Structure-based design of novel benzoxazinorifamycins with potent binding affinity to wild-type and rifampin-resistant mutant Mycobacterium tuberculosis RNA polymerases

Article abstract of DOI:10.1021/jm201716n

By utilization of three-dimensional structure information of rifamycins bound to RNA polymerase (RNAP) and the human pregnane X receptor (hPXR), representative examples (2b-d) of a novel subclass of benzoxazinorifamycins have been synthesized. Relative to rifalazil (2a), these analogues generally display superior affinity toward wild-type and Rif-resistant mutants of the Mycobacterium tuberculosis RNAP but lowered antitubercular activity in cell culture under both aerobic and anaerobic conditions. Lowered affinity toward hPXR for some of the analogues is also observed, suggesting a potential for reduced Cyp450 induction activity. Mouse and human microsomal studies of analogue 2b show it to have excellent metabolic stability. Mouse pharmacokinetics in plasma and lung show accumulation of 2b but with a half-life suggesting nonoptimal pharmacokinetics. These studies demonstrate proof of principle for this subclass of rifamycins and support further expansion of structure-activity relationships (SARs) toward uncovering analogues with development potential.

Full text of DOI:10.1021/jm201716n

Article
pubs.acs.org/jmc
Structure-Based Design of Novel Benzoxazinorifamycins with Potent
Binding Affinity to Wild-Type and Rifampin-Resistant Mutant
Mycobacterium tuberculosis RNA Polymerases
Sumandeep K. Gill,†,# Hao Xu,‡,# Paul D. Kirchhoff,§ Tomasz Cierpicki,∥ Anjanette J. Turbiak,‡
Baojie Wan,⊥ Nan Zhang,⊥ Kuan-Wei Peng,⊥ Scott G. Franzblau,⊥ George A. Garcia,†,‡
and H. D. Hollis Showalter*,‡,§
‡
§
†Interdepartmental Program in Chemical Biology, Department of Medicinal Chemistry, Vahlteich Medicinal Chemistry Core, and
∥Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109, United States
⊥Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612-7231, United
States
S
* Supporting Information
ABSTRACT: By utilization of three-dimensional structure
information of rifamycins bound to RNA polymerase (RNAP)
and the human pregnane X receptor (hPXR), representative
examples (2b−d) of a novel subclass of benzoxazinorifamycins
have been synthesized. Relative to rifalazil (2a), these
analogues generally display superior affinity toward wild-type
and Rif-resistant mutants of the Mycobacterium tuberculosis
RNAP but lowered antitubercular activity in cell culture under
both aerobic and anaerobic conditions. Lowered affinity toward hPXR for some of the analogues is also observed, suggesting a
potential for reduced Cyp450 induction activity. Mouse and human microsomal studies of analogue 2b show it to have excellent
metabolic stability. Mouse pharmacokinetics in plasma and lung show accumulation of 2b but with a half-life suggesting
nonoptimal pharmacokinetics. These studies demonstrate proof of principle for this subclass of rifamycins and support further
expansion of structure−activity relationships (SARs) toward uncovering analogues with development potential.
taken for an extended period of time (≥18 months). The
recent emergence of virtually untreatable extensively drug-
resistant TB (XDR-TB) poses a new threat to TB control
worldwide. Furthermore, effective treatment of TB in persons
co-infected with HIV is complicated because of drug−drug
interactions. Shorter and simpler regimens that are safe, well
tolerated, and effective against drug-susceptible and drug-
resistant TB, which are appropriate for joint HIV−TB
treatment and amenable to routine clinical settings, are needed
urgently.6,6b
The rifamycins are the most commonly used drugs for TB,
and semisynthetic derivatives have been reported that show
improved antimycobacterial activities.7 These include rifampin
(1a, RMP, Figure 1), which is the cornerstone of current short-
term tuberculosis treatment. Among newer derivatives, rifalazil
(2a, RLZ, Figure 1) has proved most interesting not just
because of its excellent potency but also because of its relative
lack of toxicity in early rodent studies.8 RLZ is an exceedingly
potent rifamycin derivative, being 16−256 times more potent
than RMP,9 and is particularly effective against many of the
RMP-resistant strains of MTB.9b,10 Several studies involving
strains with various rpoB mutations clearly indicated that the
INTRODUCTION
■
Tuberculosis (TB) is a contagious and deadly disease that has
reached pandemic proportions. According to the World Health
Organization (WHO), 8−10 million new cases of TB are
diagnosed each year, making Mycobacterium tuberculosis (MTB)
a leading cause of death in adults (2−3 million/year) due to an
infectious agent.1 A high proportion of these new cases and
deaths occur in HIV-positive people with a significant number
of AIDS deaths in Africa being attributed to TB infections.
Global population growth is increasing the disease burden,
posing a continuing health and financial burden in various parts
of the world, particularly Asia and Africa.
TB is caused predominantly by MTB, an obligate aerobic
bacillus that divides at an extremely slow rate. The chemical
composition of its cell wall includes peptidoglycans and
complex lipids, in particular mycolic acids, which are a
significant determinant of its virulence.2,3 The unique structure
of the cell wall of MTB allows it to lie dormant for many years
as a latent infection, particularly as it can grow readily inside
macrophages, hiding it from the host’s immune system.
The continuing rise in multidrug-resistant strains of MTB
(MDR-TB) has further contributed to the dire need for new
TB antibiotics, as no new TB drugs have been introduced into
clinical use in the past 4 decades.4,5 Drugs that are active against
resistant forms of TB are less potent, more toxic and need to be
Received: December 20, 2011
Published: March 27, 2012
© 2012 American Chemical Society
3814
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
RESULTS AND DISCUSSION
■
Analogue Design. Our impetus to investigate the RLZ
subclass, exemplified by analogues 2b−d, was driven by its ease
of synthesis and the likelihood that compounds with lowered or
absent Cyp450 induction effects would be generated. This
latter expectation was based on the known diminution of
Cyp450 induction with the progression from RMP (1a,
hydroquinone core) to rifabutin (1b, RFB, modified quinone
core) to RLZ (2a, markedly modified quinone core) subtypes.
Within this same order of structural subclasses, there also is a
trend of RLZ possessing increased potency against drug-
susceptible isolates of slow-growing mycobacteria and better in
vivo efficacy in mice.9c
Recent determinations of the three-dimensional structures of
rifamycins bound to RNAP now provide a conceptual
framework for structure-based discovery of improved rifamy-
cins. Our analysis of the recently published structures has
revealed an approach for the chemical elaboration of the ansa-
napthalene core in a novel way that should lead to enhanced
binding affinity to both wild-type and resistant mutants.
Analysis of a recent structure of RMP (1a) bound to the
human pregnane X receptor (hPXR) suggests that these
elaborations may have the significant added benefit of reducing
the affinity of the analogues for hPXR, thereby reducing
Cyp450 induction activity.
Figure 1. Structures of reference agents (1a, 1b, 2a) and novel
benzoxazinorifamycins (2b−d).
Our modeling was based on the 2.5 Å resolution structure of
rifabutin (1b) in complex with the Thermus thermophilus RNAP
holoenzyme (PDB code 2a68).22 The rifamycin binding site is
highly conserved among bacteria; therefore, this structure
provides a good foundation for understanding how proposed
rifamycin analogues may interact with the MTB RNA
polymerase. The crystal structure (2a68) contains two
complete complexes. On the basis of observed differences
between these two complexes and what is seen in the related
complexes present in PDB code 2a69 (rifapentine in complex
with the T. thermophilus RNAP holoenzyme), these studies
suggested that, at least in the free holoenzyme, the σ factor
hairpin loop may exist in two distinct physiologically relevant
conformations.
Although relatively qualitative in nature, our modeling
studies suggested a number of analogues with a range of size,
flexibility, and spatial variation for interactions with the σ
hairpin loop and other portions of the RNAP complex. These
analogues were chosen with the goal of increasing potency by
one or more of the following: (a) making additional contacts
with the σ factor and β and/or β′ region of the RNAP and (b)
interfering with the binding of the σ factor and/or further
occluding the channel.
Figure 2 displays the modeled RLZ/RNAP complex without
bound water molecules. RLZ (2a) is shown with bright green
carbon atoms. The RNAP molecular surface is shown with the
β surface colored white and light blue, β′ in brown, and the σ
factor in dark green. Figure 3 displays the interaction surfaces at
4.5 Å between the compound tail and surrounding RNAP for
benzoxazinorifamycin analogue 2b, utilizing the same coloring
scheme as Figure 2 (similar poses for RLZ, and analogues 2c,d
are shown in Figure 1a−cSI in Supporting Information). These
illustrate that the benzoxazinorifamycins have the potential to
interact with different regions of RNAP. While the modeling
used to produce these poses is qualitative in nature because of
the sizes of the channel and the analogues, it shows that the
mutations identified with the RMP, rifapentine, and rifabutin
resistant strains retained sensitivity to RLZ.11 RLZ and its
benzoxazinorifamycin analogues also have showed excellent
activity against other organisms with RMP-resistant mutations,
including Streptococcus pyogenes, Chlamydia trachomatis, and
Chlamydia pneumonia.12
In mouse in vivo efficacy studies, RLZ has been shown to be
clearly more potent than RMP including activity against some
RMP-resistant strains,9a,c,13,14 and longer term MTB studies in
combination with other agents indicated that the same level of
cure could be achieved with shorter (at least 2-fold) duration of
treatment with RLZ compared to RMP.15 In pharmacokinetic
(PK) studies, RLZ has shown a high volume of distribution and
produced tissue levels in rats up to 200 times those in plasma. It
displayed a very long half-life (60−100 h) in human trials.16
One major downside to the rifamycins used to treat TB is
their many drug−drug interactions. This effect appears to be
diminished with RLZ, as shown in rat and the dog studies.17
This study also showed RLZ not to be an inducer of hepatic
cytochrome P450 (Cyp450). In a series of phase I18,19 and
phase II9a,18−20 clinical trials, RLZ proved to be quite toxic,
with most adverse effects associated with a flulike syndrome
and leucopenia even at lower dose levels. Hence, its
development for TB indications has been suspended.21
In this paper, we detail the structure-based design, synthesis,
and biological evaluation of representative compounds (2b−d,
Figure 1) of a novel subclass of RLZ (2a) in which ether tethers
have been installed off the 3′-position of the “southeastern” part
of the benzoxazinorifamycin template. Our expectation was that
this modification would provide analogues with equal or better
potency than RLZ against wild-type and Rif-resistant (RifR)
mutants of RNA polymerase (RNAP), the molecular target of
the rifamycins, while retaining good antitubercular activity in
vitro. We also expected our compounds to display lowered
binding toward activation of the human pregnane X receptor
(hPXR).
