Synthesis and antibacterial evaluation of novel 4′-glycyl linked quinolyl-azithromycins with potent activity against macrolide-resistant pathogens

Article abstract of DOI:10.1016/j.bmc.2016.01.055

A new azithromycin-based series of antibacterial macrolones is reported, which features the use of a 4′-ester linked glycin for tethering the quinolone side chain to the macrolide scaffold. Among the analogs prepared, compounds 9e and 22f with a quinolon-6-yl moiety were found to have potent and well-balanced activity against clinically important respiratory tract pathogens, including erythromycin-susceptible and MLS_B resistant strains of Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae. In addition, potential lead compounds 9e and 22f demonstrated outstanding levels of activity against Moraxella catarrhalis and inducibly MLS_B resistant Staphylococcus aureus. The best member of this series 22f rivals or exceeds, in potency, some of the most active ketolide antibacterial agents known today, such as telithromycin and cethromycin.

Full text of DOI:10.1016/j.bmc.2016.01.055

Accepted Manuscript
Synthesis and Antibacterial Evaluation of Novel 4“-Glycyl Linked Quinolyl-
Azithromycins with Potent Activity against Macrolide-Resistant Pathogens
Dražen Pavlović, Stjepan Mutak
PII:
S0968-0896(16)30068-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.055
BMC 12798
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
20 November 2015
26 January 2016
29 January 2016
Please cite this article as: Pavlović, D., Mutak, S., Synthesis and Antibacterial Evaluation of Novel 4“-Glycyl Linked
Quinolyl-Azithromycins with Potent Activity against Macrolide-Resistant Pathogens, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.01.055
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Synthesis and Antibacterial Evaluation of Novel 4“-Glycyl Linked
Quinolyl-Azithromycins with Potent Activity against Macrolide-Resistant
Pathogens
*
Dražen Pavlović and Stjepan Mutak
PLIVA Research Institute, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia
*
Corresponding author. Present address: Siget 18c, 10000 Zagreb, Croatia, Tel.: +385 91 5975
3
80. E-mail dpavlovic@hotmail.fr
Abbreviations: MLS , macrolide-lincosamide-streptogramin B antibiotics; HBTU,
B
O-(Benzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate; DIPEA,
diisopropylethylamine; SAR, structure-activity relationships.
1

Abstract
A new azithromycin-based series of antibacterial macrolones is reported, which features
the use of a 4”-ester linked glycin for tethering the quinolone side chain to the macrolide
scaffold. Among the analogues prepared, compounds 9e and 22f with a quinolon-6-yl moiety
were found to have potent and well-balanced activity against clinically important respiratory
tract pathogens, including erythromycin-susceptible and MLS
B
resistant strains of S.
pneumoniae, S. pyogenes, and H. influenzae. In addition, potential lead compounds 9e and
2
2f demonstrated outstanding levels of activity against M. catarrhalis and inducibly MLS
B
resistant S. aureus. The best member of this series 22f rivals or exceeds, in potency, some of
the most active ketolide antibacterial agents known today, such as telithromycin and
cethromycin.
Keywords
Macrolide antibiotics, C-4”-substituted azithromycins, quinolones, macrolide resistance
2

1
. Introduction
The development of bacterial resistance to currently available antibacterial agents is a
1
growing global health problem. To address the drug resistance problem, tremendous efforts
have been devoted to the modification of existing classes of antibacterial agents to provide
new analogues with improved attributes. As a consequence, a new generation of macrolide
antibacterial agents, so called ketolides, have been developed particularly to combat
2
respiratory tract pathogens that have acquired resistance to macrolides. The ketolides are
semi-synthetic derivatives of the 14-membered macrolide erythromycin A in which
L-cladinose from the C-3 position of the macrolide ring was replaced with a ketone
3
functional group. The most active ketolides additionally contain an 11,12 cyclic carbamate
ring or -lactone ring in place of the two hydroxyl groups of erythromycin A and an arylalkyl
or arylallyl side chain, resulting in enhanced in vitro activity especially against constitutively
MLS resistant respiratory pathogens.
B
Telithromycin (1, Figure 1) is the first member of the ketolide class of antibacterials
which has been approved for clinical use and subsequently launched on the market in the EU
4
5
in 2001 and the US in 2004, while two additional ketolides, modithromycin, and
6
solithromycin are presently in Phase II and Phase III clinical trials in Japan and the US,
7
respectively. However, due to a serious hepatoxicity issue 1 had been subsequently
8
restricted , while clinical development of structurally similar ketolide, solithromycin had
3

9
,10
been seriously compromised until this issue is fully resolved. In addition, we have recently
demonstrated that the C-11,C-12 cyclic carbamate present in telithromycin and cethromycin
11
(
2, Figure 1; ABT-773) can be replaced by a properly functionalized C-11,C-12
-amino--lactone ring. This type of modification resulted in a novel lead series exemplified
1
2,13
by the prototype ketolide antibiotic GW773546X (3) (Figure 1).
O
R2
O
R2
R1
R1
O
O
N
O
N
O
O
N
O
HO
HO
1
1
O
O
1
2
O
O
O
O
O
O
O
O
N
O
N
R = CH , R =
(1)
S
N
N
1
3
2
R = CH , R = NH
1
3
2
N
(3)
N
R =
1
, R2 = H
(2)
Figure 1. Representative examples of ketolides derived from 14-membered macrolides.
Structures of telithromycin (1), cethromycin (2), and GW773546X (3)
The ketolide series is not necessarily the only class of macrolides that can effectively
address the resistance problems. Numerous other design concepts have come forward in
response to the growing need for new macrolide antibiotics with improved antibacterial
activity against resistant pathogens. Coupling of two or more different pharmacophore
substructures in a single molecule, aimed at obtaining hybrids with a possible cooperative
effect and dual mode of action, is an approach which has been investigated by many research
4

1
4-17
groups, often with success.
In this connection, the recognition ability of nucleobases has
1
8,19
been used to design hybrids with a wide variety of molecules such as porphyrins,
2
0
21
22,23

-lactam antibiotics, steroids, or peptides.
Some of these conjugates have found
applications in fields such as molecular recognition, inhibition of gene expression, or
anticancer and antivirus therapies. Recently, one of the more novel attempts involved the
2
4-26
successful coupling of a suitable quinolone and certain cephalosporins.
In a similar
approach, quinolone-sulfonamide, quinolone-trimethoprim, and quinolone-oxazolidinone
hybrids have been synthesized and evaluated for their potential to act as inhibitors with a
2
7-29
balanced dual mode of action.
3
0
The 15-membered ring macrolide antibiotics  for example, azithromycin  are an
important series within the macrolide class of antibiotics since they offer some advantages
over 14-membered macrolides derived from erythromycin A. These advantages include better
gastrointestinal tolerance, safety profile and pharmacokinetics coupled with activity against
Gram-negative pathogens. However, one of the most important disadvantages of
azithromycin in comparison to the novel generations of macrolide antibiotics is its inactivity
against most macrolide-resistant respiratory pathogens, especially those that confer resistance
by ribosome methylation (erm) and efflux by macrolide pumps (mef).
In the search of compounds likely to overcome the problem of macrolide resistance a new
class of 15-membered ring macrolide antibacterial agents, so called “macrolones”, was
5

