Noel P Pitcher1, Jitendra R Harjani1, Yichao Zhao1, Jianwen Jin1, Daniel R Knight2,3,4,5, Lucy Li6, Papanin Putsathit2,3,4,5, Thomas V Riley2,3,4,5, Glen P Carter6, Jonathan B Baell7,1,8. 1. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. 2. School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia 6027, Australia. 3. School of Biomedical Sciences, Faculty of Health and Medical Sciences, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia. 4. Medical, Molecular and Forensic Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia. 5. Department of Microbiology, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia. 6. Department of Microbiology & Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria 3000, Australia. 7. School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People's Republic of China. 8. Australian Translational Medicinal Chemistry Facility (ATMCF), Monash University, Parkville, Victoria 3052, Australia.
Abstract
Colonization of the gastrointestinal (GI) tract with pathogenic bacteria is an important risk factor for the development of certain potentially severe and life-threatening healthcare-associated infections, yet efforts to develop effective decolonization agents have been largely unsuccessful thus far. Herein, we report modification of the 1,2,4-oxadiazole class of antimicrobial compounds with poorly permeable functional groups in order to target bacterial pathogens within the GI tract. We have identified that the quaternary ammonium functionality of analogue 26a results in complete impermeability in Caco-2 cell monolayers while retaining activity against GI pathogens Clostridioides difficile and multidrug-resistant (MDR) Enterococcus faecium. Low compound recovery levels after oral administration in rats were observed, which suggests that the analogues may be susceptible to degradation or metabolism within the gut, highlighting a key area for optimization in future efforts. This study demonstrates that modified analogues of the 1,2,4-oxadiazole class may be potential leads for further development of colon-targeted antimicrobial agents.
Colonization of the gastrointestinal (GI) tract with pathogenic bacteria is an important risk factor for the development of certain potentially severe and life-threatening healthcare-associated infections, yet efforts to develop effective decolonization agents have been largely unsuccessful thus far. Herein, we report modification of the 1,2,4-oxadiazole class of antimicrobial compounds with poorly permeable functional groups in order to target bacterial pathogens within the GI tract. We have identified that the quaternary ammonium functionality of analogue 26a results in complete impermeability in Caco-2 cell monolayers while retaining activity against GI pathogens Clostridioides difficile and multidrug-resistant (MDR) Enterococcus faecium. Low compound recovery levels after oral administration in rats were observed, which suggests that the analogues may be susceptible to degradation or metabolism within the gut, highlighting a key area for optimization in future efforts. This study demonstrates that modified analogues of the 1,2,4-oxadiazole class may be potential leads for further development of colon-targeted antimicrobial agents.
The gastrointestinal
(GI) tract serves as an important site of
colonization for many pathogenic bacteria prior to the initiation
of infection. The “healthy” microbiota that resides
in the GI tract is normally considered to offer “colonization
resistance” against invading pathogens but this can be compromised
by exposure to antimicrobials or in certain illnesses.[1−3] Such disruption can lead to the overgrowth of GI pathogens, which
is a major risk factor in the development of several different healthcare-associated
infections.[4−8] Among the most significant nosocomial pathogens that colonize the
GI tract is Clostridioides difficile, which causes a range of colonic infections collectively referred
to as C. difficile infections (CDI).
CDI may lead to mild to severe diarrhoea and can lead to potentially
fatal complications such as pseudomembranous colitis and toxic mega-colon.[9,10] Indeed, C. difficile has been classified
as an urgent threat to public health by the United States Centers
for Disease Control and Prevention (CDC).[11] Traditional first-line treatments including metronidazole and vancomycin
are effective for most CDI cases but approximately 15–30% of
patients will suffer a recurrence of infection after cessation of
treatment.[12,13] More recently, fidaxomicin, a
narrow spectrum macrocyclic antimicrobial from the tiacumicin family,
has become a recommended treatment for CDI. This antimicrobial is
associated with lower rates of recurrence than vancomycin or metronidazole.[14,15] Also, metronidazole is no longer recommended as a first line therapy
of CDI due to treatment failures in the US.[14]Another important nosocomial pathogen is Enterococcus
faecium.[16] Colonization
of the GI tract by E. faecium is recognized
as a major risk factor for the onset of potentially life-threatening
diseases such as bacteraemia and infective endocarditis.[8,17]E. faecium has a high propensity
for the development of antimicrobial resistance due to its malleable
genome which has resulted in the emergence of multidrug-resistant
(MDR) strains,[18] most notably vancomycin-resistant E. faecium (VREfm). The CDC has declared VREfm a
serious threat to human health[11] and it
has been included in the 2017 World Health Organization priority pathogen
list for the development of new antimicrobials,[19] which highlights the limited therapeutic options available
for treating this pathogen. High rates of VREfm infection have rendered
the traditional first-line treatment of vancomycin increasingly ineffective
against E. faecium infections, with
clinicians now increasingly using linezolid and daptomycin.[17] Reports documenting the emergence of linezolid
and daptomycin resistance in VREfm are therefore of great clinical
concern.[20,21] Furthermore, the development of decolonization
agents that can be used to eradicate E. faecium from the gut has been an aim for several years without success.[22−25]Despite exhibiting different clinical manifestations, the
disease
states caused by both of these pathogens arise following initial colonization
of the GI tract. There is therefore an urgent need for novel antimicrobials
that selectively target pathogens within the GI tract while maintaining
the integrity of gut microbiota. Such agents would undoubtedly improve
treatment outcomes for difficult cases of C. difficile and E. faecium infections.The 1,2,4-oxadiazole antimicrobial compound class was reported
in 2014 by Chang and Mobashery et al. and displayed potent activity
against important Gram-positive pathogens in the ESKAPE panel.[26,27] Analogue 1 emerged as a lead compound in the study
with activity against Staphylococcus aureus, Enterococcus faecalis, and E. faecium, and this activity was maintained across
multiple strains, including those exhibiting vancomycin resistance.
Analogue 75b was another leading compound. Significantly,
there was no activity observed against the Gram-negative bacteria
in the ESKAPE group, potentially demonstrating a selectivity profile
that may be sparing of important members of the GI tract microbiota.
Chang and Mobashery et al. have advanced several analogues in the
class with a focus on developing effective treatments for methicillin-resistant S. aureus (MRSA) (Figure a).[28−30]
Figure 1
(a) Reported antimicrobials of a new oxadiazole
class, with leading
compounds 1 and 75b; (b) modifications reported
here, leading to 26a that exhibits potent anti-C. difficile and anti-VREfm activity. (a) Previous
work by Chang and Mobashery et al.[26,28−30] (b) This work.
(a) Reported antimicrobials of a new oxadiazole
class, with leading
compounds 1 and 75b; (b) modifications reported
here, leading to 26a that exhibits potent anti-C. difficile and anti-VREfm activity. (a) Previous
work by Chang and Mobashery et al.[26,28−30] (b) This work.Here, we explore the
utility of the 1,2,4-oxadiazole compound class
for the treatment of GI pathogens by attaching substituents that are
predicted to reduce permeability in the GI tract. The idea is that
such compounds may therefore reach the colon in relatively higher
concentrations (Figure b). In an important proof-of-concept, several analogues modified
to be less Caco-2 permeable maintained antimicrobial activity. Quaternary
ammonium derivative 26a is of particular note with complete
impermeability to Caco-2 cells and notable anti-C.
difficile and anti-VREfm activity.
Results and Discussion
Minimum
Inhibitory Concentration and Time-Kill Assays of Lead
Compound 1
The lead 1,2,4-oxadiazole compound 1 was assessed by minimum inhibitory concentration (MIC) assay
and exhibited a modest potency of 6 μg/mL against C. difficile, comparable to that of vancomycin (2
μg/mL). Compound 1 also showed concentration-dependent
killing kinetics in time-kill assays and rapid bactericidal activity
when using ≥2 × MIC (12–48 μg/mL), which
was similar to the activity observed for the positive control compound
benzalkonium chloride (a quaternary ammonium compound with known membrane
lysing activity) and superior to the bacteriostatic activity of vancomycin
(Figure ).
Figure 2
Time-kill analysis
of 1 against C.
difficile strain EDN0008. Blue circles are DMSO-containing
untreated cultures (vehicle), red squares are cultures treated with
6 μg/mL 1, green triangles are cultures treated
with 12 μg/mL 1, purple triangles are cultures
treated with 24 μg/mL 1, orange diamonds are cultures
treated with 48 μg/mL 1, turquoise circles are
cultures treated with 8 μg/mL vancomycin, and black squares
are cultures treated with 24 μg/mL benzalkonium chloride. Compounds
were tested using three independent biological replicates with the
data shown representing the mean ± SEM. VAN—vancomycin,
BZK—benzalkonium chloride, and CFU—colony-forming units.
Time-kill analysis
of 1 against C.
difficile strain EDN0008. Blue circles are DMSO-containing
untreated cultures (vehicle), red squares are cultures treated with
6 μg/mL 1, green triangles are cultures treated
with 12 μg/mL 1, purple triangles are cultures
treated with 24 μg/mL 1, orange diamonds are cultures
treated with 48 μg/mL 1, turquoise circles are
cultures treated with 8 μg/mL vancomycin, and black squares
are cultures treated with 24 μg/mL benzalkonium chloride. Compounds
were tested using three independent biological replicates with the
data shown representing the mean ± SEM. VAN—vancomycin,
BZK—benzalkonium chloride, and CFU—colony-forming units.Our previous findings demonstrated that compound 1 also displayed potent activity against a range of clinically
relevant E. faecium strains, including
both VREfm and daptomycin-resistant
strains.[31] Compound 1 was
not toxic against the human cell line HepG2 at the E. faecium MIC90 (2 μg/mL); however,
reduced viability was observed at higher concentrations. This was
deemed to represent a potentially narrow therapeutic window, but analogues
of compound 1 have been reported by Spink et al. in 2015
with activity against S. aureus and
reduced toxicity against HepG2 cells.[28] Thus, we hypothesized that optimization of the therapeutic index
for this compound class would be highly feasible.