3815
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
predict hPXR activation. However, the most exciting develop-
ments have been the determination of a number of X-ray crystal
structures of activators bound to the ligand-binding domain of
hPXR. Particularly important for our work, the structure of
RMP bound to hPXR has been determined.24
RMP (1a) is one of the, if not the most, potent activators of
hPXR.24 RMP fills the ligand-binding pocket very well, and
differences in RMP activation of human versus mouse PXR
have been interpreted in light of structural information. For
example, Leu308 in the human PXR is replaced by Phe in the
mouse, and Ser247 by Trp, leading to the suggestion that these
changes impair the binding of RMP to mouse PXR, accounting
for the differential RMP activation of these PXRs. The relative
potency of CYP3A4 induction is RMP > rifapentine > RFB >
RLZ. Figure 4 displays the modeled hPXR binding site with
these four rifamycins in spatial relation to resolved hPXR
residues (see Experimental Section for details of modeling). In
particular, there are seven hPXR residues, namely, Phe237,
Ser238, Leu239, Leu240, Pro241, His242, and Met243, very
close to the synthetic branch point for our synthesized
analogues. These residues are resolved in each of the five
hPXR structures and, other than one of the Phe237 rings, have
fairly conserved relative coordinates. This, along with the
presence of Pro241, would suggest a more rigid region of the
hPXR ligand-binding pocket.
Figure 2. Modeled RLZ/RNAP complex without bound water
molecules. RLZ (2a) is shown with bright green carbon atoms. The
RNAP molecular surface is shown with β colored white and light blue,
β′ in brown, and the σ factor in dark green.
The hPXR ligand-binding pocket is large, flexible, and
capable of adapting itself to bind a large variety of ligands. What
our modeling suggested is that our benzoxazinorifamycin
analogues, which are designed to target RNAP σ factors, may
have the added benefit of overwhelming the normally
promiscuous hPXR binding pocket. It appears that the tails
of these analogues may prevent binding to hPXR by projecting
into rigid, sterically encumbered regions of hPXR. Diminished
binding to hPXR would then presumably reduce induction of
CYP450s.
Chemistry. The synthetic route utilized to make our target
“one-armed” compounds 2b−d is shown in Scheme 1. The
RLZ literature25 suggested a strategy of annulating the
benzoxazino moiety onto rifamycin S (12) with a suitably
protected monoether (e.g., TBS) of 2-aminoresorcinol,
followed by ether deprotection and then side chain installation
off the nascent phenol by any number of alkylation method-
ologies. This was investigated, but yields were very poor and
the scope of alkylation possibilities was quite limited (data not
shown). We opted instead to annulate a fully tethered 2-
aminoresorcinol monoether onto the rifamycin S framework in
Figure 3. Interaction surfaces at 4.5 Å between the compound tail and
surrounding RNAP for benzoxazinorifamycin 2b, in the same coloring
scheme as Figure 2.
analogues can pick up additional contacts, which would be
expected to translate to different potency profiles.
As noted above, Cyp450 induction effects (due to activation
of the hPXR) decrease with a progression from the RMP to the
RLZ scaffold. A recent review discusses developments to
predict and attenuate enzyme induction and drug−drug
interactions that are mediated via activation of hPXR.23 Much
work has been done to develop computational algorithms to
Figure 4. Stereoview of clinical rifamycins modeled into the hPXR binding pocket.
3816
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of 2b−d
a
Reagents and conditions: (a) benzyl bromide (for 3) and 1,4-dibromobutane (for 5), Cs2CO3, DMF, 25 °C, 12−16 h, 93−95%; (b) BCl3, DCM,
−78 °C, 1 h, 82%; (c) Et2NCH2CH2Cl·HCl, Cs2CO3, acetone, 50 °C, 3 h, 93%; (d) 20% Pd/C, H2 (40 psi), 25 °C, 20−40 h, MeOH/HOAc (9:1)
for 6; MeOH/10% aqueous HCl (9:1) for 9a, 10; 81 − 98%; (e) 1-[2-(1H-imidazol-1-yl)ethyl]piperazine or 1-Boc-piperazine, DIPEA, CH3CN,
reflux, 12−18 h, 68−99%; (f) TFA, DCM, 25 °C, 3 h; (g) 2-(1H-imidazol-1-yl)acetic acid, DIPEA, EDC·HCl, HOBT, DMF, 25 °C, 16 h, 72% from
9b; (h) 7, 11a, or 11b; p-dioxane or 1,2-DCE, MnO2, 25 °C to reflux, 35−74%.
a single step. This allowed us to consider a wide range of
tethers off the “southeastern” part of the rifalazil-type template
and, more importantly, minimized difficult synthetic trans-
formations and product purifications involving the complex
rifamycin S core to a single last step. Thus, we set our sights
initially on developing a robust procedure to intermediate 5,
which would serve as a key starting material for introduction of
our chosen tethers. While there are two reports for the
synthesis of this compound,26,27neither was deemed practical
for our needs. Instead, we pursued a two-step procedure.
Accordingly, dialkylation of 2-nitroresorcinol (3), similar to the
literature procedure,28 gave a 95% yield of dibenzyl ether 4
which was then cleanly monodebenzylated to nitrophenol 526
in 82% yield. With 5 now in hand, we were ready to install our
target tethers. Phenolic alkylation with 2-(diethylaminoethyl)-
ethyl chloride hydrochloride under standard conditions
provided 6 in 87% yield. Hydrogenation of 6 utilizing
Pearlman’s catalyst simultaneously reduced the nitro function
and hydrogenolyzed the benzyl protecting group to give the 2-
aminoresorcinol ether 7 in 81% yield. A similar sequence of
reactions was followed to provide ethers 11a,b. Alkylation of 5
with 1,4-dibromobutane gave 8 in 93% yield, which was
subsequently aminated with two monosubstituted piperazines
to afford compounds 9a and 9b in 68% and 99% yields,
respectively. t-Boc deprotection of 9b followed by acylation of
9c with 2-(1H-imidazol-1-yl)acetic acid provided 10, the amide
congener of 9a, in 72% yield. Hydrogenation of 9a and 10 was
conducted as described for 6 to provide the remaining 2-
aminoresorcinol ethers 11a and 11b, respectively, in nearly
quantitative yields. The structure of each 2-aminoresorcinol
ether (7, 11a, 11b) is supported by mass spectrometry along
with 1H and 13C NMR spectra (Supporting Information, Figure
2SI). Each was then annulated onto rifamycin S (12) to provide
target compounds 2b−d in 35−74% yields following a two-
stage purification utilizing medium pressure and then
preparative plate silica gel chromatography. No effort was
made to optimize the condensation reaction with rifamycin S,
but we feel that this has the potential to be an efficient
transformation.
The structures of 2b−d were verified by the NMR spectra
(Supporting Information, Figure 3SI) and by chemical
ionization (CI) and high resolution (HR) mass spectrometry
(Supporting Information, Figure 4SI). We found that peaks in
1D NMR spectra of 2b−d showed significant broadening,
which was dependent on the solvent utilized. The spectra in
CDCl3 were of higher quality and were selected for the
assignment of chemical shifts. For each compound (2b−d), we
obtained complete and unambiguous assignment for the side
chain protons and carbons, verifying their point of attachment
onto the benzoxazinorifamycin core as shown (Supporting
3817
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
a
Information, Tables 1SI and 2SI). In addition, nearly complete
assignments were made for rifamycin core proton and carbon
atoms. Lack of complete assignment is due to the absence of
several peaks as a result of broadening observed in the NMR
spectra. The rifamycin core has restricted conformation and the
broadening of NMR signals most likely reflects a conforma-
tional exchange process, which is further supported by
sharpening of the NMR signals upon increasing the sample
temperature. Our NMR work provides a significant contribu-
tion to the study of rifalazil-type compounds, as there is only
one prior report in the literature in which proton and carbon
peak assignments have been made.29
Table 2. MIC90 Values (μM) of RMP (1a) and
Benzoxazinorifamycins 2a−d vs MTB
1a (RMP)
2a (RLZ)
2b
2c
2d
MABA
LORA
0.13
0.46
<0.004
<0.017
0.02
0.37
0.08
0.35
0.07
0.40
a
The MIC90 is defined as the minimum concentration of the
compound required to inhibit 90% of bacterial growth: isoniazid
(MABA, 0.24; LORA, >128); moxifloxacin (MABA, 0.46); strepto-
mycin (MABA, 0.46); PA824 (LORA, 2.53).
In Vitro Inhibition of Wild-Type and RMP-Resistant
Mutant MTB RNAPs. The inhibition constants (IC50) of the
wild-type and three Rif-resistant (RifR) mutants of the MTB
RNAP by RLZ (2a) and our analogues (2b−d) were
determined via dose−response studies as previously described,
with each compound tested in duplicate at the specified
concentrations.30 The data were plotted (log of the
benzoxazinorifamycin concentration vs % activity) and then
fitted by nonlinear regression. The log(IC50) and their standard
errors (of the fit) are reported, with these roughly translating
into a 20−25% error in the IC50 values (Supporting
Information, Table 3SI). The apparent IC50 values are listed
in Table 1. Not surprisingly, all of the benzoxazinorifamycins
assay using Alamar Blue reagent (added on day 7) for
determination of growth (MABA)32 gives an assessment of
activity against replicating MTB, while the 11-day high-
throughput, luminescence-based low-oxygen-recovery assay
(LORA)33 measures activity against bacteria in a nonreplicating
state that models clinical persistence. Minimum inhibitory
concentration (MIC90) is defined as the lowest compound
concentration effecting >90% growth inhibition.
Under aerobic conditions (MABA), all newly synthesized
compounds 2b−d display superior activities (MIC90 of 0.02−
0.08 μM) relative to RMP (1a, 0.13 μM) but are less potent (5-
to 40-fold) than RLZ (2a, <0.004 μM). Under anaerobic
conditions (LORA), activity for compounds 2b−d (MIC90 of
0.35−0.40 μM) is essentially equivalent to that for RMP, and
these range from 4- to 18.5-fold higher than in the MABA.
Relative to RLZ, LORA potency for analogues 2b−d is lower
(at least 20-fold).
a
Table 1. In Vitro RNAP IC50 Values (μM) for RLZ (2a) and
Analogues (2b−d)
2a (RLZ)
2b
2c
2d
Activation of hPXR. As discussed above, one of the limiting
factors for rifamycin utility in humans is their extremely high
potency toward activation of the human pregnane X receptor
(hPXR). To probe this, we have used a commercial in vitro
assay for hPXR activation (Puracyp, Inc.). Figure 5 shows
dose−response plots for this assay. RMP, as previously known,
exhibits a high maximal degree of activation (∼12-fold) and an
EC50 of ∼2 μM. RLZ has been reported to have essentially no
ability to activate hPXR. Our data confirm that at
concentrations lower than 100 μM, RLZ exhibits no detectable
activation of hPXR. At 100 μM, RLZ does show ∼2-fold
activation; however, it also shows ∼2-fold loss of cell viability
(suggesting cytotoxicity) at this concentration.
