Structure-Activity Relationship Studies of a Series of Semisynthetic Lipopeptides Leading to the Discovery of Surotomycin, a Novel Cyclic Lipopeptide Being Developed for the Treatment of Clostridium difficile -Associated Diarrhea

Article abstract of DOI:10.1021/acs.jmedchem.5b00366

Novel cyclic lipopeptides with different acyl tails were synthesized via a semisynthetic approach. Structure-activity relationship studies revealed that lipophilicity, chain length, and the location of key aromatic functionalities of the tail modulated activity. The lead compound surotomycin exhibited significantly improved in vitro activity compared with daptomycin (MIC₉₀ 0.5 vs 2 μg/mL) against Clostridium difficile including NAP1 epidemic strains. In hamster efficacy studies, surotomycin protected animals at a dose of 0.5 mg/kg, PO.

Full text of DOI:10.1021/acs.jmedchem.5b00366

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Brief Article
Structure-Activity Relationship Studies of a Series of
Semi-synthetic Lipopeptides Leading to the Discovery of
Surotomycin, a Novel Cyclic Lipopeptide Being Developed
for the Treatment of Clostridium difficile-Associated Diarrhea
Ning Yin, Jing Li, Yong He, Prudencio S Herradura, Andre Pearson, Michael F Mesleh, Carmela
T Mascio, Karen Howland, Judith Steenbergen, Grace M Thorne, Diane Citron, Andrew
DG Van Praagh, Lawrence I Mortin, Dennis Keith, Jared Silverman, and Chester Metcalf
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00366 • Publication Date (Web): 20 May 2015
Downloaded from http://pubs.acs.org on May 27, 2015
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Journal of Medicinal Chemistry
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Structure-Activity Relationship Studies of a Series of Semi-synthetic
Lipopeptides Leading to the Discovery of Surotomycin, a Novel
Cyclic Lipopeptide Being Developed for the Treatment of Clostridium
difficile-Associated Diarrhea
Ning Yin,*,† Jing Li,† Yong He,† Prudencio Herradura,†,‡ Andre Pearson,†,§ Michael F. Mesleh,†
Carmela T. Mascio,† Karen Howland,†,‖ Judith Steenbergen,† Grace M. Thorne,† Diane Citron⊥,
Andrew D.G. Van Praagh,†,¶ Lawrence I. Mortin,†,# Dennis Keith,† Jared Silverman,† Chester
Metcalf†,††
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†Merck and Co., Inc., Kenilworth, NJ USA.
⊥R.M. Alden Research Lab, 6133 Bristol Parkway Ste 175, Culver City, CA 90230.
Supporting Information for publication
ABSTRACT: Novel cyclic lipopeptides with different acyl tails were synthesized via a semi-synthetic approach. Structure-
activity relationship studies revealed that lipophilicity, chain length, and the location of key aromatic functionalities of
the tail modulated activity. The lead compound surotomycin exhibited significantly improved in vitro activity compared
with daptomycin (MIC90 0.5 ꢀg/mL vs 2 ꢀg/mL) against C. difficile including NAP1 epidemic strains. In hamster efficacy
studies, surotomycin protected animals at a dose of 0.5 mg/kg, PO.
Even with the current arsenal of antibiotics, the need for novel and
effective therapies that can treat virulent strains with low recurrence
INTRODUCTION
rates remains. In this study we report the preparation of a series of
Clostridium difficile-associated diarrhea (CDAD) is one of the leading
daptomycin (DAP) (Figure 1) tail analogs via a semi-synthetic ap-
causes of healthcare-associated infections in the US.1,2 CDAD is
proach, and structure activity relationship (SAR) studies with the
caused by the overgrowth of the anaerobic bacillus C. difficile in the
aim of identifying a compound with potent activity against C. difficile
lower gastrointestinal tract.3 This overgrowth is often triggered by
strains.12,13 In addition to improving the potency against C. difficile,
the use of broad spectrum antibiotics, which alters the protective
we also wanted to try to minimize the potential for resistance selec-
microbiota in the intestinal tract allowing opportunistic pathogens
tion in a manner similar to that observed with DAP, therefore, we
such as C. difficile to flourish.3,4 C. difficile toxin production can cause
also tested the activity of analogs against DAP nonsusceptible iso-
epithelial damage and mucosal inflammation resulting in the clinical
lates, including Staphylococcus aureus. This approach led to the
symptoms associated with CDAD which ranges in severity from mild
discovery of surotomycin, which is currently in phase 3 development
diarrhea to severe and life-threatening pseudomembranous colitis.5
for the treatment of CDAD.