General Strategy
To address the high bioavailability
reported for the 1,2,4-oxadiazole compound class,[26] we proposed that their physicochemical properties could
be modified to reduce “drug-likeness”, thereby decreasing
systemic exposure and leading to accumulation in the colon. Amine
and carboxylic acid functional groups that are charged at physiological
pH were linked to the oxadiazole structure to induce ionization after
oral administration and reduce compound permeability across the small
intestine.[32,33] Indeed, similar approaches have
shown success previously by utilizing the poorly permeable tetramic
acid moiety to localize metronidazole in the GI tract.[34]Suitable positions for linking to the
oxadiazole structure needed to be carefully selected as to not decrease
antimicrobial activity. It was noted in similar oxadiazole analogues
reported for the treatment of MRSA[28] that
trifluoromethyl groups at the 4 position of ring A had minimal effect
on in vitro activity but improved oral bioavailability
(Figure ). Therefore,
the trifluoromethyl group was excluded from our oxadiazole analogues
and ring A was instead used to introduce the poorly permeable groups.
Our structure–activity relationship (SAR) investigation probed
all aromatic positions of ring A while exploring different linkers
to attach the ionizable functional groups. The hydroxyl in compound 1 was maintained consistent for the analogues in the series
as it was previously recognized that a hydrogen bond donor at the
position was essential for activity against MRSA.[28]
Figure 3
General structure of the oxadiazole class, consisting of four main
aromatic rings (A–D). R′ and R″ represent the
positions explored to link poorly permeable groups.
General structure of the oxadiazole class, consisting of four main
aromatic rings (A–D). R′ and R″ represent the
positions explored to link poorly permeable groups.Additionally, we explored replacement of the phenol with
an aniline
to probe modification of ring D while maintaining the critical hydrogen
bond donor. Primary and secondary aniline groups were synthesized
to investigate if substitution from ring D was tolerated and if it
could offer an alternative site to link the poorly permeable groups.
Synthetic Chemistry
Modified oxadiazole analogues were
synthesized as outlined in Scheme . Reagents 2a–h were used to prepare t-Bu protected carboxylic acid substrates 3a–h, followed by installation of a benzonitrile moiety using a known
Ullmann-type reaction procedure.[26] Hydroxylamine
addition to the nitrile yielded amidoximes 6a–h which then underwent coupling, cyclization, and dehydration with
benzoic acid 7 in a “one-pot” process to
form the 1,2,4-oxadiazole ring C.[35] An
acetyl group was used as a transient protecting group to inhibit the
ring D phenol from reacting during coupling and was subsequently cleaved
in situ at high temperature to reveal the free phenol in compounds 8a–h. Protecting group removal was achieved under acidic
conditions to afford final analogues 9a–h.
Scheme 1
Synthesis for Modified Oxadiazole Analogues 9a–h
Reagents and conditions: (a)
(i) 2-tert-butyl-1,3-diisopropylisourea, DMAP, DCM,
rt, 96 h; (ii) 1,1′-carbonyldiimidazole, 1,8-diazabicyclo[5.4.0]undec-7-ene, tert-butanol, DMF, 65 °C, 72 h; (iii) tert-butyl bromoacetate, K2CO3, DMF, rt, 16 h;
(b) 4, CuI, Cs2CO3, N,N-dimethylglycine, 1,4-dioxane, 100 °C, 48
h; (c) NH2OH·HCl, Et3N, MeOH, 60 °C,
6 h; (d) 7, HOBt, EDC·HCl, DMF, rt for 16 h to 100
°C for 16 h; (e) TFA, DCM, rt, 8 h.
Synthesis for Modified Oxadiazole Analogues 9a–h
Reagents and conditions: (a)
(i) 2-tert-butyl-1,3-diisopropylisourea, DMAP, DCM,
rt, 96 h; (ii) 1,1′-carbonyldiimidazole, 1,8-diazabicyclo[5.4.0]undec-7-ene, tert-butanol, DMF, 65 °C, 72 h; (iii) tert-butyl bromoacetate, K2CO3, DMF, rt, 16 h;
(b) 4, CuI, Cs2CO3, N,N-dimethylglycine, 1,4-dioxane, 100 °C, 48
h; (c) NH2OH·HCl, Et3N, MeOH, 60 °C,
6 h; (d) 7, HOBt, EDC·HCl, DMF, rt for 16 h to 100
°C for 16 h; (e) TFA, DCM, rt, 8 h.A
more concise scheme was also devised to introduce the derivatized
phenols at the penultimate step, as outlined in Scheme . Hydroxylamine addition to 4-iodobenzonitrile 4 and then reaction with acyl chloride 11 yielded
the key intermediate 12.[26] Optimized Ullmann-type reaction conditions were used to couple the
derivatized phenols, during which the tert-butyldimethylsilyl
(TBS) provided a transient protecting group that was cleaved in situ.
The derivatized phenol reagents 13a–i were individually
prepared from commercially available material as described in the Supporting Information. Deprotection of the boc
groups under acidic conditions yielded final analogues 15a–i.
Scheme 2
Synthesis for Modified Oxadiazole Analogues 15a–i
Reagents and conditions: (a)
NH2OH·HCl, Et3N, MeOH, 60 °C, 6 h;
(b) 11 (synthesis previously described[26]), toluene, 120 °C, 27 h; (c) 13a–i, 1,3-diphenyl-1,3-propanedione,
Cs2CO3, copper(II) acetylacetonate, DMSO, 90
°C, 24 h; (d) 4 M HCl/1,4-dioxane, rt, 7 h
Synthesis for Modified Oxadiazole Analogues 15a–i
Reagents and conditions: (a)
NH2OH·HCl, Et3N, MeOH, 60 °C, 6 h;
(b) 11 (synthesis previously described[26]), toluene, 120 °C, 27 h; (c) 13a–i, 1,3-diphenyl-1,3-propanedione,
Cs2CO3, copper(II) acetylacetonate, DMSO, 90
°C, 24 h; (d) 4 M HCl/1,4-dioxane, rt, 7 hAniline series analogues were synthesized as outlined in Scheme . Amine- and nitro-substituted
aryl carboxylate substrates 17a–d were subjected
to coupling, cyclization, and dehydration with amidoxime intermediate 18 to form the 1,2,4-oxadiazole ring C and afford analogues 19c and 19d.[35] Treatment
with 4 M HCl in 1,4-dioxane then afforded compounds 20a and 20b. Compound 22 was synthesized via sodium hydride-mediated alkylation of intermediate 19a using 2-bromoethyl methyl ether, followed by protecting
group removal. Alkylation of 20a using tert-butyl bromoacetate and subsequent acidic removal of the protecting
group was undertaken to synthesize 24.
Scheme 3
Synthesis of Aniline
Series Analogues
Reagents and conditions: (a)
Boc2O, NaHCO3, MeOH/H2O, rt, 24 h;
(b) 18 (synthesis previously described[26]), HOBt, EDC·HCl, DMF, rt for 16 h to 100 °C for
16 h; (c) 4 M HCl/1,4-dioxane, rt, 6 h; (d) NaH (60% in mineral oil),
DMF, 0 °C to rt, 1 h, 1-bromo-2-methoxyethane, 0 °C to rt,
2 h; (e) tert-butyl bromoacetate, DIPEA, acetonitrile,
80 °C, 48 h; (f) TFA, DCM, rt, 8 h
Synthesis of Aniline
Series Analogues
Reagents and conditions: (a)
Boc2O, NaHCO3, MeOH/H2O, rt, 24 h;
(b) 18 (synthesis previously described[26]), HOBt, EDC·HCl, DMF, rt for 16 h to 100 °C for
16 h; (c) 4 M HCl/1,4-dioxane, rt, 6 h; (d) NaH (60% in mineral oil),
DMF, 0 °C to rt, 1 h, 1-bromo-2-methoxyethane, 0 °C to rt,
2 h; (e) tert-butyl bromoacetate, DIPEA, acetonitrile,
80 °C, 48 h; (f) TFA, DCM, rt, 8 hDimethylamine
and quaternary ammonium substituted analogues 25a, 25b, 26a, and 26b were synthesized
from their corresponding primary amine analogues,
as outlined in Scheme . Boc-deprotection reactions of 14e and 14h were undertaken using standard conditions followed by careful neutralization
to remove the trifluoroacetic acid (TFA) salts of the amines while
also avoiding removal of the acidic phenolic protons. Treatment with
formaldehyde and NaBH(OAc)3 was undertaken to convert the
primary amine intermediates to the dimethylamine groups,[36] and subsequent reaction with iodomethane selectively
yielded the quaternary ammonium analogues without phenol methylation.[37]
Scheme 4
Synthesis of Methylated-Amine Substituted
Analogues
Reagents and conditions: (a)
TFA, DCM, rt, 7 h; (b) 37% aq formaldehyde, NaBH(OAc)3,
acetonitrile/H2O, rt, 50 min; (c) MeI, DCM/MeOH, rt, 24
h.
Synthesis of Methylated-Amine Substituted
Analogues
Reagents and conditions: (a)
TFA, DCM, rt, 7 h; (b) 37% aq formaldehyde, NaBH(OAc)3,
acetonitrile/H2O, rt, 50 min; (c) MeI, DCM/MeOH, rt, 24
h.
MIC and Time-Kill Assays of Oxadiazole Analogues
The
antimicrobial activity of the modified oxadiazole analogues against C. difficile and E. faecium was evaluated using standardized broth microdilution MIC assays.
The initial SAR exploration sought to identify the best position for
substitution from ring A and which poorly permeable groups were suitable
for maintaining potency. The attachment of certain amine and carboxylic
acid groups was tolerated in terms of C. difficile activity but was dependent on linker length and aromatic position
(Table ). Carboxylic
acid substituents with ethylene linkers 9b (4 μg/mL)
and 9d (8 μg/mL) were favored from the 2 and 3
positions, displaying similar activities to analogue 1 (6 μg/mL). However, a 4-fold loss in potency was observed
for the same substituent in the 4 position (9g, 25 μg/mL).
Decreasing the linker length from ethyl (9b, 9d, and 9g) to methyl (9a, 9c, and 9f) was not favored with at least a 2-fold loss
in potency against C. difficile for
the carboxylic acid groups at all aromatic positions on ring A. The
methyl ether linkers (9e and 9h) also caused
a loss of activity compared with their corresponding ethyl linkers
(9d and 9g), thereby demonstrating that
additional hydrogen bond accepting or hydrophilic interactions were
not favored from the benzylic site. The trend of a 2-fold increase
in potency for the ethyl linkers (15b and 15e) compared with methyl (15a and 15d) was
also observed for the amine substituents at both the 2 position and
4 position. Ethylamine substitution was favored at the 3 and 4 positions
with analogues 15c (4 μg/mL) and 15e (3 μg/mL) giving slight increases in potency against C. difficile as compared to analogue 1 (6 μg/mL).
