-Containing Drugs by Roger Tung, Innovations in


Pharmaceutical Technology March 2010 and US. Pat. Nos.

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

ability. Compounds that contain isotopes 11C, 13N, 15O and


1 8F are Well suited for positron emission tomography. Radio labeled compounds described above can generally be pre pared by methods Well knoWn to those skilled in the art.

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

this event, subsequent exclusions of species from the compass


of applicants claims are to be considered artifacts of patent prosecution and not re?ective of the inventors concept or description of their invention; the invention encompasses all of the members of the genus (I) that are not already in the

iiiNQVNHZ jgi L}
N H 2-OH PDA O 3_Ap

possession of the public. [0079] A comprehensive list of abbreviations utiliZed by


organic chemists appears in the ?rst issue of each volume of

the Journal of Organic Chemistry. The list, Which is typically


presented in a table entitled Standard List ofAbbreviations,

2i \/\ NHZ
EDA OH ISO

NHZ.

is incorporated herein by reference. The folloWing abbrevia


tions and terms have the indicated meanings throughout:

Ac:acetyl Boc?-butyloxy carbonyl


Brederecks
methane

[0084]

It may happen that residues in the substrate of inter

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

DIPEAIdiisopropylethylamine DMAPIdimethyIaminopyridine DMF:N,N-dimethylformamide


[0080] EDCII -(3-(dimethylamino)propyl)-3-ethyl-carbo diimide hydrochloride
EtOAc:ethyl acetate

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

subsequently removed to expose the original functionality.


The removal or deprotection occurs after the completion of the reaction or reactions in Which the functionality Would interfere. Thus, When a sequence of reagents is speci?ed, as it

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

textbooks in the ?eld of chemistry, such as Protective Groups

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

pressed tablets may be prepared by compressing in a suitable


machine the active ingredient in a free-?owing form such as a poWder or granules, optionally mixed With a binder, lubri

compatible With the other ingredients of the formulation and


not deleterious to the recipient thereof.

[0086]

The formulations include those suitable for oral,

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

parenteral (including subcutaneous, intradermal, intramuscu


lar, intravenous and intraarticular), rectal and topical (includ ing dermal, buccal, sublingual and intraocular) administra
tion. The mo st suitable route may depend upon the condition

sustained, delayed or controlled release of the active ingredi


ent therein.

[0089]

Formulations for parenteral administration include

and disorder of the recipient. Parenteral pharmaceutical com

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

intended recipient. Formulations for parenteral administra


tion also include aqueous and non-aqueous sterile suspen

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

sions, Which may include suspending agents and thickening


agents. The formulations may be presented in unit-dose of

multi-dose containers, for example sealed ampoules and


vials, and may be stored in a freeZe-dried (lyophiliZed) con

thereof (active ingredient) With the carrier Which consti


tutes one or more accessory ingredients. In general, the for

dition requiring only the addition of a sterile liquid carrier, for

mulations are prepared by uniformly and intimately bringing


into association the active ingredient With liquid carriers or ?nely divided solid carriers or both and then, if necessary,

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

priate fraction thereof, of the active ingredient.


[0091] It should be understood that in addition to the ingre dients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include

capsules, cachets or tablets each containing a predetermined


amount of the active ingredient; as a poWder or granules; as a solution or a suspension in an aqueous liquid or a non-aque
ous liquid; or as an oil-in-Water liquid emulsion or a Water

in-oil liquid emulsion. The active ingredient may also be


presented as a bolus, electuary or paste.