CDAD represents an increasing clinical and economic burden and
the incidence of hospitalizations and mortality remain high.5,6 Re-
ported increases in CDAD incidence may be due to more frequent
and sensitive testing and to the emergence of particular epidemic
strains, for example the BI/NAP1/027 strain.7,8 While CDAD is tradi-
tionally considered a healthcare-associated infection, it is increasing
in incidence in the community setting, and community-associated
CDAD may account for approximately 20% - 27% of cases.1
Optimal patient treatment strategies involve eradication of the
pathogen without additional release of toxin, while allowing for
minimal disruption of the intestinal microbiota, and minimization of
spore production.7 Depending on the severity of CDAD, current
treatment recommendations include metronidazole, vancomycin
and fidaxomicin.5 While initial treatment response is observed in
approximately 90% of patients, recurrence of CDAD represents a
major problem with rates of approximately 30% - 50% following
treatment with vancomycin and metronidazole.8-11 Fidaxomicin has
been shown to lower recurrence compared with vancomycin in clini-
cal trials (15.4% vs 25.3%, respectively).11
Figure 1. Structure of Daptomycin.14
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duce different hydrophobic tails. Finally, the Boc group was removed
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RESULTS AND DISCUSSION
by TFA, and the resulting compounds were purified by reverse phase
HPLC to afford the final products (3a – 3bb and surotomycin). The
in vitro antibacterial activity of the synthesized lipopeptides was
assessed by determining the minimal inhibitory concentrations
(MICs) against a panel of bacteria according to Clinical and Labora-
tory Standards Institute (CLSI) guidelines.16
General synthetic route for the preparation of cyclic lipopeptide tail
analogs is illustrated in Scheme 1. The C10 tail of DAP was replaced
with a variety of commercially available or readily-accessible aliphat-
ic/aromatic moieties through a semi-synthetic approach. The key
Boc-Deacyl-Dap intermediate (1) was prepared through chemical
and enzymatic transformations following literature procedures.15
Amide-coupling with acyl chloride/PFP ester was employed to intro-
9
Scheme 1. Representative Synthetic Schemes of Lipopeptides.
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1
2
3a- 3bb surotomycin
The screening strategy was designed to identify compounds with
potent activity against C. difficile, improved activity against isolates
with decreased susceptibility to DAP and excellent in vivo efficacy.
To support this goal, the strains tested included 30 C. difficile clinical
isolates (11 NAP1 strains), DAP susceptible S. aureus (ATCC#29213),
lab derived DAP resistant mutants of Enterococcus faecium ATCC
#6569 (DAPR E. faecium) and Enterococcus faecalis ATCC #49452
(DAPR E. faecalis), and 28 DAP nonsusceptible S. aureus strains.
Antimicrobial activities of the tested compounds against C. difficile
are represented as a MIC90 where 90% of tested strains had this MIC
(ꢀg/mL) or lower (Table 1 and 2).