Table 1
Structure and Activity of Oxadiazole
Analogues
n.d.—not determined.
n.d.—not determined.The replacement of the phenol ring D with an unsubstituted
aniline
(20a, >50 μg/mL) was detrimental to C. difficile activity. Interestingly, the activity
was able to be somewhat restored upon functionalization of the aniline
with methyl (20b, 8 μg/mL), methoxyethyl (22, 8 μg/mL), acetic acid (24, 13 μg/mL),
or conversion to nitro groups (19d, 8 μg/mL). The
analogue bearing a nitro substituent (19d) had similar
potency to the unsubstituted phenol analogue (1), and
it was apparent that a hydrogen bond donor on ring D was not essential
for activity against C. difficile.Modification of the oxadiazole structure was less tolerated in
terms of E. faecium activity, with
analogue 1 remaining the most potent at 2 μg/mL.
Carboxylic acid substitution led to a complete loss of potency, regardless
of the aromatic position or linker variant. The decrease in activity
was less pronounced when attaching the amine substituents, with only
a 2-fold loss in activity at the 4 position (15d, 15e, 4 μg/mL) and a 4-fold loss at the 2 position (15a, 15b, 8 μg/mL). Linking with methyl
or ethyl groups had no effect on the activity for these analogues.
There was a complete loss of potency for all analogues from the aniline
series which suggested that the hydroxyl on ring D was important for
activity against E. faecium.The initial SAR exploration suggested that modification with amino
groups could be made while maintaining antimicrobial activity. Specifically,
analogue 15e with ethylamine substitution from the 4-position
of ring A appeared to be suitable, exhibiting modest activity against
both C. difficile (3 μg/mL) and E. faecium (4 μg/mL). Further, analogue 15e displayed rapid killing when assessed in time-kill assays,
with a 100% kill of C. difficile at
4× MIC (12 μg/mL) within 3 h and VREfm at 2× MIC (8
μg/mL) within 1 h (Figure ). This rapid rate of killing was comparable to the
positive control benzalkonium chloride. When benchmarked against 1, we observed superior killing activity of 15e against C. difficile at 6 μg/mL
and indistinguishable kill kinetics at 12 μg/mL. Against VREfm, 15e displayed comparable killing kinetics to 1 at 4 μg/mL despite the 2-fold loss of activity identified
above, with both compounds killing 100% of VREfm within 24 h. Of note,
while growth of VREfm remained undetectable after exposure to 4 μg/mL
of 15e for the duration of the assays, rebounding growth
of VREfm was observed after 48 h in cultures exposed to 1. At a concentration of 8 μg/mL, however, the killing kinetics
of 1 and 15e were indistinguishable, with
100% of VREfm being killed within 1 h of exposure and no rebounding
growth observed for either compound.
Figure 4
Time-kill analyses of 15e against GI pathogens: (a) C. difficile strain EDN0008, blue circles are DMSO-containing
untreated cultures (vehicle), red squares are cultures treated with
3 μg/mL 15e, closed green triangles are cultures
treated with 6 μg/mL 15e, open green triangles
are cultures treated with 6 μg/mL 1, closed purple triangles
are cultures treated with 12 μg/mL 15e, open purple
triangles are culture treated with 12 μg/mL 1,
orange diamonds are cultures treated with 24 μg/mL 15e, turquoise circles are cultures treated with 8 μg/mL vancomycin,
and black squares are cultures treated with 24 μg/mL benzalkonium
chloride; (b) E. faecium strain Aus0085,
blue circles are DMSO-containing untreated cultures (vehicle), red
squares are cultures treated with 2 μg/mL 15e,
closed green triangles are cultures treated with 4 μg/mL 15e, open green triangles are cultures treated with 4 μg/mL
1, closed purple triangles are cultures treated with 8 μg/mL 15e, open purple triangles are cultures treated with 8 μg/mL 1, orange diamonds are cultures treated with 16 μg/mL 15e, turquoise circles are cultures treated with 16 μg/mL
daptomycin, and black squares are cultures treated with 16 μg/mL
benzalkonium chloride; (c) haemolysis assays of 15e against
human erythrocytes. All assays were performed using three independent
biological replicates with the data shown representing the mean ±
SEM. VAN—vancomycin, DAP—daptomycin, BZK—benzalkonium
chloride, and CFU—colony-forming units.
Time-kill analyses of 15e against GI pathogens: (a) C. difficile strain EDN0008, blue circles are DMSO-containing
untreated cultures (vehicle), red squares are cultures treated with
3 μg/mL 15e, closed green triangles are cultures
treated with 6 μg/mL 15e, open green triangles
are cultures treated with 6 μg/mL 1, closed purple triangles
are cultures treated with 12 μg/mL 15e, open purple
triangles are culture treated with 12 μg/mL 1,
orange diamonds are cultures treated with 24 μg/mL 15e, turquoise circles are cultures treated with 8 μg/mL vancomycin,
and black squares are cultures treated with 24 μg/mL benzalkonium
chloride; (b) E. faecium strain Aus0085,
blue circles are DMSO-containing untreated cultures (vehicle), red
squares are cultures treated with 2 μg/mL 15e,
closed green triangles are cultures treated with 4 μg/mL 15e, open green triangles are cultures treated with 4 μg/mL
1, closed purple triangles are cultures treated with 8 μg/mL 15e, open purple triangles are cultures treated with 8 μg/mL 1, orange diamonds are cultures treated with 16 μg/mL 15e, turquoise circles are cultures treated with 16 μg/mL
daptomycin, and black squares are cultures treated with 16 μg/mL
benzalkonium chloride; (c) haemolysis assays of 15e against
human erythrocytes. All assays were performed using three independent
biological replicates with the data shown representing the mean ±
SEM. VAN—vancomycin, DAP—daptomycin, BZK—benzalkonium
chloride, and CFU—colony-forming units.1 has previously been shown to possess membrane-lytic
activity, with concentrations in excess of 64 μg/mL leading
to haemolysis in human erythrocytes.[31] To
determine whether 15e displayed a similar membrane-lytic
activity as 1, the compound was tested against human
erythrocytes as before (Figure c). Limited haemolysis was observed following incubation of
human erythrocytes with 4 μg/mL 15e (approximate
MIC of C. difficile and E. faecium), with only 2.5% (range: 0–7%)
of erythrocytes undergoing lysis. As previously observed for 1, increasing concentrations of 15e resulted
in increased levels of hemolysis in a dose-dependent manner, with
a concentration of ≥64 μg/mL leading to substantial levels
of haemolysis. These data therefore provide evidence to show that 15e most likely operates through a membrane-lysing mechanism.Further SAR studies of analogue 15e were undertaken
to identify the optimal linker for use and explore functionalization
of the amine (Table ). Against C. difficile, the introduction
of a branched methyl group at the benzylic position of the linker
(15f, 16 μg/mL) saw a 5-fold loss in potency compared
with the ethyl linker (15e, 3 μg/mL), which suggested
that flexibility of the linkers may be important for activity. Conversely,
activity was retained when further restricting the linker flexibility
by adding an N-methylene bridge to the 3 position of ring A to form
a tetrahydroisoquinoline moiety (15i, 4 μg/mL).
Methylation of the ethylamine group to form both secondary (15g) and tertiary amines (25a) caused slight
decreases in activity to 4 μg/mL. The observation that the dimethylamine
group retained activity suggested that favorable interactions were
made via the amine basicity or a hydrogen bond acceptor
effect, rather than through hydrogen bond donating. The conversion
of the amine group into a quaternary ammonium (26a, 8
μg/mL) was also tolerated but with another slight drop in C. difficile activity observed. Nonetheless, the
analogue was of particular interest because it represented an active
compound that also possessed a permanently cationic group which would
greatly reduce permeability across the small intestine.[32,33] A further look at modifying the linker identified that a slight
increase in activity against C. difficile was obtained when the length was increased to a propyl (15h, 2 μg/mL). Unfortunately, this increase in potency was not
maintained when forming the tertiary amine 25b (16 μg/mL)
or quaternary ammonium 26b (8 μg/mL).
Table 2
Structure and Activity of Oxadiazole
Analogues
n.d.—not determined.
n.d.—not determined.The SAR for E. faecium was generally
similar to the trends observed for changes in activity against C. difficile. Restriction of the linkers in analogues 15f and 15i had no effect on E.
faecium activity. Methylation of the ethylamine to
form secondary and tertiary amines 15g and 25a also saw no change in potency, and quaternization (26a, 8 μg/mL) caused only a 2-fold decrease in activity compared
with the primary amine 15e (4 μg/mL). Extension
of the linker to a propylamine (15h, 2 μg/mL) gave
a 2-fold increase in activity; however, again this increased activity
was not translated when forming the quaternary ammonium (26b, 16 μg/mL).
SAR Summary
Modification of the
oxadiazole analogues
with amine substituents was generally more favored than the carboxylic
acid groups. Exploring changes to the linkers indicated that while
different variations were tolerated, they did not lead to significant
increases in activity. With the discovery of analogue 26a, we identified that a poorly permeable quaternary ammonium group
could be linked to the oxadiazole structure while retaining moderate
MICs of 8 μg/mL for both C. difficile and E. faecium.
In
Vitro Permeability Studies
We sought
to measure the effect that each modification had on compound permeability
by using differentiated Caco-2 cell monolayers. Four analogues were
assessed to represent the different types of ionizable groups and
linkers (Table ).
Analogues 9e and 9g bearing carboxylic acid
groups displayed moderate permeability with Papp (10–6 cm/s) values of 5.3 ± 0.20
and 18 ± 1.0. Measurable concentrations of the primary amine-substituted
analogue 15e could not be detected in the acceptor chamber,
prohibiting the determination of a Papp value. However, the low mass balance indicated that the analogue
was likely being retained in the Caco-2 cell monolayer rather than
having intrinsically low permeability.[38] Quaternary ammonium-substituted analogue 26a could
also not be detected in the acceptor chamber but was fully recovered
in the donor chamber and therefore displayed no detectable permeability
across the Caco-2 cell monolayer. This finding strongly suggested
that the quaternary ammonium-linked analogue would be ideally suited
to exhibit minimal absorption across the small intestine.
Table 3
Caco-2 Cell Permeability Assay of
Key Oxadiazole Analogues
Values are presented
as the mean
± SD of n = 3 transwells. C.n.d.—could
not be determined.