developed in Pliva Research Institute in the early 2000’s. The term macrolone stands for a
hybrid composed of macrocyclic lactone to which a quinolone moiety is attached via suitable
linker of different structure and length (Figure 2). Considering the two pharmacophore
substructures: macrolides and quinolones, we wondered if combining the structural elements
of the macrolides and quinolones to produce the macrolones, would incorporate the beneficial
antibacterial activities of both classes.
3
1
In our previous work , we have synthesized a range of 15-membered macrolone
analogues, which has provided us with the information toward an understanding of the
essential structural features needed for antibacterial activity. Specifically, it was recognized
that the peripheral modification of the macrolide core, particularly at the C-4” position of
cladinose, with quinolone pharmacophore substructures could significantly enhance
antibacterial activity against a variety of erythromycin-susceptible and MLS -resistant
B
3
2
Gram-positive pathogens as well as fastidious Gram-negative pathogens. However, contrary
to our expectations of their dual mode of action, these studies have also shown that the C-4”
substituted macrolones actually act as protein inhibitors in agreement with their exclusive
macrolide mode of action. In addition, this approach seems to be very promising in the
development of leads for drug discovery applications, as the antibacterial activity of several
new C-4” substituted macrolones exceeds that of the parent compounds. Interestingly, the
antibacterial activity of some of these new synthesized C-4” substituted azithromycin
6

derivatives rivaled the action of some of the most effective and most advanced
3
3-35
representatives of ketolide class of antibacterial agents
pathogens.
against all key respiratory
2
. Results and discussion
2
.1. Chemistry
In order to expand the structure-activity relationships and more thoroughly define the
scope of activity, and select the promising candidates for preclinical development we felt that
synthesis of additional 4”-substituted azithromycin analogues could even further facilitate the
construction of compound libraries for biological screening, and therefore, considered such
an endeavor. We recognized that the C-4” hydroxyl group of azithromycin offered the
possibility of alternative modes of attaching the side chain other than the ether linkage. As it
had previously been demonstrated that the secondary C-4” hydroxyl group could be relatively
easily alkylated with activated alkylating agent, such as allyl-t-butylcarbonate, we reasoned
that a series of macrolones having the side chain attached through a C-4” glycyl ester linkage
should be synthetically feasible. Our strategy was to functionalize the C-4” position of
azithromycin with a suitable small linker sidearm which contain an appropriate functionality
at the terminal position. Basically, it was anticipated that BOC protected glycin could be used
as starting material to regioselectively acylate the C-4” hydroxyl group of azithromycin.
Subsequent deprotection of BOC protecting group should afford 4”-glycyl azithromycin that
7

could be used as a key intermediate for further derivatization via amide coupling or reductive
amination. In this article, we report the successful implementation of this strategy resulting in
a series of 4”-glycyl tethered azithromycin macrolones with excellent in vitro activity. A
typical design leading to novel C-4”-glycyl linked macrolides tethered to quinolone subunit
by different linkers is shown in Figure 2.
N
N
OH
HO
1
1
HO
OH
1
2
2'
O
O
O
O
4
O
O
O
H
O
O
"
N
6
7
O
OH
O
Glycyl side
chain
Linker group
R1
N
C-4"-Ester linked macrolide
R2
R = F, Cl
1
R = cPr
2
Quinolone substructure
Figure 2. Design leading to novel C-4”-glycyl linked macrolides
In order to selectively acylate the hydroxyl group at the C-4”-position, a regioselective
protection strategy had to be employed. This approach involved selective acylation of the C-4”
hydroxyl group using BOC-protected glycin. Thus, commercially available azithromycin was
first protected as its 2’-O-acetyl-11,12-cyclic carbonate derivative 4 according to a modified
3
6
literature procedure (Scheme 1). Selective acylation with BOC glycin in the presence of a
catalytic amount of DMAP followed by subsequent deprotection of the BOC protecting group
with trifluoroacetic acid gave the expected trifluoroacetate salt of 2’-O-acetyl-4”-O-glycyl
azithromycin-11,12-cyclic carbonate 6 in 75% isolated yield. Deprotection of the 2’-acetyl
8

group was readily accomplished by stirring with aqueous ammonia in methanol at room
temperature to give 4”-glycyl azithromycin 7 in essentially quantitative yield. The structure
1
13
of 7 was confirmed via H and C NMR spectroscopy. This compound served as a key
intermediate for the preparation of a series of amide analogues according to Scheme 1.
Scheme 1. Synthesis of C-4”-ester linked macrolides by HBTU mediated coupling of key
a
intermediate 7 with selected carboxylic acids (Chart 1)
N
N
O
O
O
O
OH
AcO
N
O
O
O
N
O
OH
AcO
O
a
O
O
O
O
O
O
O
O
O
O
NHBoc
OH
O
O
O
4
5
b
N
N
O
N
c
O
O
OH
N
O
O
O
OH
OH
O
AcO
O
O
O
O
O
O
O
O
O
CF3COO
NH3
O
O
O
O
NH2
O
O
O
O
7
6
d
N
N
O
N
O
OH
O
O
OH
N
O
OH
OH
HO
O
OH
e
O
O
O
O
O
O
O
O
O
O
O
H
N
H
N
R1
R1
O
O
O
O
O
O
8
a-o
9 a-o
a
.
Reagents and conditions: a) BocGly, EDC HCl, DMAP, CH
2
Cl
2
, rt, 85%; b) TFA, CH
2
Cl ,
2
9

0
°C to rt, 95%; c) aq. NH
3
, MeOH, rt, 90%; d) R
1
CO H, HBTU, DIPEA, DMF, rt, 70-90%;
2
e) LiOH, THF/H O, rt, 85-95%.
2
HBTU mediated coupling of 7 with C-6 and C-7 substituted quinolone carboxylic acids
Chart 1) in the presence of DIPEA cleanly afforded the desired amides 8a-o in yields
(
ranging from 70-90%. Deprotection of cyclic carbonates 8a-o with LiOH in aqueous THF
finally gave azithromycin derivatives 9a-o which were purified by flash column
chromatography.
Chart 1. Quinoxalines and quinolones used as the R
reductive amination of 7
1
substituents in HBTU coupling and
N
N
N
N
O
N
O
O
O
H
H
N
R
N
R
N
OH
S
S
R
H
O
O
Cl
a, R= OH
b, R= H
c, R= OH
d, R= H
e, R= OH
f, R= H
O
O
O
N
O
O
N
O
H
N
H
N
F
F
R
O
OH
H
OH
O
OH
O
R
N
O
R
N
H
N
H
HN
N
Cl
O
g, R= OH
h, R= H
i, R= OH
j, R= H
k, R= OH
l, R= H
O
N
O
O
R
O
N
O
H
F
O
N
H
OH
N
O
OH
R
N
O
H
O
N
H
Cl
O
m, R= OH
n, R= H
o, R= OH
p, R= H
Carboxylic acid a used for HBTU coupling reaction was commercially available while
1
3
the acid c was synthesized according to our published procedure. On the other hand, the
synthesis of carboxylic acids e, g, i, k, m, and o was effected by treatment of the
10