?avoring agents.
[0092] Table 1 (below) presents representative members of
the genus of the invention:

[0088]

A tablet may be made by compression or molding,

optionally With one or more accessory ingredients. Com

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

iZe compound activity against bacteria from a pathogenic


genus in the absence of multiple resistance mechanisms. TWo strains (PAWT and ECWT) are reference strains cited Within the Clinical and Laboratory Standards Institute (CLSI) Stan

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

dards for susceptibility testing by Minimum Inhibitory Con


centration (MIC) (SXPS2) and are Well suited for quality
control. ABWT and ECWT are Widely susceptible to ami noglycoside antibiotics, Whereas PAWT and KPWT harbor a
0.5

gentalnycin
alnikacin

l
l-2

8
l

0.25-0.5
1

0.5
2-4

single aminoglycoside-modifying enZyme (AGME) that tar gets 4,6-substituted-2-deoxystreptamine AGs.

streptomycin kanalnycin B neomycin

l6 l6 32

l-2 l6 4-8

2-4 0.5-1 0.25-0.5

4 2 l

US 2013/0203693 A1
11

Aug. 8,2013

TABLE 2-continued
MIC values of reference AGs against indicator strains.

rary clinical isolates of Pseudomonas aeruginosa were 264

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

Pseudomonas aeruginosa is a prominent nosocomial patho


gen. In addition to intrinsic antibiotic resistance to several

MIC (ug/mL)

Pseudomonas aeruginosa
SXP #
SXP1212-56"-7 SXP1212-56"-8 SXP1212-56"-16 2523 2524 2554 2525

antibiotics, Pseudomonas has acquired multiple additional


mechanisms of resistance. As a result, therapeutic options for the treatment of infections caused by Pseudomonas aerugi
nosa are limited. We acquired clinical isolates of Pseudomo
PAWT
4 4 1 0.25 0.25 0.25 0.25-0.5

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

has aeruginosa (Micromyx LLC, Kalamazoo, Mich), and


used this panel to test compounds for Pseudomonicidal activ ity. The panel includes PAWT, tWo clinical isolates With inter mediate aminoglycoside resistance (PR1 and PR2), and a

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

acquired clinical isolates of Klebsiella pneumoniae and

Escherichia coli (Micromyx LLC, Kalamazoo, Mich.) and


used this panel to test compounds for anti-enteric activity. The aminoglycoside resistance pro?le of clinical Eco isolates is similar to the aminoglycoside resistance pro?le of Kpn iso lates. Indeed, MIC comparison of Eco and Kpn strains are
similar (data not shoWn). As a result, We focused on Kpn and assembled a screening panel that included Klebsiella pneu moniae Wild type (KPWT), tWo clinical isolates With inter mediate aminoglycoside resistance (KRl and KR2), and a

third clinical isolate (KR3) possessing high-level aminogly


coside resistance. No single reference AG (amikacin, genta

mycin, tobramycin, streptomycin, sisomycin, neomycin,


arbekacin, dibekacin, kanamycin B, spectinomycin or kana mycinA) demonstrated acceptable potency against all strains
tested. KAN A, KAN B, and DIB shoWed limited to no

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

We tested compounds of the invention against this panel and

noted potency against KR3 (folloWed by KR2 and KR1).


[0097] As in the case of Pseudomonas aeruginosa, We con

sidered Whether the SAR observed against select Klebsiella pneumoniae strains 1, 2 or 3 Was indicative of general

tiple sources including JMI Laboratories (North Liberty,

potency. The MIC9O values for ?fteen antibiotics (amikacin,

IoWa), P?zer (Groton, Conn.), Micromyx (Kalamazoo,


Mich.), Dr. J. ChoW (Wayne State University). Strains Were
characterized for resistance to a Wide variety of classes of

gentamycin, tobramycin, ceftazidime, cefepime, piperacillin, piperacillin/tazobactam, aztreonam, ceftriaxone, imipenem,


meropenem, doripenem, ertapenem, cipro?oxacin and levo ?oxacin) against ?fty resistant contemporary clinical isolates
of Klebsiella pneumoniae Were once again Z64 ug/mL. Using this stringent Klebsiellapneumoniae panel, We tested a
subset of compounds of the invention. The results, as Well as MIC9O results against a panel of E. coli strains, are shoWn in Table 5:

antibiotics including aminoglycosides, [3-lactams and ?uoro quinolones. The MIC9O values for ?fteen antibiotics (amika

cin, gentamycin, tobramycin, ceftazidime, cefepime, piper


acillin, piperacillin/tazobactam, aztreonam, ceftriaxone, imipenem, meropenem, doripenem, ertapenem, cipro?oxa
cin and levo?oxacin) against these ?fty resistant contempo