Values are presented
as the mean
± SD of n = 3 transwells. C.n.d.—could
not be determined.
In
Vivo Pharmacokinetic Studies
To
investigate compound exposure to the colon, the in vivo pharmacokinetic properties of analogues 15e and 26a were assessed after oral administration in healthy male
Sprague Dawley rats (Table ). Importantly, there were no adverse effects observed in
the rats as a result of oxadiazole compound administration. Plasma
concentrations of the oxadiazole analogues were measurable but low
after oral administration, with AUC0-inf values
of only 0.90 and 0.28 h*μM reached by analogues 15e and 26a, respectively. However, the cumulative recoveries
of unchanged compounds in the faeces were also very low for both compounds.
Of the oral doses, only 0.42% of 15e and 1.5% of 26a were recovered in the faeces over a 48 h period and even
less detected in the urine. Based on the poor recovery levels, it
was thought that the compounds may have been degraded or metabolized
in the GI lumen by chemical or enzymatic processes.
Table 4
Plasma Exposure Parameters and Urinary
and Faecal Recovery from Rats of Unchanged Analogues after Oral Administration
entry
t1/2 (h)a
Cmax (μM)a
Tmax (h)a
AUC0-inf (h*μM)a
% dose in urine (0–48 h)a
% dose in faeces (0–48 h)a
15e
2.0 ± 0.30
0.16 ± 0.038
2.5 ± 0.0
0.90 ± 0.22
0.08 ± 0.03
0.42 ± 0.47
26a
5.3 ± 2.1
0.039 ± 0.046
2.8 ± 2.0
0.28 ± 0.27
0.06 ± 0.03
1.5 ± 0.98
Values are presented
as the mean
± SD of n = 3. Measured dose—30 mg/kg.
Values are presented
as the mean
± SD of n = 3. Measured dose—30 mg/kg.
Conclusions
The
synthesis and evaluation of colon-targeted antimicrobial agents
were accomplished via the attachment of poorly permeable
functional groups to the 1,2,4-oxadiazole class of compounds. Modified
oxadiazole analogues were produced which displayed antimicrobial activity
against two important Gram-positive pathogens. Rapid bactericidal
activity was a hallmark feature of these analogues. Our study identified
that modification with the quaternary ammonium moiety at ring A of
the oxadiazole scaffold led to a suitable physicochemical profile
for colon-targeted delivery, as these compounds were completely impermeant
toward Caco-2 cell monolayers yet maintained good antimicrobial potency.
The recovery of analogues 15e and 26a after
oral administration in rats was low and this was identified as a key
target for future optimization in order to progress the analogues
toward exhibiting in vivo efficacy. Analysis of the
resulting metabolites may be a reasonable approach toward identifying
the structural changes needed to avoid such metabolism or degradation.
In summary, we believe our results contribute important data toward
the future development of decolonization agents with utility against
difficult-to-treat infections caused by C. difficile and E. faecium.
Experimental Section
General
Chemistry
Reactions were monitored by analytical
thin-layer chromatography (TLC) using silica gel 60/F254 pre-coated
aluminium sheets (0.25 mm, Merck). Flash column chromatography was
performed with silica gel 60, 0.63–0.20 mm (70–230 mesh,
Merck). The solvents; ethyl acetate (EtOAc); dichloromethane (DCM);
dimethyl formamide (DMF); 1,4-dioxane; dimethyl sulfoxide (DMSO);
methanol (MeOH); tetrahydrofuran; and diethyl ether (Et2O) were of analytical grade or distilled laboratory grade. 1H and 13C NMR spectra were recorded at 400 and 100 Hz,
respectively, using an Avance III Nanobay 400 MHz Bruker spectrometer
coupled to the BACS 60 automatic sample changer. Chemical shifts (δ,
ppm) are reported relative to the solvent peak [CDCl3:
7.26 (1H NMR), 77.16 (13C NMR), DMSO-d6: 2.50 (1H NMR), 39.52 (13C NMR), methanol-d4: 3.31 (1H NMR), 49.00 (13C NMR)]. Each proton resonance was assigned
with the following annotations; chemical shift (δ, ppm), singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m), broad signal
(br), coupling constant (J, Hz), and number of protons.
Analytical HPLC was performed on an Agilent 1260 Infinity analytical
HPLC coupled with a G1322A degasser, G1312B binary pump, G1367E high-performance
autosampler, and G4212B diode array detector. The conditions were
Zorbax Eclipse Plus C18 Rapid Resolution column (4.6 × 100 mm)
with UV detection at 254 and 214 nm, 30 °C; the sample was eluted
using a gradient system. Solvent A: water 0.1% TFA. Solvent B: acetonitrile
0.1% TFA. PP-gradient method—gradient: 5-95% B over 9 min and
100% B over 1 min. Detection: 254 or 214 nm. Hydrophobic PP method—gradient:
5–80% B over 0.1 min, 80–100% B from 0.6 to 9 min, and
100% B for 1 min. The PP-gradient method was used for all characterization
unless otherwise specified. LCMS was obtained on an Agilent 6100 Series
Single Quad LC/MS coupled with an Agilent 1200 Series HPLC, 1200 Series
G1311A quaternary pump, 1200 series G1329A thermostatted autosampler
and 1200 series G1314B variable wavelength detector. The liquid chromatography
conditions were reverse-phase HPLC analysis fitted with a Phenomenex
Luna C8(2) 5 μm (50 × 4.6 mm) 100 Å column; column
temperature: 30 °C; injection volume: 5 μL; solvent: 99.9%
acetonitrile, 0.1% formic acid; gradient: 5–100% of solvent
over 10 min; detection: 254 nm. The mass spectrometry conditions were
quadrupole ion source; ion mode: multimode-ES; drying gas temp: 300
°C; vaporizer temperature: 200 °C; capillary voltage: 2000
V (positive), 4000 (negative); scan range: 100–1000 m/z; step size: 0.1 s Acquisition time:
10 min. HRMS were obtained on an Agilent 6224 TOF LC/MS mass spectrometer
coupled to an Agilent 1290 Infinity (Agilent, Palo Alto, CA). All
data were acquired, and reference mass was corrected via a dual-spray
electrospray ionization (ESI) source. Each scan or data point on the
total ion chromatogram (TIC) is an average of 13,700 transients, producing
a spectrum every second. Mass spectra were created by averaging the
scans across each peak and background subtracted against the first
10 s of the TIC. Acquisition was performed using the Agilent Mass
Hunter Data Acquisition software version B.05.00 Build 5.0.5042.2
and analysis was performed using MassHunter Qualitative Analysis version
B.05.00 Build 5.0.519.13. Mass spectrometer conditions: ionization
mode: ESI. Drying gas flow: 11 L/min; nebulizer: 45 psi; drying gas
temperature: 325 °C; capillary voltage (Vcap): 4000 V; fragmentor: 160 V; skimmer: 65 V; OCT RFV: 750
V; and scan range acquired: 100–1500 m/z. Internal reference ions: positive ion mode = m/z = 121.050873 & 922.009798. Chromatographic separation
was performed using an Agilent Zorbax SB-C18 Rapid Resolution HT 2.1
× 50 mm, 1.8 μm column (Agilent Technologies, Palo Alto,
CA) using an acetonitrile gradient (5–100%) over 3.5 min at
0.5 mL/min. Solvent A = aqueous 0.1% formic acid. Solvent B = acetonitrile/0.1%
formic acid. Compounds 6a–h were obtained as unpurified
intermediates and were used directly in subsequent reactions. Therefore,
these intermediates were not fully characterized. Characterization
and syntheses of the derivatized phenol reagents 13a–i in Scheme are described
in the Supporting Information. The purity
of all compounds submitted for biological testing was ≥95%
as determined using HPLC analysis.
General Procedure A
A solution of phenol (1.2 equiv)
in DCM (0.27 M) was treated with 2-tert-butyl-1,3-diisopropylisourea
(1 equiv) and a catalytic amount of DMAP and then stirred at rt for
96 h. The reaction mixture was concentrated in vacuo, diluted with
a 10% solution of aq Na2CO3, and then, Et2O was added to extract the product. The separated organic
layer was dried over anhydrous MgSO4 and concentrated in
vacuo. The residue was purified using flash chromatography eluting
with 15% EtOAc in petroleum ether to yield the product.
General Procedure
B
Phenol (1 equiv), 4-iodobenzonitrile
(1 equiv), Cs2CO3 (2 equiv), and N,N-dimethylglycine (0.3 equiv) were dissolved in
1,4-dioxane (0.59 M) in a sealed tube and purged with N2 for 15 min. CuI (0.2 equiv) was then added, the reaction vessel
was sealed, and the solution was stirred at 100 °C for 48 h.
The reaction mixture was filtered, washed with Et2O, and
the collected filtrate was evaporated in vacuo. The residue was purified
using flash chromatography eluting with 2–15% EtOAc in petroleum
ether to yield the product.
General Procedure C
NH2OH·HCl (2 equiv)
and Et3N (2 equiv) were added to a solution of benzonitrile
(1 equiv) in MeOH (0.2 M). The solution was heated to 60 °C and
allowed to stir for 6 h. The solvent was removed in vacuo, and the
residue was diluted with water and then extracted with DCM. The organic
layers were combined, dried over MgSO4, and the solvent
removed in vacuo to yield the product that was used in the subsequent
step without further purification.
General Procedure D
A solution of amidoxime (1 equiv)
in DMF (0.22 M) was treated with 4-acetoxybenzoic acid 7 (1.5 equiv), HOBt (2 equiv), and EDC·HCl (2 equiv) then the
solution was stirred at rt for 16 h. The temperature was increased
to 100 °C, and the stirring was continued for 16 h. The reaction
mixture was concentrated in vacuo, diluted with a 10% solution of
aq Na2CO3, and then, DCM was added to extract
the product. The separated organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo. The residue was purified
using flash chromatography eluting with 0–15% EtOAc and 1%
acetic acid in petroleum ether to yield the product.
General Procedure
E
A solution of the tert-butyl ester substrate
in DCM (1 mL per 100 mg substrate) was treated
with TFA (0.1 mL per 100 mg substrate) and stirred at rt for 8 h.
The reaction mixture was concentrated in vacuo, and the remaining
residue suspended in water and sonicated. The solid was filtered,
washed with water, and air-dried to yield the pure product.
