Structure-activity relationship of new anti-tuberculosis agents derived from oxazoline and oxazole benzyl esters

Article abstract of DOI:10.1016/j.ejmech.2009.12.074

During the syntheses and studies of natural iron chelators (mycobactins), we serendipitously discovered that a simple, small molecule, oxazoline-containing intermediate 3 displayed surprising anti-tuberculosis activity (MIC of 7.7?μM, average). Herein we

Full text of DOI:10.1016/j.ejmech.2009.12.074

European Journal of Medicinal Chemistry 45 (2010) 1703–1716
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure–activity relationship of new anti-tuberculosis agents derived from
oxazoline and oxazole benzyl esters
Garrett C. Moraski a, Mayland Chang a, Adriel Villegas-Estrada a, Scott G. Franzblau b, Ute Mo¨llmann c,
,
Marvin J. Miller a
*
a Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46656, USA
b Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA
c Leibniz Institute for Natural Product Research and Infection Biology – Hans Knoell Institute, Beutenbergstrasse 11a, 07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
During the syntheses and studies of natural iron chelators (mycobactins), we serendipitously discovered
Received 29 July 2009
Received in revised form
21 December 2009
Accepted 23 December 2009
Available online 14 January 2010
that a simple, small molecule, oxazoline-containing intermediate 3 displayed surprising anti-tuberculosis
activity (MIC of 7.7
syntheses and evaluation of a hundred oxazoline- and oxazole-containing compounds derived from an
efficient three step process: 1) formation of -hydroxy amides with serine or threonine; 2) cyclization to
mM, average). Herein we report elaboration of SAR around this hit as well as the
b
afford oxazolines; and 3) dehydration to give the corresponding oxazoles. A number of compounds
prepared by this method were shown to possess impressive activity against Mycobacterium tuberculosis,
extremely low toxicity and therefore high therapeutic indexes, as well as activity against even the more
recalcitrant non-replicating form of M. tuberculosis. The uniqueness of their structures and their
simplicity should allow them to be further optimized to meet ADME (absorption, distribution, metab-
olism, excretion) requirements. The syntheses of eight of the most potent in vitro compounds were scaled
up and the compounds were tested in an in vivo mouse infection model to evaluate their efficacy before
engaging upon more elaborate compound design and optimization.
Keywords:
Aryl oxazoline
Aryl oxazole
Benzyl ester
Tuberculosis agents
Structure–activity relationships
Mycobactins
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
multidrug-resistant (MDR) and, more recently, extensively drug
resistant (XDR) forms of TB have further stimulated research efforts
The battle against tuberculosis (TB, caused by Mycobacterium
tuberculosis) has raged for millennia. [1,2] This disease has claimed
the lives of over one billion people and currently infects one third of
the world’s population. With 1.7 million deaths in 2006, TB as
a single causative agent is the leading killer among infectious
diseases [3]. The spread of TB was temporarily interrupted with the
advent of several chemotherapeutic agents [2]. However, since the
1980s TB has been on the rise. Presently, over 9 million new cases
are reported annually [3]. The rise in TB/HIV co-infection incidence
and the spread of strains of TB that are resistant to at least two
frontline anti-TB drugs (multidrug-resistant TB or MDR-TB) [1–7]
has compounded the challenge for treating new cases. Today only
sustained aggressive chemotherapy treatment with a combination
of drugs can stop the disease [8]. This increase in infection has
become so alarming that in 1993 the World Health Organization
(WHO) [9] declared TB a global emergency. The development of
globally [10]. More than ever, there is an urgent need to develop
new anti-TB drugs to combat the spread of TB, particularly in its
hard-to-kill multidrug-resistant, persistent and latent forms.
One particular strategy to combat TB involves exploiting its need
for iron as illustrated by our design, syntheses and studies of
siderophore analogs and siderophore-drug conjugates [11–15]. For
example, we have demonstrated that synthetic mycobactin S (1),
with an (S)-methyl group configuration in the butyrate linker
region, inhibits the growth of Mycobacteria tuberculosis H37Rv
while mycobactin T, with an (R)-methyl group configuration,
promotes growth (Fig. 1) and substitution of the butyrate linker of
the natural mycobactins with
a
a-Boc-protected
a,b-dia-
minopropionate linker (2) resulted in potent inhibition of TB
growth.
While effective, this approach involves the syntheses of the
structurally complex mycobactin-family [16–18] of siderophores in
order to exploit TB’s essential iron assimilation processes.
Throughout the syntheses of these siderophore natural products,
protected and non-protected intermediates were routinely tested
for anti-TB activity. This led to the discovery of ND-005859, 3,
* Corresponding author. Tel.: þ574 631 7571; fax: þ574 631 6652.
E-mail address: mmiller1@nd.edu (M.J. Miller).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.074

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G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
2. Results and discussion
The goal of this study was to first produce a series of derivatives
of compound 3 (Fig. 2, Series 1) to explore structure–activity rela-
tionships (SAR) around the head group of the oxazoline core fol-
lowed by conversion of all active oxazoline analogs to the
corresponding oxazoles and comparison of their activity.
Initially, focus was placed on modifications of the head group
region (‘‘series 1’’ analogs, Fig. 2) in order to determine possible SAR
trends, increase overall potency and identify an appropriate
scaffold for eventual further elaboration of SAR by ‘‘tail’’ region
(series 2) modifications. The ultimate goal is to combine the most
potent head groups, as described in this document, with the most
potent tail groups of the series 2 compounds (work to be reported
in a future publication) and produce potentially the most potent
and metabolically stable, ‘‘drug like’’ series 3 compounds. Thus,
a relatively small set of series 1 compounds was prepared while
also considering drug-like parameters [molecular weights under
500, a predicted LogP between 2.5 and 4.5, <5 hydrogen bond
donors and <10 hydrogen bond acceptors]. It should be noted that
this was not a combinatorial approach. Rather, it was intended to be
a methodical parallel approach to create a precise, well character-
ized set of compounds with properties intended to fall within the
calculated range of the molecular physiochemical profile of the
currently used anti-TB drugs [26]. Syntheses of all compounds were
Fig. 1. Mycobactins T and S and the synthetic anti-TB analogs (diaminoproprionate
siderophore, ND-005859, and ND-005862).
a simple, non-iron binding dibenzyl-protected oxazoline, which is
a precursor in the syntheses of Mycobactin S and T, as well as the
diaminoproprionate siderophore analog (2, Fig. 1). When tested in
vitro, the structurally simple compound 3 displayed notable anti-TB
activity (MIC of 7.7
mM [19]), and furthermore was non-toxic
(>128 M) for VERO cells (green monkey kidney cell line). Indeed
m
the potency of this serendipitous ‘‘hit’’ (compound 3) compared
favorably to the natural Mycobacterium smegmatis siderophore
Mycobactin S [20] (1) or the amide analog 2 which boast MIC’s of
3.1
mM and 0.2 mM, respectively [21]. However, after benzyl
deprotection of compound 3, the resulting iron binding analog 4
(ND-005862) lost all activity. This suggested that iron assimilation
might not be involved in the anti-TB mode of action of this core
structure, though it is possible that the benzyl ester of 3 could serve
as a TB membrane permeable ‘‘prodrug’’ of the corresponding
carboxylic acid which, in turn, might competitively inhibit side-
rophore biosynthesis [22–25]. While the mechanism of action is
currently under investigation, the initial goal of this research was to
explore structure–activity relationships of this structurally simple
lead compound in order to improve activity while also providing
additional compounds for future mode of action studies.