Initially, a series of aliphatic tail containing lipopeptides were pre-
pared (Table 1). Among all the straight chain analogs (DAP, and 3a–
3d), the 10 carbon tail (DAP) gave the optimal C. difficile MIC90. As
the chain length is extended, there is a trend of reduced potency
against C. difficile, and an improved activity against DAP resistant
E faecium and E. faecalis. It was also found that analogs with a ter-
minal branching methyl chain (3e and 3f) demonstrated improved
C. difficile MIC90 potency relative to their straight-chain homologs
(3b and 3c). Cyclohexyl ring containing analog 3g displayed a 4-fold
improved MIC90 against C. difficile vs DAP. In comparison to 3g,
analogs with cyclohexyl at the terminal position (3i-3k) were less
potent. Compound 3h with a bicyclo-octane ring is 2-fold more po-
tent than DAP against C. difficile, while cyclododecane analog (3l)
showed weak anti-C. difficile activities.
over DAP, and it was also >8 fold more potent against DAP resistant
E. faecium and E. faecalis.
In comparison to surotomycin, shortening the tail by 1 carbon (3t)
reduced potency against C. difficile and the other tested
strains. For the phenyl alkyl series, the C7 alkyl substitution (3p)
provided an optimum C. difficile MIC90 (0.5 µg/mL). When the phe-
nyl ring of 3p was replaced by a thiophene, the resulting analog 3r
displayed reduced C. difficile activities (MIC90 2 vs 0.5 µg/mL). Ana-
logs with a terminal phenyl ring (3m and 3n) had significantly weak-
er antibacterial activities (MIC90 8-16 µg/mL). For the biphenyl ether
alkyl series, the C7 ether analog (D27) was shown to be the most
potent (MIC90 = 1 µg/mL); while the biaryl tail analog without alkyl
substitution (D28) had reduced activities against C. difficile (MIC90
=
16 µg/mL) and the other tested strains. This study also aimed to
identify compounds with superior activity against DAP nonsuscepti-
ble isolates. In comparison with DAP, surotomycin showed a 2- to
16-fold improvement in activity against 28 DAP nonsusceptible
S. aureus strains (Table 3).
Compounds that passed the screening criteria for in vitro potency
were further evaluated in a hamster C. difficile infection model using
a very low dose, 0.5 mg/kg once a day, to identify the most potent in
vivo compounds.17 The survival rates from day 1 to day 25 after oral
dosing once daily for 5 consecutive days of prepared cyclic lipopep-
tides or vancomycin are presented in Figure 2. Surotomycin demon-
strated superior in vivo efficacy compared with other analogs when
tested in the CDAD hamster model. It fully protected infected ani-
mals at 0.5 mg/kg. This result is comparable to or better than van-
comycin. Other lipopeptide analogs (3g, 3h, 3p, 3q and 3r) demon-
strated partial protection at 0.5 mg/kg.
To further explore SAR of the tail structures, analogs were prepared
with aromatic ring containing acyl tails. As indicated in Table 2,
lipophilicity, chain length, and the location of key aromatic func-
tionalities of the tail all impacted activity. Among all the analogs
synthesized, surotomycin displayed the best overall in vitro biologi-
cal profile. It exhibited a 4-fold improvement of C. difficile MIC90
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Table 1. In Vitro Antibacterial Activity of Aliphatic Tail-containing Lipopeptides (MICs, µg/mL). MIC90 Values are Provided for 30 C. difficile
Isolates Tested and MIC Values are Provided for S. aureus, E. faecium and E. faecalis Isolates Tested
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4
S. aureus
ATCC
S. aureus
ATCC
C. difficilea
MIC90
DAPR
DAPR
C. difficilea
MIC90
DAPR
DAPR
Cpd Structure
c Cpd Structure
E. faeciumb E. faecalis
E. faeciumb E. faecalisc
#29213
#29213
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6
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DAP
3a
2
4
0.5
2
>32
>32
>32
32
3g
3h
0.5
1
0.5
16
16
>32
32
0.25
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3b
8
0.5
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8
3i
4
1
32
>32
3c
3d
3e
16
16
1
1
4
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4
4
3j
3k
3l
1
0.5
8
16
1
2
4
1
1
16
32
>32
>32
0.25
0.125
>32
16
3f
.
1
a30 C. difficile clinical isolates were tested for MIC90.
bLab derived DAP resistant mutant of E. faecium ATCC# 6569.
cLab derived DAP resistant mutant of E. faecalis ATCC# 49452.
