Synthesis and structure-activity relationships of novel substituted 8-amino, 8-thio, and 1,8-pyrazole congeners of antitubercular rifamycin S and rifampin

Article abstract of DOI:10.1016/j.bmcl.2011.08.054

A series of rifamycin S and rifampin analogues incorporating substituted 8-amino, 8-thio, and 1,8-pyrazole substituents has been synthesized. The compounds were made by activation of the C-8 phenol as a sulfonate ester, followed by displacement with selec

Full text of DOI:10.1016/j.bmcl.2011.08.054

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6094–6099
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of novel substituted 8-amino,
8-thio, and 1,8-pyrazole congeners of antitubercular rifamycin S and rifampin
Yafei Jin a, Sumandeep K. Gill b, Paul D. Kirchhoff a, Baojie Wan d, Scott G. Franzblau d, George A. Garcia b,c
,
H.D. Hollis Showalter a,c,
⇑
a Vahlteich Medicinal Chemistry Core, University of Michigan, Ann Arbor, MI 48109-1065, USA
b Interdepartmental Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109-1065, USA
c Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, USA
d Institute for Tuberculosis Research, College of Pharmacy, University of Illinois, Chicago, IL 60612-723, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of rifamycin S and rifampin analogues incorporating substituted 8-amino, 8-thio, and 1,8-pyra-
zole substituents has been synthesized. The compounds were made by activation of the C-8 phenol as a
sulfonate ester, followed by displacement with selected nitrogen and sulfur nucleophiles. The analogues
were screened in assays to quantify their antitubercular activity under both aerobic and anaerobic con-
ditions, and for inhibition of wild-type Mycobacterium tuberculosis (MTB) RNAP and rifamycin-resistant
MTB RNAP (S450L) via an in vitro rolling circle transcription assay. Additionally, the MIC90 values were
determined for these analogues against Escherichia coli strains. Although none of the analogues displayed
superior enzymatic or microbiological activity to their parent scaffolds, the results are consistent with the
Rif C-8 hydroxyl acting as a hydrogen bond acceptor with S450 and that Rif resistance in the S450L
mutant is due to loss of this hydrogen bond. Representative analogues were also evaluated in the human
pregnane X receptor (PXR) activation assay.
Received 27 May 2011
Revised 9 August 2011
Accepted 11 August 2011
Available online 19 August 2011
Keywords:
Rifamycin
MTB
Escherichia coli
MIC90
RNAP
Ó 2011 Elsevier Ltd. All rights reserved.
With nearly one-third of the global population infected by
Mycobacterium tuberculosis (MTB), tuberculosis (TB) is still a major
cause of death. Indeed, in 2006 over nine million new cases and
1.7 million deaths occurred due to TB, and there is now significant
concern about the emergence of multi-drug resistant (MDR) strains
of TB with an estimated 0.5 million cases worldwide.1 Despite
these appalling statistics, there have been relatively few new
agents discovered in the past 40 years to treat this disease.
The rifamycins are members of the ansamycin family of antibiot-
ics, consisting of an ansa, poly-hydroxylated bridge connecting two
ends of a naphthoquinone (or naphthohydroquinone) core. They
display activity against mycobacteria as well as Gram-positive
bacteria and, to a lesser extent, Gram-negative bacteria. Particularly
attractive with the rifamycins is their sterilizing activity. This effect
also applies to slowly metabolizing extra-cellular bacilli (persisters),
a feature possessed by few other TB drugs.2,3 Mechanistically, the
rifamycins exhibit specific inhibition of DNA-dependent RNA syn-
thesis3–9 with binding constants for prokaryotic RNA polymerases
in the range of 10À8 M whereas those for eukaryotic enzymes are
at least 10,000 times weaker. Amongst the clinically utilized rifa-
mycins, rifampin (RMP, 1) is the most widely utilized worldwide.