US 2013/0203693 A1

Aug. 8,2013

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

pounds for anti-Acinetobacter activity. We ?rst tested refer


ence AGs against our panel of Aba strains. As expected,
clinical Aba isolates Were signi?cantly more resistant to ref erence AGs than Acinelobacler baumannii Wild type

(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.

the ?fteen antibiotics against ?fty resistant contemporary


clinical isolates of Acinelobacler baumannii were 232

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

ug/mL. Using this stringent Acinelobacler baumannii panel,


We tested a subset of compounds of the invention. The results are shoWn in Table 6:
TABLE 6
MIC values against Acinelobacler baumannii

tively. 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 Were euthanized at initiation of treatment and thigh CFUs Were determined

(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

Were aseptically removed, Weighed, homogenized, serially


AB3
2-4 8 8 4-8

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]

The compound identi?ed as SXP 2523 Was selected


Total Drug
Concentration

for further testing in vivo. TWenty male Sprague DaWley rats


Were pre-treated With cyclopho sphamide to render them neu

Change
Log
CFU/

Change
from 24 hr.

from
T = RX

tropenic on day 4 and day 1 With 100 mg/kg and 75 mg/kg


respectively. Rats Were infected With R aeruginosa 6294

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

It can be seen that SXP-2523 is effective against amikacin

resistant R aeruginosa in an appropriate animal model.

processed (TIRX). TWenty-four hours post infection rats Were euthanized by C02 inhalation. The right thigh muscles

[0101]

Synthesis of Examples 1-25 is shoWn in schematic

form in Scheme 1.As shoWn in Scheme 1, amikacin Was ?rst

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

lated. Displacement With various amines R8-A-NH(R7) gave


the penultimate product Which Was cleaved under acidic con ditions to provide 5F-amikcin-NH-AHB-R6"-R8ANR7 ana

(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

2 . T osy1 ation '

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

cooled to 200 C. A solution of deoxo-?uor [bis-(2-methoxy

trated. The residue Was thoroughly Washed With Water (10 ml)
and dried in vacuum at 400 C. to give compounds dd

ethyl)aminosulfur tri?uoride] (DAST) is then added drop


Wise over 15 min. The reaction is stirred overnight at 20 C

(~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

and the excess reagent is quenched by the addition of solid

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

propyl ether (2><10 mL) folloWed by CH2Cl2 (2><10 mL) and


drying at 400 C. under vacuum fumished the ?nal compounds modi?ed at CiR5 position.

ml) and mesitylene sulfonyl chloride (0.25 moles) is added in


the presence of catalytic amount of DMAP. The reaction mixture is stirred at room temperature for 5-6 hours and after completion the product is concentrated under vacuum to yield a mesitylene sulfonate intermediate. For chlorides, the inter

[0109] The general synthetic method for incorporation of


guanidine groups at all amines of aminoglycosides involves the conditions reported in the literature [Hoshi H, Aburaki S, limura S, Yamasaki T, Naito T, KaWaguchi H The Journal of

mediate is dissolved in DMF (10 mL) and LiCl (0.5 moles) is


added. The reaction is re?uxed at 100 C. for 6-8 hours. After

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,

CH2Cl2 (3><25 mL). The organic layer is Washed With brine


and dried over anhydrous Na2SO4 folloWed by concentration
under vacuum to furnish 5-deoxy 5-chloro derivatives. For

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

Scheme 1 can be employed With the appropriate starting


material aa replacing amikacin. Alternatively, one can fol loW the procedures of Schemes 2 and 3 beloW.