commercially available 6-fluoro-7-chloro-1-cyclo-propyl-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (10) with appropriate amine and K
2
CO in refluxing 1-methyl-2-pyrolidone,
3
3
7
followed by Michael addition of the resulting separated C(7)-chloro and C(6)-fluoro
regioisomers 11a-13a and 11b-13b to acrylonitrile according to a modified published
3
8
procedure (Scheme 2). Michael addition proceeded in a large excess of acrylonitrile and a
catalytic amount of DBU (0.5 molar equiv.) at 70°C for 15 h.
a
Scheme 2. Synthesis of C-6 and C-7 substituted quinolone carboxylic acids (e-o)
O
O
O
N
O
H
N
O
N
O
H2N
F
F
a
OH
OH
X
+
OH
X
N
Cl
H2N
N
Cl
H
Separated C(7)-Cl and
C(6)-F regioisomers
10
b
b
1
1
1
1a/11b (x= CH CH )
2 2
2a/12b (x= CH CH OCH CH )
2
2
2
2
3a/13b (x= CH CH OCH CH OCH CH )
2
2
2
2
2
2
O
O
H
H
O
O
N
F
N
OH
NC
X
OH
NC
X
N
N
N
N
H
Cl
H
1
4a/14b (x= CH CH )
2 2
c
c
1
5a/15b (x= CH CH OCH CH )
2 2 2 2
1
6a/16b (x= CH CH OCH CH OCH CH )
2
2
2
2
2
2
H
O
O
H
O
O
N
HO
N
F
X
O
OH
OH
O
X
Cl
N
HO
N
N
N
H
H
e/g (x= CH2CH2)
i/k (x= CH2CH2OCH2CH2)
m/o (x= CH2CH2OCH2CH2OCH2CH2)
a
Reagents and conditions: a) ethylenediamine or 2-(2-aminoethoxy)ethanamine or
2
2
-(2-(2-aminoethoxy)ethoxy)ethanamine, K
2
CO
3
, 1-methyl-2-pyrolidone, 100°C, 8 h,
SO /H O (1:1), 70°C, 5 h, 100%.
5-40%; b) acrylonitrile, DBU, 70°C, 15 h, 60-70%; c) H
2
4
2
The regioisomeric nitriles 14a-16a and 14b-16b thus obtained were then subjected to acidic
11

hydrolysis in aqueous H
2
SO (1:1) at 70°C to afford the corresponding carboxylic acids e-m
4
and g-o in essentially quantitative yield after acidic workup at pH=3.
The synthesis of aldehydes used for reductive amination is illustrated in Scheme 3.
Treatment of the regioisomerically pure quinolone amines 11a-13a and 11b-13b with 5 equiv.
3
9
of 3-acryloyl oxazolidin-2-one 17 in i-PrOH at 70°C produced a series of Michael addition
adducts 18a-20a and 18b-20b which were immediately used in the next step without further
purification. Subsequent reduction of the oxazolidinones 18a-20a and 18b-20b with
diisobutylaluminum hydride at -78°C in THF afforded the desired aldehydes f-n and h-p in
acceptable yields (60-80%).
a
Scheme 3. Synthesis of C-6 and C-7 substituted quinolone aldehydes (f-p)
O
O
H
H
H
N
O
N
O
H
N
O
N
O
H
N
O
N
O
H2N
N
N
H
N
a
b
X
OH
X
OH
X
OH
O
O
O
Cl
Cl
Cl
O
O
N
7
1
f (x= CH CH )
1
1
1
1a (x= CH CH )
2a (x= CH CH OCH CH )
3a (x= CH CH OCH CH OCH CH )
18a (x= CH2CH2)
19a (x= CH2CH2OCH2CH2)
20a (x= CH2CH2OCH2CH2OCH2CH2)
2
2
2
2
j
(x= CH CH OCH CH )
2 2 2 2
2
2
2
2
n (x= CH CH OCH CH OCH CH )
2
2
2
2
2
2
2
2
2
2
2
2
O
N
O
O
N
O
O
O
a
F
b
F
OH
F
OH
O
O
O
OH
X
X
X
N
O
N
N
N
H
N
N
H2N
N
H
H
H
H
H
1
1
1
1b (x= CH2CH2)
2b (x= CH2CH2OCH2CH2)
3b (x= CH2CH2OCH2CH2OCH2CH2)
18b (x= CH2CH2)
19b (x= CH2CH2OCH2CH2)
20b (x= CH2CH2OCH2CH2OCH2CH2)
h (x= CH2CH2)
l (x= CH2CH2OCH2CH2)
p (x= CH2CH2OCH2CH2OCH2CH2)
a
Reagents and conditions: a) 3-acryloyl oxazolidin-2-one (17), DIPEA, i-PrOH, 70°C, 16 h,
5-85%; b) DIBAL-H, THF, -78°C, 3 h, 60-80%.
6
Additionally, an alternate route had to be developed to synthesize aldehydes b and d. Thus,
12

1
3
the known methyl esters of carboxylic acids a and c were subjected to DIBAL-H reduction
at -78°C in THF to afford the corresponding aldehydes b and d in 68% and 75% isolated
yield, respectively.
Next we focused our attention on the effects of modification of the linker-glycyl side
chain connection moiety on the antibacterial activity. From 4”-O-glycyl azithromycin
11,12-cyclic carbonate (7), another series of azithromycin analogues was prepared by
reductive amination with aldehydes (Chart 1), as outlined in Scheme 4.
Scheme 4. Synthesis of C-4”-ester linked macrolides by reductive amination of amine 7 with
a
selected aldehydes (Chart 1)
N
N
O
O
O
OH
N
O
O
OH
O
O
OH
N
O
O
a
OH
O
O
O
O
O
O
O
O
O
O
O
NH2
H
O
N
O
R1
O
O
7
21 b-p
b
N
N
OH
OH
HO
OH
O
O
O
O
O
O
O
H
N
O
R1
O
22 b-p
a
Reagents and conditions: a) R
1
CHO, NaBH(OAc) , AcOH, MeOH, rt, 20 h, 60-90%; b)
3
LiOH, THF/H O, rt, 15 h, 85-95%.
2
13

4
0
Reductive amination of 7 with C-6 and C-7 substituted quinolone aldehydes (Chart 1) using
sodium triacetoxyborohydride proceeded smoothly to afford the corresponding azithromycin
analogues 21b-p in generally acceptable isolated yields ranging from 60-90%. Subsequent
deprotection of cyclic carbonates 21b-p was carried out with LiOH in aqueous THF to afford
azithromycin derivatives 22b-p in 85-95% isolated yield.
2
.2. Antibacterial activity
The antibacterial activity of the C-4”substituted azithromycins was tested against a panel
of representative pathogens selected from the Pliva Research Institute culture collection. The
in vitro antibacterial activities are reported as minimum inhibitory concentrations (MICs) that
4
1
were determined by the agar microdilution method according to NCCLS standards. Table 1
shows the in vitro activity of the azithromycin analogues and the reference compounds,
azithromycin, telithromycin (1), cethromycin (2), and ciprofloxacin.
The results tabulated in Table 1 show that although azithromycin is very potent against
erythromycin susceptible strains it is only weakly active against efflux resistance (mef) and
inactive against MLS resistance. On the other hand, telithromycin (1) and cethromycin (2)
B
showed activity against all key resistant pathogens tested except constitutively MLS
B
resistant
S. aureus. In general, the macrolones were all inactive against constitutively MLS
B
-resistant
strain of Staphylococcus aureus (MICs  64 g/mL). They were, however, in few cases (9e and
14