This agent totallyblocks the translocation step that normally follows
formation of the first phosphodiester bond by the polymerase, sug-
gesting that rifamycins sterically block the extension of the RNA
chain at a very early stage.10 The binding site for the rifamycins on
RNA polymerase (RNAP) has been shown to be at the b subunit en-
coded by the rpoB gene. A key breakthrough has been the determina-
tion of the X-ray crystal structures of several different rifamycins
bound to RNAP.11 These studies show the polymerase to form key
hydrogen bond interactions with the four hydroxyl groups (at C-1,
C-8, C-21, and C-23) of rifampin, consistent with the known struc-
ture–activity relationships (SAR) of the rifamycins, as well as the
carbonyl oxygen of the C-25 acetoxy group, and help explain the
high resistance that results from single point mutations of amino
acids found to have direct interactions with the naphthalene
backbone.
We have recently published a comprehensive review on the
rifamycins, which includes an extensive analysis of prior SAR
reports.12 While initial SAR studies indicated that most modifica-
tions of the ansa bridge led to less active compounds, those on
the naphthalene ring were much better tolerated, with the signif-
icant exception that acetylation of the C-8 hydroxyl group elimi-
nated activity.13 A wide variety of substitutions at C-3 were
found to be well tolerated, which has been exploited toward the
development of several clinical agents.13–15
Due to the general paucity of C-8 functionalized rifamycin ana-
logues, we decided to explore the SAR of this position in greater
⇑
Corresponding author.
E-mail address: showalh@umich.edu (H.D.H. Showalter).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.054

Y. Jin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6094–6099
6095
Me Me
OH
Me Me
HO
Me
HO
Me
Tos
OH
AcO
Me
O
AcO
Me
O
HO
O
1
Me
O
O
Me
Me
Me
MeO
NH
MeO
NH
(i)
(ii)
8
O
O
O
O
O
O
O
O
2
3
Me
Me
Rifamycin S (Rif S)
Me Me
HO
Me Me
OH
HO
Me
Me
(RS)
R2N
AcO
Me
O
AcO
Me
O
OH
O
Me
RN
N
Me
Me
Me
MeO
NH
MeO
NH
or
O
O
O
O
O
O
O
O
Me
4a-j
Me
5
C-1, C-8 fused pyrazoles
C-8 substituted amines, thiols
Scheme 1. Synthesis of C-8 analogues of rifamycin S. (i) p-toluenesulfonyl chloride, diisopropylethylamine, acetonitrile; (ii) R2NH, or RNHNH2, or RSNa in acetonitrile (5–80%
yield).
depth by applying an ‘enabling reaction’ that we had reported on
several years ago.16 This involved the facile activation of the 1-
hydroxy function of 9,10-anthracendiones toward displacement
by nitrogen and sulfur nucleophiles, which we believed could also
be applied to the napthoquinone core of rifamycin S (Rif S, 2) and
related congeners.
unable to secure a highly sought for analogue derived from ammo-
nia displacement of tosylate 7. Utilizing the same conditions that
gave 4a led to a product in 46% yield that both mass spec and
NMR data showed incorporation of the NH2 function, but without
loss of the C-8 tosyloxy moiety. The NMR spectrum was too com-
plex to make a definitive structural assignment. Candidate possi-
bilities include Michael addition of ammonia to the dienone
function or to the imino double bond of the side chain hydrazone.
MIC testing of this compound versus two MTB strains (vide infra)
showed it to be completely inactive. The quinone function of each
RMP S amine adduct was then reduced with ascorbic acid to the
hydroquinone form, providing corresponding RMP C-8 amine
congeners 10a–10f in 51–93% yield. The full range of synthesized
compounds is shown in Table 1.
No effort was made to optimize the reaction conditions that
provided amine, thiol, or pyrazole adducts from tosylates 3 or 7,
and product yields in general represent a single run for each target
compound. All compounds were rigorously purified by preparative
silica gel chromatography, and their structural assignments were
supported by diagnostic peaks in the 1H NMR spectra and by mass
spectrometry.
