Discovery of dap-3 polymyxin analogues for the treatment of multidrug-resistant gram-negative nosocomial infections

Article abstract of DOI:10.1021/jm400416u

We report novel polymyxin analogues with improved antibacterial in vitro potency against polymyxin resistant recent clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa. In addition, a human renal cell in vitro assay (hRPTEC) was used to inform structure-toxicity relationships and further differentiate analogues. Replacement of the Dab-3 residue with a Dap-3 in combination with a relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3- carbonyl side chain as a fatty acyl replacement yielded analogue 5x, which demonstrated an improved in vitro antimicrobial and renal cytotoxicity profiles relative to polymyxin B (PMB). However, in vivo PK/PD comparison of 5x and PMB in a murine neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting a lack of improved therapeutic index in spite of apparent in vitro advantages.

Full text of DOI:10.1021/jm400416u

Article
pubs.acs.org/jmc
Discovery of Dap‑3 Polymyxin Analogues for the Treatment of
Multidrug-Resistant Gram-Negative Nosocomial Infections
Thomas V. Magee,*,†,# Matthew F. Brown,† Jeremy T. Starr,† David C. Ackley,† Joseph A. Abramite,†
Jiri Aubrecht,† Andrew Butler,† Jared L. Crandon,‡ Fadia Dib-Hajj,† Mark E. Flanagan,† Karl Granskog,†
Joel R. Hardink,† Michael D. Huband,†,▽ Rebecca Irvine,† Michael Kuhn,† Karen L. Leach,† Bryan Li,†
Jian Lin,† David R. Luke,† Shawn H. MacVane,‡ Alita A. Miller,†,▽ Sandra McCurdy,†,○
James M. McKim, Jr.,§ David P. Nicolau,‡ Thuy-Trinh Nguyen,† Mark C. Noe,† John P. O’Donnell,†,▽
Scott B. Seibel,† Yue Shen,† Antonia F. Stepan,† Andrew P. Tomaras,† Paul C. Wilga,§ Li Zhang,∥
Jinfeng Xu,⊥ and Jinshan Michael Chen†
†Pfizer Worldwide Research & Development, Pfizer, Inc., Groton, Connecticut 06340, United States
‡Center for Anti-Infective Research and Development, Hartford Hospital, Hartford, Connecticut 06102, United States
§CeeTox, Inc., Kalamazoo, Michigan 49008, United States
∥WuXi AppTech Co., Ltd., Shanghai, P.R. China
⊥CPC Scientific, Hangzhou, P.R. China
ABSTRACT: We report novel polymyxin analogues with
improved antibacterial in vitro potency against polymyxin
resistant recent clinical isolates of Acinetobacter baumannii and
Pseudomonas aeruginosa. In addition, a human renal cell in vitro
assay (hRPTEC) was used to inform structure−toxicity
relationships and further differentiate analogues. Replacement
of the Dab-3 residue with a Dap-3 in combination with a
relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbonyl
side chain as a fatty acyl replacement yielded analogue 5x, which
demonstrated an improved in vitro antimicrobial and renal
cytotoxicity profiles relative to polymyxin B (PMB). However,
in vivo PK/PD comparison of 5x and PMB in a murine
neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting
a lack of improved therapeutic index in spite of apparent in vitro advantages.
INTRODUCTION
■
Multidrug-resistant Gram-negative pathogens such as Pseudo-
monas aeruginosa and Acinetobacter baumannii are an increasing
menace, particularly in hospital intensive care units, leading to
an impending healthcare crisis with fewer and fewer treatment
options available.1 Although over 50 years old, the polymyxin
class has made a resurgence of late in response to this crisis.2
The polymyxins are polycationic lipodecapeptides (Figure 1),
exemplified by polymyxins B (PMB) and E (more commonly
known as colistin), natural product mixtures first isolated from
Bacillus polymyxa.3 Polymyxin B and colistin are differentiated
only by the D-form amino acid in position 6 of the cyclic
peptide portion of the molecule, D-Phe-6 and D-Leu-6,
respectively. The major component of both mixtures contains
a (S)-6-methyloctanoyl (polymyxin B1 and colistin A,
respectively) lipophilic attachment at the α-amine of the
diaminobutyric acid (Dab) residue at the N-terminal or 1-
position (Dab-1).4 Both drugs are currently used in clinical
practice for treatment of serious Gram-negative infections,
Figure 1. The major components of polymyxin B (PMB) and colistin,
respectively: X = phenyl (polymyxin B1, 1) and X = 2-propyl (colistin
A, 2).
although colistin is almost exclusively administered in nonactive
prodrug form as the pentamethanosulfonate sodium salt.5
Received: March 21, 2013
Published: June 4, 2013
© 2013 American Chemical Society
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The attraction of the polymyxins is their persistent potency
against the multidrug-resistant nonfermenting (P. aeruginosa, A.
baumannii) and enteric (Klebsiella pneumoniae, Escherichia coli)
pathogens in the face of widespread carbapenem resistance.6
Unlike β-lactams, which must penetrate the bacterial outer
membrane to reach their target, survive β-lactamase hydrolysis,
and avoid the activity of efflux pumps to be effective, the
polymyxins act initially on the outer leaf of the outer membrane
by first binding the lipid A portion of lipopolysaccharide.7 What
happens after binding lipid A is not entirely clear, although
modeling suggests that polymyxins promote the exchange of
phospholipids between the outer and inner membranes,
creating osmotic instability and resulting in cell death.8 What
is clear is that a certain number of cationic residues are
required, along with lipophilic projections from both the N-
terminus and within the cyclic peptide (residues 6 and 7),
which likely provide membrane anchoring and are essential for
antimicrobial activity.9 The amphipathic nature of the charge-
lipophilicity distribution is an essential feature of their mode of
action, although the selectivity of polymyxins for (LPS-
containing) Gram-negative over Gram-positive bacteria sug-
gests a lack of general detergent qualities often associated with
amphipathic molecules. The major challenge with polycationic
lipopeptides such as polymyxin is their tendency to disrupt
renal and, in rare cases, neuromuscular function. Nephrotox-
icity, the main concern for the polymyxins, can be quite variable
and dependent upon the state and history of the patient but
presents limited therapeutic and dosing options to the treating
clinician. For this reason, these agents are often used as a “last
resort”.10 Underdosing of polymyxins owing to these safety
concerns presents the prospect of failure to eradicate the
infecting pathogen for the patient and the potential for
generating polymyxin resistant strains which could spread to
others. To address these issues, Vaara et al. have taken the
innovative approach of significantly modifying the side chain of
the lipopeptide such that it no longer contains any basic
residues. The resulting molecules, with an overall positive
charge count reduced from five to three, act on the outer
membrane to potentiate the entry of Gram-positive agents such
as macrolides that would otherwise not penetrate the outer
membrane of Gram-negative bacteria.11 Interestingly, these
“potentiators” still retain some intrinsic antimicrobial potency
as well. Polymyxin B nonapeptide (PMBN) is the result of
enzymatic cleavage of PMB, removing the fatty acyl and Dab-1
units and leaving amino acid residues 2−10 intact.12 PMBN is
also known to have relatively poor intrinsic MICs but exhibits
potentiator properties.13 Sato et al. have also modified the side
chain with a des-fatty acyl approach and reduced the basicity to
give compounds that appear to retain good intrinsic
antibacterial potency against P. aeruginosa (but lose E. coli
potency; A. baumannii and K. pneumoniae data not reported)
with an apparent lower toxicity liability.14 Other approaches to
improving upon the polymyxins have included replacing the
fatty acyl side chain with more compact entities such as an aryl
urea.15 Researchers at Monash University disclosed a patent
application16 concentrating upon extending the lipophilic
residues in the cyclic peptide region, particularly at Leu-7,
with long-chain hydrocarbons or biphenyl moieties and capping
the N-terminus with biphenyl amide to improve potency
against polymyxin resistant strains. Such resistant strains are
currently rare; however, they are known17 and are likely to
grow in importance as physicians increasingly turn to
polymyxins to treat these infections.
SYNTHETIC CHEMISTRY
■
Solid phase peptide synthesis on 2-chlorotrityl chloride (CTC)
resin was used to synthesize our target compounds (Scheme 1).
Scheme 1. Synthesis of Dap-3 Polymyxin Analogues with
Variable N-Termini (R) on Template 5
a
a
Conditions: (a) (i) CTC resin, Fmoc-Leu-OH, DIPEA, DCM,
MeOH; (ii) sequential removal of Fmoc (piperidine, DMF) followed
by amidation (Fmoc-AA-OH, HOBt, DIC, DMF) using the following
residues (resin filtration between steps): Fmoc-DPhe-OH, Fmoc-
Dab(Boc)-OH, Fmoc-Dab(Dde)-OH, Fmoc-Dap(Boc)-OH, Fmoc-
Thr(tBu)-OH, Fmoc-Dab(Boc)-OH; (iii) Z-OSu, NMM, DMF. (b)
(i) 3% hydrazine in DMF to remove Dde; (ii) sequential amidation/
Fmoc removal as above with: Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-
OH, Fmoc-Dab(Boc)-OH; (iii) 1.5% TFA in DCM (resin cleavage);
(iv) cyclization in DMF at 0.002 M dilution with EDC-HCl, DIPEA,
70% yield for this step; (v) H2 (1.1 atm), cat. Pd/C, DCM, MeOH,
NH4OH. (c) (i) For amidation, carboxylic acid coupling partner,
HATU, TEA, DMSO; for sulfonylation, sulfonyl chloride coupling
partner, NMM, DMF; for urea formation, isocyanate coupling partner,
NMM, DMF; (ii) global deprotection using TFA:H2O:iPr3SiH
(95:2.5:2.5), followed by HPLC reverse phase purification.
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Figure 2. N-Termini (R) on template 5 (see Scheme 1) resulting in final analogues 5a−x.
Starting with Fmoc-protected Leu attachment to the resin and
standard coupling/deprotection methodology, a linear chain
was constructed sequentially. With our focus on polymyxin B
analogues, the D-Phe unit was first coupled to the N-terminus
of the Leu-CTC resin. The L-2,4-diaminobutyric acid (Dab)
residue with Boc protection on the 4-amino group was then
added, followed by a Dab with 1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene)-ethyl (Dde) on its 4-amino position. The next
residue was, significantly, not the Dab traditionally found in
polymyxins at this position but the L-2,3-diaminopropanoic acid
(Dap) variant (3-amino Boc protected). The implications of
this change will be discussed in the next section. The Thr
(OtBu) and Dab (4-amino Boc) were subsequently coupled,
and the N-terminus finally capped with benzyloxycarbonyl (Z)
to yield 3. The Dde was then selectively removed with
hydrazine and the 4-amino group of this Dab unit extended
sequentially, as above with Thr (OtBu) and two Dab (Boc)
residues. The peptide was then cleaved off the resin with dilute
TFA in DCM and subsequently cyclized in 70% yield (0.002M,
EDC-HCl). Hydrogenolysis of the Z group gave the key
intermediate 4, which was then used for N-terminal
modification using standard coupling procedures (with
emphasis on amidation), followed by global deprotection
with TFA, to give analogues 5a−x (Figure 2).
RESULTS AND DISCUSSION
■
One of our first observations in this effort was that PMB was
already very potent against the key pathogens of interest,
namely P. aeruginosa, A. baumannii, K. pneumoniae, and E. coli.
To differentiate analogues, we needed to include resistant
strains in our primary screening cascade. Furthermore, we
observed that the standard method of screening antibacterial
potency for these resistant isolates, namely minimum inhibitory
concentration (MIC), was inadequate for distinguishing
between compounds and found that minimum bactericidal
concentration (MBC) greatly augmented SAR interpretation by
providing a broader dynamic range, in particular for P.
aeruginosa. Thus we included, in addition to more common
sensitive strains we had on hand, recent clinical isolates with a
polymyxin resistant (MIC ≥ 8 mg/L) phenotype for the
nonfermenters P. aeruginosa (PA-1646)18 and A. baumannii
(AB-1649) and ran MBC as well as MIC tests (Table 1). Our
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Table 1. Minimum Bactericidal Concentrations (MBC, mg/L) and Minimum Inhibitory Concentrations (MIC, mg/L) against
Polymyxin Resistant and Sensitive Strains of P. aeruginosa (PA), A. baumannii (AB), K. pneumoniae (KP), and E. coli (EC)
a
PA-1646
MBC
PA-1646
MIC
PA-01
MIC
AB-1649
MBC
AB-1649
MIC
AB-3167
MIC
KP-3700
MIC
EC-1
MIC
hRPTEC TC50
b
compd
(μM)
cLogD ΔcLogD
PMB
PMBN
1
32
>64
16
2
8
>64
4
0.5
>64
0.5
0.5
0.5
0.5
0.5
0.25
0.5
1
8
>64
4
8
>64
4
0.25
>64
0.25
0.125
0.25
0.25
0.25
1
0.25
>64
0.5
0.125
22
>100
27
−9.21
−10.69
−9.21
−9.30
−9.70
−8.53
−9.53
−7.90
−8.75
−10.13
−8.89
−8.53
−8.73
−8.64
−8.99
−8.23
−9.41
−8.91
−9.13
−9.42
−9.31
−8.49
−9.76
−9.06
−8.74
−8.66
−11.96
0
>64
−1.48
0
0.125
0.125
0.125
0.25
0.25
0.25
0.25
0.25
0.5
5a
1
4
4
0.125
0.125
0.5
72
−0.09
−0.49
0.68
−0.32
1.31
0.46
−0.92
0.32
0.68
0.48
0.57
0.22
0.98
−0.2
0.3
5b
5c
16
2
4
2
2
51
1
2
2
21
5d
5e
5f
1
1
4
4
0.25
0.5
15
4
2
>64
1
64
1
42
1
0.5
4
0.25
0.25
0.25
0.25
0.5
0.25
0.25
0.5
28
5g
5h
5i
16
4
4
2
61
2
1
1
1
32
4
2
0.25
0.5
0.5
0.5
1
0.5
2
0.5
2
0.25
0.25
0.25
0.25
0.25
0.25
0.5
0.125
0.25
0.25
0.5
39
5j
2
1
27
5k
5l
4
2
8
4
0.25
0.25
0.5
26
2
2
4
4
19
5m
5n
5o
5p
5q
5r
1
0.5
4
1
1
0.25
0.25
0.5
14
16
4
1
4
4
0.25
0.5
72
1
1
4
4
20
4
2
0.25
0.5
0.5
0.5
0.25
0.5
0.5
0.5
0.5
1
1
0.25
0.25
0.25
0.5
0.25
0.25
0.25
0.25
0.125
0.25
0.5
0.125
0.125
0.25
0.25
0.125
0.125
0.125
0.25
0.125
82
0.08
−0.21
−0.1
0.72
−0.55
0.15
0.47
0.55
−2.75
4
2
4
4
56
8
2
2
2
78
5s
1
1
4
4
24
5t
8
2
4
2
0.25
0.25
0.25
0.25
84
5u
5v
5w
5x
16
1
4
1
1
86
1
0.5
1
0.5
1
34
1
1
0.25
0.25
30
8
4
2
2
0.25
b
>100
a
Human renal proximal tubule epithelial cell line toxic concentration 50%. cLogD − cLogDPMB
.