2
2f), about one dilution more potent than azithromycin against H. influenzae strain. In contrast
3
2
to 4”-ether macrolones , most of these compounds were active against inducibly resistant S.
aureus strain. The most interesting feature of these new compounds was their effectiveness
against efflux resistant staphylococci and pneumococci as well as the activity of selected
analogues against inducibly and constitutively MLS -resistant pneumoccoci.
B
Table 1. In Vitro Antibacterial Activity of C-4”-Ester Linked Macrolides against Selected
a,b
Pathogens
S. aureus
iMLS cMLS
S. pneumoniae
S. pyogenes
c
d
Compound
7
M. cat. H. inf.
Ery-S
32
B
B
M
>64
8
Ery-S
cMLS
>64
16
16
0.5
4
B
M
32
Ery-S iMLS
B
cMLS
>64
32
16
0.5
4
B
M
>64
0.5
0.25
0.25
2
>64
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.25
32
8
>64
>64
8
9a
9c
9e
9g
4
0.25
0.125
0.25
1
16
1
0.5
1
2
4
4
4
0.125
0.25
0.5
2
0.125
0.125
0.125
0.125
0.125
0.125
0.25
1
0.125
0.5
4
8
1
0.5
1
9i
0.125
0.25
2
1
1
0.5
4
0.5
4
2
1
2
9k
4
4
8
16
8
8
4
9m
0.125
0.5
4
0.5
16
2
4
1
1
2
1
4
9
o
32
0.5
0.25
0.25
1
8
4
4
32
16
8
16
2
8
2
2
2b
2d
2
16
8
0.125 0.125
0.125 0.125
4
0.25
0.125
1
4
8
1
1
8
8
22f
0.125
0.125
0.125
0.25
0.25
0.5
0.5
1
0.125 0.125 0.125 0.125 0.125 0.125 0.125 0.125
0.5
1
22h
0.125
0.125
0.125
0.125
0.125
0.5
0.25
2
0.125 0.125
0.25
0.25
0.5
1
1
0.5
0.5
2
0.25
0.25
1
2
2j
2l
0.5
2
0.25
0.25
0.5
1
0.125
0.125
0.125
0.125
2
2
4
2
2
2
2n
2p
1
0.125
0.125
0.125
0.125
1
1
1
1
0.25
0.5
2
1
0.5
2
4
2
2
4
1
2
4
0.5
0.25
>64
16
0.25
0.25
32
0.125 0.125
0.5
0.125 0.125
8
0.25
0.125
1
2
0.125 0.125 0.125 0.125 0.125
1
0.125 0.125
16 0.25
1
Azi
0.125
>64
8
0.125
>64
>64
0.5
1
Cipro
≤0.125
≤0.125 ≤0.125 ≤0.125
0.25
0.25
2
0.25
0.125 0.125 0.125
15

a
b
Minimum inhibitory concentration (MIC) values are given in g/mL. All quinolones (a-p) were
c
d
inactive (MIC’s˃ 64 g/mL) against cMLS
B
S. aureus strain. M. catarrhalis. H. influenzae. Ery-S:
: inducible resistance to MLS antibiotics; cMLS
resistance; M: efflux mediated macrolide resistance. Azi: azithromycin. Cipro:
erythromycin-susceptible strains; iMLS
B
B
B
:
constitutive MLS
ciprofloxacin.
B
2
.2.1. Structure–activity relationships of 4”-glycyl linked azithromycin macrolones
The key intermediate used for further derivatization, 4”-glycyl azithromycin 7 was
basically inactive against all the strains tested except erythromycin-sensitive S. pneumoniae
and S. pyogenes (MIC 0.125 g/mL). The presence of a quinoxaline ring in the macrolones
9
a, 9c, 22b, and 22d resulted in marked increases in activity against inducibly MLS resistant
B
S. aureus and S. pyogenes as well as constitutively MLS resistant strains of S. pneumoniae
B
and S. pyogenes. Moreover, the compounds were active against both efflux-resistant S.
pneumoniae and S. pyogenes.
3
2
By analogy to 4”-ether macrolone series developed previously , it was anticipated that
attaching a quinolone side chain to 7 would lead to an additional improvement in activity,
particularly against inducibly and constitutively MLS
B
resistant S. pneumoniae and S.
pyogenes. Initially, a series of quinolone-substituted compounds 9e-o was prepared to
identify the optimum linker between the quinolone ring and the C-4” position of
azithromycin. It was found that compounds with the shorter sidechains such as 9e and 9g
were superior to those with the longer sidechains as in 9i, 9k, 9m, and 9o. In particular, 9e
and 9g showed good activity against constitutively MLS resistant strains of S. pneumoniae
B
16

and S. pyogenes and the inducibly resistant S. aureus strain, whereas 9i, 9k, 9m, and 9o were
basically less potent against all of these strains. All six of the compounds exhibited improved
activity against both the erythromycin-susceptible and the efflux-resistant S. pneumoniae
strains, when compared to 4”-glycyl azithromycin 7. As with telithromycin, none of the
compounds showed measurable activity against the constitutively resistant S. aureus. Overall,
the macrolone 9e appeared to have the best antibacterial profile, being the most potent within
the series of 6 amide analogues against S. aureus, H. influenzae, and constitutively MLS
resistant S. pneumoniae and S. pyogenes.
B
Having identified the linker in macrolone 9e as optimal, we synthesized a series of
macrolones in which amide bond connecting the macrolide and quinolone pharmacophores
was replaced with an amine bond as in compounds 22f, 22j, 22n, 22h, 22l, and 22p. The
4
”-glycyl amine series (22f-p) demonstrated much higher levels of in vitro activity in
comparison to the glycyl amide series (9e-o) as the MIC’s were consistently lower. Thus, the
activity of the macrolone with the longest side chain 22n was 2 to 4-fold better to its amide
counterpart 9m, with the exception of 2-fold lower potency against the efflux resistant S.
aureus strain. The antibacterial activity of the macrolone 22j was in general 2 to 4-fold better
in comparison to 9i, while the macrolone with the shortest side chain 22f also showed 2 to
4
-fold better activity when compared to 9e against most of the strains tested. In fact,
compound 22f in vitro profile compares favorably with telithromycin and cethromycin.
17

