Reduced pulmonary surfactant interaction of daptomycin analogs via tryptophan replacement with alternative amino acids

Article abstract of DOI:10.1016/j.bmcl.2012.08.013

Daptomycin was shown to interact in vitro with pulmonary surfactant leading to reduction of its antibacterial activity. We report herein the preparation and anti-staphylococcal activity of a series of daptomycin analogs with reduced pulmonary surfactant interaction by replacing tryptophan with various amino acids.

Full text of DOI:10.1016/j.bmcl.2012.08.013

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6248–6251
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Reduced pulmonary surfactant interaction of daptomycin analogs
via tryptophan replacement with alternative amino acids
⇑
Yong He , Jing Li, Nin Yin, Prudencio S. Herradura, Larry Martel, Yanzhi Zhang, Andre L. Pearson,
Vidya Kulkarni, Carmela Mascio, Karen Howland, Jared A. Silverman, Dennis D. Keith, Chester A. Metcalf
Cubist Pharmaceuticals, 65 Hyden Ave, Lexington, MA 02421, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Daptomycin was shown to interact in vitro with pulmonary surfactant leading to reduction of its antibac-
terial activity. We report herein the preparation and anti-staphylococcal activity of a series of daptomycin
analogs with reduced pulmonary surfactant interaction by replacing tryptophan with various amino acids.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 15 April 2012
Revised 20 July 2012
Accepted 1 August 2012
Available online 9 August 2012
Keywords:
Daptomycin
Pulmonary surfactant
Antibacterial
Daptomycin, a fermentation product of Streptomyces roseospo-
rus, is the first in a new class of antibiotics known as lipopeptides.1
Daptomycin has a cyclic depsipeptide containing 10 amino acids
and an exocyclic three-amino acid side chain with an n-decanoyl
lipid tail attached to the N-terminus of tryptophan ([Trp1];
Fig. 1). Daptomycin is rapidly bactericidal against a variety of
Gram-positive bacteria, including antibiotic resistant isolates, such
as MRSA, through a unique mechanism of action.2 Daptomycin ap-
pears to bind to Gram-positive cell membrane by its lipid tail, and
subsequent calcium dependent insertion and oligomerization
cause disruption of bacterial membrane potential leading to rapid
cell death. Daptomycin has been approved for the treatment of
complicated skin and skin structure infections caused by Staphylo-
coccus aureus including strains resistant to methicillin (MRSA)3 and
for the treatment of bacteremia and right-sided endocarditis.4
Phase 3 clinical trials were also conducted for the treatment of hos-
pitalized patients with community-acquired pneumonia (CAP). De-
To identify novel lipopeptides with activity in pulmonary infec-
tions while maintaining the favorable physiochemical and safety
profiles of daptomycin, our efforts were focused on generating
daptomycin analogs with reduced surfactant interactions. Synthet-
ically, there are three positions in daptomycin that are readily
accessible: [Orn6], [Ser11] and the lipid tail. Although some orni-
thine analogs6 and serine analogs maintained daptomycin-like po-
tency, none of them exhibited reduced pulmonary surfactant
interaction in vitro. Similarly, lipid tail modifications resulted in
a subset of analogs with improved antibacterial activity, but none
of them showed reduced pulmonary surfactant interaction (data
not shown). Our efforts were then directed towards the modifica-
tion of synthetically less accessible [Trp1] position. Herein we re-
port syntheses and SAR studies of a novel series of daptomycin
analogs with reduced pulmonary surfactant interactions.
The general synthesis of [Trp1] analogs of daptomycin (4a–4v)
is illustrated in Scheme 1.7 The synthesis began with a Cbz protec-
tion of the [Orn6] amine. Then the C10 lipid tail was removed via an
enzymatic deacylation reaction.7 The resulting amine 1 was con-
verted to a thiourea which upon treatment with TFA led to forma-
tion of the Edman degradation product 2 in 58% yield. Compound 2
reacted readily with pentafluorophenyl esters of a variety of Boc
protected amino acids and subsequent Boc-deprotection provided
amine 3a–3v in good yields. Acylation of 3a–3v with decanoic acid
pentafluorophenyl ester followed by final removal of Cbz group
afforded the daptomycin analogs 4a–4v (Table 1). All final products
were purified by reverse phase prep-HPLC8 and characterized by
LC-MS and 1H-NMR.
spite potent in vitro activity (MIC90
, 0.06 lg/mL) against
Streptococcus pneumoniae, the most common Gram-positive patho-
gen in CAP, daptomycin did not display adequate efficacy in these
Phase 3 clinical studies. Subsequent research suggested that the
lack of efficacy in lung tissues may be due to the binding of dapto-
mycin to pulmonary surfactants leading to reduced antibacterial
activity.5
⇑
Corresponding author. Tel.: +1 781 860 8217; fax: +1 781 861 1117.
E-mail address: yong.he@cubist.com (Y. He).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.013