primary goal was to differentiate from the standard-of-care
polymyxins against the nonfermenter (P. aeruginosa, A.
baumannii) rather than the enteric strains (K. pneumoniae, E.
coli). In addition to having this potency screening strategy
described above, we included a high-throughput in vitro screen
for gauging renal toxicity potential, as demonstrated potency
advantages would be of very little interest if the renal liabilities
increased correspondingly. Attempting to separate the anti-
microbial potency from nephrotoxicity by achieving the right
balance of physical properties remains the key challenge to
improving on the polymyxin class. The multibasic polymyxins
accumulate in the kidney, resulting in acute tubular necrosis, a
dose-limiting toxicity. This limitation consequently impacts the
therapeutic utility of the polymyxins as well as raises the specter
of incomplete pathogen eradication and greater potential for
the spread resistant strains. In vivo screening for improved renal
toxicity profiles with preclinical animal models is not practical
in discovery research mode, and while the rabbit brush border
membrane assay has been cited as useful for potentiator-mode
polymyxins with fewer basic amines,11 the dynamic range of
this assay was found to be insufficient for our purposes. In place
of this, we utilized primary human renal proximal tubule
epithelial cells (hRPTEC)19 for in vitro kidney toxicity
screening, with lactate dehydrogenase release as a cell viability
end point. We were thus able to generate cytotoxicity
concentration curves, and from this estimate the concentration
at 50% cell viability for each analogue (TC50). As of this
writing, the translatability of this assay to human or preclinical
species renal toxicity is not clear, however, it served as a useful
guide for avoiding excessively cytotoxic analogues in human
primary cells from a known target organ and for making
decisions on which analogues to progress into in vivo studies.
Prescription grade PMB, a fermentation mixture, was used as a
reference for all assays. We observed that the MBC of PMB was
4-fold higher than the MIC (32 vs 8 mg/L) against the
polymyxin resistant P. aeruginosa strain (PA-1646). The
resistant A. baumannii strain (AB-1649) did not demonstrate
this MBC/MIC shift, both being 8 mg/L. As expected, PMB
was very potent (MICs ≤ 1 mg/L) against strains identified as
susceptible in our screening panel (PA-01, AB-3167, KP-3700,
and EC-1). Because PMB is a fermentation product and as such
a mixture of components, we synthesized the major constituent
(1) and demonstrated dilution shift in potency versus the
resistant strains PA-1646 and AB-1649. The hRPTEC TC50
values for both PMB and 1 were generally ≤30 μM, which we
used as a point of reference for novel analogues. PMBN, which
has little or no intrinsic antimicrobial activity, had a TC50 > 100
μM in this assay as expected. We then set out to modulate the
basicity of the polymyxin peptide template, to eliminate
cationic charge if possible, or to lower the pKa of the Dab
residues. In so doing, we found that a very simple change,
namely replacing the 2,4-diaminobutyric acid at position-3 of
the peptide (Dab-3) with a 2,3-diaminoproprionic acid (Dap-3)
residue (amounting to a single methylene excision), yielded an
interesting and useful result in compound 5a in which the
antimicrobial potency improved significantly while simulta-
neously decreasing the hRPTEC cytotoxicity by well over 2-
fold. Improving activity by shortening the Dab residue was
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contrary to expectation9 and worked only at the 3-position with
the Dap residue. NMR studies7b have suggested that Dab-3 was
not directly interacting with the lipid A disaccharide but rather
may be involved in KDO contacts. Lack of detailed structural
knowledge of how these molecules interact with LPS and affect
the outer and inner membranes in living, dynamic bacteria
makes speculation about the nature of our SAR difficult. The
multifactorial components of what constitutes polymyxin whole
cell potency do suggest that differential flexibilities within
different parts of the molecule are important and that the
insertion of the side chain into membranes plays a crucial
role.20 Removing degrees of freedom at the Dab-3 position, by
removing a methylene in the residue side chain and bringing
the charged amine closer to the backbone, might also influence
the orientation or dynamics of insertion in situ. The distinct
lack of rigidity in the fatty acyl portion of the side chain marked
this as a further point of potential SAR and toxicity modulation.
Continuing on in the Dap-3 series, replacement of the fatty acyl
with a 2-napthyl acyl side chain yielded a compound (5b)
which was less potent against PA-1646 but was a dilution better
against AB-1649. The more extended (nonfused) biphenyl
amide 5c was consistently potent against both PA and AB-
resistant strains, however, the cytotoxicity was comparable to
PMB. Different linkers such as urea (5d) or sulfonyl (5e)
yielded very mixed results, the sulfonyl in particular
demonstrating a dramatic loss in AB-1649 activity. The more
extended p-biphenyl amide 5f appeared to have a slight potency
advantage, at least for the simple case relative to the m-biphenyl
5c. Heteroatom substitutions were then explored in order to
test the limits of lipophilicity requirements and SAR tolerance
for such changes and to monitor how polarity impacted
hRPTEC cytotoxicity. Installation of more polar functional
groups such as the O-phenylpyridone 5g, with heteroatoms
bridging the biaryl systems, generally lost activity and required
us to tread somewhat carefully. Nitrile functionality in the
proximal biphenyl (5h, 5i) held on to potency but showed no
cytotoxicity advantage. A methoxy scan (5j, 5k, and 5l) of the
proximal ring of the m-biphenyl system was much the same,
only AB-1649 dropping off with this substituent. The m-biaryl
amides appeared to tolerate substituents (nitrile, methoxy,
halogen) or heteroatom replacements best at the p-position of
the proximal ring (viz. 5i, 5j, 5m, and 5p). The potency
difference between analogues 5m and 5n, and even 5o, again
highlights the general intolerance for heteroatoms in the distal
rings of these biaryl systems. Moving the nitrogen from the p-
position of the proximal ring proved unproductive (5p vs 5q),
as were heteroatom substituents in the distal ring (e.g., 5r).
Pyridone 5s showed improved P. aeruginosa activity relative to
5p, however, the AB-1649 activity was lower and the hRPTEC
cytotoxicity increased significantly. Pyridazine 5t had a
cytotoxicity profile similar to 5p but lost potency against
both PA-1646 and AB-1649, consistent with the activity loss
observed in 5q. Chloro substitution in the distal ring of 5p
yielded 5u, 5v, and 5w, the latter two analogues displaying
superb potency profiles although cytotoxicities within the range
of PMB, and therefore not necessarily representing improve-
ments in overall properties. Because measured Log D values
were difficult to obtain with these extremely polar molecules we
utilized calculated Log D values (cLogD) and in particular the
difference between analogue cLogDs and that of PMB (labeled
ΔcLogD, Table 1) for purposes of comparison. There was a
general trend of compounds with more polar side chains (i.e.,
more negative ΔcLogD) having higher hRPTEC TC50 values
relative to PMB (5b, 5g, 5m, 5q, 5r, 5t), however, there were
several examples where the ΔcLogD values calculated out to be
more or less isolipophilic (5a, 5p, 5u) or even more lipophilic
(5e) and still showed a higher TC50 outside of the standard
deviations observed.21 At this stage, we sought to make a more
radical structural change in the side chain to even further
reduce the hRPTEC cytotoxicity liability while maintaining or
perhaps improving on our potency relative to PMB so as to test
the utility of our toxicity assay for screening analogues
preclinically. We capitalized on the observation that the p-
position of the proximal aromatic ring tolerated polarity
reasonably well and that chloride substitution at this position
yielded extremely potent compound, albeit a cytotoxic one
(5m). We reasoned that an N-phenyl pyridone (5x) might
introduce significant polarity into the side chain without
entirely losing potency. Indeed, in vitro microbiological
potencies of 5x, in particular the MBCs verus PA-1646 and
AB-1649, were found to still demonstrate a slight advantage
when compared to PMB. Consistent with these results were the
MIC50 and MIC90 values of 5x and PMB against larger
subpopulations of P. aeruginosa and A. baumannii (Table 2). In
Table 2. MIC50 and MIC90 Values of 5x Compared with
PMB versus Susceptible and Resistant Subpopulations of P.
aeruginosa, A. baumannii, E. coli, and K. pneumoniae
5x
PMB
strain subgroup (n = number of strains) MIC50 MIC90 MIC50 MIC90
susceptible P. aeruginosa (n = 96)
susceptible A. baumannii (n = 96)
susceptible E. coli (n = 101)
1
2
2
2
2
2
2
2
1
2
1
2
susceptible K. pneumoniae (n = 100)
resistant P. aeruginosa (n = 6)
resistant A. baumannii (n = 17)
0.5
2
1
1
2
8
4
16
>64
4
16
16
addition, in vitro nephrotoxicity as measured by the hRPTEC
assay indicated a significantly lower cytotoxicity liability of 5x
relative to PMB or any of the other analogues.22 Notice for 5x
the ΔcLogD = −2.75 represented the largest increase in
(calculated) polarity in comparison to the other analogues, with
the pyridone dipole making the obvious difference as planned.
A scatter plot of the hRPTEC data and ΔcLogD values shows a
general trend toward lower cytotoxicity liability with increas-
ingly negative ΔcLogD, 5x being clearly different in both
respects from PMB (Figure 3). Taken together, these data
supported in vivo profiling to determine if 5x represented the
therapeutic advantage over PMB that we sought. We then
moved into extensive comparative in vivo safety studies in rat
and dog. A 7-day exploratory toxicology study (ETS) in rat
demonstrated differentiation in the incidence of necrotic kidney
lesions (Table 3). At a free exposure of 13 mg·h/L per day, 5x
exhibited no incidence of necrotic kidney lesions, whereas at a
matched free exposure PMB produced necrotic kidney lesions
in every animal in the dose group, albeit graded to be
minimal.23 Attempts to dose PMB at the top dose (8 mg/kg·d)
failed owing to severe tolerability issues, whereas 5x was well-
tolerated and furthermore yielded no lesions, reaching a fAUC
= 29 mg·h/L. Encouraged by these results and apparent safety
differentiation observed in the rat, we then ran 7-day ETS
studies in dog. The dog proved to be a more sensitive species,
for although the low dose (fAUC = 20 mg·h/L) of 5x was well
tolerated and produced minimal nephrotoxicity, the middle
dose (fAUC = 64 mg·h/L) and high dose (fAUC = 114 mg·h/
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Figure 3. Scatter plot of hRPTEC TC50 data and ΔcLogD highlighting PMB and 5x. Note that the TC50 > 100 μM for 5x (and PMBN) in this assay,
however, they are indicated as equal to 100 μM on this plot for purposes of illustration.
Table 3. Rat (7d) and Dog (7d) Exploratory Toxicology Study (ETS) Results of 5x and PMB
5x
PMB
histological
histological
d
a
b
c
d
a
e
c
dose (mg/kg·d) fAUC (mg·h/L)
N
assessment
dose (mg/kg·d) fAUC (mg·h/L)
N
assessment
Rat
low dose not
examined
low dose not
examined
low dose not
examined
low dose not
examined
0.4
0.72
13
3
3
0/3
4
8
13
29
3
3
0/3
0/3
4
8
3/3 all minimal
high dose not
tolerated
high dose not
tolerated
high dose not
tolerated
Dog
5
20
64
8
8
8/8 all minimal
8/8
1
6
10
42
2
8
0/2
8/8
11
5 moderate
3 marked
2 moderate
6 marked
20
114
2
2/2 all marked
high dose not
examined
high dose not
examined
high dose not
examined
high dose not
examined
a
b
c
Doses given BID (Q12) intravenously. Free AUC per day for 5x: plasma fu in rat and dog = 0.81 and 0.58, respectively. Number of animals tested
d
per dose group. Kidney proximal tubules were evaluated for the presence of tubular epithelial degeneration/regeneration, and the resulting
assessments were graded in severity from none to marked; the number of animals in which lesions appeared out of the total number tested are
indicated as a ratio. Free AUC per day for PMB: plasma fu in rat and dog = 0.30 and 0.35, respectively.
e
L) both produced moderate or marked lesions in every animal.
The PMB mid dose was targeted to mimic a fAUC in human
for an efficacious exposure level and powered with eight
animals to compare to 5x at its mid dose. Although there were
differences in the severity of the lesions slightly in favor of 5x,
and although the fAUC of 5x was approximately 1.5-fold higher
than PMB (64 vs 42 mg·h/L) at this dose, we deemed that
there was no significant safety margin in favor of 5x as
measured by this study, at least in the dog. This result
demonstrates that the substantial in vitro differentiation
observed in the hRPTEC assay for 5x versus PMB did not
translate in definitive dog in vivo safety studies. Ideally, in
vitro−in vivo correlations would be investigated with cells
derived from the same species. Therefore, further work on
species-specific in vitro assays that predict for the sensitive
(nonrodent) renal toxicities in preclinical models may be
warranted for selection and progression of novel polymyxin
analogues to the clinic. However, we also profiled 5x and PMB
in a murine neutropenic thigh infection model24 against
composited strains of P. aeruginosa and observed that the
PK/PD of 5x, when matched for fAUC/MIC relative to PMB,
did not perform as well as the latter (Table 4). The resulting
pharmacokinetics of 5x and PMB were best described using a
one-compartment model with first-order input and elimination.