Analogue 2d was fit to a dose−response curve (Figure 5)
that revealed a 6-fold maximal activation of hPXR and an EC50
of ∼6 μM, both parameters within 2- to 3-fold of those for
RMP. This analogue also starts to exhibit loss of cell viability at
25 μM (Table 4SI in Supporting Information and Figure 6)
such that the 100 μM data point was not used in the dose−
response curve fit, again similar to RMP. Analogues 2b and 2c
were essentially identical with ∼3-fold hPXR activation at 6.25
μM and dramatic loss of cell viability above 6.25 μM masking
any further hPXR activation, as seen in Figure 5.
Our modeling suggests that the additional bulk of our
analogues should reduce binding to hPXR due to steric clashes
within the binding pocket. It seems likely that the flexible side
chains may allow for the side chains to adopt a conformation
that minimizes this clash. We have no explanation for the
apparent toxicities (in this cell line under the conditions of the
hPXR activation assay) of 2b and 2c. Further studies in this and
other cell lines are needed to confirm this toxicity and to probe
the associated mechanism. At least for the wild-type RNAP,
there is ∼1000-fold difference between the RNAP IC50 and the
threshold for apparent cytotoxicity.
WT RNAP (−σA)
WT RNAP (+σA)
D435 V (+σA)
H445Y (+σA)
0.0115
<0.01
541
<0.01
<0.01
20
<0.01
<0.01
9
<0.01
<0.01
13
172
171
16
437
18
574
S450L (+σA)
117
122
a
IC50 is the concentration of rifamycin resulting in 50% inhibition of
transcription. Errors of the log(IC50) values are reported as described
in the Experimental Section. As a control, the mutant RNAPs (D435
V, H445Y, and S450L) without SigA were tested against RMP where
the IC50 values are as follows: 313 μM (D435 V), 830 μM (H445Y),
and 126 μM (S450L).
(2a−d) inhibit the wild-type MTB RNAP in the 10−9 M (nM)
range. We note that the lower limit of detection of this assay is
IC50 ≈ 5 nM. It is quite possible that these compounds have
true IC50 values much lower than this. A more sensitive assay to
determine these very low IC50 values is currently being
developed in our lab. The IC50 values for RLZ (2a) against
the RifR mutants of MTB were much higher, in the 10−4
M
(∼100 μM) range. The most frequently observed MTB RifR
mutant in clinical isolates, S450L,31 was inhibited at ∼7-fold
lower concentration of 2b and 2c relative to RLZ with 2d being
essentially the same as RLZ. The MTB D435V mutant was
inhibited at 26- to 60-fold lower concentrations of 2b−d
relative to RLZ. Interestingly, the H445Y mutant appears to
retain resistance to all tested benzoxazinorifamycins. The
results within this very limited series of our designed analogues
provide proof of principle that the potency of rifamycins toward
RifR MTB RNAPs can be substantially improved.
Activity against M. tuberculosis (H37RV) in Cell Culture.
The compounds were screened in assays to quantify their
antitubercular activity under both aerobic and anaerobic
conditions (Table 2). Briefly, the 8-day microplate-based
3818
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
Figure 5. Plots from human pregnane X receptor (hPXR) activation assay.
Microsome Stability and Pharmacokinetics. RLZ (2a)
and analogue 2b were evaluated for metabolic stability in
human microsomes. Each is relatively stable with estimated
half-lives of 65 and 54 min, respectively (Table 3). Similarly,
the estimated half-life of 2a in mouse microsomes is 53 min
while that of 2b is 141 min.
5 consecutive days with a Cmax of 1.74 μM at a Tmax of 2 h,
which is 100-fold higher than that observed after a single oral
dose (Supporting Information, Figure 6SI). In the lung tissue of
these mice, 2b was also detected at 1.79 μg/g (around 0.4 μM)
but only exceeded the MIC of 0.02 μM for ∼4 h (Supporting
Information, Figure 7SI).
The PK of analogue 2b was assessed using a suspension
prepared in carboxymethylcellulose (0.05% CMC). In the
single dose study, 2b was detected in the blood but the signal
was below the lower limit of quantitation. The Cmax was 0.0185
μM at a Tmax of 1 h. However, analogue 2b appears to
accumulate in the blood of mice that were dosed once daily for
CONCLUSIONS
■
We have utilized recent determinations of the three-dimen-
sional structures of rifamycins bound to RNAP to design and
synthesize representative examples (2b−d) of a novel subclass
of benzoxazinorifamycins possessing a range of size, flexibility,
3819
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
S framework in a single step is efficient and allows for a wide
range of tethers off the 3′-position of the rifalazil-type template,
which augurs well for a rapid expansion of the SAR within this
novel subclass.
EXPERIMENTAL SECTION
■
Computational Modeling of Rifamycin−RNAP Complexes.
The structure of 2a68 was used as the starting point in our modeling
studies. In brief, modifications made to the structure prior to its use in
the modeling were as follows: All water molecules and metals greater
than 12 Å from rifabutin (1b, RFB) were removed. Four magnesium
ions within 12 Å were converted to water molecules. Partially missing
residues were repaired. Connection points for completely missing
residues were greater than 35 Å from RFB and were kept fixed in space
during the energy minimizations. All N and C termini, either real or as
a result of missing residues, were acetylated and amidated, respectively.
RFB was removed from the complex. Hydrogen atoms were added to
the proteins, and the force field was set to AMBER99 and charged.
A series of energy minimizations were then conducted to relax the
positions of the modified atoms using the AMBER99 force field to
gradients of 0.01. Positions of hydrogen atoms were first relaxed with
energy minimization. Repaired residues except for their Cα atoms,
termini, and hydrogen atoms were then relaxed. Lastly, the complete
repaired residues, termini, and hydrogen atoms were relaxed. Resulting
confirmations of the repaired residues and termini were checked. Bond
orders for RFB were corrected. Hydrogen atoms were added, and the
force field was set to MMFF94x and charged. Positions of hydrogen
atoms were relaxed with energy minimization using the MMFF94x
force field to a gradient of 0.01. RFB was returned to the complex in its
original pose.
A second series of energy minimizations were then conducted using
the MMFF94x force field to gradients of 0.01. Positions of hydrogen
atoms were first relaxed followed by positions of hydrogen atoms and
all water molecules. Atoms of the RFB were then included in the
minimizations. Lastly, positions of all RFB, water, and hydrogen atoms
and protein residues having one or more atoms within 12 Å from RFB
were relaxed with energy minimization. The naphthalene ring of RFB
drifted approximately 1 Å toward the cleft of the complex from their
crystallographic positions with relatively minor movements of the
protein residues well within the 2.5 Å resolution of the starting
structure.
Complexes for proposed analogues were generated from the
modified RFB complex in the following way: All water molecules
present in the structure were removed. All residues not having one or
more atoms within approximately 20 Å of the RFB were deleted. The
RFB structure was modified to generate the proposed analogue. Atoms
of the RFB, which were not modified in the generation of the
proposed analogue, were initially fixed in space and treated as part of
the RNAP holoenzyme. Modifications were made to the RFB structure
to produce the proposed analogue while keeping the unmodified
atoms of RFB fixed in relation to the RNAP complex. A
LowModeMD35 conformational search algorithm with energy
minimization was then employed to generate plausible poses
(conformations) of the modified portions of RFB. The LowModeMD
search was conducted in MOE36 using default settings. Hydrogen
atoms, modified portions of RFB, and protein side chains within ∼16
Å of the modified portions were allowed to move during the
conformational search and energy minimizations. Generated poses
were ranked by interaction energies and duplicate poses based on a
rmsd cutoff removed.
The lowest energy pose was then selected and the truncated RNAP
complex soaked with water to a surrounding distance of 6 Å. As
described above, a series of energy minimizations were then conducted
using the MMFF94x force field to relax the complex. First, hydrogen
atoms and then the water molecules were allowed to relax while the
entire analogue and all of the RNAP atoms were held fixed. Second,
the modified portions of RFB and side chains of RNAP within 16 Å
were also allowed to relax with the water molecules and hydrogen
atoms. Finally, the entire analogue molecule, residues of RNAP within
Figure 6. Effect of rifamycins on DXP2 cell growth as measured by
CellTiter-Fluor.
Table 3. Half-Life of Selected Benzoxazinorifamycins in
Human and Mouse Microsomes
% remaining compd after
30 min of incubation in
microsomes
t1/2 (min)
compd
mouse
human
mouse
human
2a (RLZ)
67
86
73
68
53
65
54
2b
141
and spatial variation to interact with the σ hairpin loop and
other regions of the RNAP complex. Relative to RLZ (2a),
these analogues generally display superior affinity toward wild-
type and Rif-resistant mutants of the MTB RNAP but lowered
antitubercular activity in cell culture under both aerobic and
anaerobic conditions. The lowered activity in cell culture may
be due to poorer permeability and is under investigation. We
have also utilized information from the crystal structure of RMP
(1a) bound to hPXR as part of our design strategy toward
analogues 2b−d. Unfortunately, these analogues exhibit both
hPXR activation and cytotoxicity at a concentration above 25
μM.
Mouse and human microsomal studies of analogue 2b show
it to have excellent metabolic stability relative to RLZ (2a). The
pharmacokinetics of 2b showed accumulation of the compound
in plasma after multiple dosing with an apparent half-life of
∼1−2 h, suggesting that compound levels are above the MIC
for ∼7−8 h if the decay is linear. This is corroborated by
studies in lung tissue where levels of 2b exceeded the MIC of
0.02 μM for only ∼4 h. These studies suggest nonoptimal
pharmacokinetics for this compound and that further SAR will
be necessary to find a compound to take to in vivo efficacy
studies.
Apart from an obscure report on the synthesis of a small
series of simple benzoxazinorifamycin C-3′ monoethers,34 there
are no examples of more elaborate ethers that have been
incorporated onto a rifamycin S scaffold utilizing the strategy
we outline. Furthermore, our work is the first example that we
are aware of where structure-based design has been utilized to
target binding to specific σ factors within the complex RNA
polymerase machinery. Our synthetic strategy of annulating a
fully tethered 2-aminoresorcinol monoether onto the rifamycin
3820
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
16 Å, and the water molecules and hydrogen atoms were allowed to
relax. The relaxed complexes were then examined to determine how
the proposed analogue may interact with the σ factor or other portions
of RNAP (specifically the β and β′ subunits).