In general, the structure-activity relationships of both series were quite similar to that of
C-4” ether macrolones developed previously with the exception of being more potent against
3
2
inducibly and efflux resistant strains of S. aureus. The compounds most active against
constitutively resistant streptococcal strains (9e, 22f) are linked through the C-6 position of
the quinolone nucleus and the C-4”-position of azithromycin by a spacer of ten atoms in
length. In an amide series, antibacterial results show a linker-length-dependent activity which
peaked with compound 9e, a macrolide analogue having a ten carbon-heteroatom linker
separating the quinolone ring from the C-4” ester oxygen on the cladinose ring. Compounds
9
i and 9m, analogues having longer carbon-heteroatom linkers than those of 9e, show a
reduction in antibacterial activity with an increase in the linker length. In a similar fashion
compounds within an amine series also show gradual reduction in activity, the analogue with
the shortest, ten carbon-heteroatom linker in the series 22f being the most active, following
by the macrolones 22j and 22n having 13 and 16 carbon-heteroatom spacers, respectively.
The antibacterial activity of the C-6 substituted quinolones within an amide series 9e, 9i, and
9
9
m were in general 2-8-fold better than that of the corresponding C-7 substituted analogues
g, 9k, and 9o, respectively, against most of the strains tested. In the same way, the
antibacterial activity of the C-6 substituted amine analogues 22f, 22j, and 22n were found to
be superior to their corresponding C-7 substituted counterparts, 22h, 22l, and 22p,
respectively. In addition, potential lead compounds 9e and 22f demonstrated outstanding
18

levels of activity against H. influenzae and constitutively MLS resistant S. pyogenes. In
B
particular, 9e and 22f were more potent than telithromycin against H. influenzae strain (MICs
0
.5 g/mL) and cMLS S. pyogenes (MICs of 0.125).
B
3
. Conclusion
In summary, a novel series of azithromycin derivatives with potent activity against key
respiratory pathogens, including those resistant to macrolide antibiotics, has been identified.
These compounds are characterized by having C(6) or C(7)-substituted quinolonyl moiety
tethered to the C-4”-position of the azithromycin skeleton through an ester bond. Biological
evaluation of these analogues revealed several highly potent compounds effective against
both erythromycin-susceptible and MLS -constitutively resistant pathogens. In general,
B
amide-linked quinolone analogues (9e-o) were not as active as macrolones (22f-p)
incorporating quinolone substructures linked via an amine group. On the other hand,
compounds containing the quinoxaline ring showed weak activity against constitutively
MLS resistant S. pneumoniae and S. pyogenes. However, they did show excellent activity
B
against inducibly MLS resistant S. aureus and both mef-resistant S. pneumoniae and S.
B
pyogenes.
4
. Experimental section
.1. General experimental methods
4
19

1
13
Proton ( H) and carbon ( C) nuclear magnetic resonance spectra were recorded on either a
Bruker 300 or a Bruker 500 MHz spectrometer. Chemical shifts (δ) were recorded in parts per
million (ppm) relative to tetramethylsilane as an internal standard. Low-resolution electron
impact mass spectra (MS) were recorded on a Varian MAT CH5 spectrometer. Fast atom
bombardment (FAB) mass spectra were run on a Finnigan MAT 312 double focusing mass
spectrometer, operating at an accelerating voltage of 3kV. The samples were ionized by
bombardment with xenon atoms produced by a saddle-field ion source from Ion Tech operating
with a tube current of 2 mA at energy of 6 keV. Electrospay positive ion mass spectra were
acquired using a Micromass Q-Tof 2 hybrid quadrupole time-of-flight mass spectrometer,
equipped with a Z-spray interface, over a mass range of 100-2000 Da, with a scan time of 1.5 s
and an interscan delay of 0.1 s in a continuum mode. Reserpine was used as the external mass
+
calibrant lock mass ([M+H] = 609.2812 Da). The elemental composition was calculated using
+
a MassLynx v4.1 for the [M+H] and the mass error quoted within 5 ppm range. Thin-layer
chromatography (TLC) was performed with Merck silica gel 60 F254 0.2-mm plates. The
plates were visualized by using an acid-based stain, prepared from p-anisaldehyde (5 mL),
concentrated sulfuric acid (5 mL), and glacial acetic acid (0.5 mL) in 95% ethanol (90 mL) and
warming on a hot plate. Flash chromatography was carried out using Merck silica gel 60
(
230-400 mesh). Solvent systems are reported as volume percent mixtures. Concentration in
vacuo refers to the removal of solvent using a Büchi rotary evaporator and an aspirator pump.
20

All chromatography solvents were reagent grade. Tetrahydrofuran (THF) was distilled from
sodium benzophenone ketyl, and dichloromethane was distilled from calcium hydride. All
other reagents were purified by literature procedures. All reactions were performed under an
inert atmosphere of dry argon. The course of the reaction was followed by chromatography on
a thin layer (TLC) of silica gel (Merck 60 F254) in solvent systems methylene
chloride-methanol-ammonium hydroxide 25% (90:9:1.5, system A), (90:9:0.5), system A1) or
methylene chloride-acetone (8:2, system B) (7:3, System C) unless otherwise stated. The
separation of the reaction products and the purification of the products for the purpose of
spectral analyses were performed on a silica gel column (Merck 60, 230-400 mesh, or 60-230
mesh in solvent systems A, B or C unless otherwise stated. Purity of tested compounds was
determined by elemental analysis.
The structure of all novel compounds was confirmed by HRMS, and/or NMR spectroscopic
1
13
methods. Complete and unambiguous assignments for all H and C resonances could be
1
3
achieved on the basis of chemical shift considerations, coupling information (APT C NMR
spectra), and COSY, HSQC, and HMBC spectra.
Reagents were generally purchased in the highest purity available and used without further
3
0
3
11
purification except otherwise stated. Azithromycin , telithromycin (1) and cethromycin (2)
were synthesized in-house according to literature procedures and their in vitro evaluation were
performed to allow direct comparison with 15-membered azalides presented in this paper.
21

4
.2. Determination of Minimum Inhibitory Concentration (MICs)
The MICs of all antibiotics were determined by the agar microdilution method as
recommended by National Committee of Clinical Laboratory Standards (NCCLS) guidelines.
Differences in MIC values were considered significant only when the dilution is more than a
factor of 2 apart. Zone sizes were measured with a Fisher Zone Reader, and antibiotic
concentrations were calculated from the standard curve for the appropriate compound.
4
4
.3. Experimental Procedures
.3.1. 2’-O-Acetyl-4”-O-tert-butyloxycarbonylglycyl-azithromycin-11,12-cyclic carbonate (5)
To a solution of 2’-O-acetyl azithromycin-11,12-cyclic carbonate 4 (817.0 mg, 1.0 mmol, for
1
H NMR spectrum of 4 see the Supporting Information, page S3) in dry dichloromethane (40
mL) was added Bocglycin (437.5 mg, 2.5 mmol), EDC HCl (639.0 mg, 3.35 mmol), and
4
-DMAP (102 mg, 0.85 mmol) under argon atmosphere. The resulting mixture was stirred at
room temperature for 20 hours. After evaporation to dryness, the residue was dissolved in
ethyl acetate (150 mL), washed sequentially with saturated aqueous solution of NaHCO
3
(
2x100 mL) and saturated aqueous NaCl solution (2x100 mL), and dried over anhydrous
magnesium sulfate. The solvent was then evaporated in vacuo and the residue purified by
flash chromatography on silica gel column (eluens: 90/10 CH
2
Cl
2
/MeOH) to yield 720.9 mg
+
(
74%) of 5 as a colorless solid. HRMS (ES) calcd for C48
H
83
N
3
O
17 (M+H) 974.5722, found
1
9
74.5715. H NMR (500 MHz, CDCl
3
)  8.00 (bs, 1H, tert-BuOCONH), 4.85 (d, 1H, H-1"),
22