During bacterial density studies, doses of 5x and PMB resulted
in fAUCs (mg·h/L) ranging from 7.2 to 775 and 18 to 51,
respectively. The average bacterial densities at the start of
therapy were 5.37 0.12 log10CFU and increased to 8.85
0.28 log10CFU after 24 h in untreated control animals. The R2
value for the respective Emax models was 0.83 for 5x and 0.97
for PMB. The pharmacodynamic direct comparison, i.e., the
fAUC/MIC values required for similar efficacy targets, was
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MeOH:water. Samples were diluted by factors ranging from 33:1 to
100:1. Data was processed using the MassHunter software that was
provided with the instrument. All test peptides were analyzed using a
Bruker 500 MHz spectrometer. Small molecule intermediates were
analyzed with a Varian Inova 400 MHz spectrometer.
Table 4. Comparative Pharmacodynamics of 5x and PMB
against a Composited Data Set of P. aeruginosa 1401 and
1402 (MICs = 0.5 mg/L) in the Neutropenic Thigh
Infection Model
Synthesis of Protected Template 5: H-Dab(Boc)-Thr(tBu)-
Dap(Boc)-cyclo[Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-
Thr(tBu)]. Step 1: Fmoc-Leu-CTC Resin. CTC resin (950g, 988
mmol) was suspended in DCM (8L) in a peptide synthesis vessel
equipped with a mechanical stirrer. To the suspension was added
Fmoc-Leu-OH (349 g, 988 mmol) and DIPEA (863 mL, 4940 mmol).
The mixture was stirred gently at RT for 3h. To the reaction was
added 0.95 L of MeOH. The resulting mixture was stirred at RT for
0.5 h and then filtered. Solid was washed with DCM (3 × 15L) and
subsequently MeOH (2 × 10L). The resulting solid was dried under
vacuum overnight to furnish 1250 g of Fmoc-Leu-CTC resin.
The loading level of the resin product thus obtained was established
to be 0.46 mmol/g via standard UV absorption method (Shimadzu
UV-1601, wavelength 289.5 nm) upon Fmoc cleavage of small aliquots
of the product resin.
Step 2: Fmoc-DPhe-Leu-CTC Resin. Fmoc-Leu-CTC resin (152g,
0.46 mmol/g, 70 mmol) was suspended in DMF (1 L) at RT
overnight in a peptide synthesis vessel equipped with a mechanical
stirrer. DMF was then removed via filtration. To the solid was added
20% piperidine in DMF (1.0 L), and the resulting mixture was stirred
at 18 °C for 0.5 h. Mixture was then filtered. Solid resin H-Leu-CTC
was washed with DMF (6 × 1 L).
Fmoc-DPhe-OH (54.2g, 140 mmol) and HOBt (18.9g, 140 mmol)
were dissolved in DMF (0.25L) at 0 °C. To the mixture was added
DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and mixture was
kept at 0 °C for 15 min. The activated AA solution was then added
into the H-Leu-CTC resin, and the mixture was stirred at 18 °C for 1
h, at which point Kaiser ninhydrin test indicated reaction completion.
The mixture was filtered and solid was washed with DMF (5 × 1 L).
The Fmoc-DPhe-Leu-CTC resin product was used in the subsequent
step without further treatment.
Step 3: Fmoc-Dab(Boc)-DPhe-Leu-CTC Resin. To the Fmoc-DPhe-
Leu-CTC resin in a peptide synthesis vessel equipped with a
mechanical stirrer, obtained from step 2, was added 20% piperidine
in DMF (1 L), and the resulting mixture was stirred gently at 20 °C for
0.5 h. The mixture was then filtered. Solid resin H-DPhe-Leu-CTC
was washed with DMF (6 × 1 L).
fAUC/MIC
efficacy parameter
5x
PMB
a
EI80
157.55
87.92
59.00
37.38
37.07
44.95
a
EI50
stasis
85.26
1 Log decrease
109.63
a
Exposure index required for 80% (EI80) and 50% (EI50) of maximal
efficacy.
greater for 5x on all four efficacy parameters (EI80, EI50, Stasis,
and 1 Log decrease) as compared with PMB.
CONCLUSION
■
We have made structural modifications to the polymyxin
lipopeptide, in particular replacing the Dab with the truncated
Dap residue at position-3 in the exocyclic peptide region.25 It
was surprising that such a subtle change would affect the
potency as dramatically as it did. We were also able to replace
the fatty acid side chain with a variety of biaryl amides which,
when combined with the Dap-3 modification, led to further
potency improvements, in particular against P. aeruginosa and
A. baumannii resistant strains. Our primary goal was to improve
the therapeutic index with a novel polymyxin analogue by
reducing the nephrotoxicity liability, and to that end we utilized
an in vitro human cell line for estimating the risk of renal
cytotoxicity (hRPTEC). We observed that a combination of the
Dap-3 substitution and a pyridone-biaryl amide (5x) provided a
significantly different signature in hRPTEC without compro-
mising potency. Exploratory toxicity studies (ETS) suggested
that 5x was better tolerated in rat with respect to renal lesions,
however, this advantage collapsed in the dog, pointing to the
failure of the hRPTEC assay to predict for that species. The
human translatability of these safety data is not clear, however,
this provides a significant barrier to further development.
Furthermore, 5x proved to be inferior in the direct PK/PD
comparison with PMB in the murine neutropenic thigh model,
suggesting that the overall therapeutic index of this analogue is
not likely to surpass that of PMB and highlighting the
importance of conducting rigorous, free exposure matched
efficacy comparisons when making changes to the polymyxin
structure.
Fmoc-Dab(Boc)-OH (61.6g, 140 mmol) and HOBt (18.9 g, 140
mmol) were dissolved in DMF (0.25 L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into H-DPhe-Leu-CTC resin, and the mixture was stirred
at 19 °C for 1 h, at which point Kaiser ninhydrin test indicated
reaction completion. The mixture was filtered and solid was washed
with DMF (5 × 1 L). The resulting Fmoc-Dab(Boc)-DPhe-Leu-CTC
resin product was used in the subsequent step without further
treatment.
Step 4: Fmoc-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC Resin. To the
Fmoc-Dab(Boc)-DPhe-Leu-CTC resin in a peptide synthesis vessel
equipped with a mechanical stirrer, obtained from step 3, was added
20% piperidine in DMF (1 L), and the resulting mixture was stirred at
19 °C for 0.5 h. The mixture was then filtered. Solid H-Dab(Boc)-
DPhe-Leu-CTC resin was washed with DMF (6 × 1 L).
Fmoc-Dab(Dde)-OH (70.4 g, 140 mmol) and HOBt (18.9g, 140
mmol) were dissolved in DMF (0.25L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into H-Dab(Boc)-DPhe-Leu-CTC resin, and the mixture
was stirred at 21 °C for 1 h, at which point Kaiser ninhydrin test
indicated reaction completion. The mixture was filtered and solid was
washed with DMF (5 × 1 L). The resulting Fmoc-Dab(Dde)-
Dab(Boc)-DPhe-Leu-CTC resin product was used in subsequent step
without further treatment.
EXPERIMENTAL SECTION
■
All final peptide analogues were isolated by preparative HPLC to
≥98% purity and analyzed by high resolution mass spectrometry.
Pharmaceutical grade PMB (Bedford Laboratories) was used in all
experiments. MS data were collected on an Agilent (Wilmington, DE)
6220 Accurate Mass TOF LC/MS operating in the electrospray
ionization mode. The chromatography system consisted of an Agilent
(Wilmington, DE, USA) 1200 binary pumping system with the
addition of an extra isocratic system and a dynamic splitter for dilution
of samples as they passed between the UV detector and the mass
spectrometer. The binary pump operated at 1.1 mL/min of A = 10
mM ammonium formate adjusted to pH 3.5 in water and B = 50:50
acetonitrile:methanol. Sample injections were typically 0.5 μL.
Separations were effected using an Agilent Zorbax Eclipse Plus C-18
(3.0 mm × 50 mm, 1.8 μm) column operating at 60 °C. As samples
eluted the UV detector, they were diluted in the dynamic splitter with
an isocratic pump flowing at 0.5 mL/min and using a solvent of 50:50
Step 5: Fmoc-Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC
Resin. To the resin Fmoc-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC in a
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peptide synthesis vessel equipped with a mechanical stirrer, obtained
from the step 4, was added 20% piperidine in DMF (1.5 L), and the
resulting mixture was stirred at 22 °C for 0.5 h. Mixture was then
filtered. Solid H-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC resin was
washed with DMF (6 × 1.5 L).
Fmoc-Dap(Boc)-OH (59.6 g, 140 mmol) and HOBt (18.9g, 140
mmol) were dissolved in DMF (0.25 L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into H-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC resin, and
mixture was stirred at 22 °C for 1 h, at which point the Kaiser
ninhydrin test indicated reaction completion. The mixture was filtered
and the solid was washed with DMF (5 × 1 L). The resulting Fmoc-
Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC resin product was
confirmed by peptide cleavage and subsequent HPLC and LCMS
analysis outlined below.
A small portion of the resin product (1 mL) was washed with
MeOH (3 × 5 mL) and then dried overnight under vacuum. The resin
was then treated with 5 mL of solution F at RT for 2.5 h. The reaction
mixture was filtered, and the filtrate was quenched with cold ether (50
mL). A white solid formed, and this solid was collected via removal of
the solvent after the mixture was centrifuged. The solid residue was
washed with cold ether (2 × 50 mL), dried under vacuum overnight,
and analyzed using an HP1090 system connected to a SepaxGP-C18
(5 μm, 120A, 4.6 mm × 150 mm) reversed-phase HPLC column.
Gradient method: 15−75% solvent B over 20 min at a flow rate of 1.0
mL/min (solvent A,: 0.1% TFA in water; solvent B, 0.09% TFA, 20%
water, and 79.91% eMeCN). Major peak with retention time 13.8 min,
78.1% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) = 951.4
([M + H]+), 476.4 ([M + 2H]2+/2).
Step 6: Fmoc-Thr(tBu)-Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-Leu-
CTC Resin. To the resin Fmoc-Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-
Leu-CTC in a peptide synthesis vessel equipped with a mechanical
stirrer, obtained from step 5, was added 20% piperidine in DMF (1.5
L), and the resulting mixture was stirred kept at 21 °C for 0.5 h. The
mixture was then filtered. The solid H-Dap(Boc)-Dab(Dde)-Dab-
(Boc)-DPhe-Leu-CTC resin was washed with DMF (6 × 1.5 L).
Fmoc-Thr(tBu)-OH (55.6g, 140 mmol) and HOBt (18.9g, 140
mmol) were dissolved in DMF (0.25L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into H-Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC
resin, and the mixture was stirred for 1 h, at which point the Kaiser
ninhydrin test indicated reaction completion. The mixture was filtered
and the solid was washed with DMF (5 × 1L). The resulting Fmoc-
Thr(tBu)-Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC resin
product was confirmed by peptide cleavage and subsequent HPLC
and LCMS analysis outlined in step 5.
DPhe-Leu-CTC resin product was confirmed by peptide cleavage
and subsequent HPLC and LCMS analysis outlined in step 5.
Gradient method: 15−75% solvent B over 20 min at a flow rate of
1.0 mL/min (solvent A, 0.1% TFA in water; solvent B, 0.09% TFA,
20% water, and 79.91% MeCN). Major peak with retention time 17.5
min, 78.7% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) =
1152.5 ([M + H]+), 577.9 ([M + 2H]+/2).
Step 8: Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab(Dde)-Dab(Boc)-
DPhe-Leu-CTC Resin. To the resin Fmoc-Dab(Boc)-Thr(tBu)-Dap-
(Boc)-Dab(Dde)-DPhe-Leu-CTC in a peptide synthesis vessel
equipped with a mechanical stirrer, obtained from the step 7, was
added into 20% piperidine in DMF (2.0 L), and the resulting mixture
was stirred at RT for 0.5 h. The mixture was then filtered, and the solid
H-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab(Dde)- Dab(Boc)-DPhe-Leu-
CTC resin was washed with DMF (5 × 2L). To the resin was then
added a solution of Z-OSu (52.3g, 210 mmol) and NMM (46.2 mmol,
420 mmol) in DMF (0.50 L). The resulting mixture was stirred for 1
h. Kaiser ninhydrin test indicated reaction completion. The mixture
was filtered after stirred at RT for another 50 min. Solid was washed
with DMF (5 × 2 L), and the product, Z-Dab(Boc)-Thr(tBu)-
Dap(Boc)- Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC resin product, was
confirmed by peptide cleavage and subsequent HPLC and LCMS
analysis outlined in step 5.
Gradient method: 15−75% solvent B over 20 min at a flow rate of
1.0 mL/min (solvent A, 0.1% TFA in water; solvent B, 0.09% TFA,
20% water, and 79.91% MeCN). Major peak with retention time 11.7
min, 73.7% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) =
1064.5 ([M + H]+), 533.0 ([M + 2H]+/2).
Step 9: Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Fmoc]-Dab-
(Boc)-DPhe-Leu-CTC Resin. To the resin Z-Dab(Boc)-Thr(tBu)-
Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-Leu-CTC in a peptide syn-
thesis vessel equipped with a mechanical stirrer, obtained from step 8,
was added a solution of 3% hydrazine in DMF (2L). The resulting
mixture was stirred at 20 °C for 0.5h. The mixture was then filtered
and the solid Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[-H]- Dab(Boc)-
DPhe-Leu-CTC resin was washed with DMF (5 × 2L).
Fmoc-Thr(tBu)-OH (55.6g, 140 mmol) and HOBt (18.9 g, 140
mmol) were dissolved in DMF (0.3L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into the resin Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[-H]-
Dab(Boc)- DPhe-Leu-CTC and the mixture was stirred at 18 °C for
1h at which point Kaiser ninhydrin test indicated reaction completion.
The mixture was stirred for an additional 1h then filtered. The solid
was washed with DMF (5 × 1L) and the resin product Z-Dab(Boc)-
Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Fmoc]-DPhe-Leu-CTC was con-
firmed by peptide cleavage and subsequent HPLC and LCMS analysis
outlined in step 5.
Gradient method: 15−75% solvent B over 20 min at a flow rate of
1.0 mL/min (solvent A, 0.1% TFA in water; solvent B, 0.09% TFA,
20% water, and 79.91% MeCN). Major peak with retention time 13.8
min, 91.1% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) =
1223.5 ([M + H]+), 612.6 ([M + 2H]+/2).