Table 2. In Vitro Antibacterial Activity of Aromatic Tail-containing Lipopeptides (MICs, µg/mL). MIC90 Values are Provided for 30 C. difficile
Isolates Tested and MIC Values are Provided for S. aureus (Sa42), E. faecium (Efm384) and E. faecalis (Efs312) Isolates Tested
aS. aureus
ATCC
#29213
aS. aureus
ATCC
#29213
C. difficile
MIC90
DAPR
DAPR
C. difficile
MIC90
DAPR
DAPR
Cpd Structure
Cpd Structure
E. faeciumb E. faecalisc
E. faeciumb E. faecalisc
DAP
3m
3n
2
16
8
0.5
16
>32
>32
>32
32
>32
>32
>32
>32
8
SUR
3u
0.5
8
0.25
8
4
16
2
4
>32
8
16
3v
4
0.5
0.5
0.25
0.5
3o
4
1
3w
3x
4
32
2
>32
4
3p
0.5
1
0.25
0.5
4
2
3q
4
8
3y
2
1
2
3r
2
0.125
16
32
3z
2
0.25
1
4
3s
3t
4
2
4
1
>32
32
>32
>32
3aa
1
0.125
4
4
16
3bb
16
>32
>32
a30 C. difficile clinical isolates were tested for MIC90.
bLab derived DAP resistant mutant of E. faecium ATCC# 6569.
cLab derived DAP resistant mutant of E. faecalis ATCC# 49452.
SUR, surotomycin.
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Table 3. In Vitro Activity of DAP and Surotomycin Against DAP Nonsusceptible S. aureus strains (MICs, µg/mL).
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Strain ID#
DAP
Sa42
0.5
Sa399
NDa
Sa278
Sa1145 Sa1146 Sa1179 Sa1326 Sa1616 Sa5003 Sa5004 Sa5005 Sa5006 Sa5008 Sa5010
16
1
1
1
2
4
16
2
2
1
4
1
2
2
0.5
6
Surotomycin 0.125
0.125
0.25
0.25
0.25
0.5
0.25
0.25
0.5
0.5
0.25
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8
9
Strain ID#
DAP
Sa5012 Sa5014 Sa5016 Sa5017 Sa5030 Sa5033 Sa5034 Sa5059 Sa5060 Sa5063 Sa5076 Sa5080 Sa5081 Sa5091
2
0.5
4
4
8
1
1
2
4
1
4
1
8
1
2
4
1
4
1
2
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Surotomycin 0.25
0.25
0.5
0.5
0.25
0.5
0.5
0.5
aMultiple end points.
100
90
80
70
60
50
40
30
20
10
0
Survival at day 25–26, % (n/N)
Sentinels, sham dosed
100 (4/4)
100 (8/8)
80 (4/5)
60 (3/5)
60 (3/5)
60 (3/5)
25 (2/8)
20 (1/5)
0 (0/5)
Surotomycin
3p
3h
3g
3r
Vancomycin
Dosing:
qd x5
3q
Deionized water, 0.5 mL
-2 -1 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Study day
Figure 2. In Vivo Efficacy Study of Lipopeptide Analogs in Hamster CDAD Model (lipopeptide analog and vancomycin administrations were 0.5
mg/kg, PO qd x5 days, deionized water administration was 0.5 mL, PO qd x5)
potent and selective antimicrobial activity and is currently in phase 3
clinical development for the treatment of CDAD.