Given the simplicity of the lead compound, its effectiveness
against TB in vitro, and a possibly new mechanism of action, we
sought to explore further the potential of compounds of this class
as anti-TB agents. Here we report results from the syntheses,
characterization, and screening of over 100 compounds against TB.
A series of eight compounds were further examined for their
toxicity and efficacy in an in vivo mouse model of TB. These simple
compounds show promise for future development as a new class of
anti-TB agents.
Fig. 2. Derivatization strategy of 3, an anti-TB ‘‘hit’’ compound.

G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
1705
easily scaled up for in vivo testing due to a short straight forward
synthetic plan that involved an EDC mediated coupling of the
desired benzoic acid 5 with -serine benzyl ester and then dehy-
drative cyclization of the -hydroxy amide 7 with bis(2-methox-
L
b
yethyl)amino-sulfur trifluoride (DAST) as reported by Wipf et al.
[27] to form the oxazoline core 8 as products 12–56 (Scheme 1).
Additionally, analog 57 was easily prepared by reduction of nitro
analog 28 with indium, ammonium chloride and ethanol at 100 ꢀC.
Using this effective procedure, over 45 oxazolines (8) were
prepared in overall good yields (as described in the experimental
section).
Additionally, when an oxazoline compound displayed some
activity against TB, it was immediately converted to the corre-
sponding oxazole by a mild BrCCl3/DBU oxidation [28] procedure
(Scheme 2). Overall, the oxazole analogs, 58–100, were, on average,
more potent than the corresponding oxazolines despite having
a simple, ‘‘unoptimized’’, benzyl ester tail moiety. Additionally,
when scale up was required for in vivo testing the initial amide
coupling was performed with an easily prepared or purchased acid
chloride 6 resulting in fast, efficient production of multi-gram
quantities of 15, 29 and others.
To evaluate the importance of substitution on the oxazoline and
oxazole cores a small set of threonine derived analogs were
prepared utilizing the EDC coupling of the desired benzoic acid 5
with threonine benzyl ester. Then cyclization of general structure 9
with DAST gave the methyl substituted oxazolines 10 as analogs
101–105. Lastly, the corresponding oxazoles (11) were produced by
the BrCCl3/DBU oxidation to give final compounds 106–111
(Scheme 3).
Scheme 2. Conversion of oxazoline analogs to oxazoles. aReagents: (a) DBU, BrCCl3,
DCM, 0 ꢀC to RT, 1–14 h.
substitution (17) compared to the 2-chloro (16) and 4-chloro (18)
analogs (1.80
mM, 15.2 mM and 14.9 mM in the GAST medium,
respectively). However, analogs with other electron withdrawing
groups like the 3-fluoro (25), 3-bromo (54), and 3-cyano (53) were
all less active (6.86, 52.5, and 75.1
respectively) than those with the 3-chloro substitution (17, 1.80
in the GAST medium). Incorporation of the electron donating,
3-methoxy group (52, 42.5 GAST medium) significantly
decreased activity relative to the 3-chloro analog (17, 1.80 M in
GAST medium). Similarly, analog 57 with the 4-amino group
(26.8 M in the GAST medium) was considerably less active than 4-
nitro analog 28 (7.59 M in the GAST medium). It was also evident
mM in the GAST medium,
mM
mM
m
m
m
that the benzyl ester was important for activity since the corre-
sponding methyl ester analogs 12,19, and 27 were considerably less
active. Similarly, homologation between the oxazoline core and the
phenyl head group was not tolerated as shown by diminished
activity of analogs 43 and 44. The most potent oxazoline
compounds contained two substituents on the phenyl ring. For
instance, 4-fluoro, 3-nitro analog 29 was very active with an MIC of
0.95
substituents relative to 29, was 3-fold less active (3.7
GAST medium) and further displacement, as in analogs 32 and 33,
resulted in additional reduction of activity (40.4 and 8.7 M in the
GAST medium, respectively). It should also be noted that
compounds 29 and 30 showed moderate activity (26 M and
59 M, respectively) in the LORA assay. This assay was specifically
designed to simulate non-replicating M. tuberculosis during
persistence and/or latency. Analog 51 with the 4-benzyl and 3-
fluoro substituents was the most active in this series with MIC’s of
m
M in the GAST medium! Compound 30, with a reversal of the
To determine structure–activity relationships (SAR) around our
hit compound 3, the previously mentioned rational design
approach was carried out with variation of the head group to
produce 47 oxazoline compounds which were tested for inhibition
of M. tuberculosis H37Rv (Table 1) in two different culture media,
GAS and GAST. GAST contains Tween 80 and is a more iron deficient
medium than the GAS (which has added iron but lacks the Tween
80). Surprisingly, these different media had statistically relevant
affects upon the MIC of our compounds as well as on the positive
mM in the
m
m
m
control rifampin (MIC of 0.11 mM and <0.01 mM in the GAS to GAST
0.77
validate the importance of the heterocyclic core, all oxazoline
precursors of general structure 7 (non-cyclized -serine amides)
were screened and the overwhelming majority of compounds were
found to be inactive (>128 M).
mM and 1.4 mM in the GAS and GAST assays. Additionally, to
assays, respectively). Interpretation of the anti-TB data along with
the VERO cytotoxicity assay data assisted in our selection of
compounds to advance for testing in vivo.
L
The SAR of the oxazoline compounds showed an overwhelming
m
increase in potency when the phenyl ring had
a 3-chloro
Scheme 3. Preparation of threonine-derived oxazoline and oxazole analogs.
aReagents: (a) L-Threonine-OBn, EDC, DMAP, ACN, room temp, 14 h; (b) Oxalyl chloride,
DCM, DMF (catalytic), 4 h, room temp; (c) L-Threonine-OBn, DIPEA, DCM, 14 h, 50 ꢀC
(d) DAST, K2CO3, ꢁ78 ꢀC, 1 h (e) DBU, BrCCl3, DCM, 0 ꢀC to RT, 1 h.
Scheme 1. Synthesis of oxazoline analogs. aReagents: (a) L-Serine-OBn, EDC, DMAP,
ACN, room temp, 14 h; (b) Oxalyl chloride, DCM, DMF (catalytic), 4 h, room temp; (c)
L-Serine-OBn, DIPEA, DCM, 14 h, 50 ꢀC; (d) DAST, K2CO3, ꢁ78 ꢀC, 1 h.

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G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
Table 1
SAR of oxazoline (series 1) compounds.