Gradient method: 15−75% solvent B over 20 min at a flow rate of
1.0 mL/min (solvent A, 0.1% TFA in water; solvent B, 0.09% TFA,
20% water, and 79.91% MeCN). Major peak with retention time 19.3
min, 89.6% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) =
1052.5 ([M + H]+), 527.0 ([M + 2H]+/2).
Step 10: Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Dab(Boc)-
Fmoc]-Dab(Boc)-DPhe-Leu-CTC Resin. To the resin Z-Dab(Boc)-
Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Fmoc]-Dab(Boc)-DPhe-Leu-
CTC in a peptide synthesis vessel equipped with a mechanical stirrer,
obtained from step 9, was added 20% piperidine in DMF (2 L), and
the resulting mixture was stirred at 19 °C for 0.5 h. The mixture was
then filtered, and the solid Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab-
[Thr(tBu)-H)]-Dab(Boc)-DPhe-Leu-CTC resin was washed with
DMF (5 × 2 L).
Fmoc-Dab(Boc)-OH (61.6 g, 140 mmol) and HOBt (18.9 g, 140
mmol) were dissolved in DMF (0.3 L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-H]-
Dab(Boc)-DPhe-Leu-CTC, and the mixture was stirred at 19 °C for 1
h at which point the Kaiser ninhydrin test indicated reaction
completion. The mixture was stirred for an additional 1 h and then
Step 7: Fmoc-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab(Dde)-Dab(Boc)-
DPhe-Leu-CTC Resin. To the resin Fmoc-Thr(tBu)-Dap(Boc)-Dab-
(Dde)-DPhe-Leu-CTC in a peptide synthesis vessel equipped with a
mechanical stirrer, obtained from step 6, was added 20% piperidine in
DMF (1.5 L), and the resulting mixture was stirred at 21 °C for 0.5 h.
The mixture was then filtered and the resin H-Thr(tBu)-Dap(Boc)-
Dab(Dde)-Dab(Boc)-DPhe Leu-CTC was washed with DMF (6 × 1.5
L).
Fmoc-Dab(Boc)-OH (61.6 g, 140 mmol) and HOBt (18.9g, 140
mmol) were dissolved in DMF (0.25 L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into H-Thr(tBu)-Dap(Boc)-Dab(Dde)-Dab(Boc)-DPhe-
Leu-CTC resin, and the mixture was stirred at 22 °C for 1 h, at which
point the Kaiser ninhydrin test indicated reaction completion. The
mixture was filtered and the solid was washed with DMF (5 × 1 L).
The Fmoc-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab(Dde)-Dab(Boc)-
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filtered. The solid was washed with DMF (5 × 2 L). The resin product
Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Dab(Boc)-Fmoc]-
Dab(Boc)-DPhe-Leu-CTC was confirmed by peptide cleavage and
subsequent HPLC and LCMS analysis outlined in step 5.
Gradient method: 15−75% solvent B over 20 min at a flow rate of
1.0 mL/min (solvent A, 0.1% TFA in water; solvent B, 0.09% TFA,
20% water, and 79.91% MeCN). Major peak with retention time 16.3
min, 79.2% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) =
1323.5 ([M + H]+), 662.5 ([M + 2H]+/2).
Dab(Boc)-Thr(tBu)-Dap(Boc)-cyclo[Dab-Dab(Boc)-DPhe-Leu-Dab-
(Boc)-Dab(Boc)-Thr(tBu)] (obtained from step 12, 130 g, assuming
70 mmol) in DCM/MeOH (1.5 L/1.5 L) in a round-bottom-flask was
added concentrated ammonium hydroxide (10 mL). This mixture was
then purged with N2, and 10% Pd/C (dry, 15g) was added under
nitrogen. The reaction mixture was stirred at 15 °C under 1.1
atmospheric pressure hydrogen for 4 d. The hydrogen source was
removed, and the system was purged with nitrogen. The mixture was
then filtered through a pad of Celite, and filtrate was concentrated in
vacuum to furnish a yellow solid, 123 g (crude yield 94%).
HPLC and LCMS analysis gradient method: 70−100% solvent B
over 20 min at a flow rate of 1.0 mL/min (solvent A, 0.1% TFA in
water; solvent B, 0.09% TFA, 20% water, and 79.91% MeCN). Major
peak with retention time 13.4 min, 78.7% by UV220; ESI-MS
(Thermo LCQ advantage) (m/z) = 1662.6 ([M + H]+), 831.43 ([M +
2H]+/2).
Coupling and Deprotection of Template 5 to Generate Final
Test Analogues. General Procedure for Preparation of Amide
Analogues: 5p. Step 1: A solution of H-Dab(Boc)-Thr(tBu)-
Dap(Boc)-cyclo[Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr-
(tBu)] (127 g, 76.5 mmol), 2-phenylisonicotinic acid (15.2 g, 76.5
mmol), HATU (35 g, 91.8 mmol), and NMM (25.3 mL, 230 mmol)
in DMF (1700 mL) in a 3 L round-bottom flask was stirred at RT
overnight. TLC indicated complete consumption of the carboxylic acid
starting material (DCM/MeOH/AcOH: 40/1/0.5); MS confirmed
reaction was complete. The mixture was concentrated in vacuum to
remove the majority of DMF. The residue was poured into water (9
L). A white solid formed, which was collected via filtration. The filter
cake was washed with water (2 L) and dried under vacuum to give 125
g of the amide intermediate as a yellow solid (89% yield), which was
used in the next step without further purification.
Step 2: Into a 3 L round-bottom flask at 0 °C was charged, with the
yellow solid amide intermediate from step 1 (125g, 76.8 mmol),
solution F (TFA:water:iPr3SiH 95:2.5:2.5) (1000 mL). The mixture
was stirred at 0 °C for 0.5 h and RT for 2 h. The reaction mixture was
quenched with cold ether (10 L), whereupon a white precipitate
formed and was collected by removing the supernatant after
centrifugation. This solid crude product was then purified by
preparative HPLC to give the desired final amide target analogue as
a white solid (TFA salt). The TFA salt thus obtained was converted to
the sulfate salt by loading onto to a Novasep-C18 10 μm 100A 50 mm
× 450 mm column and eluted, at a flow rate of 400 mL/min, initially
with 90% A and 10% B for 3 column volumes, then 10% B and 90% C
for 3 column volumes, and finally 40% B and 60% C for 1.5 column
volumes (A, 0.2% H2SO4 in water; B, 100% MeCN; C, 100% water).
The fractions containing product were combined and lyophilized to
furnish 24 g of a white solid (overall yield 29%).
General Procedure for Preparation of Urea Analogues: 5d. Step
1: A solution of H-Dab(Boc)-Thr(tBu)-Dap(Boc)-cyclo[Dab-Dab-
(Boc)-DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)] (425 mg, 0.256
mmol), the isocyanate (50 mg, 0.256 mmol), and NMM (0.078 mL,
0.768 mmol) in DMF (10 mL) was stirred at RT for 4 h. MS indicated
the reaction was complete. The mixture was concentrated in vacuum.
The residue was dissolved in DCM (10 mL) and water (10 mL). The
mixture was concentrated to remove DCM. White precipitate formed,
and the solid was collected and dried under high vacuum to give a
yellow solid (300 mg, yield 64%).
Step 2: The crude product from step 1 (300 mg, 0.162 mmol) was
dissolved in solution F (TFA:water:iPr3SiH 95:2.5:2.5) (3 mL) and
stirred at RT for 2 h. The reaction mixture was quenched with cold
ether (30 mL). White precipitate formed, and the solid was isolated
upon removal of the supernatant after the mixture was centrifuged.
The solid was then purified by preparative HPLC to give pure product
(62 mg, yield 30%) as a white solid. Preparative HPLC method: crude
product dissolved in MeCN/water (5/95), loaded onto a 50 mm ×
250 mm 10 miron Luna C18 column and purified using a 25−50% B
in 60 min at a flow rate of 60 mL/min (A, 0.1% TFA in water; B,
MeCN with 0.1% TFA). Product thus obtained was 85% pure and was
resubjected to preparative HPLC purification again using an isocratic
34% B method, and the desired product was obtained in >97% purity.
Step 11: Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Dab(Boc)-
Dab(Boc)-Fmoc]-Dab(Boc)-DPhe-Leu-CTC Resin. To the resin Z-
Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Dab(Boc)-Fmoc]-
Dab(Boc)-DPhe-Leu-CTC in a peptide synthesis vessel equipped with
a mechanical stirrer, obtained from step 10, was added 20% piperidine
in DMF (2 L), and the resulting mixture was stirred at 21 °C for 0.5 h.
The mixture was then filtered. Solid Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-
Dab[Thr(tBu)-Dab(Boc)-H)]-Dab(Boc)-DPhe-Leu-CTC resin was
washed with DMF (5 × 2L).
Fmoc-Dab(Boc)-OH (61.6 g, 140 mmol) and HOBt (18.9 g, 140
mmol) were dissolved in DMF (0.3L) at 0 °C. To the mixture was
added DIC (22.1 mL, 140 mmol) at 0 °C in one portion, and the
mixture was kept at 0 °C for 15 min. The activated AA solution was
then added into Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-
Dab(Boc)-H]-Dab(Boc)-DPhe-Leu-CTC, and the mixture was stirred
at 22 °C for 1 h, at which point Kaiser ninhydrin test indicated
reaction completion. The mixture was stirred for 1 more hour and
then filtered. The solid was washed with DMF (5 × 2L), and the Z-
Dab(Boc)-Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Dab(Boc)-Dab(Boc)-
Fmoc]-Dab(Boc)-DPhe-Leu-CTC was confirmed by peptide cleavage
and subsequent HPLC and LCMS analysis outlined in step 5.
Gradient method: 15−75% solvent B over 20 min at a flow rate of
1.0 mL/min (solvent A, 0.1% TFA in water; solvent B, 0.09% TFA,
20% water, and 79.91% MeCN). Major peak with retention time 14.9
min, 84.2% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) =
1423.6 ([M + H]+), 712.5 ([M + 2H]+/2).
Step 12: Z-Dab(Boc)-Thr(tBu)-Dap(Boc)-cyclo[Dab-Dab(Boc)-
DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)]. To the resin Z-Dab(Boc)-
Thr(tBu)-Dap(Boc)-Dab[Thr(tBu)-Dab(Boc)-Dab(Boc)-Fmoc]-
Dab(Boc)-DPhe-Leu-CTC in a peptide synthesis vessel equipped with
a mechanical stirrer, obtained from step 11, was added 20% piperidine
in DMF (2 L), and the resulting mixture was stirred at 23 °C for 0.5 h.
The mixture was then filtered. The solid resin Z-Dab(Boc)-Thr(tBu)-
Dap(Boc)-Dab[Thr(tBu)-Dab(Boc)-Dab(Boc)-H]-Dab(Boc)-DPhe-
Leu-CTC was washed with DMF (5 × 2 L).
To the resin was then added 6 L of 1.5% TFA in DCM, and the
resulting mixture was stirred at RT for 0.5 h. The mixture was then
filtered. The filtrate was neutralized with the addition of DIPEA
(approximately 180 mL) to neutral pH. The mixture was then
concentrated to remove DCM. The residue was dissolved in 35 L of
DMF to make up a solution of the cleaved acyclic peptide in 0.002 M
concentration. To this solution was added DIPEA (61.3 mL, 350
mmol) and EDC-HCl (26.8g, 140 mmol). The resulting mixture was
stirred at RT. LCMS indicated cyclization reaction completed after
overnight stirring. DMF was removed on the rotary evaporator with
water bath temperature no higher than 40 °C. The residue was
dissolved in DCM (1.5 L), and the DCM solution was washed with
water (4 × 0.7 L). DCM layer was concentrated and dried under high
vacuum to furnish 130 g of the desired cyclic peptide Z-Dab(Boc)-
Thr(tBu)-Dap(Boc)-cyclo[Dab-Dab(Boc)-DPhe-Leu-Dab(Boc)-Dab-
(Boc)-Thr(tBu)] as a light-yellow solid.
HPLC and LCMS analysis gradient method: 70−100% B over 20
min at a flow rate of 1.0 mL/min (A, 0.1% TFA in water; B, 0.1% TFA
in 80% MeCN and 20% water). Major peak with retention time 17.24
min, 70.5% by UV220; ESI-MS (Thermo LCQ advantage): (m/z) =
1795.7 ([M + H]+), 898.6 ([M + 2H]+/2). High resolution mass
spectrometry result indicated the presence of M + 1, M + 2, M + 3
isotope distribution pattern consistent with the desired monocyclic
peptide product.
Step 13: H-Dab(Boc)-Thr(tBu)-Dap(Boc)-cyclo[Dab-Dab(Boc)-
DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)]. To a stirred solution of Z-
5087
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Article
Analytical method: 32% B to 42% B in 20 min at a flow rate of 1.0
mL/min on a SepaxGP-C18, 3 μm, 120 A, 4.6 mm × 150 mm column
using UV 220 nm (solvent A, 0.1% TFA in water; B, 0.1% TFA, 20%
water, 80% MeCN). Final product retention time 10.5 min; ESI-MS
(m/z) = 1243.92 (100%, [M + H]+).
General Procedures for the Preparation of Sulfonamide
Analogues: 5e. Step 1: A solution of compound A or the decapeptide
template, H-Dab(Boc)-Thr(tBu)-Dab(Boc)-cyclo(Dab-Dab(Boc)-
DPhe-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)) (4.37g, 2.63 mmol), bi-
phenyl-3-sulfonyl chloride (1.00 mg, 3.95 mmol), and NMM (0.44
mL, 4.0 mmol) in DMF (40 mL) was stirred at RT overnight. MS
indicated the reaction was complete. The mixture was poured into
water (400 mL), and a white solid formed. The solid was collected via
filtration, washed with water (3 × 50 mL), and dried under high
vacuum to give crude material as a white solid, 4.51 g (92% yield),
which was used in the next step without purification.
Step 2: The crude product from step 1 (4.51g, 2.4 mmol) was
dissolved in solution F (TFA:water:iPr3SiH 95:2.5:2.5) (40 mL) and
stirred at RT for 2 h. The reaction mixture was quenched with cold
ether (400 mL), and white solid precipitate formed. The solid was
isolated by removal of the supernatant after the mixture was
centrifuged. The solid was dried and purified by preparative HPLC
to give pure desired product as a white solid (1.394g, yield 45%).