CONCLUSIONS
A series of DAP tail analogs were prepared, and the relationship
between the tail structures and their anti-C. difficile activities is dis-
cussed. Surotomycin was identified as an antibacterial development
candidate. Surotomycin is a new member of the cyclic lipopeptide
family of antibiotics and has been shown to cause cell membrane
depolarization of the S. aureus cell membrane without increasing
membrane permeability, resulting in loss of cell viability and negli-
gible cell lysis.18-20 It exhibited significantly improved in vitro activi-
ties compared with DAP against C. difficile including NAP1 strains. In
addition, surotomycin showed an 8-fold increase in potency against
lab derived DAP resistant E. faecium and E. faecalis and enhanced
activity against DAP nonsusceptible S. aureus isolates. Surotomycin
has also been shown to have good activity, MIC90 = 0.125 and 0.5,
against C. difficile strains with reduced susceptibility to vancomycin
(MIC of ≥4 μg/mL) and metronidazole (MIC of ≥8 μg/mL).21 Further
in vitro data also indicate that surotomycin is less disruptive to the
gastrointestinal microbiota than vancomycin with MIC90
≥8,192μg/mL for the Bacteroides fragilis group and Prevotella spp.22
In the hamster CDAD model efficacy studies, surotomycin protected
animals at a dose of 0.5 mg/kg. Surotomycin is a new agent with
EXPERIMENTAL SECTION
Synthesis of surotomycin. Deacylated BOC-protected DAP (3.50
g, 2.23 mmol) and sodium bicarbonate (1.13 g, 61.0 mmol) were dis-
solved in THF (130 mL) and water (50 mL). The resulting solution
was cooled to o°C and a solution of (E)-3-(4-pentylphenyl) but-2-
enoyl chloride (1.96 g, 7.82 mmol) in THF (20 mL) was then intro-
duced. The reaction mixture was warmed to room temperature and
stirred for 4 h. The mixture was concentrated in vacuo to remove
THF. The remaining aqueous solution was loaded onto a C18 flash
chromatography column (35 mm× 300 mm, Bondesil HF C18 resin
purchased from Varian). The column was first washed with water to
remove salt and then with methanol to wash out product. Crude
product of compound 2 (3.46 g) was afforded as a white solid after
removal of methanol. MS m/z 1780.8 (M + H)+. TFA (10 mL) was
added to a solution of crude compound 2 (3.46 g,) in DCM (50 mL)
at room temperature. The reaction mixture was stirred vigorously for
45 min and added slowly to vigorously stirring diethyl ether (100
mL). The resulting yellow precipitate was collected by filtration. The
crude product was purified by preparative HPLC to afford the TFA
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Journal of Medicinal Chemistry
salt of surotomycin (0.75 g). MP carbonate resin (purchased from
Biotage) was added to the solution of surotomycin TFA salt (0.70 g,
0.39 mmol) in anhydrous methanol (30 mL). The reaction mixture
was stirred at room temperature for 4 h. The resin was removed by
filtration and rinsed with methanol. The methanol solution was
concentrated under vacuum to give surotomycin as an off-white
solid (408 mg). MS m/z 1680.7185 (M + H)+, calcd 1680.7176. Purity
of the final product was ≥95% as assessed by HPLC and NMR.
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ACKNOWLEDGMENT
The authors thank You Seok Hwang for HR-LCMS testing. Medical
writing and editorial assistance was provided by Amy E. Ramsden,
PhD and Cara L. Hunsberger of StemScientific, Lyndhurst, NJ, an
Ashfield Company, part of UDG Healthcare plc. This assistance was
funded by Merck and Co, Inc., Kenilworth, NJ.
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ABBREVIATIONS
MIC assays. The in vitro antibacterial activity of the synthesized
lipopeptides was assessed by determining the MICs against a panel
of bacteria according to CLSI guidelines.16 The strains tested includ-
ed 30 C. difficile clinical isolates (11 NAP1 strains), DAP susceptible
S. aureus (ATCC#29213), and lab derived DAP resistant E. faecium
and E. faecalis. Briefly, C. difficile MICs were determined by the agar
dilution method where serial two-fold dilutions of the test com-
pounds were prepared, and added to molten agar (Brucella agar
supplemented with Vitamin K1, hemin, 5% lysed sheep blood and
adjusted to contain 50 mg/L Ca2+), mixed, and poured into Petri
plates. C. difficile, equal to the turbidity of the 0.5 McFarland, was
prepared in Brucella broth and used to inoculate the agar plates.