Apart from a previous report of the synthesis of Rif S ammonia
adduct 4a in the patent literature,17 no other analogues have ever
been reported in which C-8 substituents derived from amine or
sulfur nucleophiles have been incorporated onto a rifamycin scaf-
fold. Furthermore, despite many elaborations of the parent rifamy-
cin scaffold onto the ‘southeastern’ part of the naphthoquinone
template, including ring annulations (e.g., rifalazil18), we are una-
ware of any ring fusions onto the 1,8-carbon positions. Hence our
synthesis of pyrazoles 5, 9a, and 9b represents the first report of
such a core modification.
Our strategy was to first activate the C-8 peri-phenolic func-
tionality of readily available Rif S (2) as a sulfonate ester, and then
displace this with selected nitrogen nucleophiles through an
addition–elimination mechanism (Scheme 1). Given that the rifa-
mycin framework is embellished with so much functionality,
including free hydroxyl groups at C-21 and C-23 (Fig. 1), we first
needed to work out conditions for selective functionalization of
the C-8 phenolic moiety. Thus, the reaction of Rif S (2) with sulfo-
nyl chlorides representing a range of reactivity (p-toluenesulfonyl,
p-chlorosulfonyl, methanesulfonyl) and triflic anhydride was eval-
uated under different temperature and solvent conditions. Of
these, only p-toluenesulfonyl and p-chlorosulfonyl sulfonate esters
formed cleanly under optimum conditions (Hunig’s base, acetoni-
trile, room temperature) with tosylate 3 formed in 84% yield. Sub-
sequent reactions of these with amines and hydrazines indicated
that the p-chlorosulfonate ester was susceptible to cleavage to
Rif S, whereas the p-toluenesulfonate ester provided the desired
C–O bond displacement. With this finding, condensation of 3 was
then carried out with a range of amines to provide adducts 4a–4i
in 11–51% yields, and with (2-hydroxyethyl)hydrazine to give
annulated pyrazole product 5 in very low (5%) yield. We also tested
tosylate displacement with a single thiol (methanethiol sodium
salt), which provided analogue 4j in poor yield. Having developed
the chemistry for Rif S, we then applied it toward making related
analogues of RMP (1, Scheme 2). Thus, oxidation of RMP with
potassium ferricyanide provided rifampin S (RMP S, 6) in 65% yield.
Tosylation of the 8-hydroxyl function was conducted as described
above to give key intermediate 7 in nearly quantitative yield. Ami-
nation of 7 was then carried out with several of the same amines
utilized above to give adducts 8a–8f in 35–60% yields and pyra-
zoles 9a (33% yield) and 9b (40% yield). Disappointingly, an
attempt to make the simple unsubstituted pyrazole congener de-
rived from hydrazine was unsuccessful. Similarly, we were
Results and discussion
The compounds were screened in assays to quantify their anti-
tubercular activity under both aerobic and anaerobic conditions
(Table 1). Briefly, the 8 day microplate-based assay using Alamar
blue reagent (added on day 7) for determination of growth
(MABA)19 gives an assessment of activity against replicating MTB,

6096
Y. Jin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6094–6099
MABA. A single thioether derivative (4j) within the Rif S series
shows a unique profile with modest, albeit essentially equivalent,
potency in both MABA and LORA (2.0 vs 1.3 M, respectively).
l
Amine adducts within the RMP S (8a–8f) and RMP (10a–10f) series
display similar potencies as the Rif S series in the MABA (MIC90s
3.6–4.7 lM) and the LORA (MIC90s 13.7–19.2 lM), with a 3.5- to
5-fold differential between the two assays. Core-modified pyrazole
analogues within both Rif S (5) and RMP S (9a, 9b) series are
weakly to moderately active with MIC90s of 3.9–5.4
MABA and >18.6 M in the LORA. Surprising and especially note-
worthy are the potencies of the tosylated intermediates. Rif S tos-
ylate (3) shows MIC90s of 0.20 M in the MABA and 3.0 M in the
LORA, and RMP S tosylate (7) 0.24 M and 4.8 M, respectively, in
lM in the
l
l
l
l
l
the two assays. To determine if this was due to a prodrug effect
from simple sulfonate hydrolysis to parent compounds 2 and 6,
respectively, we subjected each tosylate to conditions utilized for
MIC determinations (buffer, 37 °C, 24 h) and the RNAP assay (vide
infra; buffer, 37 °C, 2 h). At the end of the assay period, the medium
was extracted with ethyl acetate and the organic extract was ana-
lyzed by thin-layer chromatography for the presence of either par-
ent phenol or corresponding tosylate. In all cases, only the
presence of tosylate was observed. In summary, none of the amine
or pyrazole derivatives show superior microbiological properties to
their parent scaffolds in the MABA and LORA. The tosylated (3 and
7) and thioether (4j) derivatives display surprising low MIC90s,
which are corroborated by excellent potency toward the target
RNAP enzyme (vide infra).