Preparative HPLC method: crude product dissolved in MeCN/water
(5/95), loaded onto a 50 mm × 250 mm 10 micron Luna C18 column
and purified using a 20−55% B in 60 min at a flow rate of 60 mL/min
(A, 0.1% TFA in water; B, MeCN with 0.1% TFA). Product thus
obtained was 80% pure and was resubjected to preparative HPLC
purification again using an isocratic 40% B, and the desired product
was obtained in 97% purity. Analytical method: 33% B to 43% B in 20
min at a flow rate of 1.0 mL/min on a SepaxGP-C18, 3 μm, 120 A, 4.6
mm × 150 mm column using UV 220 nm (solvent A, 0.1% TFA in
water; B, 0.1% TFA, 20% water, 80% MeCN). Final product retention
time 7.77 min; ESI-MS (m/z) = 633.8 (100%, [M + 2H]+/2).
HRMS and NMR Data for 5a−x. Compound 5a. HRMS: (M +
2H)+2 595.3749, calcd 595.3744; (M + 2Na)+2 617.3563, calcd
617.3564. 1H NMR (500 MHz, D2O) δ 7.33 (t, J = 7.23 Hz, 2H), 7.28
(t, J = 7.27 Hz, 1H), 7.20 (d, J = 7.09 Hz, 2H), 4.79−4.73 (m, 1H),
4.51 (t, J = 8.24 Hz, 1H), 4.48−4.41 (m, 2H), 4.36 (d, J = 3.81 Hz,
1H), 4.28−4.11 (m, 7H), 3.45 (dd, J = 4.97, 13.49 Hz, 1H), 3.33−3.22
(m, 2H), 3.13−2.96 (m, 9H), 2.86−2.77 (m, 1H), 2.77−2.67 (m, 1H),
2.27 (t, J = 7.35 Hz, 2H), 2.23−1.75 (m, 10H), 1.59−1.47 (m, 2H),
1.47−1.39 (m, 1H), 1.38−1.29 (m, 1H), 1.30−1.17 (m, 5H), 1.17−
1.10 (m, 6H), 1.10−0.99 (m, 2H), 0.80−0.73 (m, 7H), 0.70 (d, J =
6.37 Hz, 3H), 0.63 (d, J = 6.27 Hz, 3H).
= 6.40 Hz, 3H), 0.88−0.77 (m, 1H), 0.75 (d, J = 6.36 Hz, 3H), 0.68
(d, J = 6.26 Hz, 3H).
Compound 5d. HRMS: (M + 2H)+2 622.8486, calcd 622.8486; (M
1
+ 2Na)+2 644.8296, calcd 644.8305. H NMR (500 MHz, D2O) δ
7.66−7.60 (m, 2H), 7.57 (s, 1H), 7.47 (t, J = 7.61 Hz, 2H), 7.44−7.36
(m, 3H), 7.32 (t, J = 7.22 Hz, 2H), 7.29−7.24 (m, 2H), 7.19 (d, J =
7.10 Hz, 2H), 4.73−4.71 (m, 1H), 4.50 (t, J = 8.24 Hz, 1H), 4.44−
4.36 (m, 3H), 4.32−4.25 (m, 1H), 4.24−4.11 (m, 5H), 4.10 (d, J =
4.60 Hz, 1H), 3.39 (dd, J = 4.97, 13.43 Hz, 1H), 3.30−3.16 (m, 2H),
3.16−2.94 (m, 9H), 2.85−2.75 (m, 1H), 2.74−2.64 (m, 1H), 2.28−
1.99 (m, 6H), 1.99−1.71 (m, 4H), 1.47−1.37 (m, 1H), 1.38−1.28 (m,
1H), 1.17 (d, J = 6.41 Hz, 3H), 1.08 (d, J = 6.38 Hz, 3H), 0.84−0.72
(m, 1H), 0.70 (d, J = 6.38 Hz, 3H), 0.63 (d, J = 6.26 Hz, 3H).
Compound 5e. HRMS: (M + 2H)+2 633.3266, calcd 633.3266; (M
1
+ Na)+ 1287.6269, calcd 1287.6279. H NMR (500 MHz, D2O) δ
8.12−8.09 (m, 1H), 8.03−7.98 (m, 1H), 7.88−7.83 (m, 1H), 7.77−
7.68 (m, 3H), 7.56 (t, J = 7.53 Hz, 2H), 7.49 (t, J = 7.37 Hz, 1H), 7.37
(t, J = 7.24 Hz, 2H), 7.32 (t, J = 7.28 Hz, 1H), 7.24 (d, J = 7.06 Hz,
2H), 4.74−4.70 (m, 1H), 4.55 (t, J = 8.26 Hz, 1H), 4.47 (dd, J = 5.46,
8.89 Hz, 1H), 4.31−4.13 (m, 6H), 4.10 (dd, J = 4.66, 9.67 Hz, 1H),
4.03 (d, J = 3.65 Hz, 1H), 3.94−3.86 (m, 1H), 3.47 (dd, J = 4.95,
13.43 Hz, 1H), 3.33−3.19 (m, 2H), 3.18−2.96 (m, 9H), 2.91−2.80
(m, 1H), 2.79−2.69 (m, 1H), 2.30−2.05 (m, 5H), 2.04−1.75 (m, 5H),
1.53−1.43 (m, 1H), 1.43−1.34 (m, 1H), 1.16 (d, J = 6.41 Hz, 3H),
0.85 (d, J = 6.42 Hz, 3H), 0.83−0.77 (m, 1H), 0.75 (d, J = 6.32 Hz,
3H), 0.68 (d, J = 6.22 Hz, 3H).
1
Compound 5f. HRMS: (M + 2H)+2 615.343, calcd 615.3431. H
NMR (500 MHz, D2O) δ 7.89−7.83 (m, 2H), 7.80−7.75 (m, 2H),
7.73−7.67 (m, 2H), 7.53−7.47 (m, 2H), 7.46−7.40 (m, 1H), 7.36−
7.30 (m, 2H), 7.29−7.25 (m, 1H), 7.19 (d, J = 6.77 Hz, 2H), 4.78−
4.73 (m, 2H), 4.51 (t, J = 8.22 Hz, 1H), 4.45−4.39 (m, 2H), 4.32−
4.25 (m, 1H), 4.25−4.12 (m, 5H), 4.10 (d, J = 4.50 Hz, 1H), 3.47 (dd,
J = 5.06, 13.48 Hz, 1H), 3.32−3.21 (m, 2H), 3.20−2.95 (m, 9H),
2.86−2.76 (m, 1H), 2.76−2.66 (m, 1H), 2.37−2.27 (m, 1H), 2.26−
2.01 (m, 5H), 2.01−1.74 (m, 4H), 1.47−1.38 (m, 1H), 1.38−1.29 (m,
1H), 1.17 (d, J = 6.40 Hz, 3H), 1.09 (d, J = 6.35 Hz, 3H), 0.84−0.73
(m, 1H), 0.70 (d, J = 6.44 Hz, 3H), 0.63 (d, J = 6.30 Hz, 3H).
Compound 5g. HRMS: (M + 2H)+2 623.8382, calcd 623.8382; (M
+ Na)+ 1268.6505, calcd 1268.6511. 1H NMR (500 MHz, D2O) δ 8.51
(d, J = 2.12 Hz, 1H), 8.22 (dd, J = 2.50, 8.76 Hz, 1H), 7.51 (t, J = 7.98
Hz, 2H), 7.42−7.28 (m, 4H), 7.25 (d, J = 7.09 Hz, 2H), 7.20 (d, J =
7.66 Hz, 2H), 7.11 (d, J = 8.77 Hz, 1H), 4.84−4.78 (m, 2H), 4.56 (t, J
= 8.27 Hz, 1H), 4.48 (dd, J = 5.47, 8.93 Hz, 1H), 4.45 (d, J = 3.86 Hz,
1H), 4.36−4.11 (m, 7H), 3.51 (dd, J = 4.94, 13.43 Hz, 1H), 3.37−3.25
(m, 2H), 3.22−2.98 (m, 9H), 2.93−2.82 (m, 1H), 2.82−2.72 (m, 1H),
2.40−2.28 (m, 1H), 2.27−2.09 (m, 5H), 2.07−1.78 (m, 4H), 1.54−
1.43 (m, 1H), 1.43−1.34 (m, 1H), 1.20 (d, J = 6.40 Hz, 3H), 1.17 (d, J
= 6.40 Hz, 3H), 0.87−0.77 (m, 1H), 0.75 (d, J = 6.16 Hz, 3H), 0.67
(d, J = 6.15 Hz, 3H).
Compound 5b. HRMS: (M + 2H)+2 602.3358, calcd 602.3353; (M
+ Na)+ 1225.6447, calcd 1225.6452. 1H NMR (500 MHz, D2O) δ 8.41
(s, 1H), 8.09−8.03 (m, 2H), 8.01 (d, J = 8.03 Hz, 1H), 7.85 (dd, J =
1.62, 8.63 Hz, 1H), 7.74−7.61 (m, 2H), 7.37 (t, J = 7.25 Hz, 2H), 7.32
(t, J = 7.29 Hz, 1H), 7.24 (d, J = 7.15 Hz, 2H), 4.84−4.77 (m, 2H),
4.55 (t, J = 8.24 Hz, 1H), 4.51−4.43 (m, 2H), 4.38−4.30 (m, 1H),
4.30−4.22 (m, 3H), 4.22−4.15 (m, 2H), 4.13 (d, J = 4.53 Hz, 1H),
3.52 (dd, J = 4.96, 13.43 Hz, 1H), 3.39−3.26 (m, 2H), 3.26−2.98 (m,
9H), 2.93−2.82 (m, 1H), 2.81−2.72 (m, 1H), 2.45−2.34 (m, 1H),
2.34−2.06 (m, 5H), 2.06−1.78 (m, 4H), 1.52−1.43 (m, 1H), 1.42−
1.34 (m, 1H), 1.22 (d, J = 6.39 Hz, 3H), 1.12 (d, J = 6.39 Hz, 3H),
0.91−0.76 (m, 1H), 0.75 (d, J = 6.28 Hz, 3H), 0.67 (d, J = 6.19 Hz,
3H).
Compound 5h. HRMS: (M + 2H)+2 627.8409, calcd 627.8407; (M
+ 2Na)+2 649.8210, calcd 649.8227. 1H NMR (500 MHz, D2O) δ 8.29
(d, J = 1.78 Hz, 1H), 8.13 (dd, J = 1.91, 8.21 Hz, 1H), 7.74 (d, J = 8.24
Hz, 1H), 7.65 (dd, J = 1.75, 7.73 Hz, 2H), 7.62−7.55 (m, 3H), 7.36 (t,
J = 7.21 Hz, 2H), 7.31 (t, J = 7.26 Hz, 1H), 7.23 (d, J = 7.07 Hz, 2H),
4.85−4.77 (m, 2H), 4.55 (t, J = 8.24 Hz, 1H), 4.51−4.44 (m, 2H),
4.38−4.12 (m, 7H), 3.52 (dd, J = 5.00, 13.43 Hz, 1H), 3.38−3.26 (m,
2H), 3.25−2.99 (m, 9H), 2.94−2.83 (m, 1H), 2.83−2.72 (m, 1H),
2.42−2.31 (m, 1H), 2.31−2.08 (m, 5H), 2.08−1.79 (m, 4H), 1.52−
1.42 (m, 1H), 1.42−1.33 (m, 1H), 1.22 (d, J = 6.38 Hz, 3H), 1.15 (d, J
= 6.41 Hz, 3H), 0.90−0.76 (m, 1H), 0.74 (d, J = 6.24 Hz, 3H), 0.67
(d, J = 6.18 Hz, 3H).
Compound 5c. HRMS: (M + 2H)+2 615.3439, calcd 615.3431; (M
1
+ Na)+ 1251.6610, calcd 1251.6609. H NMR (500 MHz, D2O) δ
Compound 5i. HRMS: (M + 2H)+2 627.8407, calcd 627.8407; (M
+ 2Na)+2 649.8219, calcd 649.8227. 1H NMR (500 MHz, D2O) δ 8.00
(d, J = 8.12 Hz, 1H), 7.96 (d, J = 1.28 Hz, 1H), 7.90 (dd, J = 1.67, 8.11
Hz, 1H), 7.65 (dd, J = 1.58, 7.87 Hz, 2H), 7.61−7.53 (m, 3H), 7.37 (t,
J = 7.23 Hz, 2H), 7.32 (t, J = 7.26 Hz, 1H), 7.24 (d, J = 7.08 Hz, 2H),
4.87−4.76 (m, 2H), 4.56 (t, J = 8.25 Hz, 1H), 4.51−4.44 (m, 2H),
4.36−4.17 (m, 6H), 4.15 (d, J = 4.54 Hz, 1H), 3.51 (dd, J = 4.97,
13.43 Hz, 1H), 3.37−3.24 (m, 2H), 3.18 (t, J = 7.75 Hz, 2H), 3.14−
2.99 (m, 7H), 2.91−2.83 (m, 1H), 2.82−2.72 (m, 1H), 2.41−2.29 (m,
8.08−8.04 (m, 1H), 7.95−7.89 (m, 1H), 7.83−7.78 (m, 1H), 7.76−
7.70 (m, 2H), 7.63 (t, J = 7.81 Hz, 1H), 7.55 (t, J = 7.59 Hz, 2H), 7.47
(t, J = 7.39 Hz, 1H), 7.37 (t, J = 7.22 Hz, 2H), 7.32 (t, J = 7.30 Hz,
1H), 7.24 (d, J = 7.06 Hz, 2H), 4.83−4.76 (m, 2H), 4.56 (t, J = 8.25
Hz, 1H), 4.50−4.42 (m, 2H), 4.38−4.29 (m, 1H), 4.29−4.16 (m, 5H),
4.14 (d, J = 4.55 Hz, 1H), 3.52 (dd, J = 4.99, 13.43 Hz, 1H), 3.38−3.25
(m, 2H), 3.24−2.98 (m, 9H), 2.92−2.82 (m, 1H), 2.81−2.71 (m, 1H),
2.42−2.31 (m, 1H), 2.31−2.07 (m, 5H), 2.06−1.77 (m, 4H), 1.52−
1.43 (m, 1H), 1.43−1.34 (m, 1H), 1.21 (d, J = 6.42 Hz, 3H), 1.14 (d, J
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1H), 2.29−2.08 (m, 5H), 2.07−1.77 (m, 4H), 1.53−1.44 (m, 1H),
1.43−1.33 (m, 1H), 1.20 (d, J = 6.40 Hz, 3H), 1.15 (d, J = 6.40 Hz,
3H), 0.88−0.77 (m, 1H), 0.75 (d, J = 6.25 Hz, 3H), 0.67 (d, J = 6.19
Hz, 3H).