4
.80 (dd, 1H, H-13), 4.75 (dd, 1H, H-2’), 4.65 (d, 1H, H-4”), 4.42 (d, 1H, H-1’), 4.30 (m, 1H,
H-11), 4.25 - 4.10 (m, 2H, H-5'', H-3), 3.93 (m, 2H, tert-BuOCONHCH ), 3.70 (m, 1H, H-5'),
2
3
.45 (m, 1H, H-5), 3.28 (s, 3H, 3''-OMe), 3.13 (m, 1H, H-10), 2.70 (m, 1H, H-2), 2.50-2.30
m, 2H, H-3', H-9a), 2.25-2.20 (m, 2H, H-2''b, H-9b), 2.15 (s, 6H, 3'-NMe ), 2.13 (s, 3H,
-NMe), 2.10 (s, 3H, 2’-OCOMe), 2.01 (m, 1H, H-9b), 1.92-1.85 (m, 3H, H-4, H-7b, H-8),
.75-1.60 (m, 2H, H-14b, H-14a), 1.56-1.49 (m, 2H, H-4'b, H-2''a), 1.48 (s, 3H, 12-Me), 1.45
(
2
9
1
(
m, 1H, H-7a), 1.40 (s, 9H, tert-BuOCONH), 1.23 (d, 3H, 5''-Me), 1.19-1.14 (m, 6H, 6-Me,
''-Me), 1.12 (d, 3H, 2-Me), 1.10 (m, 1H, H-4'a), 1.05 (d, 3H, 5'-Me), 1.00-0.93 (m, 6H, 4-Me,
3
1
1
13
0-Me), 0.88 (d, 3H, 8-Me), 0.85 (t, 3H, 15-Me); C NMR (125 MHz, CDCl
54.3 (tert-BuOCONH), 152.4 (11,12-C=O), 171.8 (4”-C=O), 170.0 (2’-OCOMe), 102.2
C-1'), 94.7 (C-1''), 85.9 (C-4''), 85.2 (C-11), 84.5 (C-12), 82.3 (C-5), 77.0 (C-3), 75.1 (C-13),
9.2 (tert-BuOCONH, s, C), 73.5 (C-6), 72.9 (C-3''), 70.2 (2C; C-2', C-5'), 66.5 (C-9), 64.6
C-3'), 59.8 (C-5''), 59.5 (C-10), 47.4 (3''-OMe), 44.6 (C-2), 41.8 (C-4), 41.1 (C-7), 40.4
3'-NMe ), 34.5 (C-2''), 34.0 (9a-NMe), 30.4 (C-4'), 28.4 (tert-BuOCONH, q, CH ), 27.2
3
)  177.7 (C-1),
(
7
(
(
(
(
2
3
6-Me), 25.2 (C-8), 22.6 (8-Me), 21.8 (5'-Me), 21.5 (C-14), 21.0 (3''-Me), 18.8 (5''-Me), 14.7
2-Me), 13.5 (12-Me), 10.6 (C-15), 9.2 (4-Me), 5.6 (10-Me). Anal. Calcd. For C48
H
83
N
3
17
O :
C, 59.18; H, 8.59; N, 4.31 Found: C, 59.35; H, 8.81; N, 4.10.
.3.2. 2’-O-Acetyl-4”-O-glycyl-azithromycin-11,12-cyclic carbonate trifluoroacetate salt (6)
To a stirred solution of 2’-O-Acetyl-4”-O-Bocglycyl-azithromycin 5 (974.2 mg, 1.0 mmol) in
4
23

dichloromethane (20 mL) cooled in an ice-water bath was added trifluoroacetic acid (456.1
mg, 4.0 mmol). The mixture was allowed to warm to room temperature for 6 hours followed
by evaporation of the volatile materials under reduced pressure. The residue was crystallized
from diethyl ether/dichloromethane mixture (1:1) and filtered to afford trifluoroacetate salt 6
(
859.6 mg, 87%) of sufficient purity to be used in subsequent reaction step without further
+
purification. HRMS (ES) calcd for C45
H
76
F
3
N
3
O
17 (M+H) 988.5127, found 988.5144. Anal.
Calcd. For C45
H
76
F
3
3
N O17: C, 54.70; H, 7.75; N, 4.25 Found: C, 55.34; H, 7.81; N, 4.11.
4
.3.3. 4”-O-glycyl-azithromycin-11,12-cyclic carbonate (7)
To a solution of 6 (889.3 mg, 0.9 mmol) in 20 mL of methanol was added 10 mL of aqueous
NH (25%). The solution was stirred at room temperature for 18 h. The solvent was removed
3
in vacuo, water (100 mL) was added, and the pH was adjusted to 8 with concentrated aqueous
ammonium hydroxide. The solution was extracted with ethyl acetate (2x100 mL), and the
organic extracts were dried over K
2
CO to give 850.0 mg of white foam. The product was
3
purified by flash column chromatography eluting with methylene chloride-methanol-
ammonium hydroxide 25% (90:9:0.5) solvent system, to afford 688.9 mg (92%) of the title
+
compound 7 as colorless foam. HRMS (ES) calcd for C41
H
73
N
3
O
14 (M+H) 832.5093, found
1
8
1
3
32.5105. H NMR (500 MHz, CDCl
3
) : 5.20 (dd, 1H, H-1”), 4.71(m, 1H, H-13), 4.65 (m,
H, H-4”), 4.57 (d, 1H, H-1’), 4.25 (m, 1H, H-3), 3.81 (m, 1H, H-5”), 3.70 (s, 1H, H-11),
.66 (m, 1H, H-5’), 3.63 (d, 1H, H-5), 3.32 (s, 3H, 3”OMe), 3.23 (dd, 1H, H-2’), 2.76 (m, 1H,
24