HCl and brine. The organic phase was dried and concentrated to leave
a yellow oil, which was diluted with 2-propanol to precipitate pure
product. The solids were collected to leave 4 (4.11 g, 95%) as light
yellow crystals: mp 87.5−88 °C (lit.28 80 °C); Rf = 0.26 (hexanes/
ethyl acetate, 5:1); 1H NMR (CDCl3) δ 7.3 (m, 10 H), 7.23 (t, J = 8.5
Hz, 1 H), 6.64 (d, J = 8.5, 2 H), 5.16 (s, 4 H); 13C NMR (CDCl3) δ
150.9, 135.6, 130.9, 128.7, 128.2, 127.0, 106.2, 71.0; MS (ES+) m/z
358.1 (M + Na)+.
Computational Modeling of Rifamycin−hPXR Complexes.
The 3D structures in Figure 4 were generated using the 2.5 Å
resolution crystal structure for Thermus thermophilus RNAP in
complex with rifabutin (1b, RFB) obtained from the Protein Data
Bank (PDB code 2a68).22 As described above, coordinates for RFB
were relaxed in the presence of the RNAP using a series of energy
minimizations with decreasing constraints on the surrounding protein
atoms, and water molecules. Coordinates for RMP, rifapentine, and
RLZ were created and relaxed in the same fashion after modifying
RFB. A 2.8 Å resolution crystal structure of hPXR in complex with
rifampin is available from the Protein Data Bank (PDB code 1skx).24
Unfortunately, the 1-amino-4-methylpiperazine tail of RMP and three
hPXR loops adjacent to the binding pocket (residues 178−209, 229−
235, and 310−317) are disordered and unresolved in the structure.
The missing residues in this and other published hPXR structures
make accurate modeling of the hPXR and the tails of rifamycins
difficult. Figure 4 was created by overlaying the naphthalene portions
of the four rifamycins generated from the 2a68 structure onto the
naphthalene portion of RMP in complex with hPXR structure 1skx.
The relative location of synthetic branch point for the analogues
described in Figure 1 is also indicated in Figure 4. In addition to the
complex with RMP, four other relatively complete hPXR structures are
available. The hPXR apo structure (PDB code 1ilg)37 and hPXR
complexes with SR12813 (PDB code 1ilh),37 hyperforin (PDB code
1m13),38 and colupulone (PDB code 2qnv)39 were obtained from the
Protein Data Bank. These four hPXR complexes were superimposed
onto the hPXR structure of 1skx containing the four modeled in
rifamycins. Coordinates were not relaxed with energy minimization
because of the many missing residues.
3-(Benzyloxy)-2-nitrophenol (5). A solution of dibenzyl ether 4
(3.0 g, 8.95 mmol) in dichloromethane (80 mL) at −78 °C was
treated dropwise with boron trichloride (13 mL, 1 M in heptane)
during which the color changed to dark purple. The reaction was
monitored by TLC, and the mixture was stirred at −78 °C until all
starting material was consumed (1 h). Methanol (5 mL) was added
dropwise, and the mixture was brought to room temperature, diluted
with water, and then extracted with dichloromethane (2×). The
combined extracts were dried and concentrated to an orange oil that
was purified by flash silica gel chromatography, eluting with hexanes/
ethyl acetate (5:1). Product fractions were pooled and concentrated to
give 5 (1.79 g, 82%) as a bright yellow solid: mp 67−67.5 °C; Rf =
1
0.24 (hexanes/ethyl acetate, 5:1); H NMR (CDCl3) δ 10.18 (brs, 1
H), 7.4 (m, 2 H), 7.3 (m, 4 H), 6.72 (d, J = 8.5 Hz, 2 H), 6.6 (d, J =
8.5 Hz, 2 H), 5.21 (s, 2 H); 13C NMR (CDCl3) δ 155.7, 154.7, 135.6,
135.4, 128.7, 128.2, 126.9, 111.0, 105.1, 71.4; MS (ES+) m/z 268.0 (M
+ Na)+.
2-(3-(Benzyloxy)-2-nitrophenoxy)-N,N-diethylethanamine (6). A
mixture of nitrophenol 5 (1.31 g, 5.4 mmol), 2-(diethylaminoethyl)-
ethyl chloride hydrochloride (1.2 g, 7 mmol), Cs2CO3 (4.37 g, 13.4
mmol), and acetone (20 mL) was stirred at 50 °C for 3 h. The mixture
was filtered and the filtrate was concentrated to a residue that was
purified by flash silica gel chromatography, eluting with hexanes/ethyl
acetate (5:1). Product fractions were pooled and concentrated to leave
6 (1.71 g, 93%) as a light yellow oil: Rf = 0.22 (CH2Cl2/methanol,
1
Chemistry. Materials and Methods. All reagents were
commercially available and used without further purification.
Rifalazil (2a, RLZ) of 98.3% purity by HPLC was synthesized
and purified as previously described.25 Melting points were
determined in open capillary tubes on a Laboratory Devices
95:5); H NMR (CDCl3) δ 7.3 (m, 6 H), 6.62, (dd, J1 = 3.6 Hz, J2 =
14.1 Hz, 2 H), 5.16 (s, 2 H), 4.1 (t, J = 10.5 Hz, 2 H), 2.8 (t, J = 10.5
Hz, 2 H), 2.6 (q, J = 11.9 Hz, 4 H), 1.0 (t, J = 11.9 Hz, 6 H); 13C
NMR (CDCl3) δ 151.2, 150.8, 135.6, 130.9, 128.6, 128.2, 127.0, 105.9,
105.6, 70.9, 68.5, 51.2, 47.9, 11.9; MS (ES+) m/z 245.1 (M + H)+.
2-Amino-3-(2-(diethylamino)ethoxy)phenol (7). 2-
(Diethylamino)ethyl ether 6 (1.8 g, 5.2 mmol) was dissolved in 10%
acetic acid in methanol (50 mL) in a 250 mL Parr hydrogenation
bottle. Catalyst (20% Pd(OH)2/C, 0.1 g) was added, and the mixture
was hydrogenated at 40 psi of H2 for ∼20 h. The reaction mixture was
rapidly filtered over Celite, and the filtrate was concentrated and
diluted with ethyl acetate. The solution was washed with 5% aqueous
sodium carbonate, dried, and concentrated to a brown solid that was
triturated in hot hexanes. The solids were collected and dried to leave
7 (0.95 g, 81%): mp 91−91.5 °C; Rf = 0.21 (CH2Cl2/methanol,
85:15); 1H NMR (CD3OD) δ 6.63 (t, J = 8.2 Hz, 1 H), 6.51 (m, 2 H),
4.34 (t, J = 5.1 Hz, 2 H), 3.60 (t, J = 5.0 Hz, 2 H), 3.35 (m, 4 H), 1.37
(t, J = 7.2 Hz, 6 H); 13C NMR (CD3OD) δ 148.5, 147.9, 124.8, 120.1,
110.4, 105.4, 64.0, 52.7, 21.0, 9.2; MS (ES+) m/z 225.1 (M + H)+.
1-(Benzyloxy)-3-(4-bromobutoxy)-2-nitrobenzene (8). To a mix-
ture of DMF (5 mL), 1,4-dibromobutane (5 mL), and Cs2CO3 (1.66
g, 5.1 mmol) was added slowly a solution of nitrophenol 5 (0.5 g, 2.0
mmol) in DMF (5 mL). The mixture was stirred at room temperature
for 16 h, and then DMF was removed in vacuo to leave an oil that was
distributed between 1% aqueous HCl and ethyl acetate. The organic
phase was dried and concentrated to a light yellow oil that was purified
by flash silica gel chromatography, eluting with hexanes/ethyl acetate
(6:1). Product fractions were pooled and concentrated to give 8
(0.702 g, 93%) as a light yellow oil: Rf = 0.45 (hexanes/ethyl acetate,
1
13
Mel-Temp apparatus and are uncorrected. Routine H and C
NMR spectra were obtained on a Bruker 500 MHz
spectrometer with CDCl3, CD3OD, or DMSO-d6 as solvent,
and chemical shifts are reported relative to the residual solvent
peak in δ (ppm). For compounds 2b−d, 1D NMR spectra were
investigated in different solvents including CDCl3, DMSO-d6,
and pyridine-d5, with CDCl3 yielding spectra of highest quality.
1
1
The H, 13C, H−13C HSQC and COSY experiments were
measured at 25 °C using a 600 MHz Bruker spectrometer
equipped with a cryogenic probe. Use of a cryogenic probe
resulted in some distortion of the 13C baseline due to the high
Q-value of the probe (see Supporting Information, Figure 3SI);
however, this did not affect the analysis of the spectra. Mass
spectrometry analysis was performed using a Waters LCT time-
of-flight mass spectrometry instrument. High resolution mass
spectrometry (HRMS) analysis was performed on an Agilent
Q-TOF system. Compound purity for target benzoxazinor-
ifamycins 2b−d was assessed by analytical HPLC, which was
performed on a Agilent 1100 series system with an Agilent
Eclipse plus C18 (4.6 mm × 7.5 mm, 3.5 mm particle size)
column. The mobile phase was a 11 min binary gradient of
acetonitrile (containing 0.1% TFA) and water (10−90%).
HPLC traces of benzoxazinorifamycins 2b−d are shown in
Supporting Information, Figure 5SI. Thin-layer chromatog-
raphy (TLC) was performed on silica gel GHLF plates (250
μm) purchased from Analtech. Extraction solutions were dried
over MgSO4 prior to concentration.
1
2:1); H NMR (CDCl3) δ 7.36 (m, 5 H), 7.26 (m, 1 H), 6.61 (m, 2
H), 5.16 (s, 2 H), 4.08 (t, J = 5.8 Hz, 2 H), 3.46 (t, J = 6.3 Hz, 2 H),
2.02 (m, 2 H), 1.92 (m, 2 H); 13C NMR (CDCl3) δ 151.1, 150.9,
135.6, 131.0, 128.7, 128.2, 127.0, 106.1, 105.6, 70.9, 68.4, 33.4, 28.9,
27.5; MS (ES+) m/z 401.9, 403.9 (M + Na)+.
1-(2-(1H-Imidazol-1-yl)ethyl)-4-(4-(3-(benzyloxy)-2-
nitrophenoxy)butyl)piperazine (9a). A solution of bromobutyl ether
8 (1.0 g, 2.6 mmol), 1-[2-(1H-imidazol-1-yl)ethyl]piperazine (0.52 g,
(((2-Nitro-1,3-phenylene)bis(oxy))bis(methylene))dibenzene (4).