(m, 1H), 2.82−2.72 (m, 1H), 2.39−2.28 (m, 1H), 2.28−2.08 (m, 5H),
2.06−1.76 (m, 4H), 1.51−1.42 (m, 1H), 1.43−1.33 (m, 1H), 1.21−
1.17 (m, 3H), 1.15 (d, J = 6.41 Hz, 3H), 0.91−0.77 (m, 1H), 0.75 (d, J
= 6.33 Hz, 3H), 0.67 (d, J = 6.24 Hz, 3H).
Compound 5j. HRMS: (M + 2H)+2 630.3493, calcd 630.3484; (M
+ Na)+ 1281.6722, calcd 1281.6715. 1H NMR (500 MHz, D2O) δ 7.87
(dd, J = 2.40, 8.69 Hz, 1H), 7.78 (d, J = 2.39 Hz, 1H), 7.57−7.47 (m,
4H), 7.47−7.41 (m, 1H), 7.37 (t, J = 7.23 Hz, 2H), 7.32 (t, J = 7.30
Hz, 1H), 7.24 (d, J = 8.64 Hz, 3H), 4.84−4.77 (m, 2H), 4.56 (t, J =
8.25 Hz, 1H), 4.51−4.41 (m, 2H), 4.37−4.29 (m, 1H), 4.29−4.23 (m,
3H), 4.23−4.16 (m, 2H), 4.15 (d, J = 4.58 Hz, 1H), 3.87 (s, 3H), 3.51
(dd, J = 4.99, 13.44 Hz, 1H), 3.40−3.26 (m, 2H), 3.23−2.98 (m, 9H),
2.93−2.82 (m, 1H), 2.82−2.71 (m, 1H), 2.39−2.29 (m, 1H), 2.29−
2.07 (m, 5H), 2.07−1.79 (m, 4H), 1.53−1.43 (m, 1H), 1.43−1.34 (m,
1H), 1.20 (d, J = 6.41 Hz, 3H), 1.14 (d, J = 6.40 Hz, 3H), 0.89−0.77
(m, 1H), 0.75 (d, J = 6.32 Hz, 3H), 0.68 (d, J = 6.22 Hz, 3H).
Compound 5k. HRMS: (M + 2H)+2 630.3494, calcd 630.3484; (M
+ 2Na)+2 652.3317, calcd 652.3303. 1H NMR (500 MHz, D2O) δ 7.71
(d, J = 7.21 Hz, 2H), 7.68−7.65 (m, 1H), 7.53 (t, J = 7.51 Hz, 2H),
7.50−7.44 (m, 2H), 7.40−7.34 (m, 3H), 7.32 (t, J = 7.28 Hz, 1H),
7.24 (d, J = 7.08 Hz, 2H), 4.83−4.76 (m, 2H), 4.55 (t, J = 8.25 Hz,
1H), 4.49−4.43 (m, 2H), 4.38−4.30 (m, 1H), 4.30−4.22 (m, 3H),
4.22−4.15 (m, 2H), 4.13 (d, J = 4.51 Hz, 1H), 3.93 (s, 3H), 3.52 (dd, J
= 5.02, 13.43 Hz, 1H), 3.37−3.26 (m, 2H), 3.23−2.98 (m, 9H), 2.92−
2.83 (m, 1H), 2.83−2.73 (m, 1H), 2.42−2.31 (m, 1H), 2.31−2.08 (m,
5H), 2.07−1.78 (m, 4H), 1.51−1.42 (m, 1H), 1.42−1.33 (m, 1H),
1.22 (d, J = 6.41 Hz, 3H), 1.13 (d, J = 6.41 Hz, 3H), 0.85−0.76 (m,
1H), 0.74 (d, J = 6.02 Hz, 3H), 0.67 (d, J = 6.05 Hz, 3H).
Compound 5p. HRMS: (M + 2H)+2 615.8399, calcd 615.8407. 1H
NMR (500 MHz, D2O) δ 8.71 (d, J = 5.20 Hz, 1H), 8.10 (d, J = 0.63
Hz, 1H), 7.89 (d, J = 7.72 Hz, 2H), 7.69 (d, J = 5.20 Hz, 1H), 7.50−
7.60 (m, 3H), 7.34 (t, J = 7.30 Hz, 2H), 7.29 (t, J = 6.90 Hz, 1H), 7.21
(d, J = 7.57 Hz, 2H), 4.77−4.81 (m, 2H), 4.42−4.49 (m, 3H), 4.30−
4.37 (m, 1H), 4.18−4.30 (m, 4H), 4.13 (dd, J = 3.39, 11.43 Hz, 1H),
4.09 (d, J = 4.57 Hz, 1H), 3.52 (dd, J = 4.97, 13.32 Hz, 1H), 3.28−3.39
(m, 2H), 2.99−3.21 (m, 8H), 2.89−2.99 (m, 2H), 2.81−2.89 (m, 1H),
2.32−2.43 (m, 1H), 2.10−2.32 (m, 5H), 1.89−2.10 (m, 3H), 1.76−
1.89 (m, 1H), 1.36−1.48 (m, 1H), 1.24−1.36 (m, 1H), 1.20 (d, J =
6.31 Hz, 3H), 1.13 (d, J = 6.46 Hz, 3H), 0.67 (d, J = 6.15 Hz, 3H),
0.59 (d, J = 5.67 Hz, 3H), 0.50−0.57 (m, 1H).
Compound 5q. HRMS: (M + 2H)+2 615.8405, calcd 615.8407; (M
+ Na)+ 1252.6556, calcd 1252.6561. 1H NMR (500 MHz, D2O) δ 9.12
(d, J = 2.00 Hz, 1H), 9.04 (d, J = 1.84 Hz, 1H), 8.89−8.81 (m, 1H),
7.78 (d, J = 6.82 Hz, 2H), 7.66−7.52 (m, 3H), 7.37 (t, J = 7.24 Hz,
2H), 7.32 (t, J = 7.29 Hz, 1H), 7.24 (d, J = 7.09 Hz, 2H), 4.85−4.77
(m, 2H), 4.55 (t, J = 8.26 Hz, 1H), 4.52−4.45 (m, 2H), 4.37−4.13 (m,
7H), 3.51 (dd, J = 4.98, 13.45 Hz, 1H), 3.38−3.26 (m, 2H), 3.26−2.98
(m, 9H), 2.92−2.82 (m, 1H), 2.82−2.72 (m, 1H), 2.42−2.08 (m, 6H),
2.07−1.79 (m, 4H), 1.53−1.43 (m, 1H), 1.43−1.34 (m, 1H), 1.22 (d, J
= 6.40 Hz, 3H), 1.16 (d, J = 6.41 Hz, 3H), 0.85−0.77 (m, 1H), 0.75
(d, J = 6.17 Hz, 3H), 0.67 (d, J = 6.13 Hz, 3H).
Compound 5r. HRMS: (M + 2H)+2 630.8468, calcd 630.8460; (M
+ Na)+ 1282.6658, calcd 1282.6667. 1H NMR (500 MHz, D2O) δ 8.81
(d, J = 6.16 Hz, 1H), 8.54 (d, J = 1.00 Hz, 1H), 8.14 (dd, J = 1.68, 6.19
Hz, 1H), 7.92 (d, J = 8.96 Hz, 2H), 7.37 (t, J = 7.23 Hz, 2H), 7.32 (t, J
= 7.28 Hz, 1H), 7.27−7.20 (m, 4H), 4.88−4.78 (m, 2H), 4.55 (t, J =
8.25 Hz, 1H), 4.52−4.46 (m, 2H), 4.37−4.14 (m, 7H), 3.93 (s, 3H),
3.51 (dd, J = 4.95, 13.44 Hz, 1H), 3.38−3.26 (m, 2H), 3.25−2.98 (m,
9H), 2.93−2.83 (m, 1H), 2.82−2.73 (m, 1H), 2.42−2.09 (m, 6H),
2.08−1.80 (m, 4H), 1.53−1.42 (m, 1H), 1.42−1.33 (m, 1H), 1.22 (d, J
= 6.32 Hz, 3H), 1.16 (d, J = 6.41 Hz, 3H), 0.85−0.75 (m, 1H), 0.74
(d, J = 5.75 Hz, 3H), 0.67 (d, J = 5.94 Hz, 3H).
Compound 5l. HRMS: (M + 2H)+2 630.3481, calcd 630.3484; (M
+ 2Na)+2 652.3315, calcd 652.3303. 1H NMR (500 MHz, D2O) δ 8.02
(d, J = 2.39 Hz, 1H), 7.87 (dd, J = 2.42, 8.71 Hz, 1H), 7.68 (d, J = 7.43
Hz, 2H), 7.52 (t, J = 7.71 Hz, 2H), 7.45−7.27 (m, 5H), 7.24 (d, J =
7.15 Hz, 2H), 4.81−4.75 (m, 2H), 4.56 (t, J = 8.24 Hz, 1H), 4.51−
4.44 (m, 2H), 4.38−4.11 (m, 7H), 3.99 (s, 3H), 3.53 (dd, J = 5.07,
13.46 Hz, 1H), 3.38−3.24 (m, 2H), 3.23−2.96 (m, 9H), 2.90−2.81
(m, 1H), 2.81−2.71 (m, 1H), 2.42−2.31 (m, 1H), 2.29−2.05 (m, 5H),
2.05−1.79 (m, 4H), 1.53−1.44 (m, 1H), 1.43−1.34 (m, 1H), 1.23 (d, J
= 6.39 Hz, 3H), 1.13 (d, J = 6.40 Hz, 3H), 0.87−0.77 (m, 1H), 0.75
(d, J = 6.21 Hz, 3H), 0.68 (d, J = 6.13 Hz, 3H).
Compound 5s. HRMS: (M + 2H)+2 630.8465, calcd 630.8460; (M
+ Na)+ 1282.6654, calcd 1282.6667. 1H NMR (500 MHz, D2O) δ 8.01
(dd, J = 1.51, 7.94 Hz, 2H), 7.75 (d, J = 0.97 Hz, 1H), 7.59−7.50 (m,
3H), 7.37 (t, J = 7.24 Hz, 2H), 7.32 (t, J = 7.26 Hz, 1H), 7.24 (d, J =
7.10 Hz, 2H), 7.14 (d, J = 0.97 Hz, 1H), 4.83−4.77 (m, 2H), 4.55 (t, J
= 8.24 Hz, 1H), 4.50−4.43 (m, 2H), 4.37−4.29 (m, 1H), 4.29−4.16
(m, 5H), 4.14 (d, J = 4.53 Hz, 1H), 4.05 (s, 3H), 3.52 (dd, J = 5.01,
13.44 Hz, 1H), 3.38−3.25 (m, 2H), 3.23−2.99 (m, 9H), 2.91−2.82
(m, 1H), 2.81−2.72 (m, 1H), 2.41−2.30 (m, 1H), 2.30−2.06 (m, 5H),
2.05−1.78 (m, 4H), 1.52−1.42 (m, 1H), 1.42−1.33 (m, 1H), 1.22 (d, J
= 6.40 Hz, 3H), 1.14 (d, J = 6.41 Hz, 3H), 0.86−0.77 (m, 1H), 0.74
(d, J = 6.31 Hz, 3H), 0.67 (d, J = 6.21 Hz, 3H).
Compound 5m. HRMS: (M + 2H)+2 632.3241, calcd 632.3236;
(M + 2Na)+2 654.3070, calcd 654.3056. 1H NMR (500 MHz, D2O) δ
7.80 (d, J = 2.17 Hz, 1H), 7.77 (dd, J = 2.26, 8.37 Hz, 1H), 7.68 (d, J =
8.36 Hz, 1H), 7.56−7.46 (m, 5H), 7.37 (t, J = 7.23 Hz, 2H), 7.32 (t, J
= 7.28 Hz, 1H), 7.24 (d, J = 7.09 Hz, 2H), 4.83−4.76 (m, 2H), 4.56 (t,
J = 8.26 Hz, 1H), 4.51−4.43 (m, 2H), 4.36−4.17 (m, 6H), 4.15 (d, J =
4.56 Hz, 1H), 3.51 (dd, J = 4.98, 13.44 Hz, 1H), 3.38−3.25 (m, 2H),
3.22−2.99 (m, 9H), 2.92−2.82 (m, 1H), 2.82−2.71 (m, 1H), 2.39−
2.28 (m, 1H), 2.28−2.07 (m, 5H), 2.07−1.77 (m, 4H), 1.53−1.43 (m,
1H), 1.43−1.33 (m, 1H), 1.19 (d, J = 6.41 Hz, 3H), 1.15 (d, J = 6.40
Hz, 3H), 0.89−0.77 (m, 1H), 0.75 (d, J = 6.32 Hz, 3H), 0.68 (d, J =
6.22 Hz, 3H).
Compound 5t. HRMS: (M + 2H)+2 616.3385, calcd 616.3384; (M
+ 2Na)+2 638.3199, calcd 638.3203. 1H NMR (500 MHz, D2O) δ 9.40
(d, J = 1.98 Hz, 1H), 8.44 (d, J = 1.99 Hz, 1H), 8.04−7.90 (m, 2H),
7.65−7.53 (m, 3H), 7.32 (t, J = 7.24 Hz, 2H), 7.27 (t, J = 7.27 Hz,
1H), 7.19 (d, J = 7.09 Hz, 2H), 4.79−4.72 (m, 2H), 4.51 (t, J = 8.25
Hz, 1H), 4.45−4.39 (m, 2H), 4.31−4.06 (m, 7H), 3.47 (dd, J = 4.99,
13.44 Hz, 1H), 3.32−3.21 (m, 2H), 3.20−2.93 (m, 9H), 2.87−2.77
(m, 1H), 2.77−2.67 (m, 1H), 2.39−2.27 (m, 1H), 2.27−2.02 (m, 5H),
2.02−1.74 (m, 4H), 1.48−1.38 (m, 1H), 1.38−1.29 (m, 1H), 1.17 (d, J
= 6.40 Hz, 3H), 1.10 (d, J = 6.40 Hz, 3H), 0.83−0.72 (m, 1H), 0.70
(d, J = 6.37 Hz, 3H), 0.62 (d, J = 6.25 Hz, 3H).