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

examples 26-29, (S)-AHB-Boc. The coupling (h>i) may be


accomplished by any of the methods Well-known in the art of

peptide synthesis. Condensing agents for reacting amines


With carboxylic acids include carbodiimides of various sorts, mixed anhydrides, EEDQ, HATU, and the like. It is also possible to pre-react the carboxylic acid of the linker With an
appropriate leaving group to form an activated ester. Acti vated esters denote esters Which are capable of undergoing a

reaction is completed, the reaction mixture is partially con

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;

the compounds in which R50 is the deshydroxy residue of the


aminoacid.

O-esters of N-hydroxyimides and N-hydroxy heterocycles; speci?c examples include S-t-butyl esters, S-phenyl esters,

S-2-pyridyl esters, N-hydroxypiperidine esters, N-hydrox


ysuccinimide esters, N-hydroxyphthalimide esters and N-hy
droxybenZotriaZole esters.
[0112] The TFA group Was then cleaved and replaced With a Boc-group. Tosylation or mesitylation of the R6"-OH fol

[0106]

For compounds in Which R5 is an alkyne, the mesi

tylene sulfonate intermediate can be reacted With a metal salt

of the appropriate alkyne, such as sodium acetylide.

[0107] General procedure for deprotection of acetyl


groups: To a solution of cc (80 mg) in 5 mL of 30% NaOMe
in MeOH Was added, and the mixture Was stirred for over night. The solution Was neutraliZed With aq HCl and concen

loWed by displacement With various amines R8-A-NH(R7)


gave the penultimate product Which Was cleaved under acidic

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]

An alternative route is shown in Scheme 3, Wherein

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.

deprotected sequentially, ?rst With NaOCH3 and then With


HCl to remove the Boc-groups:

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

In a speci?c example, SXP-2523 Was synthesized as follows:

[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.

The dried Zn(OAc)2.2H2O and tobramycin free base (25.0 g,


53.5 mmol) Were added to a 3 L round-bottom ?ask and

stirred With methanol (350 mL) at room temperature for 2

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

removed by rotary evaporation (bath temp 40 C.). The solid


residue Was dissolved in n-butanol (500 mL) and Washed With

is removed in vacuo. The resulting solid is Washed With Water

NH4OHzbrine (2: 1, 1 L. 3><330 mL), followed by brine


(2><330 mL). Then an equal volume of Water (500 mL) Was
added to the butanol layer. The combined Water and butanol

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

?oWing White solid (45% yield.


[0117] A round-bottomed ?ask (oven-dried) equipped With
an Argon inlet and a magnetic stir bar Was charged With a

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

solution ofTob-N1-AHB(Boc)5 (q) (45.11 g, 42.20 mmol) in


dry pyridine (120 mL). The reaction solution Was stirred at room temperature and 2-mesitylenesulfonyl chloride (10.08
g, 46.20 mmol) Was added. This solution Was stirred at room

Genevac Rocket Evaporator (45 C., 18 h). The solid


obtained Was re-dissolved With methanol, and the insoluble

White crystalline solid (NaCl) Was discarded by ?ltration. The


methanol layer Was concentrated under vacuum to yield 40 g

crude product. The major impurity in Tob-Boc3 is Tob-Boc5.