A mixture of 2-nitroresorcinol (3, 2.0 g, 12.9 mmol), Cs2CO3 (10.5
g, 32.2 mmol), benzyl bromide (3.39 mL, 28.4 mmol), and DMF (35
mL) was stirred at room temperature for 12 h. The mixture was
diluted with ethyl acetate and washed sequentially with 1% aqueous
3821
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
2.9 mmol; Oakwood Products Inc.), N,N-diisopropylethylamine (5
mL), and acetonitrile (18 mL) was heated at reflux overnight. The
solution was concentrated, and the residue was distributed between
dichloromethane and 5% aqueous sodium carbonate. The organic
phase was dried and concentrated to an orange oil that was purified by
flash silica gel chromatography, eluting with dichloromethane:
methanol/NH4OH (90:10:0.5). Product fractions was pooled and
concentrated to leave 9a (0.86 g, 68%) as an oil: 1H NMR (CDCl3) δ
7.53 (s, 1 H), 7.36−7.22 (m, 6 H), 7.03 (s, 1 H), 6.97 (d, J = 1.1 Hz, 1
H), 6.61 (m, 1 H), 5.15 (s, 1 H), 4.05 (t, J = 6.3 Hz, 2 H), 4.01 (t, J =
6.5 Hz, 2 H), 2.67 (t, J = 6.5 Hz, 2 H), 2.48 (bs, 8 H), 2.36 (t, J = 6.5
Hz, 2 H), 1.79 (m, 2 H), 1.61 (m, 2 H); 13C NMR (CDCl3) δ 151.3,
150.8, 137.4, 135.6, 132.8, 130.9, 129.2, 128.7, 128.2, 127.0, 119.3,
105.8, 105.6, 70.9, 69.3, 58.6, 57.8, 53.3, 53.0, 44.7, 26.9, 23.1; MS
(ES+) m/z 480.1 (M + H)+.
1-(4-(4-(2-Amino-3-hydroxyphenoxy)butyl)piperazin-1-yl)-2-(1H-
imidazol-1-yl)ethanone (11b). Compound 10 (0.37 g, 0.75 mmol)
was dissolved in a mixture of 10% aqueous HCl (5 mL) and methanol
(45 mL) in a Parr hydrogenation bottle. Catalyst (20% Pd(OH)2/C,
0.02 g) was added, and the mixture was hydrogenated at 40 psi of H2
for ∼40 h. Workup as described above for the synthesis of 11a gave
1
11b (0.27 g, 98%) as a brown solid: H NMR (CD3OD) δ 7.78 (s, 1
H), 7.13 (s, 1 H), 7.06 (s, 1 H), 6.58 (m, 1 H), 6.43 (m, 2 H), 5.08 (s,
2 H), 4.03 (t, J = 5.9 Hz, 2 H), 3.66 (s, 2 H), 3.62 (s, 2 H), 2.75 (s, 2
H), 2.65 (m, 4 H), 1.82 (m, 4 H); 13C NMR (CD3OD) δ 174.8, 165.8,
148.0, 145.8, 138.1, 126.0, 123.0, 121.3, 118.2, 107.8, 103.5, 67.7, 57.3,
52.1, 51.8, 43.5, 40.9, 26.8, 22.2, 20.1; MS (ES+) m/z 374.1 (M + H)+.
Benzoxazinorifamycin (2b). A mixture of aminophenol 7 (0.336 g,
1.5 mmol), rifamycin S (12, 2.085 g, 3 mmol), and 1,4-dioxane (20
mL) was stirred at room temperature overnight. The mixture was then
concentrated to a black solid that was dissolved in 20 mL of methanol
and treated with MnO2 (0.3 g, 3.45 mmol). The mixture was stirred at
room temperature for 30 min, filtered over Celite and the filtrate
concentrated to a dark residue that was purified by flash silica gel
chromatography, eluting with dichloromethane/methanol (95:5 to
90:10). Product fractions were pooled and concentrated to give
partially purified 2b as a deep purple solid. Further purification by
preparative TLC was conducted on a 20 mg scale, eluting with
dichloromethane/methanol (90:10). The yield was ∼55%: Rf = 0.58
(dichloromethane/methanol, 85:15); HPLC tR = 9.4 min (98.1%
tert-Butyl 4-(4-(3-(Benzyloxy)-2-nitrophenoxy)butyl)piperazine-1-
carboxylate (9b). A solution of bromobutyl ether 8 (0.4 g, 1.05
mmol), 1-Boc-piperazine (0.282 g, 1.514 mmol) N,N-diisopropylethyl-
amine (4 mL), and acetonitrile (10 mL) was heated at reflux for 12 h.
The solution was concentrated and distributed between ethyl acetate
and brine. The organic phase was dried and concentrated to residue
that was purified by flash silica gel chromatography, eluting with ethyl
acetate. Product fractions were pooled and concentrated to leave 9b
1
(0.505 g, 99%): H NMR (CDCl3) δ 7.36 (m, 4H), 7.31 (m, 1 H),
7.24 (m, 1 H), 6.61 (m, 2 H), 5.16 (s, 2 H), 4.06 (t, J = 6.2 Hz, 2 H),
3.41 (t, J = 7.0 Hz, 4 H), 2.36 (t, J = 7.0 Hz, 4 H), 1.80 (m, 2 H), 1.62
(m, 2 H), 1.46 (s, 9 H), 1.26 (t, J = 7.2 Hz, 2 H); 13C NMR (CDCl3)
δ 154.9, 151.5, 150.9, 135.8, 131.0, 128.8, 128.3, 127.1, 106.0, 105.7,
79.7, 71.1, 69.4, 60.5, 58.1, 53.1, 28.6, 27.0, 23.2, 21.2, 14.3; MS (ES+)
m/z 486.1 (M + H)+.
1
purity). For H and 13C NMR (CDCl3), see Supporting Information,
Tables 1SI and 2SI. MS (ES+) m/z 900.1 (M + H)+; HRMS (MALDI)
calcd for C49H61N3O13 [(M + H)+], 900.4277; found 900.4269.
Benzoxazinorifamycin (2c). A mixture of aminophenol 11a (80
mg, 0.22 mmol), rifamycin S (12, 220 mg, 0.32 mmol), and 1,2-
dichloroethane (10 mL) was stirred at room temperature for 16 h. The
reaction mixture was then concentrated to a black solid that was
dissolved in 10 mL of methanol and treated with MnO2 (80 mg, 0.92
mmol). The mixture was stirred at room temperature for 30 min,
filtered over Celite, and concentrated to a dark residue that was
purified by flash silica gel chromatography, eluting with dichloro-
methane/methanol/NH4OH (94:6:0.5). Product fractions were
pooled and concentrated to give a solid that was further purified by
preparative TLC, eluting with dichloromethane/methanol (92:8). The
product band was processed to give 2c (170 mg, 74%) as a dark purple
1-(4-(4-(3-(Benzyloxy)-2-nitrophenoxy)butyl)piperazin-1-yl)-2-
(1H-imidazol-1-yl)ethanone (10). Trifluoroacetic acid (2 mL) was
added dropwise to a solution of 9b (0.505 g, 1.04 mmol) in
dichloromethane (8 mL), and the resultant mixture was stirred at
room temperature for 3 h. The solution was concentrated to leave 9c
(0.52 g, quantitative) as the crystalline trifluoroacetate salt. This was
then dissolved into DMF (10 mL) and N,N-diisopropylethylamine (3
mL), and the mixture was stirred at room temperature for 10 min
followed by treatment with 1-ethyl-3-[3-dimethylaminopropyl]-
carbodiimide hydrochloride (EDC·HCl, 0.22 g, 1.14 mmol), N-
hydroxybenzotriazole (HOBt, 0.175 g, 1.14 mmol), and 2-(1H-
imidazol-1-yl)acetic acid (0.197 g, 1.56 mmol; Tokyo Chemical
Industry Co. Ltd.). After the mixture was stirred under N2 for 16 h,
DMF was removed in vacuo and the residue was distributed between
dichloromethane and 5% aqueous sodium carbonate. The organic
phase was dried and concentrated to an oil that was purified by flask
silica gel chromatography, eluting with dichloromethane/methanol/
NH4OH (95:5:0.5). Product fractions were pooled and concentrated
to give 10 (0.37 g, 72%) as a yellow oil: 1H NMR (CDCl3) δ 7.49 (s, 1
H), 7.36 (m, 5 H), 7.26 (m, 1 H), 7.09 (s, 1 H), 6.95 (s, 1 H), 6.61
(m, 1H), 5.16 (s, 2 H), 4.75 (s, 2 H), 4.07 (t, J = 5.9 Hz, 2 H), 3.62
(m, 2 H), 3.44 (m, 2 H), 2.42 (t, J = 4.9 Hz, 4 H), 2.39 (t, J = 7.2 Hz, 2
H), 1.81 (m, 2 H), 1.62 (m, 2 H); 13C NMR (CDCl3) δ 164.4, 151.3,
150.8, 138.0, 135.6, 131.0, 129.5, 128.7, 128.2, 127.0, 120.1, 105.9,
105.5, 70.9, 69.1, 57.6, 52.6, 47.9, 45.1, 42.3, 26.7, 22.9; MS (ES+) m/z
494.1 (M + H)+.
3-(4-(4-(2-(1H-Imidazol-1-yl)ethyl)piperazin-1-yl)butoxy)-2-ami-
nophenol (11a). Compound 9a (0.86 g, 1.8 mmol) was dissolved in a
mixture of 10% aqueous HCl (10 mL) and methanol (90 mL) in a
Parr hydrogenation bottle. Catalyst (20% Pd(OH)2/C, 0.05 g) was
added, and the mixture was hydrogenated at 40 psi of H2 for ∼40 h.
The reaction mixture was rapidly filtered over Celite, and the filtrate
was concentrated and diluted with ethyl acetate. The solution was
washed with 5% aqueous sodium carbonate, dried, and concentrated to
give 11a (0.61 g, 95%) as a brown solid: 1H NMR (CDCl3) δ 7.56 (s,
1 H), 7.05 (s, 1 H), 6.96 (s, 1 H), 6.52 (t, J = 7.2 Hz, 1 H), 6.44 (s, 1
H), 6.37 (d, J = 8.0 Hz, 1 H), 3.98 (m, 4 H), 2.66 (t, J = 6.2 Hz, 2 H),
2.49 (bs, 8 H), 2.41 (t, J = 6.5 Hz, 2 H), 1.78 1.68 (m, 2 H); 13C NMR
(CDCl3) δ 147.6, 145.5, 128.6, 124.9, 119.4, 117.3, 108.7, 103.7, 68.1,
58.4, 58.2, 53.0, 50.4, 44.7, 27.5, 23.3; MS (ES+) m/z 360.1 (M + H)+.