Compounds
R1
R2
R3
Mol wt
ClogPb
GAS (
12.6a
>128
118
>128
>128
m
M)
GAST (
7.69a
>128
>128
>128
>128
m
M)
VERO Cells: IC50 (
m
M)
NRP-TB: LORA (
67.3
mM)
3
4
H
H
H
H
H
H
H
H
2-OBn
2-OH
2-OBn
H
2-OH
H
2-Cl
3-Cl
H
3-Cl
3-Cl
H
H
3-Cl
3-F
3-F
3-F
Bn
H
Me
H
387.44
207.19
311.34
191.19
221.21
281.31
315.75
315.76
315.75
239.66
225.63
311.34
325.36
345.78
329.32
299.3
333.74
250.21
326.31
344.29
344.29
421.87
344.29
344.29
5.77
4.06
4.06
1.93
1.71
4.08
4.79
4.79
4.79
3.09
3.09
4.00
4.53
4.62
4.08
4.22
4.94
2.12
3.82
3.67
3.67
6.39
3.97
3.97
>128
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Me
Bn
Bn
Bn
Bn
Me
H
Bn
Bn
Bn
Bn
Bn
Bn
Me
Bn
Bn
Bn
Bn
Bn
Bn
7.20a
49.9
11.6
61.0
>128
>128
26.1
60.6
60.7
50.5
60.7
52.0
4.42a
15.2
1.80
14.9
68.7
106
2.29
30.5
7.58
>128
>128
63
4-Cl
H
H
4-OMe
4-OEt
4-OMe
4-OMe
H
4-Cl
4-NO2
4-NO2
4-F
>128
3.91
6.86
6.49
H
H
>128
30.6
6.24
30.2
6.30
>128
7.59
0.95
3.68
34.0
40.4
8.70
>128
>128
>128
3-NO2
3-F
2-OBn
2-NO2
2-F
26
59
4-NO2
3-Cl
4-F
30.7
13.0
4-NO2
34
35
36
390.45
374.39
361.19
5.50
4.68
3.53
17.8
58.8
27.6
22.9
13.4
>128
26.6
21.1
37
38
39
361.19
282.29
282.29
3.53
2.58
2.58
110
127
53.3
92.9

G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
1707
M)
Table 1 (continued)
Compounds
R1
R2
R3
Mol wt
282.29
ClogPb
2.58
GAS (
mM)
GAST (
m
M)
VERO Cells: IC50 (
m
M)
NRP-TB: LORA (m
40
55.6
81.3
41
42
283.28
341.36
1.63
3.74
61.2
39.0
>128
4-OMe
3-OMe
Bn
21.6
43
44
295.33
325.36
3.26
4.01
>128
>128
>128
>128
45
46
47
48
49
50
51
52
53
54
H
2-OMe
H
H
H
H
2-OBn
3-F
3-OMe
3-CN
3-Br
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
311.33
339.39
339.39
353.41
295.33
432.43
405.42
311.33
306.32
360.20
4.00
5.06
4.84
5.59
4.58
5.81
5.85
4.00
3.51
4.94
37.0
2.97
3.2
29.2
22.4
14.9
5.11
27.3
4.86
1.42
42.5
4-OPr
4-OiPr
4-OBu
4-CH3
4-NO2
4-OBn
H
2.72
15.5
15.6
0.77
24.8
47.2
24.2
>128
H
H
75.1
52.5
55
296.32
3.08
73.0
122
56
358.39
4.47
2.85
12.5
25.4
29.8
26.8
57
4-NH2
H
Bn
296.32
822.94
Rifampin (control)
0.06a
0.01a
125a
a
Average of assay repeats.
b
CLogP was calculated using the Cambridge Soft ChemDraw Ultra, v 8.0 software. Activity against non-replicating M. tuberculosis was determined using the Low Oxygen
Recovery Assay (LORA).
Next, since many of the oxazoline analogs showed relatively
potent anti-TB activity, the corresponding oxazole derivatives
were produced to test what effects a planer, aromatic heterocy-
clic core would have on anti-TB activity (Table 2). Remarkably, the
majority of oxazoles synthesized had improved in vitro activity,
with seven compounds 59, 65, 66, 68, 70, 78 and 89 having sub
oxazoles with significant activity (76, 77, 79 as the 2-pyridyl,
3-pyridyl, 4-pyridyl, respectively) with MIC’s between 1 and
4
m
M
in the GAS assay, whereas the analogous oxazoline
compounds (38, 39, and 40) had weak activities at best
(MIC’s > 50 M in the GAS medium). In contrast, oxazoline 51
(MIC’s w1 M in both assays) was three-fold more active than the
corresponding oxazole, 72 (MIC w3 M in both assays). As with
m
m
micromolar (<1
m
M) MIC’s! Interestingly, there was little differ-
m
ence in activity of the ortho-, meta-, and para-methoxy
substituted analogs (93, 94, and 60) and the corresponding
chloro derivatives (68, 59, and 87), though the ortho- and meta-
chloro compounds were the most active. Of these, most
compounds tested were active in the LORA non-replicating assay
the oxazoline core, removal of the benzyl ester or trans-
esterification to the methyl, ethyl or t-butyl esters resulted in
nearly complete loss of activity (data not shown). This loss of
activity after transesterification as was surprising considering
that the ethyl ester was key to the potency in a series of related
3,5-disubstituted isoxazolines reported by Lee et al. [29] and the
quinoline-isoxazole series described by Kozikowski et al. [30].
and were non-toxic in the VERO cytotoxicity assay (>121
mM).
Replacement of the phenyl ring with a pyridyl substituent gave

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G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
Next, in an attempt to determine whether simple methyl
test compounds. After two days of no treatment (in order to reduce
the probability of carry over of lung tissue-associated compound on
the plating medium), on day 85 post-infection mice were sacrificed
and lung tissue homogenates plated to determine bacterial load.
Whereas the rifampin control effected a reduction in bacterial load
of greater than 2 logs versus the untreated control, none of the test
compounds showed efficacy with this dosing regimen and mode of
administration due potentially to the metabolic liability of the
benzyl ester moiety. The stability of the benzyl ester was then
evaluated in vitro in simulated gastric fluid and rat liver micro-
somes. Not surprisingly, the benzyl esters were rapidly cleaved to
their corresponding inactive carboxylic acids in simulated gastric
fluid assay (Table 5).