As reported earlier, the activity and inhibition (by commercially
available rifamycins) of wild-type (WT) and rifamycin-resistant
MTB RNAPs has been assessed via an in vitro rolling circle tran-
scription assay.21 Twelve b subunit residues interact with rifampin
where the S450 residue is involved in directly forming a hydrogen
bond with the C-8 hydroxyl group. To determine the IC50 values
(the concentration that results in 50% inhibition of transcription)
of these analogues, the mutant MTB RNAP (S450L) was assessed
along with the WT MTB RNAP (Table 2, S-Table 1, S-Fig. 1). For
the WT MTB RNAP, RMP (1), Rif S (2), thioether 4j and RMP S (6)
show the highest activities with IC50 values in the low nM range.
The IC50 values for RMP (1) and Rif S (2) were determined to be less
than 10 nM. However, the amount of enzyme used in each reaction
was 10 nM (the lowest concentration that we could confidently
use). Under these conditions, it is possible that a 10 nM IC50 may
simply reflect the ‘titration’ of the enzyme by an inhibitor with a
much lower true IC50. We have therefore elected to report any
IC50 determined to be less than 10 nM as simply ‘<0.01’ lM.
Figure 1. RNAP is shown as a molecular surface within 5 Å of the inhibitors (carbon
atoms shown in gray) and shaded to indicate areas of lipophilicity (green),
hydrogen bonding (magenta) and mild polar (blue). (A) Rif S (2). (B) Cycloheptyla-
mino analogue (4i).
As mentioned above, the tosylated intermediates 3 & 7 have
comparable MTB MIC90 values for both MABA and LORA; however,
the IC50 value with WT MTB RNAP for 7 is ꢀseven-fold greater than
that for 3. The IC50 value of 3 (21 nM) is ꢀtwo-fold greater than the
IC50 value of Rif S (<10 nM) with the WT MTB RNAP. Owing to the
fact that the MABA MIC90 value for 3 is five-fold greater than that
for Rif S, a parallel decrease in potency is observed for both the
MIC90 and IC50 values for 3 in comparison with Rif S. However
for compound 7, this trend is not as quantitative. The IC50 value
with WT MTB RNAP for 7 (142 nM) is more than 10-fold greater
than the IC50 value for RMP S (13 nM); whereas, the MABA MIC90
value of 7 is only ꢀthree-fold greater than that for RMP S. Surpris-
ingly for the MTB RNAP (S450L) mutant, a lower IC50 value is
observed for 7 (ꢀnine-fold lower) than the IC50 value of 3. Within
the Rif S series, the amino analogues (4a, 4c, and 4i) that had exhib-
ited modest MIC90 values under aerobic conditions have much
more variable IC50 values (ꢀ70- to 2000-fold greater than Rif S).
These analogues have comparable IC50 values against the MTB
RNAP (S450L) mutant, which are still 3- to 8-fold greater than Rif
S. The thioether derivative (4j) also exhibits modest MABA MIC90
values, but the IC50 values are comparable to the IC50 values
while the 11 day high-throughput, luminescence-based low-oxy-
gen-recovery assay (LORA)20 measures activity against bacteria in
a non-replicating state that models clinical persistence. Minimum
inhibitory concentrations (MIC90s) are defined as the lowest com-
pound concentration effecting >90% growth inhibition.