1
solid: HPLC tR = 7.54 min (94.3% purity). For H and 13C NMR
(CDCl3), see Supporting Information, Tables 1SI and 2SI. MS (ES+)
m/z 1035.1 (M + H)+; HRMS (MALDI) calcd for C49H61N3O13 [(M
+ H)+], 1035.5074; found 1035.5095.
Benzoxazinorifamycin (2d). Reaction of a mixture of aminophenol
11b (30 mg, 0.08 mmol), rifamycin S (12, 102 mg, 0.15 mmol), and
1,2-dichloroethane (4 mL) and subsequent purification were carried
out exactly as described above for the synthesis of 2c to provide 2d (29
mg, 34.5%) as a dark purple solid: HPLC tR = 7.48 min (95.1%
1
purity). For H and 13C NMR (CDCl3), see Supporting Information,
Tables 1SI and 2SI. MS (ES+) m/z 1049.2 (M + H)+; HRMS
(MALDI) calcd for C49H61N3O13 [(M + H)+], 1049.4866; found
1049.4857.
Biological Materials and Methods. Expression and Purifica-
tion of MTB RNAP (WT and RifR Mutants). The wild-type MTB
RNAP and the RifR mutants were prepared as previously
described with minor alterations.30 For cell lysis, the sonication
method was preferred over the freeze/thaw method. For the
remainder of the purification steps, the protocol outlined by
Gill and Garcia30 was followed.
Cloning, Expression, and Purification of MTB SigA. The pAvitag
vector (modified pMSCG7 vector with an Avitag introduced between
BglII and KpnI sites) was linearized with SspI at 37 °C for 1 h, and the
reaction product was purified using the Qiagen PCR kit. The linearized
pAvitag vector (1.6−2.0 μg) was treated with T4 DNA polymerase in
10× T4 polymerase buffer, 5 mM DTT, 4 mM dGTP in a final
reaction volume of 60 μL. The mixture was incubated for 30 min at 22
°C and then for 20 min at 75 °C before being stored at −20 °C. PCR
primers were designed to amplify the Rv2703/sigA gene encoding
SigA from pSR01, which was provided by S. Rodrigue.40 The primers
3822
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
included an overhang sequence that complemented the vector ligation
independent cloning (LIC) overhangs. The sigA gene was purified via
Qiagen PCR kit. The purified PCR product (0.2 pmol) was incubated
with T4 DNA polymerase, 5 mM DTT, 4 mM dCTP, and 10× T4
DNA polymerase in a final reaction volume of 20 μL. The mixture was
incubated for 30 min at 22 °C and then for 20 min at 75 °C and stored
at −20 °C. The treated sigA was incubated with treated pAvitag vector
(∼0.2 pmol) for 10 min at 22 °C. Then 6.25 mM EDTA was added
followed by incubation at 22 °C for 5 min before reducing the
temperature to 4 °C. The annealed pAvitag vector containing sigA was
transformed into BL21(DE3) CodonPlus RIPL cells.
and other redox reagents such as MTT has shown excellent correlation
with colony-forming unit (CFU) based and radiometric analyses of
mycobacterial growth in many laboratories. The MIC is defined as the
lowest concentration effecting a reduction in fluorescence (or
luminescence) of 90% relative to controls. Isoniazid and rifampin
are included as positive quality control compounds with expected MIC
ranges of 0.025−0.1 and 0.06−0.125 μg/mL, respectively.
The low oxygen recovery (LORA) in vitro assay33 is designed to
detect compounds that may have the potential for shortening the
duration of therapy through (more) efficient killing of the non-
replicating persistor (NRP) population. The assay involves (1)
adaptation of MTB to low oxygen through gradual, monitored, self-
depletion of oxygen during culture in a sealed fermentor, (2) exposure
for 10 days of the low-oxygen adapted culture to test compounds in
microplates that are maintained under an anaerobic environment, thus
precluding growth, and (3) subsequent evaluation of MTB viability as
determined by the ability to recover. Recovery/viability is determined
by either (a) (aerobic) subculture onto solid, drug-free mediim and
determination of colony forming units or (b) the extent to which a
luciferase-expressing strain can recover the ability to produce
luminescence. Compounds such as isoniazid and ethambutol, which
are considered to be devoid of “sterilizing activity”, are inactive in this
assay, while the rifamycins and the more potent fluoroquinolones,
which do appear to eliminate some proportion of the persistor
population and thus can affect treatment duration, are active, albeit at
concentrations higher than the MICs for replicating cultures.
Correlation between the CFU and luminescence readout has been
good with the exception of the fluoroquinolone class for which
luminescence underestimates absolute activity but not relative activity.
Human Pregnane X Receptor (hPXR) Activation Assay. To assess
the ability of specific rifamycins to activate the human pregnane X
receptor (hPXR), the hPXR activation assay system from Puracyp, Inc.
was used. The manufacturer’s protocol was followed for the 96-well
plate assay. Briefly, the DPX2 cells were thawed in a 37 °C water bath
and mixed thoroughly with culture medium. Then 100 μL of cell
mixture was transferred into each well and the plate was incubated
overnight in a 5% CO2 incubator at 37 °C. The following day, the
dosing medium was thawed in a 37 °C water bath. The dilutions of
RLZ (2a) and analogues (2b−d) and RMP (1a, positive control) were
prepared as described in the manual. The 96-well plate was removed
from the incubator, and liquid from each well was discarded before
adding 100 μL of the dilutions to the specific wells. Each dilution of
the rifamycin derivative was tested in duplicate. The plate was placed
in the 5% CO2/37 °C incubator again for 24 h. The next day, the
CellTiter-Fluor buffer and CellTiter-Fluor were thawed at room
temperature before adding 5 μL of CellTiter-Fluor to 10 mL of
CellTiter-Fluor buffer. The wells of the 96-well plate were emptied
again, and 100 μL of CellTiter-Fluor reagent was added to each well.
The plate was incubated for 1 h in the 5% CO2/37 °C incubator. A
Synergy H1 hybrid multimode microplate reader (BioTek) was used
to measure fluorescence (λex = 390 nm; λem = 505 nm). To obtain
luminescence readings, the contents of ONE-Glo assay buffer were
added to the ONE-Glo assay substrate, and then 100 μL of mixture
was transferred into each well. The plate was read after 5 min where
the luminometer was set for 5 s of preshake with 5 s/well read time.
The relative luminescence units (RLUs) and relative fluorescence units
(RFUs) were determined as outlined under the “Quantitation of PXR
Receptor Activation” section of the manual. The normalized luciferase
activity (RLU/RFU) was divided by the normalized DMSO control to
represent the data as “fold activation” relative to the control. The
replicate data points were averaged, and both the original data points
and the average values were plotted as a function of log of
concentration versus PXR activation. The average values were then
fit by nonlinear regression to a modified four-parameter logistic
equation using Kaleidagraph (Synergy Software, Essex, VT),
For the expression of SigA protein in BL21(DE3) CodonPlus RIPL
cells, the cells were grown in 500 mL of 2xTY liquid cultures
containing 100 μg/mL carbenicillin and 30 μg/mL chloramphenicol at
37 °C with vigorous shaking until cell density reached OD600 nm = 0.5−
0.6. The protein was induced by the addition of isopropyl β-D-
thiogalactoside (IPTG) to a final concentration of 1 mM. The cultures
were allowed to incubate for an additional 20−24 h at 19 °C. The cells
were harvested by centrifugation (6000g, 15 min, 4 °C). The cell pellet
of each 500 mL culture was resuspended in 10 mL of Ni2+-NTA bind
buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH 8.0).
The freeze/thaw method was followed to lyse the cells, and it was
repeated a total of three times. The sample was supplemented with 10
μL of Lysonase bioprocessing reagent and 100 μM phenyl-
methylsulfonyl fluoride (PMSF), and then the resulting lysate was
cleared by centrifugation (21000g, 30 min, 4 °C). All further
purification steps were performed at 4 °C. The lysate was incubated
with 2 mL Ni2+-NTA His·bind resin overnight with gentle shaking.
Each supernatant−resin mixture was applied to individual columns.
The columns were washed twice with 4 mL of Ni2+-NTA wash buffer
(300 mM NaCl, 50 mM NaH2PO4, 20 mM imidazole, pH 8.0), and
the protein was then eluted in 6 mL of Ni2+-NTA elute buffer (300
mM NaCl, 50 mM NaH2PO4, 250 mM imidazole, pH 8.0). The
protein was concentrated to a final volume of ∼500 μL and then
sterile-filtered with a 0.22 μm syringe before being applied to a HiPrep
16/60 Sephacryl S-200 HR (GE Healthcare) column, and the running
buffer was RNAP storage buffer (10 mM Tris-HCl (pH 7.9), 0.1 mM
EDTA, 0.1 mM DTT, 0.1 M NaCl). The fractions containing SigA
were pooled together and concentrated to a final volume of ∼500 μL
using Amicon centrifugal filter units (MWCO = 10 kDa). The enzyme
was mixed with 1 volume of 100% glycerol and stored in liquid
nitrogen. The final concentration of the enzyme was determined via
Bradford assay using the Bio-Rad protein assay kit.
In Vitro Transcriptional Activity of MTB RNAPs and Dose
Response Curves. Dose response studies with RLZ (2a) and
analogues (2b−d) were performed via rolling circle transcription
assay as described previously30 to determine the IC50 values. Each of
the compounds was tested in duplicate (n = 2). The concentration
range used for the wild-type MTB RNAP ( SigA) was 1.56−100 nM
for RLZ and analogues (2b−d). The concentration ranges used for
MTB RNAP (D435V) with SigA were as follows: for 2a, 39.1−2500
μM; for 2b−d, 1.25−80 μM. The concentration ranges used for MTB
RNAP (H445Y) with SigA were as follows: for 2a, 20.5−5000 μM; for
2b−d, 8.2−2000 μM. The concentration ranges used for MTB RNAP
(S450L) with SigA were as follows: for 2a, 8.2−2000 μM; for 2b, 3.3−
800 μM; for 2c and 2d, 1.64−400 μM. The final concentration of the
wild-type MTB RNAP was 10 nM, whereas the final concentration of
the mutant RNAPs was 100 nM in the reactions. The core RNAP and
SigA were incubated for 30 min on ice in 1× RNAP reaction buffer (40
mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 0.01% Triton X-
100) before adding the test compound and DNA nanocircle template
(80 nM). Each reaction was initiated upon the addition of NTP
solution (500 μM each NTP). The IC50 values were determined via
nonlinear regression to a modified four-parameter logistic equation as
described previously.30 The log(IC50) values and their standard errors
(of the fit) are reported in Table 3SI (Supporting Information).