HPLC With an evaporative light scattering detector (ELSD) Rt 4.4 min (Phenomenex-C18, 50><3 mm, 4 pm, gradient elution With 95% Water (0.1% formic acid) and 100% metha nol); LC-MS: m/Z 768.33 (M+1) obtained, 767.86 calculated:

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%

dimesityl compound. Solvents Were then removed by rotary

evaporation folloWed by aZeotropic removal of pyridine With


toluene (3><100 mL). The solid (58.0 g) Was Washed With Water (3><500 mL), to remove pyridinium hydrochloride, ?l
tered to afford a dry poWder, Which Was placed in vacuum oven at 40 C. for 18 h. The solid obtained (45.0 g, 86.5%

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

tem (0 to 20% gradient) to afford the desired product (r)

Tob-Boc4-N1-AHB-Boc-6"-O-mesitylene sulfonate (11.6 g,


43% yield). The remaining 18 g Was dry-packed With silica gel and loaded onto a pre-packed 220 g silica gel to afford the

vac Rocket Evaporator (45 C.) to provide 32 g (77.9% yield)


of Tob-Boc3 as a pure White solid.

[0115] Tob-Boc3 (m) (19.5 g, 25.0 mmol) Was co-evapo


rated With anhydrous DMF (2><50 mL). To a stirred solution

desired product (r) (7.6 g, 42.2% yield). Both batches Were


dissolved in dichloromethane and the solvent Was evaporated under reduced pressure to afford a dry poWder, Which Was placed in vacuum oven at 40 C. for 18 hours to yield 17.4 g

of Tob-Boc3 in anhydrous DMF (70 mL) Was added ethyl


tri?uoroacetate (3.62 g, 25.0 mmol) dropWise at 5 C. for 30
min. The mixture Was stirred at room temperature for 10 min.

The LC-MS of the mixture shoWs completion of the reaction

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

1M solution ofAHB-Boc in DMF (76.6 mmol), HOBt (4.6 g,

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

1,3-propanediamine Was aZeotroped With toluene (3><300


mL) to provide a poWder Which Was dried under high vacuum for 4 h. The crude residue Was stirred With NH4OH (1.0 M, 300 mL) for 1 hour then ?ltered through a sintered glass

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

funnel folloWed by stirring With deioniZed Water (400 mL).


The aqueous suspension Was ?ltered and Washed With Water

(400 mL) until ?ltrate became neutral to pH paper. The solid


Was dried under high vacuum at 40 C. for 16 hours to afford

at 40 C. for 1 hour. Tob-Boc3-TFA-AHB-Boc (0) Was

obtained in 81% yield (21.5 g). HPLC (ELSD) Rt 4.4 min


(Phenomenex-C18, 50><3 mm, 4 pm, gradient elution With 95% Water (0.1% formic acid) and 100% methanol); LC-MS:

a desired product Tob-Boc4-N1-AHB-Boc-6"-propanedi

amine (s; R7:H, R8:3-aminopropyl) (12.5 g, 87.7% yield).


HPLC (ELSD) Rt 7.8 min (Phenomenex C18, 50><3 mm, gradient elution With 95% Water (0.1% formic acid) gradient 100% methanol); LC/ MS: m/Z 1125.44 (M+) observed, 1225. 30 calcd. TLC Rf 0.2 (DCM:MeOH:NH4OH:4.9514.9510.

m/Z 1065.71 (M+1) obtained, 1065.09 calculated; TLC (nin

hydrin) (CH2Cl2: MeOH:28% NH4OH, 8.5:1.0:0.5) Rf0.40.


[0116] Thirteen and ?ve-tenths grams (12.7 mmol) of Tob
Boc3 -TFA-AHB-Boc (0) Was dissolved in 150 mL of dioxane in a round bottomed ?ask under nitrogen. To this is added a solution of 640 mg of LiOH in 7 mL of Water With stirring.
The reaction is alloWed to stir at room temperature for 2.5

1).
[0119] A round-bottomed ?ask (oven-dried) equipped With
a magnetic stir bar Was charged With a solution of Tob-N1

AHB(Boc)5 -PDA-Boc (s; R7:H, R8:3 -aminopropyl),


12.30 g, 11.0 mmol) 1,4-dixane. The reaction solution Was stirred at room temperature and then triethylamine (2.22 mL,

hours and then Boc anhydride 3.3 g (30 mmol) is added.


Stirring is continued for a further 2 hours and then the solvent