Y. He et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6248–6251
6249
NH2
O
NH2
O
H
N
HO
O
CH3
O
13
N
H
O
O
11
12
H
H
N
O
O
N
HN
4
N
N
H
2
3
1
H
CO2H
O
10
O
O
O
O
HO2C
NH
HN
5
CO2H
9
HO2C
N
H
NH
O
HN
O
H
N
6
7
O
N
H
8
NH2
O
Figure 1. Structure of daptomycin.
NH2
NH2
NH2
CH3
CH3
CH3
O
O
O
O
O
O
O
O
O
O
O
H
H
N
H
H
O
O
O
N
N
NH2
N
N
H
HO 2
N
N
H
HO 2C
NH2
N
H
HO2C
N
H
1. Cbz-OSu, NaHCO3
1. PhN=C=S, pyridine, H2O
H
O
O
O
O
O
O
O
C
HN
HN
HN
2. Deacylase
80%
2. CF3COOH, CH2Cl2
58%
N
N
HN
O
HN
O
HN
O
O
O
H
H
O
Daptomycin
1
2
NHCbz
NH2
NHCbz
NH2
O
NH2
O
CH3
O
O
O
H
NH2
O
O
H
N
N
NH2
HO
1. n-CH3(CH2)8COOC6F5
DIPEA, DMF
1. L-BocNHCH(R)CO2C6 F5
N
H
HO 2
N
O
CH3
O
N
H
O
O
O
H
DIPEA, DMF
O
H
H
N
O
R
O
N
C
HN
HN
N
H
N
CO H
H
2. TFA/CH2Cl2
70 - 90%
O
2
2. Pd(OH)2, H2
HOAc, MeOH
O
O
O
O
R
HO2C
HN
O
NH
NH
HN
HN
O
CO2H
HO2C
O
O
H
N
30 - 60%
3a-3v
4a-4v
O
N
H
NHCbz
O
NH2
Scheme 1. Synthesis of daptomycin analogs.
The antibacterial activity of the semi-synthetic [Trp1] analogs of
daptomycin was determined by MIC testing against methicillin
susceptible S. aureus (ATCC #29213) in the MHBc media.9
Surfactant interactions were assessed by the MIC shift in the pres-
ence of various concentrations of surfactant.10 The protein binding
effect was also evaluated by measuring the MIC in the presence of
4% human serum albumin (HSA).11
DAP) and increase surfactant effect, reflected by the larger MIC
shift in the presence of 1% and 5% of surfactant (e.g., 4f, 4g vs
4e), whereas electron-donating groups, such as –OMe, –OEt, and
–NMe2, reduced surfactant MIC shifts, and in some cases reduced
MIC potency as well (e.g., 4i, 4m, 4n vs 4e). Compounds with
hydrogen-bond donors (–OH, –COOH, and –NH2) on the aromatic
ring were generally inactive (4o–q).
As shown in Table 1, the antibacterial activity of daptomycin
was reduced by 32-fold with just 1% of surfactant, while Ceftriax-
one’s MIC was not affected by surfactant. Initial structure-activity
relationship analysis at the [Trp1] position showed that the L-
configuration of the amino acids is essential to the antibacterial
activity. Analogs with D-Trp or other D-amino acids were less ac-
Among all the analogs synthesized, 4-MeOPhe analog 4i and
4-Me2NPhe analog 4r not only maintained good antibacterial
activities (2 lg/mL) against S. auerus in Mueller-Hinton Broth
supplemented with 50 mg/L calcium (MHBc), but also showed no
measureable MIC shifts in the presence of 1% and 5% surfactant,
a significant improvement compared to daptomycin.
tive with MIC against S. aureus greater than >8
l
g/mL. Replacing
In summary, a variety of tryptophan analogs of daptomycin
were synthesized. Some analogs showed improved in vitro MIC
activity compared to daptomycin. A number of analogs showed
reduced surfactant MIC shifts in the presence of 1% or 5% of sur-
factant, while maintaining good MIC potency. Results of further
evaluation of these analogs in vivo will be reported in due
course.
[Trp1] with non-aromatic amino acids, such as Ala, Asn, Ser,
etc., also resulted in loss of activity. Analogs with selected aro-
matic
L-amino acids as replacement to [Trp1] are shown in Table 1. In
general, electron-withdrawing groups, such as –F, –CF3, on the
aromatic ring tend to maintain or improve potency (e.g., 4a vs