Furthermore, these compounds were metabolically unstable
(Table 5). The in vitro metabolism of 15 was further studied and the
major metabolite was the corresponding carboxylic acid, providing
an additional reason why these fairly potent in vitro compounds
failed to show in vivo efficacy. As such, plans are now underway to
improve the pharmacokinetic properties of the lead compounds
through the use of more metabolically stable replacements of the
simple benzyl esters to hopefully impart enhanced in vivo stability
and possibly efficacy. The experimental section profiles the
syntheses and characterization of these eight compounds evaluated
in vivo, as well as by microsomal and simulated gastric fluid assays.
substitution on the oxazoline (Table 3) and oxazole (Table 4) cores
would affect anti-TB activity, a small focused set of threonine-
derived compounds was prepared and tested. Preliminary results
indicated that methyl substituted oxazoline compounds were
significantly less active compared to the analogous oxazoline
parents. For instance, compounds 102 and 103 were completely
inactive (>128
4.42 M in the GAST assay, respectively). The threonine-derived
oxazole compounds also had greatly diminished activity, as seen,
for example, by comparison of compound 108 (>128 M) to
compound 3 (7.69 M in the GAST assay). In fact, the more potent
mM) relative to compounds 3 and 15 (7.69 mM and
m
m
m
head group analogs, 109, 110, and 111 all had diminished anti-TB
activity as methyl substituted oxazole analogs compared to the
non-methyl bearing oxazole analogs (65, 60, 66). Furthermore,
compound 109 had greatly increased toxicity in the VERO assay. In
short, even simple methyl substitution on the oxazoline and oxa-
zole cores resulted in diminished potency and often increased
toxicity toward the VERO cells.
Encouraged by the anti-TB data, several compounds were
subsequently tested against a diverse panel of nontuberculosis
mycobacteria and other bacteria (Gram positive and negative) as
well as select strains of yeast. This panel included Bacillus subtilis
ATCC 6633, Staphylococcus aureus SG 511, Mycobacterium luteus
ATCC 10240, E. coli SG 458, Proteus vulgaris OX 19, M. smegmatis SG
987, Mycobacterium aurum SB 66, M. aurum DSM 43536, Myco-
bacterium vaccae 10670, Mycobacterium fortuitum B, Spor-
obolomyces salmonicolor SBUG 549, Candida albicans C.A.,
Glomerella, and Mycobacterium avium strains MAC 100, 101, 108,
109, 116. In all cases, compounds 1, 15, 29, 60, 63, 65, 66, and 68
were inactive (MIC’s equal to negative control) despite showing
some zones of inhibition in the agar diffusion assay against
M. vaccae 10670 and M. fortuitum B. This remarkably selective
inhibition of TB suggests a unique mode of action for the slower
4. Conclusion
On a planet in which over 1/3 of the population is infected with
tuberculosis, it is more than ever prudent to evaluate new anti-
tuberculosis agents in vitro and in vivo. Herein we described simple
serine derived oxazoline and oxazole templates with highly selec-
tive anti-TB activity. SAR studies demonstrate that, in general,
oxazoles are more potent than their oxazoline precursors and that
alkyl substitution on either the oxazoline or oxazole diminishes
anti-TB activity. Activity can also be influenced by the type and
position of substituents on the pendant phenyl group, with electron
withdrawing chloro and combination of chloro and nitro groups
being somewhat advantageous though clear correlations require
more study. The corresponding acids and simple alkyl esters are
substantially less active than the corresponding benzyl esters.
While the metabolic lability of the benzyl esters precludes their use
as orally active agents, many of these agents like compound 65
growing M. tuberculosis.
A microarray experiment [31] with
compound 3 revealed up regulation of the Mycobacterium tuber-
culosis genes (responsible for mycobacin/exochelin synthesis)
coupled with the down regulation of the bacterioferritin (bfr) genes
(data not shown). These results suggested a condition of iron stress,
possibly due to inhibition of the mycobacterial siderophore
biosynthesis pathway and in turn interference of iron trafficking
[22–25]. Further mode of action studies are ongoing and will be
reported in due course.
(with an MIC of <0.5
mM against replicating M. tuberculosis H37Rv)
can be made on multiple gram scale in just three steps, thus further
studies to extend both SAR and ADME properties are merited.
3. Animal testing
In vivo studies to assess toxicity were performed using
compounds 3, 15, 29, 60, 63, 65, 66, and 68. Mice were given
5. Experimental section
100 mg/kg po once a day in 100
m
l 0.5% carboxymethylcellulose
All anhydrous solvents, reagent grade solvents for chromatog-
raphy and starting materials were purchased from either Aldrich
Chemical Co. (Milwaulkee, WI) or Fisher Scientific (Suwanee, GA).
Water was distilled and purified through a Milli-Q water system
(CMC) for five days, followed by 2 days of rest and then 200 mg/kg
for five days. All compounds were well tolerated with respect to
body weight, gross appearance and behavior with the exception of
compound 66 where the average weight loss was 12% at the end of
the two week period (from 21 g to 18.5 g). Subsequently, all
compounds were tested for efficacy in BALB/c mice infected with M.
tuberculosis. At days 5, 60 and 85 following a lose dose aerosol
infection with M. tuberculosis Erdman, mean cfu (SD) in mouse
lungs of untreated controls were 2.3 (2.5) ꢂ 102, 1.8 (1.0) ꢂ 106 and
4.7 (2.3) ꢂ 106, respectively. Beginning on day 60 post-infection,
groups of 8 mice received a single oral dose at 200 mg/kg (except
compound 66 which was dosed at 50 mg/kg due to a 10% loss in
(Millipore Corp., Bedford, MA). L-Glutathione reduced form (GSH)
and nicotinamide adenine dinucleotide phosphate reduced form
(NADPH) were obtained from Sigma–Aldrich (St. Louis, MO). Male
Sprague–Dawley rat liver microsomes were purchased from BD
Gentest (Woburn, MA). General methods of purification of
compounds involved the use of silica cartridges purchased from
AnaLogix, Inc. (Burlington, WI; www.ana-logix.com) and/or
recrystallization. The reactions were monitored by thin-layer
chromatography (TLC) on precoated Merck 60 F254 silica gel plates
and visualized using UV light (254 nm) reported in Rf values. All
compounds were analyzed for purity and characterized by 1H and
13C NMR using Varian 300 MHz NMR and/or Varian 500 MHz NMR
body weight during tolerance testing) in 200 mL of 0.5% CMC 5 days
per week for three weeks. Untreated controls received the CMC
vehicle only and the positive drug control consisted of rifampin
(RMP) at 10 mg/kg dosed in an identical manner and vehicle as the
spectrometers. Chemical shifts are reported in ppm (d) relative to

G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
1709
Table 2
SAR of oxazole compounds.