H-2), 2.75 (m, 2H, CH
2
NH ), 2.71 (m, 1H, H-10), 2.55 (m, 1H, H-3’), 2.55 (m, 1H, H-9a),
2
2
.39 (d, 1H, H-2”a), 2.32 (s, 3H, 9-NMe), 2.31 (s, 6H, 3’-NMe
m, 1H, H-8), 2.01 (m, 1H, H-4), 1.91 (m, 1H, H-14a), 1.78 (d, 1H, H-7a), 1.68 (m, 1H,
H-4’a), 1.61 (dd, 1H, H-2”b), 1.47 (m, 1H, H-14b), 1.31 (s, 3H, 6-Me), 1.29 (m, 1H, H-7b),
2
), 2.07 (m, 1H, H-9b), 2.04
(
1
3
8
.26 (m, 1H, H-4’b), 1.21 (d, 3H, 2-Me), 1.19 (d, 3H, 5”-Me), 1.16 (d, 3H, 5’-Me), 1.15 (s,
H, 3”-Me), 1.11 (s, 3H, 12-Me), 1.09 (d, 3H, 10-Me), 1.06 (d, 3H, 4-Me), 0.91 (d, 3H,
1
3
-Me), 0.89 (t, 3H, 15-Me). C NMR (125 MHz, CDCl
OCOCH NH ), 152.4 (11,12-C=O), 102.3 (C-1’), 94.6 (C-1”), 83.1 (C-5), 78.7 (C-4”), 77.6
C-3), 77.3 (C-13), 74.2 (C-11), 73.5 (C-6), 73.3 (C-12), 72.9 (C-3”), 70.8 (C-2’), 70.0 (C-9),
3
) : 178.8 (C-1), 171.9
(
(
2
2
6
4
2
8.2 (C-5’), 67.7 (C-5”), 65.6 (C-3’), 62.4 (C-10), 53.3 (CH
2
NH ), 49.3 (3”OMe), 45.1 (C-2),
2
2.1 (C-7), 42.0 (C-4), 40.2 (3’-NMe ), 36.2 (9N-Me), 34.9 (C-2”), 28.8 (C-4’), 27.5 (6-Me),
2
6.7 (C-8), 22.8 (5’-Me), 21.9 (8-Me), 21.4 (C-14), 21.2 (3”-Me), 17.7 (5”-Me), 16.1
(
12-Me), 14.4 (2-Me), 11.2 (15-Me), 8.9 (4-Me), 7.3 (10-Me). Anal. Calcd. For C41
H
73
N
3
14
O :
1
C, 59.19; H, 8.84; N, 5.05. Found: C, 59.37; H, 8.90; N, 4.75. For H and COSY NMR
spectra of 7 in CD OD see the Supporting Information S4-S5.
3
4
4
.3.4. HBTU coupling. General procedure.
.3.4.1. 4”-O-(glycyl-(6-(2-(2-carboxyethylamino)ethylamino)-7-chloro-1-cyclopropyl-1,4-
dihydro-4-oxoquinolin-6-yl-3-carboxy)-azithromycin-11,12-cyclic carbonate (8e)
25

DIPEA (201.5 L, 1.4 mol. equiv.) was added dropwise via syringe at 0°C to a solution of
6
3
-(2-(2-carboxyethylamino)ethylamino)-7-chloro-1-cyclopropyl-1,4-dihydro-4-oxoquinoline-
-carboxylic acid e (98.5 mg, 0.25 mmol) and HBTU (92.8 mg, 0.25 mmol) in dry DMF (3.0
mL). The mixture was stirred for 15 min before 4”-O-glycyl-azithromycin-11,12-cyclic
carbonate 7 (149.8.3 mg, 0.18 mmol) was added over a period of 30 min. The reaction mixture
was stirred at room temperature overnight (20 h), and then diluted with water (40 mL). The
aqueous phase was extracted twice with EtOAc (2x50 mL), and the combined organic phases
were washed sequentially with saturated aqueous NaHCO (40 mL), and brine (40 mL). Drying
3
with Na
2
SO and evaporation afforded 157.5 mg (70%) of the title compound 8e as a colorless
4
1
solid. HRMS (ES) calcd for C59
H
91ClN O18 (M+H) 1207.6078, found 1207.6080. H NMR
6
(
500 MHz, CDCl
H, H-1”), 4.73(m, 1H, H-13), 4.60 (m, 1H, H-4”), 4.55 (d, 1H, H-1’), 4.24 (m, 1H, H-3),
.16 (m, 2H, COCH NHCO), 3.80 (m, 1H, H-5”), 3.69 (s, 1H, H-11), 3.65 (m, 1H, H-5’),
3
) : 8.70 (s, 1H), 8.03 (s, 1H), 7.59 (s, 1H), 6.35 (bs, 1H, CONH), 5.25 (dd,
1
4
3
3
2
.60 (d, 1H, H-5), 3.50 (m, 1H, cyclopropyl CH), 3.33 (s, 3H, 3”OMe), 3.24 (dd, 1H, H-2’),
.18 (t, 2H), 2.95 (t, 2H), 2.80 (t, 2H), 2.75 (m, 1H, H-2), 2.70 (m, 1H, H-10), 2.59 (m, 1H,
H-3’), 2.53 (m, 1H, H-9a), 2.38 (d, 1H, H-2”a), 2.37 (t, 2H), 2.34 (s, 3H, 9-NMe), 2.30 (s, 6H,
3
1
2
’-NMe
2
), 2.09 (m, 1H, H-9b), 2.06 (m, 1H, H-8), 2.02 (m, 1H, H-4), 1.95 (m, 1H, H-14a),
.77 (d, 1H, H-7a), 1.68 (m, 1H, H-4’a), 1.60 (dd, 1H, H-2”b), 1.45 (m, 1H, H-14b), 1.43 (m,
H, cyclopropyl CH ), 1.32 (s, 3H, 6-Me), 1.29 (m, 1H, H-7b), 1.25 (m, 1H, H-4’b), 1.22 (d,
2
26

3
H, 2-Me), 1.20 (m, 2H, cyclopropyl CH
2
), 1.17 (m, 3H, 5”-Me), 1.15 (m, 3H, 5’-Me), 1.13
(
s, 3H, 3”-Me), 1.11 (s, 3H, 12-Me), 1.08 (d, 3H, 10-Me), 1.05 (d, 3H, 4-Me), 0.92 (d, 3H,
1
3
8
1
1
-Me), 0.88 (t, 3H, 15-Me). C NMR (125 MHz, CDCl
3
) : 177.7 (4”’-CO), 177.0 (C-1),
73.5 (CONH), 172.4 (4”-OCOCH NH ), 166.5 (COOH), 153.3 (CO cyclic carbonate),
2
2
49.0, 138.9, 134.8, 129.0, 128.0, 119.7, 118.8, 110.5 (C-Ar), 102.5 (C-1’), 94.3 (C-1”), 83.8
C-5), 79.1(C-4”), 77.5 (C-3), 76.9 (C-13), 74.5 (C-6), 73.9 (C-12), 72.3 (C-11), 72.1 (C-3”),
0.4 (C-2’), 69.6 (C-9), 68.4 (C-5’), 66.5 (C-5”), 65.4 (C-3’), 62.2 (C-10), 53.1, 49.4
3”OMe), 49.2, 47.9, 45.9, 45.0 (C-2), 42.1 (C-4), 41.4 (C-7), 40.1 (3’-NMe ), 37.9, 36.1,
(
7
(
2
3
2
6.0 (9N-Me), 34.1 (C-2”), 28.7 (C-4’), 27.3 (6-Me), 25.2 (C-8), 21.8 (8-Me), 21.5 (3”-Me),
1.0 (5’-Me), 20.5 (C-14), 18.7 (5”-Me), 16.5 (12-Me), 14.8 (2-Me), 11.5 (15-Me), 9.7, 8.8
(
4-Me), 7.3 (10-Me). Anal. Calcd. For C59
H
91ClN O18: C, 58.67; H, 7.59; N, 6.96 Found: C,
6
5
8.92; H, 7.73; N, 6.79.
According to the above general procedure following 4”-O-ester linked azithromycin-
1,12-cyclic carbonates were also prepared: 8a, 8c, 8g, 8i, 8k, 8m and 8o. These compounds
1
were immediately used in the next step without further purification. For NMR spectra of 8a
see the Supporting Information, pages S6-S10.
4
4
.3.5. LiOH hydrolysis of cyclic carbonates 8a-8o. General procedure.
.3.5.1. 4”-O-(glycyl-(6-(2-(2-carboxyethylamino)ethylamino)-7-chloro-1-cyclopropyl-1,4-
dihydro-4-oxoquinolin-6-yl-3-carboxy)-azithromycin (9e)
27