The title compound was synthesized as previously
reported
to yield the product as a yellow solid (287 mg, 84%).[26]1H NMR (400 MHz, CDCl3): δ
8.13–8.07 (m, 4H), 7.41–7.35 (m, 2H), 7.17 (t, J = 7.4 Hz, 1H), 7.12–7.06 (m, 4H), 7.01–6.96
(m, 2H); spectroscopic data consistent with the literature.[26]
tert-Butyl 2-(2-Hydroxyphenyl)acetate
(3a)
The title compound was synthesized from
2-(2-hydroxyphenyl)acetic
acid 2a (9.12 mmol, 1.39 g) according to general procedure
A and obtained as a yellow solid (800 mg, 42%). 1H NMR
(400 MHz, CDCl3): δ 8.00 (br, 1H), 7.19 (td, J = 7.9, 1.7 Hz, 1H), 7.07 (dd, J = 7.5,
1.4 Hz, 1H), 6.96 (dd, J = 8.1, 1.0 Hz, 1H), 6.87
(td, J = 7.4, 1.2 Hz, 1H), 3.60 (s, 2H), 1.47 (s,
9H); spectroscopic data consistent with the literature.[39]
tert-Butyl 3-(2-Hydroxyphenyl)propanoate
(3b)
3-(2-Hydroxyphenyl) propionic acid 2b (1 equiv, 18.1 mmol, 3.00 g) was dissolved in DMF (16 mL),
and 1,1′-carbonyldiimidazole
(1 equiv, 18.1 mmol, 2.93 g) was added carefully. The solution was
stirred for 2 h at 40 °C. 1,8-Diazabicyclo[5.4.0]undec-7-ene
(2 equiv, 36.1 mmol, 5.40 mL) and anhydrous tert-butanol
(2.5 equiv, 45.2 mmol, 4.30 mL) were added, and the solution was stirred
at 65 °C for 3 days. Solvents were removed in vacuo, and the
residue was diluted with water and aq 2 M HCl solution. The aqueous
layer was washed with EtOAc, then the organic layers were combined,
dried over MgSO4, and the solvent was removed in vacuo.
The residue was purified using flash chromatography (10% EtOAc in
petroleum ether) to yield the product as light yellow oil (804 mg,
20%). 1H NMR (400 MHz, CDCl3): δ 7.54
(br, 1H), 7.14–7.04 (m, 2H), 6.93–6.83 (m, 2H), 2.90–2.81
(m, 2H), 2.66–2.61 (m, 2H), 1.42 (s, 9H); spectroscopic data
consistent with the literature.[40]
tert-Butyl 2-(3-Hydroxyphenyl)acetate (3c)
The title compound was synthesized from 2-(3-hydroxyphenyl)acetic
acid 2c (15.9 mmol, 2.42 g) according to general procedure
A and obtained as clear oil (0.796 g, 29%). 1H NMR (400
MHz, CDCl3): δ 7.16 (t, J = 7.8
Hz, 1H), 6.81 (d, J = 7.4 Hz, 1H), 6.74 (s, 1H),
6.72 (d, J = 8.1 Hz, 1H), 5.47 (br, 1H), 3.47 (s,
2H), 1.44 (s, 9H); spectroscopic data consistent with the literature.[41]
tert-Butyl 3-(3-Hydroxyphenyl)propanoate
(3d)
3-(3-Hydroxyphenyl) propionic acid 2d (1 equiv, 18.1 mmol, 3.00 g) was dissolved in DMF (16 mL)
and 1,1′-carbonyldiimidazole
(1 equiv, 18.1 mmol, 2.93 g) was added carefully. The solution was
stirred for 2 h at 40 °C. 1,8-Diazabicyclo[5.4.0]undec-7-ene
(2 equiv, 36.1 mmol, 5.40 mL) and anhydrous tert-butanol
(2.5 equiv, 45.2 mmol, 4.30 mL) were added, and the solution was stirred
at 65 °C for 3 days. Solvents were removed in vacuo, and the
residue was diluted with water and aq 2 M HCl solution. The aqueous
layer was washed with EtOAc, then the organic layers were combined,
dried over MgSO4, and the solvent was removed in vacuo.
The residue was purified using flash chromatography (10% EtOAc in
petroleum ether) to yield the product as clear oil (1.95 g, 49%). 1H NMR (400 MHz, DMSO-d6): δ
9.26 (br, 1H), 7.04 (t, J = 7.7 Hz, 1H), 6.64–6.53
(m, 3H), 2.70 (t, J = 7.5 Hz, 2H), 2.45 (t, J = 7.5 Hz, 2H), 1.36 (s, 9H). 13C NMR (101 MHz,
CDCl3): δ 172.5, 155.8, 142.8, 129.7, 120.9, 115.4,
113.2, 80.6, 37.0, 31.1, 28.2. HPLC: tR = 6.6 min, 99% at 254 nm. HRMS m/z: calcd for 2×(C13H18O3)–H+ [2M – H+], 443.2439; found, 443.2440.
tert-Butyl 2-(3-Hydroxyphenoxy)acetate (3e)
A solution of resorcinol 2e (50
mmol, 5.50 g, 5 equiv) in DMF (50 mL) was treated with anhydrous K2CO3 (50 mmol, 6.91 g, 5 equiv) and tert-butyl bromoacetate (10 mmol, 1.48 mL, 1 equiv); then, the solution
was stirred at rt for 16 h. The reaction mixture was concentrated
in vacuo, and a 6 M solution of aq HCl was added while stirring until
the solution became until acidic. DCM was added to extract the product,
and the separated organic layer was dried over anhydrous MgSO4 and then concentrated in vacuo. The residue was purified
using flash chromatography (25% EtOAc in petroleum ether) to yield
the product as orange oil (1.18 g, 53%). 1H NMR (400 MHz,
CDCl3): δ 7.10 (t, J = 8.1 Hz, 1H),
6.50–6.35 (m, 3H), 5.76 (br, 1H), 4.48 (s, 2H), 1.49 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 168.6, 159.2,
157.2, 130.2, 109.1, 106.4, 102.8, 82.9, 65.8, 28.2. HPLC: tR = 4.8 min, 99% at 254 nm. HRMS m/z: calcd for C12H16O4 + H+ [M + H+], 225.1121; found, 225.1119.
tert-Butyl 2-(4-Hydroxyphenyl)acetate (3f)
The title compound was synthesized from 2-(4-hydroxyphenyl)acetic
acid 2f (24 mmol, 3.65 g) according to general procedure
A and obtained as a white solid (1.15 g, 28%). 1H NMR (400
MHz, CDCl3): δ 7.12–7.08 (m, 2H), 6.75–6.70
(m, 2H), 5.33 (br, 1H), 3.45 (s, 2H), 1.44 (s, 9H); spectroscopic
data consistent with the literature.[39]
tert-Butyl 3-(4-Hydroxyphenyl)propanoate (3g)
The title compound was synthesized from 3-(4-hydroxyphenyl)propanoic
acid 2g (24.0 mmol, 3.98 g) according to general procedure
A and obtained as yellow oil (1.17 g, 26%). 1H NMR (400
MHz, CDCl3): δ 7.05 (d, J = 8.4
Hz, 2H), 6.76–6.70 (m, 2H), 5.31 (br, 1H), 2.83 (t, J = 7.7 Hz, 2H), 2.50 (t, J = 7.7 Hz, 2H),
1.42 (s, 9H); spectroscopic data consistent with the literature.[40]
tert-Butyl 2-(4-Hydroxyphenoxy)acetate
(3h)
A solution of hydroquinone 2h (50
mmol, 5.50 g, 5 equiv) in DMF (50 mL) was treated with anhydrous K2CO3 (50 mmol, 6.91 g, 5 equiv) and tert-butyl bromoacetate (10 mmol, 1.48 mL, 1 equiv); then, the solution
was stirred at rt for 16 h. The reaction mixture was concentrated
in vacuo and a 6 M solution of aq HCl was added while stirring until
the solution became until acidic. DCM was added to extract the product,
and the separated organic layer was dried over anhydrous MgSO4 and then concentrated in vacuo. The residue was purified
using flash chromatography eluting with 25% EtOAc in petroleum ether
to yield the product as an off-white amorphous solid (1.75 g, 78%). 1H NMR (400 MHz, DMSO-d6): δ
8.96 (br, 1H), 6.75–6.69 (m, 2H), 6.68–6.63 (m, 2H),
4.49 (s, 2H), 1.41 (s, 9H); spectroscopic data consistent with the
literature.[42]
The title compound was synthesized from tert-butyl 2-(4-(4-cyanophenoxy)phenoxy)acetate 5h (2.53 mmol, 823 mg) according to general procedure C and obtained
as a yellow amorphous solid (860 mg, 95%). The product was used in
the next step without further purification. 1H NMR (400
MHz, CDCl3): δ 7.57–7.53 (m, 2H), 6.99–6.86
(m, 6H), 4.87 (br, 2H), 4.50 (s, 2H), 1.49 (s, 9H). 13C
NMR (100 MHz, CDCl3): δ 168.0, 159.9, 154.6, 150.1,
127.4, 126.6, 121.1, 117.5, 115.9, 82.5, 66.2, 28.1. LCMS (ESI+) m/z: 359.2 [M + H]+, tR = 4.9 min.
The title compound was synthesized
from tert-butyl 2-(4-(4-(5-(4-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)phenoxy)phenoxy)acetate 8h (0.217 mmol, 100 mg) according to general procedure E and
obtained as off-white powder (83 mg, 95%). 1H NMR (400
MHz, DMSO-d6): δ 10.56 (br, 1H),
8.03 (t, J = 9.1 Hz, 4H), 7.14–7.05 (m, 4H),
7.02–6.95 (m, 4H), 4.69 (s, 2H). 13C NMR (101 MHz,
DMSO-d6): δ 175.4, 170.2, 167.6,
162.1, 160.7, 154.7, 148.6, 130.1, 129.1, 121.4, 120.5, 117.3, 116.3,
116.0, 114.2, 64.9. HPLC: tR = 5.8 min,
99% at 254 nm. HRMS m/z: calcd for
C22H16N2O6 + H+ [M + H+], 405.1081; found, 405.1073.