Compound 5n. HRMS: (M + 2H)+2 632.8213, calcd 632.8213; (M
+ 2Na)+2 655.3051, calcd 655.3046. 1H NMR (500 MHz, D2O) δ 8.83
(d, J = 5.44 Hz, 1H), 8.58 (t, J = 7.89 Hz, 1H), 8.11 (d, J = 7.91 Hz,
1H), 8.07−7.96 (m, 3H), 7.81 (d, J = 8.30 Hz, 1H), 7.38 (t, J = 7.26
Hz, 2H), 7.32 (t, J = 7.28 Hz, 1H), 7.25 (d, J = 7.10 Hz, 2H), 4.86−
4.79 (m, 2H), 4.56 (t, J = 8.28 Hz, 1H), 4.52−4.42 (m, 2H), 4.35−
4.12 (m, 7H), 3.50 (dd, J = 4.95, 13.46 Hz, 1H), 3.39−3.26 (m, 2H),
3.24−2.99 (m, 9H), 2.94−2.82 (m, 1H), 2.82−2.72 (m, 1H), 2.39−
2.28 (m, 1H), 2.28−2.08 (m, 5H), 2.07−1.77 (m, 4H), 1.53−1.43 (m,
1H), 1.43−1.33 (m, 1H), 1.20 (d, J = 6.34 Hz, 3H), 1.17 (d, J = 6.40
Hz, 3H), 0.85−0.77 (m, 1H), 0.74 (d, J = 6.04 Hz, 3H), 0.67 (d, J =
6.08 Hz, 3H).
Compound 5u. HRMS: (M + 2H)+2 632.8214, calcd 632.8213; (M
+ 2Na)+2 654.8023, calcd 654.8032. 1H NMR (500 MHz, D2O) δ 8.86
(dd, J = 0.58, 5.55 Hz, 1H), 8.19−8.11 (m, 1H), 7.99 (dd, J = 1.67,
5.55 Hz, 1H), 7.69−7.61 (m, 1H), 7.60−7.48 (m, 3H), 7.37 (t, J =
7.23 Hz, 2H), 7.32 (t, J = 7.29 Hz, 1H), 7.24 (d, J = 7.07 Hz, 2H),
4.85−4.76 (m, 2H), 4.55 (t, J = 8.26 Hz, 1H), 4.52−4.44 (m, 2H),
4.36−4.12 (m, 7H), 3.51 (dd, J = 4.98, 13.43 Hz, 1H), 3.38−3.26 (m,
2H), 3.24−2.99 (m, 9H), 2.92−2.83 (m, 1H), 2.82−2.73 (m, 1H),
2.41−2.30 (m, 1H), 2.30−2.09 (m, 5H), 2.09−1.79 (m, 4H), 1.52−
Compound 5o. HRMS: (M + 2H)+2 644.8216, calcd 644.8213. 1H
NMR (500 MHz, D2O) δ 7.87 (d, J = 8.40 Hz, 2H), 7.84−7.79 (m,
2H), 7.70 (d, J = 8.94 Hz, 1H), 7.67 (d, J = 8.40 Hz, 2H), 7.37 (t, J =
7.25 Hz, 2H), 7.32 (t, J = 7.27 Hz, 1H), 7.24 (d, J = 7.09 Hz, 2H),
4.83−4.76 (m, 2H), 4.56 (t, J = 8.24 Hz, 1H), 4.50−4.41 (m, 2H),
4.36−4.17 (m, 6H), 4.15 (d, J = 4.55 Hz, 1H), 3.51 (dd, J = 4.96,
13.44 Hz, 1H), 3.36−3.24 (m, 2H), 3.23−2.99 (m, 9H), 2.92−2.82
5089
dx.doi.org/10.1021/jm400416u | J. Med. Chem. 2013, 56, 5079−5093

Journal of Medicinal Chemistry
Article
1.43 (m, 1H), 1.43−1.34 (m, 1H), 1.21 (d, J = 6.40 Hz, 3H), 1.16 (d, J
= 6.40 Hz, 3H), 0.86−0.76 (m, 1H), 0.74 (d, J = 6.12 Hz, 3H), 0.67
(d, J = 6.11 Hz, 3H).
dryness to afford 200 mg (41% yield) of desired product as a white
1
solid. H NMR (400 MHz, DMSO-d6) δ 13.69 (br s, 1H), 8.06−8.21
(m, 2H), 7.90 (d, J = 1.17 Hz, 1 H), 7.38−7.62 (m, 3H), 7.16 (d, J =
0.98 Hz, 1H), 4.01 (s, 3H).
Compound 5v. HRMS: (M + 2H)+2 632.8220, calcd 632.8213; (M
+ 2Na)+2 654.8033, calcd 654.8032. 1H NMR (500 MHz, D2O) δ 8.82
(d, J = 5.60 Hz, 1H), 8.31 (d, J = 0.73 Hz, 1H), 7.96 (dd, J = 1.55, 5.57
Hz, 1H), 7.93 (t, J = 1.69 Hz, 1H), 7.86−7.79 (m, 1H), 7.65−7.59 (m,
1H), 7.57 (t, J = 7.86 Hz, 1H), 7.37 (t, J = 7.23 Hz, 2H), 7.32 (t, J =
7.29 Hz, 1H), 7.24 (d, J = 7.07 Hz, 2H), 4.85−4.78 (m, 2H), 4.55 (t, J
= 8.27 Hz, 1H), 4.51−4.45 (m, 2H), 4.38−4.12 (m, 7H), 3.52 (dd, J =
4.97, 13.44 Hz, 1H), 3.39−3.27 (m, 2H), 3.25−2.99 (m, 9H), 2.94−
2.83 (m, 1H), 2.83−2.73 (m, 1H), 2.43−2.32 (m, 1H), 2.31−2.08 (m,
5H), 2.08−1.79 (m, 4H), 1.52−1.42 (m, 1H), 1.42−1.33 (m, 1H),
1.23 (d, J = 6.40 Hz, 3H), 1.15 (d, J = 6.41 Hz, 3H), 0.84−0.76 (m,
1H), 0.74 (d, J = 5.92 Hz, 3H), 0.67 (d, J = 6.03 Hz, 3H).
4-Chloro-3-pyridin-2-yl-benzoic Acid. Step 1: 5-Ethoxycarbonyl-2-
chlorophenyl boronic acid (2.05 g, 8.87 mmol, 1.0 equiv) was
dissolved in a solution of degassed 1,4-dioxane/water (5:1, 49.0 mL),
followed by 2-chloropyridine (0.917 mL, 9.69 mmol, 1.1 equiv),
potassium carbonate (3.65g, 26.4 mmol, 3.0 equiv), and Pd(PPh3)4
(1.04g, 0.881 mmol, 0.10 equiv), the reaction was stirred at 83 °C for
3.0 h. The mixture was cooled to RT. Water (30.0 mL) and ethyl
acetate (150.0 mL) were added. The organics were separated and the
aqueous layer extracted with ethyl acetate (3 × 80 mL). The combined
organics were dried over sodium sulfate, filtered, and concentrated.
The crude product was purified by column chromatography (Analogix
SF-65 400 g column) using 8−50% EtOAc/heptane gradient to yield
the desired product (1.01g, 3.86 mmol, 43.9% yield).
Step 2: The ethyl ester from step 1 (1.01g, 3.86 mmol, 1.0 equiv)
was dissolved in a solution of water:THF (1:2, 60 mL) and treated
with LiOH hydrate (0.277 g, 11.6 mmol, 3.0 equiv). The solution was
stirred at RT for 4 h, then extracted with heptanes (2 × 20 mL). The
mixture was then saturated with sodium chloride, and pH was adjusted
stepwise from 5 to 1 with 1 N HCl. This mixture was subsequently
extracted with EtOAc (9 × 50 mL). The combined EtOAc layer was
dried over sodium sulfate, filtered, and concentrated to yield the
desired product (0.650 g, 62.5% yield). 1H NMR (400 MHz, DMSO-
d6) δ 13.26 (br s, 1H), 8.55−8.85 (m, 1H), 7.87−8.17 (m, 3H), 7.63−
7.78 (m, 2H), 7.44 (ddd, J = 7.61, 4.88, 1.17 Hz, 1H).
Compound 5w. HRMS: (M + 2H)+2 632.8212, calcd 632.8213. 1H
NMR (500 MHz, D2O) δ 8.80 (d, J = 5.52 Hz, 1H), 8.28 (s, 1H),
7.93−7.84 (m, 3H), 7.62 (d, J = 8.55 Hz, 2H), 7.42−7.28 (m, 3H),
7.24 (d, J = 7.15 Hz, 2H), 4.86−4.77 (m, 2H), 4.55 (t, J = 8.23 Hz,
1H), 4.51−4.44 (m, 2H), 4.37−4.11 (m, 7H), 3.51 (dd, J = 4.97, 13.43
Hz, 1H), 3.38−3.26 (m, 2H), 3.25−2.99 (m, 9H), 2.93−2.81 (m, 1H),
2.81−2.71 (m, 1H), 2.42−2.31 (m, 1H), 2.31−2.07 (m, 5H), 2.06−
1.78 (m, 4H), 1.53−1.43 (m, 1H), 1.42−1.34 (m, 1H), 1.22 (d, J =
6.40 Hz, 3H), 1.15 (d, J = 6.39 Hz, 3H), 0.88−0.77 (m, 1H), 0.75 (d, J
= 6.29 Hz, 3H), 0.67 (d, J = 6.20 Hz, 3H).
Compound 5x. HRMS: (M + 3H)+3 416.2291, calcd 416.2279; (M
+ 2H)+2 623.8390, calcd 623.8382; (M + Na)+ 1268.65 calcd
1
1268.6511. H NMR (600 MHz, D2O) δ 8.40 (dd, J = 2.6, 0.6 Hz,
5-Methoxy-biphenyl-3-carboxylic Acid. Step 1: A mixture of 1,3-
dibromo-5-methoxy-benzene (5g, 18.8 mmol), Pd(dppf)Cl2 (4g, 5.47
mmol), and TEA (3.8g, 37.55 mmol) in MeOH/DMF (75 mL/75
mL) was stirred at 50 °C under 50 psi CO for 16 h. TLC (petroleum
ether/EtOAc 10/1) indicated little starting material remained. The
mixture was cooled to RT and filtered. The filtrate was concentrated,
and the residue was dissolved in water (100 mL) and extracted with
EtOAc (3 × 100 mL). The combined organic layer was washed with
brine, dried over sodium sulfate, filtrated, and concentrated. The
residue was purified via flash column chromatography to give an oil
product 3-bromo-5-methoxy-benzoic acid methyl ester (1.2 g, 24.5%
yield).
Step 2: A mixture of 3-bromo-5-methoxy-benzoic acid methyl ester
(830 mg, 3.39 mmol), phenylboronic acid (573 mg, 4.07 mmol),
Pd(PPh3)4 (200 mg, 0.173 mmol), and sodium carbonate (900 mg,
8.49 mmol) in DME/water (20/20 mL) was heated to reflux with
stirring for 16 h under nitrogen. TLC (petroleum ether/EtOAc 20/1)
showed the reaction was complete. The reaction mixture was filtered,
and the filtrate was extracted with EtOAc (4 × 50 mL). The combined
organic layer was washed with brine, dried over sodium sulfate, and
concentrated to give 5-methoxy-biphenyl-3-carboxylic acid methyl
ester (1.3 g), which was purified via flash chromatography to give the
product as an oil (600 mg, 73.2% yield).
Step 3: To a mixture of 5-methoxy-biphenyl-3-carboxylic acid
methyl ester (600 mg, 2.49 mmol) in THF/water (30 mL/30 mL) was
added LiOH hydrate (312 mg, 7.43 mmol) at 0−5 °C. After the
addition, the mixture was stirred at RT for 16 h. TLC (petroleum
ether/EtOAc 10/1) showed the reaction was complete. The mixture
was concentrated, and the residue was dissolved in water (150 mL)
and extracted with ether (3 × 150 mL). The aqueous layer was
acidified pH to 2−3 with 1 M HCl and extracted with EtOAc (6 × 50
mL), and the combined organic layers were dried, filtered, and
concentrated to give the desired compound (540 mg, 95.6% yield) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.14 (br s, 1H), 7.70−
7.77 (m, 3H), 7.39−7.49 (m, 5H), 3.88 (s, 3H).
6-Oxo-1-phenyl-1,6-dihydropyridine-3-carboxylic Acid. A stirred
solution of methyl 2-oxo-2H-pyran-5-carboxylate (500 mg, 3.246
mmol) and aniline (604.2 mg, 6.492 mmol) in pyridine (5 mL) was
heated to 80 °C and stirred overnight. TLC (petroleum ether/EtOAc
= 2:1) showed the starting material was consumed completely. The
reaction solution was cooled to room temperature, evaporated to
dryness, and purified via chromatography (petroleum ether/EtOAc
1H), 8.14 (dd, J = 9.6, 2.6 Hz, 1H), 7.71−7.64 (m, 3H), 7.53−7.49
(m, 2H), 7.47−7.44 (m, 2H), 7.43−7.39 (m, 1H), 7.33 (d, J = 6.9 Hz,
2H), 6.85 (dd, J = 9.6, 0.6 Hz, 1H), 4.88 (dd, J = 8.7, 5.0 Hz, 1H), 4.76
(dd, J = 9.3, 5.3 Hz, 1H), 4.64 (t, J = 8.2 Hz, 1H), 4.56 (dd, J = 8.9, 5.5
Hz, 1H), 4.52 (d, J = 3.9 Hz, 1H), 4.41−4.23 (m, 7H), 3.58 (dd, J =
13.5, 5.0 Hz, 1H), 3.42−3.35 (m, 2H), 3.27−3.09 (m, 9H), 2.99−2.91
(m, 1H), 2.89−2.81 (m, 1H), 2.42−2.18 (m, 6H), 2.13−1.88 (m, 4H),
1.59−1.52 (m, 1H), 1.51−1.43 (m, 1H), 1.27 (d, J = 6.4 Hz, 3H), 1.25
(d, J = 6.4 Hz, 3H), 0.96−0.86 (m, 1H), 0.83 (d, J = 6.5 Hz, 3H), 0.76
(d, J = 6.4 Hz, 3H).