Under aerobic conditions (MABA) within the Rif S series, ana-
logues incorporating C-8 amine substituents display modest
MIC90s (1.4–5.6 lM) relative to the parent Rif S 2 (0.03 lM). The
unsubstituted NH2 analogue is best (compare 4a vs 4b–4i),
although within this small series there is no significant variation
from the smallest to the largest substituent. In addition the instal-
lation of small polar functionality (e.g., 4e, 4g) or bulky lipophilic
moieties (e.g., 4h, 4i) seems to impart minimal effect on potency.
Activity under anaerobic conditions (LORA) mirrors much the same
pattern with only 4c displaying modest activity (5.4
lM), and with
compound MIC90s ranging from 3- to 16-fold higher than in the

Y. Jin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6094–6099
6097
Me Me
HO
Me Me
Me Me
OH
HO
Me
HO
Me
Me
Tos
O
AcO
Me
O
AcO
Me
O
AcO
Me
O
OH
OH
HO
OH
Me
HO
Me
O
Me
O
Me
Me
Me
MeO
NH
N
MeO
NH
N
MeO
NH
N
(i)
(ii)
O
O
O
O
O
O
O
O
OH
O
N
O
N
O
N
Me
NMe
Me
NMe
Me
Me
NMe
7
Rifampin S (RMP S)
6
Rifampin (RMP)
1
(iii)
Me Me
Me Me
HO
Me Me
OH
HO
Me
HO
Me
Me
AcO
Me
O
AcO
Me
O
AcO
Me
O
OH
OH
RNH
Me
RNH OH
Me
O
RN
Me
N
Me
Me
MeO
NH
N
MeO
NH
N
MeO
NH
N
or
(iv)
O
O
O
O
OH
O
O
O
O
O
N
O
N
O
N
Me
NMe
Me
NMe
Me
NMe
8a-f
9a,b
10a-f
Rifampin S
Rifampin
C-8 substituted amines
Rifampin S
C-8 substituted amines
C-1, C-8 fused pyrazoles
Scheme 2. Synthesis of C-8 analogues of rifampin and rifampin S. (i) potassium ferricyanide, pH 7.4 phosphate buffer, ethyl acetate; (ii) p toluenesulfonyl chloride,
diisopropylethylamine, acetonitrile; (iii) RNH2, or RNHNH2 in acetonitrile (33–60% yield); (iv) ascorbic acid, methanol (51–93% yield).
Table 2
Table 1
Selected rifamycins versus MTB RNAP and E. coli
Screening results of rifamycin analogues versus MTB
No. IC50
(
l
M)a (WT
IC50
RNAP (S450L))
(
l
M)a (MTB
E. coli MIC90
(
l
M)b
EC28880
No.
Class
C-8 Substituent
MTB MIC90
MABA
(
l
M)a
LORA
0.4
3.0
22.2
20.6
5.4
21.4
12.5
MTB RNAP)
TG2
DH5
12.5
a
2
3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
5
6
7
8a
8b
8c
8e
8f
8g
9a
9b
10a
10b
10c
10d
10e
10f
1 (RMP)
Isoniazid
PA824
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S
Rif S pyrazole
RMP S
RMP S
RMP S
RMP S
RMP S
RMP S
RMP S
RMP S
RMP S pyrazole
RMP S pyrazole
RMP
Hydroxyl
Tosyloxy
Amino
0.03
0.20
1.41
>5.6
1.8
5.5
3.8
5.2
5.32
2.4
1
2
3
4a
4c
4i
4j
6
<0.01
<0.01
0.021
4.4
0.503
15.4
0.015
0.013
0.142
0.139
52.1
36.0
982
186
270
127
37.5
46.5
111
29.9
12.5
100
0.1
0.78
3.125
>100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
Methylamino
Dimethylamino
Ethylamino
Methoxyamino
N-(2-propenyl)amino
(2-Hydroxyethyl)amino
Benzylamino
Cycloheptylamino
Methylthio
2-Hydroxyethyl
Hydroxyl
Tosyloxy
Methylamino
Ethylamino
N-(2-propenyl)amino
(2-Hydroxyethyl)amino
Benzylamino
Cycloheptylamino
Methyl
2-Hydroxyethyl
Methylamino
Ethylamino
12.5
12.5
>12.5
>12.5
0.1
12.5
>12.5
18.3
12.5
25
>21.7
>20.4
>20.2
1.3
>21.7
0.5
7
9b
>100 >100
>100 >100
2.2
2.0
a
The IC50 is defined as the concentration that results in 50% inhibition of tran-
scription. (The experimental conditions are reported in the Supplementary data).