(Z)-N′-Hydroxy-4-iodobenzimidamide
(10)
The title compound was synthesized from
4-iodobenzonitrile 4 (5.24 mmol, 1.20 g) according to
general procedure C and obtained as off-white crystals (1.16 g, 84%). 1H NMR (400 MHz, DMSO-d6): δ
9.72 (br, 1H), 7.77–7.68 (m, 2H), 7.49–7.42 (m, 2H),
5.84 (br, 2H); spectroscopic data consistent with the literature.[43]
A suspension of 4-((tert-butyldimethylsilyl)oxy)benzoyl chloride 11 (1 equiv,
9.11 mmol) in toluene (30 mL) was added to a suspension of (Z)-N′-hydroxy-4-iodobenzimidamide 10 (1 equiv, 9.11 mmol, 2.39 g) in toluene (20 mL) and allowed
to stir at reflux (130 °C) for 48 h. The solution was filtered
while still hot, and the filtrate was concentrated in vacuo. The residue
was recrystallized from EtOH to yield the product as off-white crystals
(2.62 g, 60%). 1H NMR (400 MHz, CDCl3): δ
8.12–8.07 (m, 2H), 7.92–7.83 (m, 4H), 7.00–6.95
(m, 2H), 1.02–0.99 (m, 9H), 0.28–0.24 (m, 6H). 13C NMR (101 MHz, CDCl3): δ 176.0, 168.4,
160.2, 138.2, 130.2, 129.2, 126.9, 120.9, 117.4, 97.9, 25.7, 18.4,
−4.2. HPLC: (hydrophobic PP method) tR = 5.0 min, 92% at 254 nm. HRMS m/z: calcd for C20H23IN2O2Si + H+ [M + H+], 479.0646; found, 479.0661.
General Procedure F
5-(4-((tert-Butyldimethylsilyl)oxy)phenyl)-3-(4-iodophenyl)-1,2,4-oxadiazole 12 (1 equiv), phenol (4–5 equiv), K2CO3 (2 equiv) and 1,3-diphenyl-1,3-propanedione (1 equiv) were
dissolved in DMSO (0.93 M) in a sealed tube and purged with N2 several times. Copper(II) acetylacetonate (0.5 equiv) was
then added, the reaction vessel was sealed, and the solution was stirred
at 90 °C for 24 h. The reaction mixture was diluted with EtOAc
and filtered through Celite. The filtrate was washed with aq NH4Cl solution and brine; then, the organic layers were dried
over MgSO4 and solvent was removed in vacuo. The desired
product was isolated using flash chromatography eluting with 10–35%
EtOAc in petroleum ether. 1,3-Diphenyl-1,3-propanedione impurities
that were present with the obtained product were removed after the
subsequent reaction.
General Procedure G
The boc-protected
substrate was
dissolved in 4 M HCl/1,4-dioxane (∼1 mL/30 mg substrate) and
stirred at rt for 6 h. After reaction completion as determined by
TLC, the solution was diluted with Et2O, filtered, and
further washed with Et2O. The filtered solid was air-dried
and collected to yield the HCl salt of the product.
The title compound was synthesized
from tert-butyl (3-hydroxyphenethyl)carbamate 13c (5 equiv, 1.6 mmol, 370 mg) according to general procedure
F, and flash chromatography (5–20% EtOAc in petroleum ether)
was carried out to yield the impure product. The crude material was
dissolved in 4 M HCl/1,4-dioxane (0.5 mL) and stirred at rt for 6
h. The solution was diluted with Et2O, filtered, and further
washed with Et2O. The collected solid was air-dried, and
the products were separated using preparative HPLC. The recovered
material was treated with 1.25 M HCl/1,4-dioxane (0.50 mL) to convert
the product to the HCl salt. The reaction mixture was concentrated
in vacuo, diluted with Et2O, and the resulting solid was
collected and dried to yield the product as a white solid (7.2 mg,
6% over two steps). 1H NMR (400 MHz, DMSO-d6): δ 8.11–8.05 (m, 2H), 8.02 (d, J = 8.8 Hz, 2H), 7.75 (br, 3H), 7.42 (t, J = 7.9 Hz, 1H), 7.21–7.16 (m, 2H), 7.14 (d, J = 7.7 Hz, 1H), 7.07 (s, 1H), 7.05–6.99 (m, 3H), 3.10–3.03
(m, 2H), 2.93–2.86 (m, 2H). 13C NMR (101 MHz, methanol-d4): δ 177.4, 169.4, 163.5, 161.5, 158.1,
140.4, 131.7, 131.2, 130.3, 125.7, 123.2, 121.1, 119.6, 117.2, 116.4,
41.8, 34.4 (1 peak missing). HPLC: tR =
6.1 min, 99% at 254 nm. HRMS m/z: calcd for C22H19N3O3 + H+ [M + H+], 374.1499; found, 374.1503.
The title compound was synthesized
from tert-butyl 7-(4-(5-(4-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)phenoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate 14i (0.068 mmol, 33 mg) according
to general procedure G and obtained as a tan solid (21 mg, 75%). 1H NMR (400 MHz, DMSO-d6): δ
10.61 (br, 1H), 9.28 (br, 2H), 8.11–8.06 (m, 2H), 8.04–8.00
(m, 2H), 7.32 (d, J = 8.2 Hz, 1H), 7.17–7.12
(m, 2H), 7.10–7.04 (m, 2H), 7.03–6.98 (m, 2H), 4.26
(s, 2H), 3.42–3.34 (m, 2H), 3.01 (t, J = 6.1
Hz, 2H). 13C NMR (101 MHz, methanol-d4): δ 177.4, 169.4, 163.5, 161.3, 156.6, 132.0, 131.2,
131.1, 130.3, 128.5, 123.4, 120.8, 119.5, 118.7, 117.2, 116.4, 45.7,
43.0, 25.6. HPLC: tR = 5.7 min, 100% at
254 nm. HRMS m/z: calcd for C23H19N3O3 + H+ [M
+ H+], 386.1499; found, 386.1508.
4-((tert-Butoxycarbonyl)amino)benzoic Acid
(17a)
4-Aminobenzoic acid 16a (1
equiv, 21.9 mmol, 3.00 g) was solubilized in MeOH (24 mL) and water
(24 mL) and NaHCO3 (2 equiv, 43.8 mmol, 3.72 g) was added.
Boc2O (1.5 equiv, 32.8 mmol, 7.15 g) dissolved in MeOH
(24 mL) was added dropwise, and the solution was stirred at rt overnight.
The solvent was removed in vacuo, and the residue was extracted with
EtOAc and then washed with aq 1 M HCl solution and brine. The organic
layers were combined, dried over MgSO4, and the solvent
was removed in vacuo to yield the product as a white solid (4.47 g,
86%). 1H NMR (400 MHz, CDCl3): δ 8.06–8.01
(m, 2H), 7.46 (d, J = 8.8 Hz, 2H), 6.73 (br, 1H),
1.54 (s, 9H); spectroscopic data consistent with the literature.[44]
4-(Methylamino)benzoic acid 16b (1 equiv, 13.2 mmol, 2.0 g) was solubilized in MeOH (16
mL) and water (16 mL) and NaHCO3 (2 equiv, 26.5 mmol, 2.25
g) was added. Boc2O (1.5 equiv, 19.9 mmol, 4.32 g) dissolved
in MeOH (16 mL) was added dropwise, and the solution was stirred at
rt overnight. The solvent was removed in vacuo, and the residue was
extracted with EtOAc and then washed with aq 1 M HCl solution and
brine. The organic layers were combined, dried over MgSO4, and the solvent was removed in vacuo to yield the product as a
white solid (2.61 g, 79%). 1H NMR (400 MHz, DMSO-d6): δ 7.95–7.82 (m, 2H), 7.47–7.33
(m, 2H), 3.22 (s, 3H), 1.41 (s, 9H); spectroscopic data consistent
with the literature.[45]
(Z)-N′-Hydroxy-4-phenoxybenzimidamide
(18)
The title compound was synthesized as previously
reported and obtained as a white solid (1.06 g, 80%).26 1H NMR (400 MHz, CDCl3): δ 7.63–7.56 (m, 2H),
7.40–7.33 (m, 2H), 7.18–7.12 (m, 1H), 7.06–6.98
(m, 4H), 4.84 (br, 2H); spectroscopic data consistent with the literature.[26]
General Procedure H
A solution of
(Z)-N′-hydroxy-4-phenoxybenzimidamide 18 (1 equiv) in DMF (0.11 M) was treated with benzoic acid
(1.5 equiv), HOBt (2 equiv), and EDC·HCl (2 equiv); then, the
solution was stirred at rt for 16 h. The temperature was increased
to 100 °C, and the stirring was continued for 16 h. The reaction
mixture was concentrated in vacuo, diluted with a 10% solution of
aq Na2CO3, and then, EtOAc was added to extract
the product. The separated organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo; then, the residue was
purified using flash chromatography eluting with 5–20% EtOAc
in petroleum ether to yield the product.
A solution of (Z)-N′-hydroxy-4-phenoxybenzimidamide 18 (1.5 equiv, 2.07
mmol, 473 mg) in DMF (5 mL) was treated with 4-nitrobenzoic acid 17d (1 equiv, 1.38 mmol, 231 mg), HOBt (2 equiv, 2.76 mmol,
373 mg), and EDC·HCl (2 equiv, 2.76 mmol, 531 mg); then, the
solution was stirred at rt for 16 h. The temperature was increased
to 100 °C, and the stirring was continued for 16 h. The reaction
mixture was concentrated in vacuo, diluted with a 10% solution of
aq Na2CO3, and then, EtOAc was added to extract
the product. The separated organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo; then, the residue was
purified using flash chromatography (20% EtOAc in petroleum ether)
to yield the product as a yellow solid (290 mg, 58%). 1H NMR (400 MHz, DMSO-d6): δ 8.28–8.23
(m, 1H), 8.20–8.15 (m, 1H), 8.06 (d, J = 8.7
Hz, 2H), 8.02–7.96 (m, 2H), 7.47 (t, J = 7.7
Hz, 2H), 7.27–7.21 (m, 1H), 7.19–7.12 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ
172.4, 167.8, 160.0, 155.3, 148.2, 134.2, 133.9, 131.6, 130.3, 129.3,
124.9, 124.5, 120.1, 119.7, 118.4, 117.5. HPLC: tR = 6.9 min, 99% at 254 nm. HRMS m/z: calcd for C20H13N3O4 + H+ [M + H+], 360.0979; found, 360.0980.