6250
Y. He et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6248–6251
Table 1
In vitro antibacterial activities of daptomycin [Trp1] analogs (MICs;
l
g/mL).
S. aureus ATCC 29213
1% Surf 5% Surf
Compd
–NHCH(R)CO–
Cmpd
–NHCH(R)CO–
S. aureus ATCC 29213
1% Surf 5% Surf
MHBc
2
4% HSA
MHBc
2
4% HSA
O
HN
Ceftri-axone
—
2
2
8
4k
2
4
4
OMe
O
O
NH
HN
DAP
4a
1
16
16
16
>32
>32
32
2
1
8
4l
2
8
2
8
4
8
8
16
32
MeO
N
H
O
O
HN
HN
F
0.25
4m
4n
MeO
OMe
N
H
O
O
HN
HN
OMe
OMe
OMe
4b
4
16
16
16
N
H
O
O
NH
HN
4c
4d
4e
2
4
2
8
8
4
16
16
8
4
32
4
4o
4p
4q
>32
>32
16
>32
>32
16
>32
>32
32
>32
>32
32
OH
S
O
O
NH
HN
S
CO2H
NH2
N
O
O
HN
HN
O
O
HN
HN
4f
2
2
8
16
8
8
4r
4s
2
1
2
4
2
4
4
F
O
O
HN
HN
4g
32
>32
32
CF3
N
O
O
H
N
HN
4h
1
2
4
2
2
4
8
2
8
8
8
4t
1
2
4
8
8
32
32
4
8
O
O
H
N
HN
4i
4u
4v
OMe
OMe
O
O
H
N
HN
4j
16
32
>32
32
O
OEt

Y. He et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6248–6251
6251
8. Prep-HPLC method: column, 250 Â 21.2 mm SunFireÒ
5
l
C8 column; Solvents:
References and notes
A: water with 0.01% TFA; B: acetonitrile with 0.01% TFA. Gradient: 20–60% of
B%. Purity and structural confirmation were obtained by LC-MS and NMR
(600 MHz).
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9. Minimum inhibitory concentration (MIC) was determined by broth
microdilution according to Clinical and Laboratory Standards Institutes (CLSI)
guidelines, except that Mueller-Hilton Broth was supplemented with 50 mg/L
Ca2+ and all assays were performed at 37 °C as previously described.
10. For surfactant MIC testing, MHBc was supplemented with SurvantaÒ to the
indicated concentration and the assay followed as above. SurvantaÒ contains
25 mg/mL phospholipids, including 11.0–15.5 mg/mL disaturated phosphati-
dylcholine, 0.5–1.75 mg/mL triglycerides, 1.4–3.5 mg/mL free fatty acids, and
<1.0 mg/mL protein in 0.9% NaCl.
11. For human serum albumin (HSA) MIC testing, MHB was supplemented with 4%
HSA and 75 mg/L Ca2+. In the presence of serum or serum proteins (e.g.,
albumin), free calcium (Ca2+
)
is added at 75 mg/L, roughly equivalent to
physiological blood levels once chelation by albumin is factored in.