Compounds
R1
R2
R3
MolWt
ClogPb
GAS (
m
M)
GAST (
42.3
0.99
1.80
m
M)
VERO Cells: IC50 (
mM)
NRP-TB: LORA (mM)
58
59
60
4-NO2
H
4-OMe
H
3-Cl
H
OBn
OBn
OBn
324.29
313.74
309.32
3.49
4.19
3.49
1.79
1.25
1.72
>128
>128
61
62
383.36
372.8
3.13
4.09
35.8
49.5
>128
118
48.1
10.3
63
64
65
66
67
68
69
70
71
72
H
3-Cl
H
4-F
4-NO2
H
2-OBn
2-OBn
H
3-NO2
3-F
2-Cl
2-NO2
2-F
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
385.41
419.86
279.29
342.28
342.28
313.74
342.28
342.28
430.41
403.4
4.69
5.26
3.45
3.34
3.34
3.94
3.64
3.64
4.68
5.27
62.6
5.40
0.47
1.10
>128
>128
0.49
121
>128
>128
>128
>128
>128
3.80
31.9
0.48
2.66
1.69
9.90
1.65
35.4
0.73
2.40
0.70
11.4
11.6
4-F
4-NO2
4-NO2
4-OBn
2-OBn
3-F
>128
>128
3.03
3.86
73
74
359.17
359.17
2.96
2.96
2.37
2.24
>128
>128
>128
2.54
1.84
25.1
75
76
77
294.30
280.28
280.28
2.55
2.25
2.04
3.01
2.00
3.82
10.2
2.47
6.20
(continued on next page)

1710
Table 2 (continued)
G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
Compounds
R1
R2
R3
MolWt
356.37
ClogPb
3.93
GAS (
m
M)
GAST (
m
M)
VERO Cells: IC50 (
m
M)
NRP-TB: LORA (mM)
78
79
80
1.49
1.42
0.98
6.10
>128
280.28
420.44
2.04
3.74
66.2
>128
81
82
83
388.44
389.44
281.27
4.98
3.59
1.29
8.00
6.86
7.32
>128
91.4
22.5
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
H
2-PO(Ph)2
3-OMe
H
H
H
3-F
3-Cl
3-F
3-F
2-OMe
3-OMe
3-CN
3-Br
H
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
479.86
339.34
323.34
313.74
294.3
297.28
343.76
331.73
327.31
309.32
309.32
304.3
358.19
293.32
337.37
337.37
351.40
822.94
4.22
3.20
4.02
3.99
2.63
3.59
3.87
4.15
3.47
3.49
3.49
2.92
4.34
3.97
4.54
4.32
5.07
61.8
61.5
17.5
1.97
7.77
3.67
1.42
5.33
4-OMe
4-OEt
4-Cl
4-NH2
H
4-OMe
4-Cl
4-OMe
H
H
H
H
4-CH3
4-OPr
4-OiPr
4-OBu
16.0
15.5
18.9
>128
>128
>128
>128
>128
29.7
56.2
68.6
1.98
0.91
2.35
2.87
5.55
3.88
6.04
22.4
6.99
7.54
3.76
>128
25.2
60.5
1.93
12.84
3.48
6.08
7.68
1.94
3.21
3.83
0.01a
H
H
H
1.95
1.76
Rifampin (control)
0.06a
125a
a
Average of assay repeats.
b
CLogP was calculated using the Cambridge Soft ChemDraw Ultra, v 8.0 software. Activity against non-replicating M. tuberculosis was determined using the Low Oxygen
Recovery Assay (LORA).
the residual solvent peak in the corresponding spectra; chloroform
7.26 ppm and 77.23 ppm, methanol 3.31 ppm and 49.00 ppm,
methylene chloride 5.32 ppm and 54.00 ppm and coupling
constants (J) are reported in hertz (Hz) (where, s ¼ singlet,
bs ¼ broad singlet, d ¼ doublet, dd ¼ double doublet, bd ¼ broad
doublet, ddd ¼ double doublet of dublet, t ¼ triplet, tt – triple
triplet, q ¼ quartet, m ¼ multiplet) and analyzed using MestReC
NMR data processing.High resolution mass spectra (‘‘HRMS’’) by
fast atom bombardment (‘‘FAB’’) were taken on a JEOL AX505HA
magnetic sector mass spectrometer and values are reported as m/z
Melting points (‘‘mp’’) were measured on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. FTIR(‘‘IR’’)
spectra were obtained using a Thermo-Nicolet IR200 spectrometer
and absorption frequencies were reported in reciprocal centimeters
(cmꢁ1). The high pressure liquid chromatography (‘‘HPLC’’) anal-
yses were carried out on a Waters 2695 instrument consisting of
a photodiode array detector 996, using the Waters symmetry C18
5
mm reverse phase column (Waters, Milford, MA, www.waters.
com). Mobile phases: (a) 50% HPLC grade acetonitrile in Millipore
purified water at a flow rate of 1.0 mL minꢁ1 and UV diction at

G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
1711
Table 3
SAR of threonine-derived oxazoline.
Compounds
R1
R2
R3
Mol Wt
ClogPb
GAS (
mM)
GAST (
m
M)
VERO Cells: IC50 (
m
M)
NRP-TB: LORA (mM)
101
102
103
104
H
H
H
H
H
2-OBn
H
2-NO2
OH
205.21
219.24
401.45
325.35
358.32
822.94
2.44
2.89
6.29
4.51
4.18
>128
>128
>128
>128
>128
>128
37.8
4.11
0.01a
OMe
OBn
OBn
OBn
4-OMe
4-F
54.1
12.5
105
38.1
125a
Rifampin (control)
0.06a
a
Average of assay repeats.
b
CLogP was calculated using the Cambridge Soft ChemDraw Ultra, v 8.0 software. Activity against non-replicating M. tuberculosis was determined using the Low Oxygen
Recovery Assay (LORA).
254 nm (b) 60% HPLC grade acetonitrile in Millipore purified water
at a flow rate of 1.0 mL minꢁ1 and UV diction at 254 nm. A typical
run time was 20 min. The liquid chromatography mass spectrum
(‘‘LC/MS’’) analyses were carried out on Waters ZQ instrument
consisting of chromatography module Alliance HT, photodiode
array detector 2996, and mass spectrometer Micromass ZQ, using
a 3 ꢂ 50 mm Pro C18 YMC reverse phase column. Mobile phases:
10 mM ammonium acetate in HPLC grade water (A) and HPLC
grade acetonitrile (B). A gradient was formed from 5% to 80% of B
in 10 min at 0.7 mL/min. The MS electrospray source operated
at capillary voltage 3.5 kV and a desolvation temperature 300 ꢀC.
Elemental analyses were performed by Midwest Microlabs, LLC
(Indianapolis, IN). All reactions were conducted under argon
unless otherwise noted. Solvents were removed in vacuo on a rotary
(ACN) were treated with N-(3-Dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride (EDC-HCl, 2.9 g, 14.9 mmol, 2 equiv)
and N,N-dimethylaminopyridine (DMAP, 2.3 g, 18.6 mmol,
2.5 equiv). The resulting solution was stirred for 12 h at room
temperature. The reaction mixture was diluted with ethyl acetate
(EtOAc), washed with 0.5 N citric acid (2ꢂ), water and 10% aqueous
NaHCO3 (2ꢂ). The organic phase was collected and dried over
sodium sulfate (Na2SO4), filtered and then concentrated in vacuo.
Crude material obtained was purified by silica gel column chro-
matography with a 1:10–1:1 ethyl aceteate: dichloromethane
(DCM) gradient solvent system.