See footnote a, Table 1. (The experimental conditions are reported in the Sup-
plementary data).
>5.4
0.07
0.24
3.64
>4.7
>4.7
>4.6
>4.4
3.9
b
4.8
>19.2
>18.9
>18.6
>18.5
>17.6
13.7
>19.3
>18.6
>19.1
>18.8
>18.6
>18.5
>17.5
>17.4
1.9
observed for the controls (1, 2, 6) with both WT MTB RNAP and the
mutant. Although the pyrazole analogue (9b) is weakly active
microbiologically with higher MTB MIC90 values, the IC50 value is
comparable to that observed for 7 (both about 10- to 20-fold high-
er than those for RMP, RMP S and Rif S). All rifamycin analogues
have MTB MIC90 values that are higher than their IC50 values for
the MTB RNAP with the exception of 4i. This differential between
IC50 and MTB MIC90 may be due in some cases (where the differen-
tial is large) to the molecules not equilibrating efficiently across
the cell envelop or to the more trivial explanation (in cases where
the differential is small) that the IC50 is a 50% inhibition point
whereas the MIC90 is a 90% inhibition point. In the case of 4i, the
analogue has an MTB MIC90 that is approximately eight-fold smal-
ler than the IC50. One possibility is that the cycloheptyl moiety of 4i
may help to concentrate the analogue within the MTB cell. No
analogues tested here result in a more potent rifamycin derivative
against the MTB RNAP (S450L) mutant.
>4.8
3.9
4.0
RMP
RMP
RMP
RMP
>4.7
>4.6
>4.6
>4.4
3.8
0.13
0.39
0.66
0.64
N-(2-propenyl)amino
(2-Hydroxyethyl)amino
Benzylamino
Cycloheptylamino
Hydroxyl
RMP
N/Ab
N/A
N/A
>128
2.3
4.3
Moxifloxacin
a
The MIC90 is defined as the minimum concentration of the compound required
to inhibit 90% of bacterial growth.
b
N/A = not applicable.

6098
Y. Jin et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6094–6099
Table 3
The IC50 values have also been previously determined for the
Escherichia coli RNAP.21 It was observed that both MTB and E. coli
enzymes exhibited similar trends, with IC50 values in the 10À9
PXR activity of selected rifamycin analogues
b
M)a
EMAX (fold increase)
M
No.
EC50
(l
(nM) range for commercially available rifamycins tested. This
observation is consistent with previous postulates that the lower
sensitivity of Gram-negative bacteria to rifamycins is due to the
removal of rifamycins from the interior of the cell via TolC-depen-
dent efflux pumps. To probe that same efflux activity with our
novel analogues, we have determined the MIC90 values of a subset
1
5.6
9.5
6.9
nda
8.9
8a
9a
10a
2.1
ndac
4.1
a
EC50 is defined as the half maximal effective concentration.
ECMAX is the maximal effective concentration of the compound. (The experi-
mental conditions are reported in the Supplementary data).
b
of our compounds using WT E. coli strains (TG2 and DH5a) and a
c
nda: no detectable activation of hPXR at concentrations up to 100 lM.
mutant E. coli strain (EC2880-‘permeable’ strain, tolCÀ and impÀ).
Generally, the mutant E. coli strain is more sensitive than the WT
strains to tested rifamycins. Interestingly, the MIC90 value for com-
pound 3 is only four-fold greater than that observed for Rif S. This
parallels compound 3’s IC50 value, which is 3–4 higher than that for
Rif S. The results in Table 2 indicate that the rifamycins tested are
subject to Gram-negative efflux pump activity.