Synthesis of Noncommercial Biaryl Acid Intermediates. 2-
Methoxy-6-phenyl-isonicotinic Acid. Step 1: Citrazinic acid (2.00 g,
12.9 mmol, 1.0 equiv) and phosphorus oxybromide (11.19 g, 3.0
equiv) were heated at 180 °C under nitrogen for 2 h. The reaction
mixture was cooled to RT and ice water added cautiously, and the
resulting mixture was left at RT for 12 h before extracting with EtOAc
(4 × 100 mL). The combined organic layer was dried over sodium
sulfate, filtered, and concentrated to dryness to afford 3.0 g (83%
yield) of 2,6-dibromo-isonicotinic acid as a light-brown solid.
Step 2: 2,6-Dibromo-isonicotinic acid (3.00 g, 1 equiv) and sodium
methoxide (2.88 g, 5 equiv) were dissolved in anhydrous MeOH (21
mL) and heated to reflux for 48 h. The mixture was cooled to RT,
dissolved in 20 mL water, and adjusted to pH ∼ 3 with 3 N HCl. This
was then extracted with EtOAc (3 × 50 mL). The combined organic
layer was dried over sodium sulfate, filtered, and concentrated to
dryness to afford 2.3 g (93% yield) of 2-bromo-6-methoxy-isonicotinic
acid as a light-brown solid.
Step 3: To a mixture of phenylboronic acid (394 mg, 1.5 equiv) and
crude acid of step 2 (500 mg, 1.0 equiv) in dioxane (5 mL) was added
2 M Na2CO3 (3.76 mL, 3.5 equiv) and Pd(PPh3)4 (248 mg, 0.1 equiv)
in a 35 mL Q-tube (Sigma-Aldrich). The mixture was stirred at 180 °C
for 20 min. The crude product was then filtered through Celite. The
Celite was subsequently rinsed several times with water (2 × 5 mL),
and the filtrate was poured into a separatory funnel and extracted with
heptane (3 × 20 mL) and then with EtOAc (1 × 20 mL) to remove
some of the unwanted organic materials. The aqueous layer was
acidified to pH ∼ 2 with 1 N HCl and extracted with EtOAc (3 × 20
mL). The combined EtOAc layers were dried over anhydrous sodium
sulfate, filtered, and concentrated to dryness to afford 412 mg of crude
yellow solid. The crude product was purified by flash chromatography
using an Analogix SF25 40 g column with a gradient of 0−5% of
MeOH/DCM. Relevant fractions were combined and concentrated to
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gradient) to give methyl 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbox-
ylate (290 mg, 39% yield) as a yellow solid. This material was
subsequently dissolved in MeOH (3 mL) and to this was added
aqueous LiOH (159.6 mg, 3.799 mmol, dissolved in 1 mL water).
After the addition, the resulting solution was stirred at RT overnight.
LC-MS showed the starting material was consumed completely. The
bulk of the solvent was removed in vacuo, and the resulting residue
was diluted with water (20 mL) and then extracted with MTBE (20
mL). The aqueous layer was adjusted to pH = 2−3 with 1 M HCl
solution and extracted with EtOAc (3 × 20 mL). The combined
EtOAc extracts were evaporated to dryness to give the title compound
(156 mg, 57.3% yield) as a yellow solid. 1H NMR: (400 MHz,
methanol-d4): δ 8.317−8.311 (d, 1H), 8.068−8.038 (m, 1H), 7.593−
7.521 (m, 3H), 7.450−7.429 (m, 2H), 6.648−6.624 (d, 1H).
MIC and MBC Determinations. The in vitro antibacterial activity
of test compounds was evaluated by MBC and MIC testing according
to Clinical and Laboratory Standards Institute (CLSI). The following
bacterial strains were used in these MIC determinations: PA-1646 and
AB-1649 are polymyxin-resistant clinical isolates of P. aeruginosa and
A. baumannii, respectively, obtained from JMI laboratories (www.
jmilabs.com); PA01 is a P. aeruginosa lab strain (www.fems-
microbiology.org); AB-3167 is clinical isolate of A. baumannii obtained
from IHMA (www.ihmainc.com); KP-3700 is an ESBL+ clinical isolate
of K. pneumoniae obtained in 2006; EC-1 is a mouse virulent strain of
E. coli.
hRPTEC Cytotoxicity Assay. Human renal proximal tubule
epithelial cells (hRPTEC) were obtained from Lonza (Walkersville,
MD) and cultured in REGM (Renal Epithelial Cell Basal Medium
supplemented with human epithelial growth factor, hydrocortisone,
epinephrine, insulin, triiodothyronine, transferrin, gentamycin/ampho-
tericin-B, and fetal bovine serum). The hRPTEC cells were seeded
into 96-well plates and incubated at 37 °C in 5% CO2. Following a
growth period of 72 h, cells were treated with compounds at 100, 67,
44, 30, 20, and 13 μM aqueous solution for 24 h at 37 °C in 5% CO2.
The cell supernatant was then harvested for determination of LDH
(lactate dehydrogenase) production. The Cytotoxicity Detection Kit
from Roche was used for the measurement of LDH activity released
from damaged cells. The toxic concentration 50% (TC50, μM) values
were determined for each compound and compared to the control
compounds (PMB was used as a positive control and PMBN was used
as negative control).
Toxicology Studies in Rats and Dogs. All studies were
conducted in accordance with the current guidelines for animal
welfare (ILAR Guide for the Care and Use of Laboratory Animals,
1996; Animal Welfare Act, 1966, as amended in 1970, 1976, and 1985,
9 CFR Parts 1, 2, and 3). The procedures used in these studies were
reviewed and approved by the Institutional Animal Care and Use
Committee of the testing facility.
Rat Studies. Rat studies were conducted utilizing male Wistar Han
IGS rats (CRL:WI [HAN]) with an age range of 6−9 weeks at study
initiation. Rats were administered intravenous bolus doses twice daily
to achieve total daily doses of 0 (0.9% sterile saline), 0.4, 4, or 10 mg/
kg/day polymyxin B (PMB) or 0 (0.9% sterile saline), 4 or 8 mg/kg/
day 5x with at least 6 h between each dose for 7 days. Plasma samples
were collected on days 1 and 7 for toxicokinetic analysis. Animals were
euthanized and necropsied the day after the last dose. After gross
examination, the kidneys were weighed, collected, processed, and
examined microscopically. Microscopic lesions were graded on a scale
of 1−5 as minimal, mild, moderate, marked, or severe.
In Vivo PK/PD Studies. The comparative in vivo pharmacody-
namics of 5x and PMB (X-Gen Pharmaceuticals Inc., Big Flats, NY)
were evaluated using the well described neutropenic thigh infection
model.24 The study protocol was reviewed and approved by the
Hartford Hospital Institutional Animal Care and Use Committee.
Briefly, female ICR mice weighing approximately 25 g were
acquired from Harlan Sprague−Dawley, Inc. (Indianapolis, IN) and
utilized throughout these experiments. Mice were rendered
neutropenic with 100 and 150 mg/kg intraperitoneal injections of
cyclophosphamide (Cytoxan; Bristol-Myers Squibb, Princeton, NJ)
given 1 and 4 days prior to inoculation, respectively. Each thigh of the
mice was inoculated with 107 colony forming units (CFU)/mL of the
infecting organism. For these studies, P. aeruginosa 1401 (PA01) and
1402 (UC12120) were utilized. 5x and PMB minimum inhibitory
concentrations were 0.5 mg/L for both organisms as determined using
the broth microdilution methodology described by the Clinical and
Laboratory Standards Institute. Two hours after inoculation, groups of
three mice were administered subcutaneous doses of 5x and PMB as
total daily doses of 5−400 mg/kg given 1, 2 (q12h) or 4 (q6h) times
over a 24 h period. After 24 h, mice were euthanized by CO2
asphyxiation followed by cervical dislocation; thigh tissue was
harvested, homogenized in normal saline, and bacterial density
quantified using serial dilution and plating techniques. Efficacy was
evaluated as the change in bacterial density in treated mice as
compared with 0 h (initiation of treatment) control animals.
Pharmacokinetic studies of 5x and PMB were conducted in infected,
neutropenic mice. For these analyses, mice were infected and
inoculated as described above. Next animals were administered single
subcutaneous doses of the test compounds ranging from 12.5 to 75
mg/kg. Groups of six mice were euthanized as described above, and
blood was collected via cardiac puncture at eight time points over a 12
h interval. Plasma was collected via centrifugation, and samples were
stored at −80 °C until concentration determination via a validated
liquid chromatography−mass spectrometry assay.
Pharmacokinetic parameters for single doses of 5x and PMB were
calculated by nonlinear least-squares techniques (WinNonlin version
5.0.1, Pharsight, Mountain View, CA). The mean pharmacokinetic
parameters derived from single dose studies were used to construct
concentration−time profiles for all doses evaluated in the bacterial
density studies. The area under the concentration−time profile (AUC)
was calculated using the trapezoidal rule. For free drug assessments,
protein binding values of 46% and 83% were utilized for 5x and PMB,
respectively.
For pharmacodynamic analyses, composite plots of the change in
log10CFU for both P. aeruginosa isolates versus fAUC/MIC (ratio of
the fAUC to MIC of the infecting organism) were constructed and fit
to a sigmoidal Emax inhibitory model with baseline effects (WinNonlin
version 5.0.1, Pharsight, Mountain View, CA). The exposure index
required for stasis, 1 log reduction, 50% of maximal efficacy (EI50), and
80% of maximal (EI80) efficacy, was calculated from the respective Emax
profile for 5x and PMB.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 617-551-3513. E-mail: thomas.v.magee@pfizer.com.
Present Addresses
Dog Studies. Dog studies were conducted utilizing male and female
Beagle dogs with a body weight range of 5−11 kg at study initiation.
Dogs were administered intravenous bolus doses twice daily to achieve
total daily doses of 0 (0.9% sterile saline), 1, or 6 mg/kg/day PMB or
0 (0.9% sterile saline), 5, 11, or 20 mg/kg/day 5x with at least 6 h
between each dose for 7 days. Plasma samples were collected on days
1 and 7 for toxicokinetic analysis. Animals were euthanized and
necropsied the day after the last dose. After gross examination, the
kidneys were weighed, collected, processed, and examined micro-
scopically. Microscopic lesions were graded on a scale of 1−5 as
minimal, mild, moderate, marked, or severe.
#Pfizer Cardiovascular, Metabolic, and Endocrine Diseases
(CVMED) Chemistry, Cambridge, Massachusetts 02139,
United States.
▽Astra-Zeneca Research Center, Waltham, Massachusetts
02451, United States.
○Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington,
Massachusetts 02421, United States.
Notes
The authors declare no competing financial interest.
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(5) The prodrug is known as either colistimethate sodium, colistin
methanesulfonate, or simply CMS. Each of the five γ amine nitrogens
of the Dab residues at positions 1, 3, 5, 8, and 9 has a
methanosulfonate moiety which decomposes in serum to the
corresponding free base. See Gobin, P.; Lemaitre, F.; Marchand, S.;
Couet, W.; Olivier, J.-C. Assay of colistin and colistin methanesulfo-
nate in plasma and urine by liquid chromatography−tandem mass
spectrometry. Antimicrob. Agents Chemother. 2010, 54, 1941−1948.
(6) Molina, J.; Cordero, E.; Pachon, J. New information about the
polymyxin/colistin class of antibiotics. Expert Opin. Pharmacother.
2009, 10, 2811−2828.
(7) (a) HsuChen, C.-C.; Feingold, D. S. The mechanism of
polymyxin B action and selectivity toward biological membranes.
Biochem. 1973, 12, 2105−2111. (b) Mares, J.; Kumaran, S.; Gobbo,
M.; Zerbe, O. Interactions of lipopolysaccharide and polymyxin
studied by NMR spectroscopy. J. Biol. Chem. 2009, 284, 11498−
11506. (c) Srimal, S.; Surolia, N.; Balasubramanian, S.; Surolia, A.
Titration calorimetric studies to elucidate the specificity of the
interactions of polymyxin B with lipopolysaccharides and lipid A.
Biochem. J. 1996, 315, 679−686. (d) Bruch, M. D.; Cajal, Y.; Koh, J.
T.; Jain, M. K. Higher-order structure of polymyxin B: the functional
significance. J. Am. Chem. Soc. 1999, 121, 11993−12004. (e) Bhatta-
charjya, S.; David, S. A.; Mathan, V. I.; Balaram, P. Polymyxin B
nonapeptide: conformations in water and in lipopolysaccharide-bound
state determined by two-dimensional NMR and molecular dynamics.
Biopolymers 1997, 41, 251−265. (f) Pristovsek, P.; Kidric, J. Solution
structure of polymyxins B and E and effect of binding to
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with cationic compounds. Eng. Life Sci. 2008, 8, 523−529.
ACKNOWLEDGMENTS
■
We thank Justin G. Stroh for performing high resolution mass
spectrometry analyses and the staff of the Center for Anti-
Infective Research and Development for their assistance with in
vivo experimentation.
ABBREVIATIONS USED
■
PMB, polymyxin B; PMBN, polymyxin B nonapeptide; LPS,
lipopolysaccharide; MIC, minimum inhibitory concentration;
MBC, minimum bactericidal concentration; hRPTEC, human
renal proximal tubule epithelial cell; TC50, toxicity concen-
tration 50%; Dab, L-2,4-diaminobutyric acid; Dap, L-2,3-
diaminoproprionic acid; PA, Pseudomonas aeruginosa; AB,
Acinetobacter baumannii; KP, Klebsiella pneumoniae; EC,
Escherichia coli; hRPTEC, human renal proximal tubule
epithelial cell; AA, amino acid; KDO, 3-deoxy-D-mannooctulo-
sonic acid; DCM, dichloromethane; Fmoc, fluorenylmethylox-
ycarbonyl; Dde, 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)-
ethyl; ETS, exploratory toxicity study
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