6.2. Method 2
evaporator. Abbreviations: DCM
¼
dichloromethane; DMF
¼
Benzoyl chloride (4.15 g, 3.4 mL, 28.6 mmol, 1 equiv) was dis-
solved in 120 mL of dichloromethane (DCM, anhydrous) and then
dimethylformamide; ACN ¼ acetonitrile EtOAc ¼ ethyl acetate;
HOAc ¼ acetic acid; DAST ¼ Bis(2-methoxyethyl)amino-sulfur tri-
fluoride; EDC ¼ N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride.
L-serine benzyl ester hydrochloride (7.2 g, 30.1 mmol, 1.05 equiv)
and diisopropylamine (DIPEA, 9.3 g, 12.5 mL, 71.6 mmol, 2.5 equiv)
was added very slowly to the chilled reaction mixture (0 ꢀC, ice bath
temperature). The reaction was warmed to room temperature and
then heated to reflux, 50 ꢀC (bath temperature) for 14 h. The
reaction mixture was cooled, washed with water and then 10%
aqueous NaHCO3 solution (2ꢂ). The organic phase was collected
and dried over sodium sulfate (Na2SO4), filtered and then concen-
trated in vacuo. Crude material obtained was purified by silica gel
column chromatography with a 1:10–1:1 ethyl acetate: DCM
gradient solvent system.
6. General procedure for preparation of serine amides
(7, open L-serine form)
6.1. Method 1
Benzoic acid (1 g, 8.2 mmol, 1.1 equiv) and L-serine benzyl ester
hydrochloride (1.7 g, 7.4 mmol, 1 equiv) in 30 mL of acetonitrile
Table 4
SAR of threonine-derived oxazole.
Compounds
R1
R2
R3
Mol wt
ClogPb
GAS (
m
M)
GAST (
mM)
VERO Cells: IC50 (
mM)
NRP-TB: LORA (mM)
106
107
108
109
110
111
H
H
H
H
H
OH
OH
OBn
OBn
OBn
OBn
203.2
219.2
399.45
293.32
323.34
356.3
2.46
1.49
4.94
3.74
3.76
3.34
>128
>128
28.3
3.92
>128
>128
>128
15.2
15.8
2-OH
2-OBn
H
H
2-NO2
110
35.0
>128
>128
4-OMe
4-F
47.4
15.0
4.62
0.01a
>128
Rifampin (control)
822.94
0.06a
125a
a
Average of assay repeats.
b
CLogP was calculated using the Cambridge Soft ChemDraw Ultra, v 8.0 software. Activity against non-replicating M. tuberculosis was determined using the Low Oxygen
Recovery Assay (LORA).

1712
G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
solid. Rf ¼ 0.44 (DCM); mp 69–70 ꢀC; IR (neat) 1740 (C]O), 1638
(C]N), 1498, 1450, 1257, 1177 cmꢁ1; 1H NMR (300 MHz, CDCl3) 7.82
(1H, dd, J ¼ 8.0, 1.8 Hz), 7.50–7.47 (2H, m), 7.42–7.26 (9H, m), 6.99
(2H, dd, J ¼ 7.7, 6.2 Hz), 5.25 (4H, dd, J ¼ 25.1, 11.8 Hz), 5.01 (1H, dd,
Table 5
Compound stability to simulated gastric juices and microsomes.
Compounds
Gastric stability
Microsomes
% Remaining (2 h)
% Remaining (15 min)
J ¼ 10.6, 8.0 Hz), 4.61 (2H, m); 13C NMR (126 MHz, CDCl3)
d171.03,
3
1.4
0.4
2.2
0.4
4.2
0.5
2.1
4.2
8.4
5.7
13
14
13
15
8.1
8.8
165.65, 157.56, 136.79, 135.40, 132.64, 131.62, 128.50, 128.36, 128.29,
128.25, 127.53, 126.69, 120.69, 120.67, 117.12, 113.75, 70.55, 69.16,
68.77, 67.12; FABMS: 388 (M þ 1); HRMS calcd. for C24H21NO4
15
29
60
63
65
66
68
387.1471, found 387.1458. Elemental calcd. for C24H21NO4 C, 74.40;
25
H, 5.46; N, 3.62; found C, 74.35; H, 5.32; N, 3.60. [
(c ¼ 1.545 g/100 mL CHCl3).
a
]
þ 1.02
D
6.4.1. (S)-Benzyl 4,5-dihydro-2-phenyloxazole-4-carboxylate (15)
Benzoyl chloride (4.15 g, 3.4 mL, 28.6 mmol) was dissolved in
120 mL of dichloromethane (DCM, anhydrous) and then L-serine
6.3. General procedure for preparation of oxazoline products
(compounds 3, 4, 8, 12–57)
benzyl ester hydrochloride (7.2 g, 30.1 mmol) and diisopropylamine
(DIPEA, 9.3 g, 12.5 mL, 71.6 mmol) was added very slowly to the
chilled reaction mixture (0 ꢀC, ice bath temperature). The reaction
mixture was warmed to room temperature and then heated to
reflux, 50 ꢀC (bath temperature) for 14 h. The reaction mixture was
cooled, washed with 10% aqueous NaHCO3 solution (2ꢂ), water,
then washed with 0.5 N citric acid (2ꢂ) and then brine. The organic
phase was collected and dried over sodium sulfate (Na2SO4),
filtered and then concentrated in vacuo to give 7.15 g (83%) of (S)-
benzyl 2-(benzamido)-3-hydroxypropanoate as an oil which
became an off white solid upon standing. 1H NMR (300 MHz, CDCl3)
L
-serine amide 7 (204 mg, 0.68 mmol, 1 equiv) was dissolved in
DCM (5 mL) and cooled to ꢁ78 ꢀC (bath temp, dry ice-acetone).
Bis(2-methoxyethyl)amino-sulfur trifluoride (DAST, 0.1 mL,
0.78 mmol, 1.15 equiv) was added slowly to the cold mixture under
argon and the reaction was stirred for 30 min. Next, K2CO3,
(254 mg, 1.8 mmol, 2.7 equiv) was added and then the reaction was
allowed to warm to room temperature. Once the reaction appeared
to be complete by TLC analysis, the reaction mixture was poured
into 10% aqueous NaHCO3 solution and was extracted with DCM
(2ꢂ). The organic layer washed again with the NaHCO3 solution,
brine, dried over Na2SO4 , filtered and then concentrated in vacuo.
Crude material obtained was purified by silica gel column chro-
matography with a 1:10–1:1 ethyl acetate: DCM gradient solvent
system.
d
7.85–7.79 (2H, m), 7.56–7.30 (7H, m), 7.14 (1H, d, J ¼ 7.1 Hz), 5.25
(2H, s), 4.91 (1H, td, J ¼ 7.1, 3.5, 3.5 Hz), 4.15–4.01 (2H, m).