A solution of tert-butyl (4-(3-(4-phenoxyphenyl)-1,2,4-oxadiazol-5-yl)phenyl)carbamate 19a (0.50 mmol, 214 mg) in 4 M HCl/1,4-dioxane (4.0 mL) was
stirred at rt for 6 h. After reaction completion as determined by
TLC, the solution was diluted with Et2O, filtered, and
further washed with Et2O. The recovered precipitate was
suspended in saturated aq sodium bicarbonate solution and washed with
EtOAc. The organic layers were combined and dried over MgSO4 and then concentrated in vacuo to yield the product as yellow powder
(128 mg, 78%). 1H NMR (400 MHz, CDCl3): δ
8.15–8.08 (m, 2H), 8.04–7.97 (m, 2H), 7.42–7.35
(m, 2H), 7.20–7.14 (m, 1H), 7.12–7.06 (m, 4H), 6.78–6.73
(m, 2H), 4.13 (br, 2H). 13C NMR (101 MHz, CDCl3): δ 176.1, 168.3, 160.1, 156.4, 150.8, 130.2, 130.1, 129.4,
124.2, 122.2, 119.8, 118.5, 114.7, 114.2. HPLC: tR = 7.3 min, 99% at 254 nm. HRMS m/z: calcd for C20H15N3O2 + H+ [M + H+], 330.1237; found, 330.1245.
A solution of tert-butyl
methyl(4-(3-(4-phenoxyphenyl)-1,2,4-oxadiazol-5-yl)phenyl)carbamate 19b (0.23 mmol, 100 mg) in 4 M HCl/1,4-dioxane (2.0 mL) was
stirred at rt for 6 h. After reaction completion as determined by
TLC, the solution was diluted with Et2O, filtered, and
further washed with Et2O. The collected precipitate was
air-dried to yield the product as a white solid (66 mg, 86%). 1H NMR (400 MHz, DMSO-d6): δ
8.10–8.01 (m, 2H), 7.93–7.85 (m, 2H), 7.49–7.42
(m, 2H), 7.26–7.20 (m, 1H), 7.17–7.10 (m, 4H), 6.73–6.66
(m, 2H), 2.77 (s, 3H). 13C NMR (101 MHz, methanol-d4): δ 176.5, 169.7, 162.0, 157.5, 147.1,
131.2, 131.1, 130.3, 125.5, 122.6, 121.1, 120.9, 120.0, 119.3, 34.6.
HPLC: tR = 7.9 min, 99% at 254 nm. HRMS m/z: calcd for C21H17N3O2 + H+ [M + H+], 344.1394;
found, 344.1399.
tert-Butyl (4-(3-(4-phenoxyphenyl)-1,2,4-oxadiazol-5-yl)phenyl)carbamate 19a (0.12 mmol, 50 mg) was dissolved in anhydrous DMF (3 mL)
under N2 atmosphere and NaH (60% in mineral oil, 1.2 equiv,
0.14 mmol, 5.8 mg) was added in small portions at 0 °C. The reaction
mixture was stirred for 1 h at rt. 1-Bromo-2-methoxyethane (1.5 equiv,
0.18 mmol, 16 μL) was added slowly at 0 °C; then, the mixture
was stirred at rt for 2 h. The reaction mixture was diluted with aq
NH4Cl solution and extracted with EtOAc. The organic layers
were combined, washed with aq NH4Cl solution and brine,
then dried over MgSO4, and concentrated in vacuo. The residue
was purified using flash chromatography (5–10% EtOAc in petroleum
ether) to yield the product as opaque oil (16 mg, 28%). 1H NMR (400 MHz, CDCl3): δ 8.19–8.11 (m, 4H),
7.52–7.46 (m, 2H), 7.42–7.36 (m, 2H), 7.18 (ddd, J = 8.5, 2.2, 1.1 Hz, 1H), 7.12–7.07 (m, 4H), 3.86
(t, J = 5.7 Hz, 2H), 3.59 (t, J =
5.7 Hz, 2H), 3.34 (s, 3H), 1.47 (s, 9H). 13C NMR (101 MHz,
CDCl3): δ 175.4, 168.7, 160.3, 156.3, 154.2, 147.3,
130.1, 129.5, 128.8, 127.2, 124.3, 121.8, 121.4, 119.9, 118.6, 81.3,
70.56, 58.9, 49.8, 28.5. HPLC: tR = 9.3
min, 94% at 254 nm. HRMS m/z: calcd
for C28H29N3O5 + H+ [M + H+], 488.2180; found, 488.2186.
A solution of tert-butyl
(2-methoxyethyl)(4-(3-(4-phenoxyphenyl)-1,2,4-oxadiazol-5-yl)phenyl)carbamate 21 (0.031 mmol, 15 mg) in 4 M HCl/1,4-dioxane (1.5 mL) was
stirred at rt for 6 h. After reaction completion as determined by
TLC, the solution was diluted with Et2O, filtered, and
further washed with Et2O. The obtained precipitate was
dissolved in acetonitrile and washed with petroleum benzines; then,
the solvent was removed in vacuo. The residue was dissolved in EtOAc
and washed with water; then, the organic layer dried over MgSO4 and concentrated in vacuo to yield the product as a yellow
solid (7.4 mg, 87%). 1H NMR (400 MHz, methanol-d4): δ 8.10–8.05 (m, 2H), 7.95–7.90
(m, 2H), 7.41 (tt, J = 7.6, 2.2 Hz, 2H), 7.22–7.17
(m, 1H), 7.11–7.05 (m, 4H), 6.78–6.72 (m, 2H), 3.61
(t, J = 5.5 Hz, 2H), 3.39 (s, 3H), 3.37 (t, J = 5.6 Hz, 2H). 13C NMR (101 MHz, methanol-d4): δ 177.9, 169.2, 161.7, 157.6, 154.4,
131.1, 130.8, 130.2, 125.4, 123.1, 120.8, 119.3, 113.1, 112.3, 72.0,
59.0, 43.7. HPLC: tR = 8.4 min, 95% at
254 nm. HRMS m/z: calcd for C23H21N3O3 + H+ [M
+ H+], 388.1656; found, 388.1656.
tert-Butyl (4-(4-(5-(4-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)phenoxy)phenethyl)carbamate 14e (0.42 mmol, 200 mg) was dissolved in DCM (2.5 mL), and
TFA (0.2 mL) was added to stir at rt for 6 h. The solvent was removed
in vacuo, and the residue was treated with saturated aq sodium bicarbonate
solution and then sonicated until a precipitate formed. The suspension
was treated with 10% aq citric acid solution until pH 6 and then extracted
with EtOAc three times. The organic layers were combined, dried over
MgSO4, and the solvent was removed in vacuo to yield 4-(3-(4-(4-(2-aminoethyl)phenoxy)phenyl)-1,2,4-oxadiazol-5-yl)phenol 15e as the neutral species (158 mg, 100%). 1H NMR
(400 MHz, methanol-d4): δ 8.11–8.06
(m, 2H), 8.06–8.01 (m, 2H), 7.38–7.27 (m, 2H), 7.14–7.03
(m, 4H), 7.00–6.92 (m, 2H), 3.25–3.15 (m, 2H), 3.03–2.94
(m, 2H).To a suspension of 15e (1 equiv, 0.13
mmol, 50 mg) in acetonitrile (2 mL) and water (0.5 mL) was added 37%
aq formaldehyde solution (12 equiv, 1.6 mmol, 130 μL). NaBH(OAc)3 (4.8 equiv, 0.64 mmol, 140 mg) was added slowly, and the
reaction was stirred at rt for 50 min. The reaction mixture was quenched
with saturated aq sodium bicarbonate solution and then extracted several
times with a 9:1 solution of EtOAc/isopropanol. The organic layers
were combined and concentrated in vacuo. The residue was treated with
small amounts of EtOAc and saturated aq sodium bicarbonate solution
until a precipitate formed. A sodium citrate buffer of pH ∼
6.5 was added slowly until the solution became neutral and the precipitate
dissolved. The aqueous layer was washed with EtOAc; then, the organic
layers were combined and set aside. The aqueous layer was concentrated
in vacuo to obtain cruder residue. The sequential process of saturated
aq sodium bicarbonate solution and sodium citrate buffer additions
followed by EtOAc extraction was repeated until no further precipitation
was observed. The combined organic layers were dried over MgSO4, and the solvent was removed in vacuo to yield the product
as an off-white solid (39 mg, 74%). 1H NMR (400 MHz, methanol-d4): δ 8.11–8.01 (m, 4H), 7.32–7.27
(m, 2H), 7.10–7.05 (m, 2H), 7.05–7.01 (m, 2H), 6.99–6.94
(m, 2H), 2.90–2.82 (m, 2H), 2.77–2.71 (m, 2H), 2.44
(s, 6H). 13C NMR (101 MHz, methanol-d4): δ 177.4, 169.5, 163.7, 162.0, 155.9, 136.8, 131.4,
131.2, 130.2, 122.8, 121.1, 119.1, 117.2, 116.4, 62.0, 45.1, 33.6.
HPLC: tR = 6.0 min, 99% at 254 nm. HRMS m/z: calcd for C24H23N3O3 + H+ [M + H+], 402.1812;
found, 402.1812.