MTB MIC90 Assays. All compounds were evaluated for MIC90 vs
MTB H37RV using the microplate Alamar Blue assay (MABA) as
previously described32 except that 7H12 medium is now used
(replacing 7H9 + glycerol + casitone + OADC). The use of this
y = 1 + [(M3 − 1)/(1 + 10[(M1−M0)M2])]
where M3 is the ECMAX, 1 is the lower limit of the assay, M0 is the log
of the rifamycin concentration, M1 is the log of the EC50, and M2 is
3823
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
1
2b−d (1D and 2D NMR spectra along with H and 13C
assignments and HRMS and HPLC traces), PK plots of
analogue 2b in plasma and lung tissue, table of log(IC50) values
of 2a−d against RNAP, and table of relative fluorescence units
of 1a and 2a−d in the hPXR activation assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
the Hill slope. The data were normalized such that the lower limit was
set to 1. M1, M2, and M3 were fit by the regression.
Microsome Stability. Test compound stock solutions were
prepared at 200 μM in acetonitrile. An amount of 2 μL was added
to 198 μL of PBS containing 1 mg/mL human or mouse microsome.
After mixing, 25 μL aliquots were dispensed in triplicate in 96-well
plates. Control and reaction wells received 25 μL of PBS and of 2 mM
NADPH in PBS, respectively. Plates were incubated for 30 min at 37
°C with shaking at 600 rpm. Internal standard solution (150 μL) was
added to each well to quench the reaction. For controls, quenching
was done prior to incubation. Plates were centrifuged at 4000g for 30
min at 4 °C, and the supernatant was collected for analysis. The
percentage of compound remaining and half-life of compound in
microsomes were calculated according to the following formula:
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 734-764-5504. E-mail: showalh@umich.edu.
Author Contributions
#These authors contributed equally.
Notes
reaction
control
percentage remaining =
× 100
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(incubation time)[ln(2)]
ln(percentage remaining/100)
■
half‐life (min) = −
We acknowledge generous support by the University of
Michigan College of Pharmacy Ella and Hans Vahlteich and
UpJohn Research Funds. We also acknowledge additional
funding by the University of Michigan Office of the Vice
President for Research and the Rackham Graduate School. We
thank Dr. Paul Aristoff for stimulating discussions.
Pharmacokinetics of Analogue 2b. Previously described methods
were followed.41,42
Single Dose Study. Analogue 2b was prepared in 0.5% CMC at 1
mg/mL. Healthy female BALB/c mice were administered 10 mg/kg
suspension via oral gavage. Two mice per time point were used, and
0.4 mg/kg fentanyl was given 15 min prior to bleeding by
intraperitoneal injection. For each mouse, at least 100 μL of venous
blood was collected via retro-orbital bleeding in BD Vacutainer spray-
coated K2EDTA tubes at 0.5, 1, 2, 4, 8, and 24 h postdose. Tubes were
inverted several times and kept on ice. Blood was transferred to
polypropylene tubes and centrifuged at 4000g for 30 min at 4 °C. The
harvested plasma was transferred to new polypropylene tubes and
stored at −80 °C until analysis. To each sample, 3× volume of chilled
acetonitrile was added containing 0.2 μM internal standard (IS). The
solution was vortexed and then subsequently centrifuged at 10000g for
15 min. Calibration standard samples were prepared by spiking the
stock solution of analogue 2b in acetonitrile into mouse plasma to
yield the following concentrations: 0.097656, 0.195313, 0.390625,
0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 μM. Supernatant was
injected into an LC−MS/MS instrument for analysis. In addition, a
blank (blank plasma extracted with 3× volume of IS) and a double
blank (blank plasma extracted with 3× volume of pure acetonitrile)
were prepared. The concentration of 2b in blood sample for each time
point was then determined.
Multiple Dose Study. Mice were dosed once daily for 5 consecutive
days by oral gavage. Blood samples at time points 0.5, 1, 2, 4, 8, and 24
h were collected and analyzed in the same way as the single dose study.
After collection of the blood, the mice were sacrificed by carbon
dioxide asphyxiation. Lung tissue was aseptically removed, rinsed in 3
mL of PBS, air-dried on sterilized gauze pads, weighed, and suspended
in 4× (solvent/tissue, w/v) PBS buffer. Lung tissue was homogenized,
mixed, and extracted with 3× acetonitrile containing internal standard
at 0.2 μM and centrifuged at 10000g for 15 min at 4 °C. The
supernatant was collected for LC−MS/MS analysis. Calibration
standard lung samples were prepared by spiking the stock solution
of compound (in methanol or acetonitrile) into homogenized mouse
lungs and extracting with 3× volume acetonitrile to yield the following
concentrations: 0.024, 0.049, 0.098, 0.195, 0.39, 0.78, 1.56, 3.12, 6.25,
12.5, 25, 50 μM. In addition, a blank (blank lung tissue extracted with
3× volume of IS) and a double blank (blank lung tissue extracted with
3× volume of pure acetonitrile) were prepared. The concentration of
2b in lung tissue for each time point was then determined.
ABBREVIATIONS USED
■
RNAP, RNA polymerase; hPXR, human pregnane X receptor;
SAR, structure−activity relationship; TB, tuberculosis; MTB,
Mycobacterium tuberculosis; MDR-TB, multidrug-resistant tu-
berculosis; XDR-TB, extensively drug-resistant TB; Rif,
rifamycin; RMP, rifampin; RFB, rifabutin; RLZ, rifalazil; PK,
pharmacokinetic; RifR, rifamycin-resistant; MABA, microplate
Alamar Blue assay; LORA, low oxygen recovery assay; MIC,
minimum inhibitory concentration; RFB, rifabutin
REFERENCES
■
(1) WHO Report 2008. Global Tuberculosis Control: Surveillance,
Planning, Financing; World Health Organization: Geneva, Switzerland,
2008.
(2) Barrera, L. The Basics of Clinical Bacteriology. In Tuberculosis
2007: From Basic Science to Patient Care; Palomino, J. C., Leao, S. C.,
̃
Ritacco, V., Eds.; Amedeo Challenge, 2007; pp 93−112.
(3) Godreuil, S.; Tazi, L.; Banuls, A.-L. Pulmonary Tuberculosis and
̃
Mycobacterium Tuberculosis: Modern Molecular Epidemiology and
Perspectives. In Encyclopedia of Infectious Diseases: Modern Method-
ologies; Tibayrenc, M., Ed.; Wiley-Liss: Hoboken, NJ, 2007; pp 1−30.
(4) Garcia, A. B.; Palacios, J. J.; Ruiz, M. J.; Barluenga, J.; Aznar, F.;
Cabal, M. P.; Garcia, J. M.; Diaz, N. Strong in vitro activities of two
new rifabutin analogs against multidrug-resistant Mycobacterium
tuberculosis. Antimicrob. Agents Chemother. 2010, 54 (12), 5363−5365.
(5) Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. The
challenge of new drug discovery for tuberculosis. Nature 2011, 469
(7331), 483−490.
(6) (a) Siddiqi, M. I.; Kumar, A. Review of knowledge for rational
design and identification of anti-tubercular compounds. Expert Opin.
Drug Discovery 2009, 4 (10), 1005−1015. (b) Lienhardt, C.; Vernon,
A.; Raviglione, M. C. New drugs and new regimens for the treatment
of tuberculosis: review of the drug development pipeline and
implications for national programmes. Curr. Opin. Pulm. Med. 2010,
16 (3), 186−193.
(7) Aristoff, P. A.; Garcia, G. A.; Kirchhoff, P. D.; Showalter, H. D. H.
Rifamycins: obstacles and opportunities. Tuberculosis 2010, 90 (2),
94−118.
(8) Saito, H.; Tomioka, H.; Sato, K.; Emori, M.; Yamane, T.;
Yamashita, K.; Hosoe, K.; Hidaka, T. In vitro antimycobacterial
activities of newly synthesized benzoxazinorifamycins. Antimicrob.
Agents Chemother. 1991, 35, 542−547.
ASSOCIATED CONTENT
■
S
* Supporting Information
Plots of 2a,c,d with interaction surfaces of RNAP, stereoview of
1
superimposed compounds 2a−d, H and 13C NMR digital
spectra of 2-aminoresorcinol ethers (7, 11a, 11b) and
precursors, detailed digital data for benzoxazinorifamycins
3824
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
(9) (a) Lounis, N.; Roscigno, G. In vitro and in vivo activities of new
rifamycin derivatives against mycobacterial infection. Curr. Pharm. Des.
2004, 10, 3229−3238. (b) Luna-Herrera, J.; Venkata Reddy, M.;
Gangadharam, P. R. J. In vitro activity of the benzoxazinorifamycin
KRM-1648 against drug-susceptible and multidrug-resistant tubercle
bacilli. Antimicrob. Agents Chemother. 1996, 39, 440−444. (c) Tomioka,
H. Current status of some antituberculosis drugs and the development
of new antituberculous agents with special reference to their in vitro
and in vivo antimicrobial activities. Curr. Pharm. Des. 2006, 12, 4047−
4070.
J.; O’Connor, T. M. Novel agents in the management of
Mycobacterium tuberculosis disease. Curr. Med. Chem. 2007, 14 (18),
2000−2008.
(21) Portero, J.-L.; Rubio, M. New anti-tuberculosis therapies. Expert
Opin. Ther. Pat. 2007, 17, 617−637.
(22) Artsimovitch, I.; Vassylyeva, M. N.; Svetlov, D.; Svetlov, V.;
Perederina, A.; Igarashi, N.; Matsugaki, N.; Wakatsuki, S.; Tahirov, T.
H.; Vassylyev, D. G. Allosteric modulation of the RNA polymerase
catalytic reaction is an essential component of transcription control by
rifamycins. Cell 2005, 122 (3), 351−63.
(10) Yamamoto, T.; Amitani, R.; Suzuki, K.; Tanaka, E.; Murayama,
T.; Kuze, F. In vitro bactericidal and in vivo therapeutic activities of a
new rifamycin derivative, KRM-1648, against Mycobacterium tuber-
culosis. Antimicrob. Agents Chemother. 1996, 40, 426−428.
(11) (a) Moghazeh, S.; Pan, X.; Arain, T.; Stover, C. K.; Musser, J.
M.; Kreiswirth, B. N. Comparative antimycobacterial activities of
rifampin, rifapentine and KRM-1648 against a collection of rifampin-
resistant Mycobacterium tuberculosis isolates with known rpoB
mutations. Antimicrob. Agents Chemother. 1996, 40, 2655−2657.