(S)-Benzyl 2-(benzamido)-3-hydroxypropanoate (7.15 g,
28.5 mmol) was dissolved in DCM (240 mL) and cooled to ꢁ78 ꢀC
(bath temp, dry ice-acetone). Bis(2-methoxyethyl)amino-sulfur
trifluoride (DAST, 4.4 g, 3.6 mL, 27.5 mmol) was added slowly to the
cold mixture under argon where it stirred for 30 min. Then K2CO3
(8.9 g, 64.4 mmol) was added and the reaction mixture was allowed
to warm to room temperature. Once the reaction appeared
complete by TLC analysis, the reaction mixture was poured into 10%
aqueous NaHCO3 solution and extracted with DCM (2ꢂ). The
organic layer was washed again with the NaHCO3 solution, brine,
and then dried over Na2SO4, filtered and concentrated in vacuo.
Crude material obtained was purified by silica gel column chro-
matography with a 1:10–1:1 ethyl acetate: DCM gradient solvent
system to give 5.7 g (85%) of 15 as a white solid. Rf ¼ 0.25 (DCM);
6.4. (S)-Benzyl 2-(2-(benzyloxy)phenyl)-4,5-dihydrooxazole-4-
carboxylate (3)
See our previous studies.[17,18] 2-(Benzyloxy)benzoyl chloride
(13.3 g, 52.3 mmol) was dissolved in 250 mL of DCM and then
-serine benzyl ester hydrochloride (13.1 g, 54.9 mmol) and diiso-
L
propylethylamine (22.8 mL, 130.7 mmol) were added while the
reaction was chilled in an ice bath (0 ꢀC bath temperature). The
reaction mixture was warmed to room temperature and then
heated to reflux, 50 ꢀC (bath temperature) for 14 h. The reaction
mixture was cooled, washed with 10% aqueous NaHCO3 solution
(2ꢂ), water, then washed with 0.5 N citric acid (2ꢂ) and then brine.
The organic phase was collected and dried over sodium sulfate
(Na2SO4), filtered and then concentrated in vacuo to give 21.2 g (99%)
of (S)-benzyl 2-(2-(benzyloxy)benzamido)-3-hydroxypropanoate
as an oil which became an off white solid upon standing. 1H NMR
mp 51–52 ꢀC; IR (neat) 1741 (C]O), 1643 (C]N), 1188 cmꢁ1
NMR (300 MHz, CDCl3) 8.04–7.92 (2H, m), 7.57–7.29 (8H, m), 5.26
(2H, dd, J ¼ 20.9, 12.3 Hz), 4.99 (1H, dd, J ¼ 10.6, 7.9 Hz), 4.80–4.42
(2H, m). 13C NMR (126 MHz, CDCl3)
170.42, 163.45, 158.40, 156.25,
;
1H
d
d
135.56, 135.48, 135.14, 128.67, 128.61, 128.49, 126.70, 124.19, 118.88,
118.71, 70.31, 68.83, 67.60. Elemental calcd. for C17H15NO3: C, 72.58;
25
(300 MHz, CDCl3)
d
8.79 (1H, d, J ¼ 7.1 Hz), 8.20 (1H, dd, J ¼ 7.8,
H, 5.37; N, 4.98; found C, 72.44; H, 5.40; N, 4.95; [
(c ¼ 1.54 g/100 mL CHCl3).
a
]
þ 1.14
D
1.8 Hz), 7.52–7.27 (10H, m), 7.07 (2H, dd, J ¼ 15.9, 8.0 Hz), 5.23–5.14
(4 H, m), 4.93–4.85 (1H, m), 3.92 (2H, dd, J ¼ 3.9, 1.6 Hz).
(S)-Benzyl 2-(2-(benzyloxy)benzamido)-3-hydroxypropanoate
(21.2 g, 54 mmol) was dissolved in DCM (450 mL) and then cooled
to ꢁ78 ꢀC (bath temp, dry ice-acetone). Bis(2-methoxyethyl)amino-
sulfur trifluoride (DAST, 8.2 mL, 62.1 mmol) was added slowly to the
cold mixture under argon where it was stirred for 30 min. Then
K2CO3 (20.2 g, 145.8 mmol) was added and the reaction mixture
was allowed to warm to room temperature. Once the reaction
appeared complete by TLC analysis, the reaction mixture was
poured into 10% aqueous NaHCO3 solution and was extracted with
DCM (2ꢂ). The organic layer washed again with the NaHCO3
solution, brine washed, and was dried over Na2SO4 , filtered and
then concentrated in vacuo. Crude material obtained was purified
by silica gel column chromatography with 1:10 to 1:1 ethyl acetate:
DCM gradient solvent system to give 13.7 g (65%) of 3 as a white
6.4.2. (S)-Benzyl 2-(4-fluoro-3-nitrophenyl)-4,5-dihydrooxazole-4-
carboxylate (29)
4-Fluoro-3-nitrobenzoic acid (6.65 g, 35.2 mmol) was dissolved
in 75 mL of anhydrous DCM and then oxalyl chloride (6.2 mL,
70.4 mmol) was added drop wise followed by the addition of 50 mL
of dimethylformamide (DMF) to catalyze the reaction. The reaction
mixture bubbled and was stirred at room temperature for 4 h.
During this time it became a homogeneous solution. The reaction
mixture was concentrated to give a yellow paste which was
subsequently concentrated in vacuo from toluene to give 4-fluoro-
3-nitrobenzoyl chloride as a crude yellow solid (7.2 g, 94%) which is
used immediately in the subsequent reaction. Rf ¼ 0.5 (EtOAc, red
streak) 1H NMR (300 MHz, CDCl3)
8.40 (1H, ddd, J ¼ 8.8, 4.0, 2.4 Hz), 7.57–7.43 (1H, m).
d
8.85 (1H, dd, J ¼ 7.0, 2.3 Hz),

G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
1713
4-Fluoro-3-nitrobenzoyl chloride (7.2 g, 34.1 mmol) was dis-
solved in 150 mL of DCM and then -serine benzyl ester hydro-
aqueous NaHCO3 solution (2ꢂ), water, with 0.5 N citric acid (2ꢂ)
and then brine. The organic phase was collected dried over sodium
sulfate (Na2SO4), filtered and then concentrated in vacuo The crude
material obtained was purified using silica gel column chroma-
tography with 5%–50% ethyl acetate: DCM as the solvent system to
give (S)-benzyl 2-(4-methoxybenzamido)-3-hydroxypropanoate
(7.65 g, 99%) as a yellow oil which solidified upon standing.