To a solution of 4”-O-glycyl-(6-(2-(2-carboxyethylamino)ethylamino)-7-chloro-1-cyclo-
propyl-1,4-dihydro-4-oxoquinolin-6-yl-3-carboxy-azithromycin-11,12-cyclic carbonate 8e
(
140.0 mg, 0.11 mmol) in THF-water mixture (1:1, 10.0 mL) was added LiOH (125.2 mg, 3.0
mmol), and the resulting reaction mixture was stirred at room temperature for 12 h. The
solvent was removed under reduced pressure, and the solid was azeotroped with toluene (5 x
5
mL) and finally dried under vacuum. The acid salt was dissolved in water and the resulting
solution was made acidic by dropwise addition of 2M aqueous HCl. The precipitate was
filtered off to give 117.0 mg (87%) of the title product as colorless solid. HRMS (ES) calcd
+
1
for C58
H93ClN
6
O
17 (M+H) 1181.6286, found 1181.6293. H NMR (500 MHz, CDCl
.65 (s, 1H), 8.07 (s, 1H), 7.51 (s, 1H), 6.38 (bs, 1H, CONH), 5.29 (dd, 1H), 4.70(m, 1H), 4.62
m, 1H), 4.53 (d, 1H), 4.30 (m, 1H), 4.15 (m, 2H), 3.75 (m, 1H), 3.70 (s, 1H), 3.64 (m, 1H),
3
) :
8
(
3
2
6
1
1
3
0
.58 (d, 1H), 3.47 (m, 1H), 3.30 (s, 3H), 3.22 (dd, 1H), 3.15 (t, 2H), 2.93 (t, 2H), 2.78 (t, 2H),
.75-2.70 (m, 2H), 2.60-2.52 (m, 2H), 2.39 (d, 1H), 2.35 (t, 2H), 2.31 (s, 3H, 9-NMe), 2.28 (s,
H, 3’-NMe ), 2.10-2.05 (m, 2H), 2.00-1.94 (m, 2H), 1.74 (d, 1H), 1.65 (m, 1H), 1.60 (dd,
2
H), 1.44 (m, 1H), 1.40 (m, 2H, cyclopropyl CH ), 1.35 (s, 3H, 6-Me), 1.30-1.25 (m, 2H),
2
.23 (d, 3H, 2-Me), 1.19 (m, 2H, cyclopropyl CH ), 1.18 (m, 3H), 1.16 (m, 3H), 1.13 (s, 3H,
2
”-Me), 1.10 (s, 3H, 12-Me), 1.09 (d, 3H, 10-Me), 1.04 (d, 3H, 4-Me), 0.93 (d, 3H, 8-Me),
1
3
.89 (t, 3H, 15-Me). C NMR (125 MHz, CDCl
3
) : 177.9 (4”’-CO), 177.8 (C-1), 173.7
(
CONH), 172.1 (4”-OCOCH NH ), 166.8 (COOH), 149.5, 139.3, 135.4, 129.5, 128.1, 119.5,
2
2
28

1
18.3, 110.7 (C-Ar), 102.8 (C-1’), 94.5 (C-1”), 83.3 (C-5), 78.9 (C-4”), 77.7 (C-3), 77.5
C-13), 74.1 (C-6), 73.7 (C-12), 73.4 (C-11), 73.0 (C-3”), 70.8 (C-2’), 70.0 (C-9), 68.7 (C-5’),
7.1 (C-5”), 65.8 (C-3’), 62.6 (C-10), 52.5, 49.7 (3”OMe), 49.0, 47.3, 46.5, 45.4 (C-2), 42.6
C-4), 42.2 (C-7), 40.4 (3’-NMe ), 37.3, 36.5, 36.1 (9N-Me), 34.6 (C-2”), 28.9 (C-4’), 27.6
(
6
(
(
(
2
6-Me), 26.9 (C-8), 21.9 (8-Me), 21.6 (3”-Me), 21.4 (5’-Me), 21.3 (C-14), 18.2 (5”-Me), 16.3
12-Me), 14.5 (2-Me), 11.2 (15-Me), 9.5, 8.9 (4-Me), 7.1 (10-Me). Anal. Calcd. For
C
58
H
93ClN O17: C, 58.94; H, 7.93; N, 7.11. Found: C, 58.90; H, 7.86; N, 6.80.
6
4
.3.5.2. Spectral data for compounds 9a-9o
The following compounds were also prepared by using the same general procedure:
9
a: Yield= 72% (for 2 step sequence starting from 7). HRMS (ES) calcd for C50
H
81
N
5
14
O S
+
1
(
M+H) 1008.5501, found 1008.5505. H NMR (500 MHz, CDCl
3
) : 8.71 (m, 1H), 8.15 (m,
H), 8.03 (m, 1H), 7.74 (m, 1H), 7.65 (m, 1H), 6.35 (bs, 1H, CONH), 5.29 (dd, 1H),
.72-4.60(m, 2H), 4.50 (d, 1H), 4.32 (m, 1H), 4.16 (m, 2H), 3.82 (m, 2H), 3.74 (m, 1H), 3.68
1
4
(
(
s, 1H), 3.64 (m, 1H), 3.58 (d, 1H), 3.30 (s, 3H), 3.22 (dd, 1H), 2.75-2.68 (m, 2H), 2.62-2.50
m, 2H), 2.39 (d, 1H), 2.32 (s, 3H, 9-NMe), 2.30 (s, 6H, 3’-NMe ), 2.11-2.04 (m, 2H),
2
2
6
3
0
.02-1.92 (m, 2H), 1.75 (d, 1H), 1.65 (m, 1H), 1.59 (dd, 1H), 1.40 (m, 1H), 1.34 (s, 3H,
-Me), 1.30-1.24 (m, 2H), 1.20 (d, 3H, 2-Me), 1.18 (m, 3H), 1.16 (m, 3H), 1.14 (s, 3H,
”-Me), 1.12 (s, 3H, 12-Me), 1.10 (d, 3H, 10-Me), 1.05 (d, 3H, 4-Me), 0.95 (d, 3H, 8-Me),
1
3
.87 (t, 3H, 15-Me). C NMR (125 MHz, CDCl ) : 178.4, 173.5, 172.1, 154.1, 144.3, 142.1,
3
29