(b) Yang, B.; Koga, H.; Ohno, H.; Ogawa, K.; Fukuda, M.; Hirakata,
Y.; Maesaki, S.; Tomono, K.; Tashiro, T.; Kohno, S. Relationship
between antimycobacterial activities of rifampicin, rifabutin and KRM-
1648 and rpoB mutations of Mycobacterium tuberculosis. J. Antimicrob.
Chemother. 1998, 42, 621−628.
(12) (a) Mullin, S.; Rothstein, D. M.; Murphy, C. K. Activity of novel
benzoxazinorifamycins against rifamycin-resistant Streptococcus pyo-
genes. Antimicrob. Agents Chemother. 2006, 50, 1908−1909. (b) Roblin,
P. M.; Reznik, T.; Kutlin, A.; Hammerschlag, M. R. In vitro activities of
rifamycin derivatives ABI-1648 (rifalazil, KRM-1648), ABI-1657, and
ABI-1131 against Chlamydia trachomatis and recent clinical isolates of
Chlamydia pneumoniae. Antimicrob. Agents Chemother. 2003, 47, 1135−
1136.
(13) (a) Brooks, J. V.; Orme, I. M. Evaluation of once-weekly therapy
for tuberculosis using isoniazid plus rifamycins in the mouse aerosol
infection model. Antimicrob. Agents Chemother. 1998, 42, 3047−3048.
(b) Klemens, S. P.; Grossi, M. A.; Cynamon, M. H. Activity of KRM-
1648, a new benzoxazinorifamycin, against Mycobacterium tuberculosis
in a murine model. Antimicrob. Agents Chemother. 1994, 38, 2245−
2248.
(14) Kelly, B. P.; Furney, S. K.; Jessen, M. T.; Orme, I. M. Low-dose
aerosol infection model for testing drugs for efficacy against
Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 1996, 40,
2809−2812.
(15) (a) Klemens, S. P.; Cynamon, M. H. Activity of KRM-1648 in
combination with isoniazid against Mycobacterium tuberculosis in a
murine model. Antimicrob. Agents Chemother. 1996, 40, 298−301.
(b) Lenaerts, A. M.; Chase, S. E.; Cynamon, M. H. Evaluation of
rifalazil in a combination treatment regimen as an alternative to
isoniazid−rifampin therapy in a mouse tuberculosis model. Antimicrob.
Agents Chemother. 2000, 44, 3167−3168. (c) Shoen, C. M.; Chase, S.
E.; DeStefano, M. S.; Harpster, T. S.; Chmielewski, A. J.; Cynamon, M.
H. Evaluation of rifalazil in long-term treatment regimens for
tuberculosis in mice. Antimicrob. Agents Chemother. 2000, 44, 1458−
1462.
(16) Rifalazil. Tuberculosis (Edinburgh) 2008, 88, 148−150.
(17) Mae, T.; Hosoe, K.; Yamamoto, T.; Hidaka, T.; Ohashi, T.;
Kleeman, J. M.; Adams, P. E. Effect of a new rifamycin derivative,
rifalazil, on liver microsomal enzyme induction in rat and dog.
Xenobiotica 1998, 28, 759−766.
(18) Rose, L. M.; Porubek, D. J.; Montgomery, A. B. Method for
Treatment of Bacterial Infections with Once- or Twice-Weekly
Administered Rifalazil. WO2000035408A2, 2000.
(23) Sinz, M. W. Pregnane X receptor: prediction and attenuation of
human CYP3A4 enzyme induction and drug−drug interactions. Annu.
Rep. Med. Chem. 2008, 43, 405−418.
(24) Chrencik, J. E.; Orans, J.; Moore, L. B.; Xue, Y.; Peng, L.;
Collins, J. L.; Wisely, G. B.; Lambert, M. H.; Kliewer, S. A.; Redinbo,
M. R. Structural disorder in the complex of human pregnane X
receptor and the macrolide antibiotic rifampicin. Mol. Endocrinol.
2005, 19 (5), 1125−1134.
(25) Yamane, T.; Hashizume, T.; Yamashita, K.; Konishi, E.; Hosoe,
K.; Hidaka, T.; Watanabe, K.; Kawaharada, H.; Yamamoto, T.; Kuze, F.
Synthesis and biological activity of 3′-hydroxy-5′-aminobenzoxazinor-
ifamycin derivatives. Chem. Pharm. Bull. (Tokyo) 1993, 41 (1), 148−
155.
(26) Dhingra, K.; Maier, M. E.; Beyerlein, M.; Angelovski, G.;
Logothetis, N. K. Synthesis and characterization of a smart contrast
agent sensitive to calcium. Chem. Commun. 2008, 29, 3444−3446.
(27) Binggeli, A.; Christ, A. D.; Green, L.; Guba, W.; Maerki, H. P.;
Martin, R. E.; Mohr, P. Preparation of Benzoxazole, Oxazolopyridine,
Benzothiazole and Thiazolopyridine Derivatives as Somatostatin
Receptor Modulators and Antidiabetic Compounds.
WO2007025897A2, 2007.
(28) Havera, H. J.; Strycker, W. G. 3-Substituted-5-phenyl-5-
pyridylhydantoins. US3994904A, 1976.
(29) Mae, T.; Konishi, E.; Hosoe, K.; Hidaka, T. Isolation and
identification of major metabolites of rifalazil in mouse and human.
Xenobiotica 1999, 29, 1073−1087.
(30) Gill, S. K.; Garcia, G. A. Rifamycin inhibition of WT and Rif-
resistant Mycobacterium tuberculosis and Escherichia coli RNA
polymerases in vitro. Tuberculosis 2011, 91 (5), 361−369.
(31) (a) Telenti, A.; Imboden, P.; Marchesi, F.; Lowrie, D.; Cole, S.;
Colston, J.; Matter, L.; Schopfer, K.; Bodmer, T. Detection of
rifampicin-resistance mutation in Mycobacterium tuberculosis. Lancet
1993, 341, 647−650. (b) Williams, D. L.; Spring, L.; Collins, L.;
Miller, L. P.; Heifets, L. B.; Gangadharam, P. R. J.; Gillis, T. P.
Contribution of rpoB mutations to development of rifamycin cross-
resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother.
1998, 42, 1853−1857.
(32) Collins, L.; Franzblau, S. Microplate Alamar Blue assay versus
BACTEC 460 system for high-throughput screening of compounds
against Mycobacterium tuberculosis and Mycobacterium avium. Anti-
microb. Agents Chemother. 1997, 41 (5), 1004−1009.
(33) Cho, S. H.; Warit, S.; Wan, B.; Hwang, C. H.; Pauli, G. F.;
Franzblau, S. G. Low-oxygen-recovery assay for high-throughput
screening of compounds against nonreplicating Mycobacterium tuber-
culosis. Antimicrob. Agents Chemother. 2007, 51 (4), 1380−1385.
(34) Wang, J.; Huang, J.; Li, H.; Zhang, S. Synthesis of
benzoxazinorifamycin derivatives and preliminary study of antibacterial
activity. Huaxi Yaoxue Zazhi 2003, 18 (4), 241−243.
(35) Labute, P. LowModeMD: implicit low mode velocity filtering
applied to conformational search of macrocycles and protein loops. J.
Chem. Inf. Model. 2010, 50, 792−800.
(36) Molecular Operating Environment (MOE); Chemical Computing
Group: Montreal, Canada, 2010.
(37) Watkins, R. E.; Wisely, G. B.; Moore, L. B.; Collins, J. L.;
Lambert, M. H.; Williams, S. P.; Willson, T. M.; Kliewer, S. A.;
Redinbo, M. R. The human nuclear xenobiotic receptor PXR:
structural determinants of directed promiscuity. Science 2001, 292
(5525), 2329−2333.
(19) Dietze, R.; Teixeira, L.; Rocha, L. M. C.; Palaci, M.; Johnson, J.
L.; Wells, C.; Rose, L.; Eisenach, K.; Ellner, J. J. Safety and bactericidal
activity of rifalazil in patients with pulmonary tuberculosis. Antimicrob.
Agents Chemother. 2001, 45, 1972−1976.
(20) (a) Rose, L. M.; Porubek, D. J.; Montgomery, A. B. Method for
Treatment of Bacterial Infections with Once or Twice-Weekly
Administered Rifalazil. US 6566354 B1, May 20, 2003. (b) Barry, P.
3825
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826

Journal of Medicinal Chemistry
Article
(38) Watkins, R. E.; Maglich, J. M.; Moore, L. B.; Wisely, G. B.;
Noble, S. M.; Davis-Searles, P. R.; Lambert, M. H.; Kliewer, S. A.;
Redinbo, M. R. 2.1 Å crystal structure of human PXR in complex with
the St. John’s wort compound hyperforin. Biochemistry 2003, 42 (6),
1430−1438.
(39) Teotico, D. G.; Bischof, J. J.; Peng, L.; Kliewer, S. A.; Redinbo,
M. R. Structural basis of human pregnane X receptor activation by the
hops constituent colupulone. Mol. Pharmacol. 2008, 74 (6), 1512−
1520.
(40) Jacques, J.-F.; Rodrigue, S.; Brzezinski, R.; Gaudreau, L. A
recombinant Mycobacterium tuberculosis in vitro transcription system.
FEMS Microbiol. Lett. 2006, 255, 140−147.
(41) (a) Jayaram, R.; Gaonkar, S.; Kaur, P.; Suresh, B. L.; Mahesh, B.
N.; Jayashree, R.; Nandi, V.; Bharat, S.; Shandil, R. K.; Kantharaj, E.;
Balasubramanian, V. Pharmacokinetics−pharmacodynamics of rifam-
pin in an aerosol infection model of tuberculosis. Antimicrob. Agents
Chemother. 2003, 47 (7), 2118−2124. (b) Jayaram, R.; Shandil, R. K.;
Gaonkar, S.; Kaur, P.; Suresh, B. L.; Mahesh, B. N.; Jayashree, R.;
Nandi, V.; Bharath, S.; Kantharaj, E.; Balasubramanian, V. Isoniazid
pharmacokinetics−pharmacodynamics in an aerosol infection model
of tuberculosis. Antimicrob. Agents Chemother. 2004, 48 (8), 2951−
2957.
(42) Shudo, J.; Pongpeerapat, A.; Wanawongthai, C.; Moribe, K.;
Yamamoto, K. In vivo assessment of oral administration of probucol
nanoparticles in rats. Biol. Pharm. Bull. 2008, 31 (2), 321−325.
3826
dx.doi.org/10.1021/jm201716n | J. Med. Chem. 2012, 55, 3814−3826