Plates were incubated anaerobically at 37°C for 48 h. The MIC was
the concentration of drug that inhibited growth or markedly reduced
growth as compared with the drug-free control plate. Broth microdi-
lution MICs were determined for S. aureus ATCC #29213 and
E. faecalis and E. faecium in Mueller-Hinton broth adjusted to con-
tain 50 mg/L Ca2+ (MHBc) per CLSI methodologies except that cul-
tures were incubated at 37°C with rotation (200 rpm).
CDAD, Clostridium difficile-associated diarrhea; DAP, daptomycin;
MIC, minimum inhibitory concentration; S. aureus, Staphylococcus
aureus; E. faecalis, Enterococcus faecalis; E. faecium, Enterococcus
faecium.
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Golan Y.; Gorbach S.; Sears P.; Shue Y. K.; OPT-80-003 Clinical Study
Group. Fidaxomicin versus vancomycin for Clostridium difficile infec-
tion. N. Engl. J. Med. 2011, 364, 422-431.
Hamster efficacy studies. All experiments utilized C. difficile ATCC
#43596, a clinical isolate from the feces of a patient with pseudo-
membranous colitis. All animal protocols were reviewed and ap-
proved by the Institutional Animal Care and Use Committee at
Merck and Co., Inc., before the studies were initiated. Male Syrian
golden hamsters (Mesocricetus auratus, Charles River Laboratories,
Wilmington, MA) were pretreated with 10 mg/kg clindamycin sub-
cutaneously 24 h before bacterial challenge. An inoculum of 20
C. difficile spores in sterile saline was administered orally. Inoculated
hamsters (n=5-8 hamsters/group) were treated beginning 4 h after
spore inoculation with vancomycin or various lipopeptide analogs
(0.5 mg/kg oral once daily for 5 days). Lipopeptide analogs and van-
comycin dosing solutions were prepared and diluted to desired con-
centration in sterile, deionized water. CDAD was confirmed as the
cause of death by observations at necropsy, including wet tail and/or
macroscopic cecal alterations.
ASSOCIATED CONTENT
Supporting Information.
Detailed experimental procedures and analytical data for surotomy-
cin and representative compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*N. Yin: Phone, +1 765-409-6677; Email, ningyin@hotmail.com
(12) Yin N.; He Y.; Herradura P.; Pearson A.; Li J.; Mascio C.T.;
Townsend-Howland K.; Silverman J.; Steenbergen J.; Thorne G.;
Citron D.; Van Praagh A.D.G.; Mortin L.I.; Pawliuk R.; Oleson R.;
Keith D.; Metcalf C. In vitro and in vivo studies of a series of aliphatic
tail-containing semi-synthetic lipopeptides against Clostridium dif-
ficile. 50th Interscience Conference on Antimicrobial Agents and
Chemotherapy Conference, Boston, MA, Abstract F1-1618, 2010.
(13) Yin N.; He Y.; Herradura P.; Pearson A.; Li J.; Mascio C.T.;
Townsend-Howland K.; Silverman J.; Steenbergen J.; Thorne G.;
Citron D.; Van Praagh A.D.G.; Mortin L.I.; Pawliuk R.; Oleson R.;
Keith D.; Metcalf C. Structure activity relationship studies of aro-
matic tail containing lipopeptides leading to CB-183,315, a novel cy-
clic lipopeptide being developed for the treatment of Clostridium
Present Addresses
‡P.H: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.
§A.P: BiotechGenerics LLC, 613 Elton Street, Brooklyn, NY 11208.
ǁK.H: Grafton High School. 24 Providence Rd, Grafton, MA, 01519.
¶A.VP: Bruker BioSpinCorp., 44 Manning Road, Billerica, MA, 08121.
#L.M: McCarthy Consultants, 8 McCarthy Circle, Framingham, MA,
01702.
††C.M: Cerulean Pharma Inc., 840 Memorial Drive, Cambridge, MA
02139.
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