tert-Butyl
(3-(4-(4-(5-(4-hydroxyphenyl)-1,2,4-oxadiazol-3-yl)phenoxy)phenyl)propyl)carbamate 14h (0.20 mmol, 100 mg) was dissolved in DCM (1 mL) and TFA
(0.1 mL) was added to stir at rt for 7 h. The solvent was removed
in vacuo, and the residue was treated with saturated aq sodium bicarbonate
solution and then sonicated until a precipitate was formed. The suspension
was treated with 10% aq citric acid solution until pH 6 and then was
extracted with EtOAc three times. The organic layers were combined,
dried over MgSO4, and the solvent was removed in vacuo
to yield 4-(3-(4-(4-(3-aminopropyl)phenoxy)phenyl)-1,2,4-oxadiazol-5-yl)phenol 15h as the neutral species (79 mg, 100%). 1H NMR
(400 MHz, methanol-d4): δ 8.11–8.03
(m, 4H), 7.30 (d, J = 8.6 Hz, 2H), 7.10–7.02
(m, 4H), 7.00–6.94 (m, 2H), 2.99–2.92 (m, 2H), 2.79–2.71
(m, 2H), 1.99 (dt, J = 15.5, 7.7 Hz, 2H). To a suspension
of 15h (1 equiv, 0.052 mmol, 20 mg) in acetonitrile (0.9
mL) and water (0.1 mL) was added 37% aq formaldehyde solution (12
equiv, 0.62 mmol, 50 μL). NaBH(OAc)3 (4.8 equiv,
0.25 mmol, 53 mg) was added slowly, and the reaction was stirred at
rt for 50 min. The reaction mixture was quenched with saturated aq
sodium bicarbonate solution and then extracted several times with
a 9:1 solution of EtOAc/isopropanol. The organic layers were combined
and concentrated in vacuo. The residue was treated with small amounts
of EtOAc and saturated aq sodium bicarbonate solution until a precipitate
formed. A sodium citrate buffer of pH ∼ 6.5 was added slowly
until the solution became neutral and the precipitate dissolved. The
aqueous layer was washed with EtOAc; then the organic layers were
combined and set aside. The aqueous layer was concentrated in vacuo
to obtain cruder residue. The sequential process of saturated aq sodium
bicarbonate solution and sodium citrate buffer additions followed
by EtOAc extraction was repeated until no further precipitation was
observed. The combined organic layers were dried over MgSO4, and the solvent was removed in vacuo to obtain the impure product
as the neutral species. The residue was dissolved in 4 M HCl/1,4-dioxane
(1.5 mL) and stirred at rt for 6 h. The solution was diluted with
Et2O, filtered, and the solid was further washed with Et2O. The filtered solid was collected to yield the product as
an off-white solid (2.4 mg, 10%). 1H NMR (400 MHz, methanol-d4): δ 8.14–8.04 (m, 4H), 7.33 (d, J = 8.5 Hz, 2H), 7.13–7.04 (m, 4H), 7.02–6.96
(m, 2H), 3.21–3.14 (m, 2H), 2.92 (s, 6H), 2.76 (t, J = 7.6 Hz, 2H), 2.13–2.03 (m, 2H). 13C NMR (101 MHz, methanol-d4): δ
177.4, 169.4, 163.6, 161.9, 156.1, 137.6, 131.2, 131.1, 130.2, 122.9,
121.2, 119.1, 117.2, 116.4, 58.6, 43.5, 32.7, 27.5. HPLC: tR = 6.3 min, 96% at 254 nm. HRMS m/z: calcd for C25H25N3O3 + H+ [M + H+], 416.1969;
found, 416.1979.
The neutral species of 4-(3-(4-(4-(3-(dimethylamino)propyl)phenoxy)phenyl)-1,2,4-oxadiazol-5-yl)phenol 25b (1 equiv, 0.072 mmol, 30 mg) was dissolved in a 2:1 solution
of DCM/MeOH (2 mL) under N2 atmosphere and iodomethane
(5 equiv, 0.36 mmol, 23 μL) was added. The reaction mixture
was stirred for 24 h at rt. The solvent was removed in vacuo; then,
the residue was suspended in Et2O and filtered. The precipitate
was washed with EtOAc, air-dried, and then collected to yield the
product as yellow powder (23 mg, 58%). 1H NMR (400 MHz,
methanol-d4): δ 8.14–8.03
(m, 4H), 7.38–7.33 (m, 2H), 7.13–7.05 (m, 4H), 7.02–6.96
(m, 2H), 3.44–3.39 (m, 2H), 3.17 (s, 9H), 2.76 (t, J = 7.6 Hz, 2H), 2.22–2.12 (m, 2H). 13C NMR (101 MHz, methanol-d4): δ
177.4, 169.4, 163.6, 161.9, 156.1, 137.4, 131.2, 131.2, 130.2, 122.9,
121.2, 119.1, 117.2, 116.4, 67.4, 67.4, 67.4, 53.7, 53.7, 53.6, 32.5,
25.8. HPLC: tR = 6.4 min, 95% at 254 nm.
HRMS m/z: calcd for C26H28N3O3 [M+], 430.2125;
found, 430.2111.
Antimicrobial Susceptibility Testing
Antimicrobial
susceptibility testing was performed using CLSI methods.[47] Specifically, MICs were determined by broth
microdilution assays as described previously for C.
difficile(48) and E. faecium.[31] In brief,
a 2-fold dilution series (from 128 to 1 μg/mL) of 1,2,4 oxadiazole
analogues, vancomycin (C. difficile comparator control), or daptomycin (E. faecium comparator control) was made in 100 μL volumes of either SDW
(C. difficile) or cation-adjusted Mueller–Hinton
broth (CAMHB) (E. faecium) purchased
from Thermo Fisher (additionally supplemented with 50 μg/mL
Ca2+ for daptomycin assays) in a 96-well plate (Corning)
and an inoculum of 100 μL from an overnight C.
difficile or E. faecium broth culture adjusted to 5 × 105 CFU/mL in supplemented,
pre-reduced Brucella broth (C. difficile) or CAMHB (E. faecium) added. After
48 h incubation under anaerobic conditions (C. difficile) or 24 h incubation under aerobic conditions (E.
faecium), the MIC was defined as the lowest antimicrobial
concentration that inhibited visible growth. All MIC testing was performed
in biological triplicate.
Time-Kill Assays
Time-kill assays
were performed with C. difficile strain
EDN0008 (NAP1/027) or E. faecium strain
Aus0085 (ST203), using supplemented
brain heart infusion broth (BHIBS) for C. difficile or CAMHB for E. faecium. BHIBS—BHIB
supplemented with 5 g/L yeast extract, 0.1% (w/v) l-cysteine,
and 0.3% (w/v) glucose. Broths were supplemented with either DMSO
(vehicle) or with 1× MIC, 2× MIC, 4× MIC, and 8×
MIC of 1,2,4-oxadiazole or vancomycin (8 μg/mL), daptomycin
(16 μg/mL) (additionally supplemented with 50 μg/mL Ca2+ for daptomycin assays) or benzalkonium chloride (24 or 16
μg/mL) as controls. Broths were inoculated with overnight bacterial
broth cultures and adjusted to 1 × 106 CFU/mL. Samples
withdrawn at 0, 1, 3, 24, and 48 h post-inoculation were serially
diluted in phosphate-buffered saline and plated onto either supplemented
brain heart infusion agar and incubated overnight at 37 °C in
a DG250 anaerobic chamber (Don Whitley Scientific) for C. difficile or on BHIA (Oxoid) and grown overnight
at 37 °C aerobically for E. faecium before colony enumeration was performed. All assays were performed
using three independent biological replicates, with each test strain
being purity plated from frozen glycerol stocks three independent
times and each bacterial culture used in the time-kill assay then
being derived from a single colony picked from one of the three separate
purity plates.
Haemolysis Assay
Fresh whole human
blood was collected
into K2-EDTA-coated Vacutainer tubes and pelleted at 500 g for 5 min.
The red blood cell pellet was washed twice in saline solution followed
by a third wash in phosphate-buffered saline (PBS) before being diluted
1:50 in PBS. Compound 15e was prepared to 20× concentration
in DMSO, and a 10 μL aliquot of each concentration was
added to 190 μL of diluted blood in a 96-well U-bottom
plate (Corning) giving a final concentration range of 0.125–128
mg/L. The plate was then incubated at 37 °C for 1 h. Negative
(vehicle) controls were incubated with DMSO, and positive controls
were incubated with 1% (v/v) Triton X-100. The plate was centrifuged
at 500g after the incubation to pellet intact erythrocytes,
and 100 μL of the supernatant from each well was then
collected and transferred to a 96-well flat bottom plate (Corning).
The absorbance of each supernatant sample was then measured at 450
nm using CLARIOstar plus (BMG LABTECH). All assays were performed
using three independent biological replicates.
Caco-2 Cell
Permeability
The apical to basolateral
apparent permeability (A–B Papp) was assessed in Caco-2 cells (passage 39) seeded onto 0.3 cm2 polycarbonate filter transwells at a density of 6 ×
104 cells/well. Experiments were conducted 23 days post-seeding.
Experiments were performed using pH 7.4 Hanks balanced salt solution
(containing 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid) with the donor solution spiked
with the test compound. Sample aliquots were taken 5–6 times
over 120 min, and the compound flux were determined by assessing compound
concentration using LCMS. The apical to basolateral apparent permeability
coefficient (A–B Papp) for each
compound was determined using the equation, Papp (cm/s) = dQ/dt × 1/(A × Cinitial donor), where dQ/dt = apparent steady-state
transport rate (μmol/s), A = surface area of
Caco-2 monolayer (0.3 cm2), and Cinitial donor = donor chamber concentration at the start
of the experiment (μmol/cm3). Transepithelial electrical
resistance (TEER) readings, high and low permeability markers, and
a multidrug resistance protein-mediated efflux marker were included
in the study to assess the integrity of the cell monolayers. The TEER
values for transwells used in the Caco-2 permeability study ranged
from 457 to 551 Ωcm2, indicating the presence of
confluent monolayers. For lucifer yellow, Papp values of <1 × 10–6 cm/s were yielded
in the acceptor solution. For propranolol, an A–B Papp value of 42 ± 2.5 × 10–6 cm/s was obtained and for rhodamine 123, an A–B Papp value of 1.6 ± 0.13 × 10–6 cm/s, B–A Papp value of 12 ±
2.7 × 10–6 cm/s, and an efflux ratio of 7.3
were obtained, all of which compared well with in-house validation
data. Values are presented as the mean ± SD of n = 3 transwells.
Pharmacokinetic Studies
Experimental
procedures are
summarized in the Supporting Information.
Authors: Glen P Carter; Jitendra R Harjani; Lucy Li; Noel P Pitcher; Yi Nong; Thomas V Riley; Deborah A Williamson; Timothy P Stinear; Jonathan B Baell; Benjamin P Howden Journal: J Antimicrob Chemother Date: 2018-06-01 Impact factor: 5.790
Authors: Konstantinos Z Vardakas; Konstantinos A Polyzos; Konstantina Patouni; Petros I Rafailidis; George Samonis; Matthew E Falagas Journal: Int J Antimicrob Agents Date: 2012-03-06 Impact factor: 5.283
Authors: Philip T Cherian; Xiaoqian Wu; Lei Yang; Jerrod S Scarborough; Aman P Singh; Zahidul A Alam; Richard E Lee; Julian G Hurdle Journal: J Antimicrob Chemother Date: 2015-08-18 Impact factor: 5.790
Authors: Jacek Czepiel; Mirosław Dróżdż; Hanna Pituch; Ed J Kuijper; William Perucki; Aleksandra Mielimonka; Sarah Goldman; Dorota Wultańska; Aleksander Garlicki; Grażyna Biesiada Journal: Eur J Clin Microbiol Infect Dis Date: 2019-04-03 Impact factor: 3.267
Figure 1
Figure 2
Figure 3
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Figure 4