To gain better understanding of the SAR, we carried out struc-
ture-based modeling studies. Modeling was based on the 2.5 Å res-
olution structure of rifabutin complexed with the Thermus
thermophilus RNAP holoenzyme (PDB ID: 2a68).22 Since the rifamy-
cin binding site is highly conserved among bacteria, this structure
provides a good foundation for understanding how proposed rifa-
mycin analogues may interact with the MTB RNA polymerase.
Preparation of the structure before modeling is described in the
Supplementary data and was conducted using MOE.23
Given the size of the rifamycins, size of the binding site, and the
flexibility of the ansa ring, accurate docking of these inhibitors to
the RNAP complex would be challenging using standard docking
approaches. Since most of the rifamycin structure remains
unchanged, modeled poses of the analogues were generated by
mutating rifamycin to the analogue and then relaxing the complex
through a series of energy minimizations as described in the
Supplementary data.
Modeled poses were generated for each of the structures listed
in Table 2. A QSAR model (not shown) using interaction energies
between the inhibitor and RNAP complex and two other ligand
descriptors produced a very good R2 of 0.90 and a cross validation
R2 of 0.79. The good fit of the QSAR model based on energies from
the structure-based modeling supports the accuracy of the latter.
The robustness of our structure-based modeling has also been sup-
ported by experimental studies where sensitivity to rifamycins
against binding site mutants qualitatively correlates with our mod-
els.21 The QSAR model will be presented elsewhere.
Figure 2. Selected rifamycin analogues versus PXR activation.
aerobic and anaerobic conditions show that our modification of the
8-OH position of the parent scaffolds results in diminished activity.
The enzymatic and microbiological data are consistent with mod-
eling and computational studies which support the C-8 hydroxyl
acting as a hydrogen bond acceptor with S450 and that Rif resis-
tance in the S450L mutant is due to loss of this hydrogen bond.
Data on representative analogues of this study suggest that only
the pyrazole modification to the rifamycin parent scaffold mini-
mizes hPXR activation.
Acknowledgments
Modeled poses for Rif S (2) and amino analogue (4i) are shown
in Figure 1. RNAP is shown as a molecular surface within 5 Å of the
inhibitors and shaded to indicate areas of lipophilicity (green),
hydrogen bonding (magenta) and mild polar (blue). This figure
illustrates how the loss of the C-8 hydroxyl is a potential loss of
a hydrogen bond contact with RNAP.
We acknowledge generous support by the University of Michi-
gan College of Pharmacy Ella and Hans Vahlteich and UpJohn
Research Funds. We would also like to acknowledge additional
funding by the University of Michigan Office of the Vice President
for Research, and the Rackham Graduate School.
One of the clinical liabilities of RMP (1) includes its induction of
cytochrome P450 3A4 (CYP3A4), which is mediated by the Human
Pregnane X Receptor (PXR).12 Therefore we utilized a commercially
available assay kit24 to examine if representative analogues (8a, 9a,
and 10a) activate the PXR. The results (Table 3, Fig. 2, S-Fig. 2)
show that the methylamino derivatives of RMP S (8a) and RMP
(10a) do activate the PXR to an extent very similar to that for
rifampin, whereas the RMP S pyrazole derivative (9a) does not
exhibit significant hPXR activation at concentrations up to
Supplementary data
Supplementary data (details of the chemical syntheses, the
in vitro RNAP assays, molecular modeling, and the human preg-
nane X receptor (PXR) activation assay) associated with this article
can be found, in the online version, at doi:10.1016/j.bmcl.2011.
08.054.
100 lM. From these data, we cannot yet address whether these
analogues are Cyp inhibitors or substrates.
References and notes
In summary, we have synthesized a novel series of rifamycin S
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and 1,8-pyrazole substituents. Screening the compounds for
inhibition of WT M. tuberculosis (MTB) RNAP and rifamycin-resis-
tant MTB RNAP (S450L) as well as antitubercular effects under both
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