L
chloride (8.56 g, 35.8 mmol) and diisopropylethylamine (14.9 mL,
85.3 mmol) were added while the reaction was chilled in an ice
bath (0 ꢀC bath temperature). The reaction mixture was warmed to
room temperature and then heated to reflux, 50 ꢀC (bath temper-
ature) for 14 h. The reaction mixture was cooled, washed with 10%
aqueous NaHCO3 solution (2ꢂ), water, then washed with 0.5 N
citric acid (2ꢂ) and then brine. The organic phase was collected and
dried over Na2SO4, filtered and then concentrated in vacuo The
crude material obtained was purified using silica gel column
chromatography with 5%–50% ethyl acetate: DCM as the solvent
system to give (S)-benzyl 2-(4-fluoro-3-nitrobenzamido)-3-
hydroxypropanoate (11.1 g, 90%) as a yellow oil which became
a yellow solid upon standing. Rf product ¼ 0.1 (DCM). 1H NMR
Rf ¼ 0.31 (DCM). 1H NMR (300 MHz, CDCl3)
d 1H NMR (300 MHz,
CDCl3)
d ppm 7.83–7.74 (2H, m), 7.44–7.30 (4H, m), 7.07 (1H, d,
J ¼ 7.0 Hz), 6.95–6.86 (2H, m), 5.24 (2H, s), 4.89 (1H, td, J ¼ 7.1, 3.6,
3.6 Hz), 4.05 (2H, dd, J ¼ 3.6, 1.9 Hz), 3.84 (3H, s).
(S)-Benzyl
2-(4-methoxybenzamido)-3-hydroxypropanoate
(7.65 g, 23.3 mmol) was dissolved in DCM (240 mL) and cooled to
ꢁ78 ꢀC (bath temp, dry ice-acetone). DAST (3.6 mL, 4.3 mmol) was
added slowly to cold mixture under argon where it stirred for
30 min. Then K2CO3 (8.73 g, 63.1 mmol) was added and the
reaction was allowed to warm to room temperature. Once the
reaction appeared complete by TLC analysis the mixture was
poured into 10% aqueous NaHCO3 solution and was extracted with
DCM (2ꢂ). The organic layer washed again with the NaHCO3
solution, brine, dried over Na2SO4 , filtered and then concentrated
in vacuo. Crude material obtained was purified by silica gel
column chromatography with a 1:10 to 1:1 ethyl acetate: DCM
gradient solvent system to give 6.74 g (93%) of (S)-benzyl 2-(4-
(300 MHz, CDCl3)
d
8.54 (1H, dd, J ¼ 7.0, 2.2 Hz), 8.13 (1H, ddd,
J ¼ 8.6, 4.1, 2.3 Hz), 7.44–7.30 (5H, m), 7.19 (1H, d, J ¼ 6.86 Hz), 5.33–
5.20 (2H, m), 4.96–4.87 (1H, m), 4.10 (2H, ddd, J ¼ 35.3, 11.2, 3.1 Hz).
(S)-Benzyl 2-(4-fluoro-3-nitrobenzamido)-3-hydroxypropanoate
(5.9 g, 16.3 mmol) was dissolved in DCM (160 mL) and cooled to
ꢁ78 ꢀC (bath temp, dry ice-acetone). DAST (2.45 mL,18.7 mmol) was
added slowly to the cold mixture under argon and the reaction was
stirred for 30 min. Then K2CO3 (6.1 g, 43.9 mmol) was added and the
reaction was allowed to warm to room temperature. Once the
reaction appeared complete by TLC analysis, the mixture was poured
into 10% aqueous NaHCO3 and extracted with DCM (2ꢂ). The organic
layer washed again with the NaHCO3 solution, brine, and then dried
over Na2SO4 , filtered and concentrated in vacuo. Crude material
obtained was purified by silica gel column chromatography with
a 1:10–1:1 ethyl acetate: DCM gradient solvent system to give 4.9 g
(87%) of 29 as a yellow solid upon standing. Rf ¼ 0.18 (DCM); mp 77–
78 ꢀC; IR (neat) 1741 (C]O), 1651, 1541 (C]O), 1350, 1190 cmꢁ1; 1H
methoxyphenyl)-4,5-dihydrooxazole-4-carboxylate,
21,
as
a white solid upon standing. Rf ¼ 0.40 (DCM). 1H NMR (300 MHz,
CDCl3)
d 7.96–7.89 (2H, m), 7.44–7.31 (5H, m), 6.94–6.87 (2H, m),
5.25 (2H, q, J ¼ 12.3, 12.3, 12.3 Hz), 4.95 (1H, dd, J ¼ 10.5, 7.8 Hz),
4.69–4.50 (2H, m), 3.84 (3H, s).
(S)-benzyl 2-(4-methoxyphenyl)-4,5-dihydrooxazole-4-carbox-
ylate (21, 6.74 g, 21.6 mmol) was dissolved in 75 mL of dry DCM and
chilled in an ice bath (0 ꢀC bath temperature). DBU (9.7 mL,
64.9 mmol) was added followed by slow addition of bromotri-
chloromethane (6.5 mL, 64.9 mmol). The reaction was stirred for
1 h cold and then allowed to warm to room temperature. Once the
reaction appeared complete by TLC analysis, the reaction mixture
was poured into 10% aqueous NaHCO3 solution and extracted with
DCM (2ꢂ). The combined organic layers were washed again with
the NaHCO3 solution and brine then dried over Na2SO4 , filtered and
concentrated in vacuo. Crude material obtained was purified by
silica gel column chromatography with a 1:10 to 1:1 ethyl acetate:
DCM gradient solvent system to give 5.0 g (75%) of 60 as a white
solid. Rf ¼ 0.55 (DCM); mp 111–112 ꢀC; IR (neat) 1716 (C]O), 1313,
NMR (300 MHz, CDCl3)
d
8.68 (1H, dd, J ¼ 7.2, 2.2 Hz), 8.28 (1H, ddd,
J ¼ 8.7, 4.2, 2.2 Hz), 7.45–7.31 (6H, m), 5.26 (2H, dd, J ¼ 20.8, 12.2 Hz),
5.02 (1H, dd, J ¼ 10.6, 8.0 Hz), 4.78–4.61 (2H, m). 13C NMR (126 MHz,
CDCl3) d 170.42, 163.45, 158.40, 156.25, 135.56, 135.48, 135.14, 128.67,
128.61, 128.49, 126.69, 124.19, 118.88, 118.71, 70.32, 68.83, 67.60.
HRMS calcd. For C17H13ClFNO3 345.0887, found 345.0901. Elemental
calcd. for C17H13ClFNO3: C, 59.30; H, 3.81; N, 8.14; found C, 59.20; H,
25
3.77; N, 8.13; [
a
]
þ 0.94 (c ¼ 1.52 g/100 mL CHCl3).
D
6.5. General procedure for preparation of oxazole products
(compounds 58–100)
1263, 1138, 1117 cmꢁ1
8.10–8.01 (2H, m), 7.51–7.30 (5H, m), 7.02–6.94 (2H, m), 5.40 (2H, s),
3.86 (2H, s). 13C NMR (126 MHz, CDCl3)
162.86, 162.17, 161.56,
; d 8.23 (1H, s),
1H NMR (300 MHz, CDCl3)
Oxazoline 8 (165 mg, 0.5 mmol,1 equiv) was dissolved in 2 mL of
dry DCM and chilled in an ice bath (0 ꢀC bath temperature). DBU
(0.22 mL, 1.5 mmol, 3 equiv) was added followed by slow addition
of bromotrichloromethane (0.15 mL, 1.5 mmol, 3 equiv). The reac-
tion was stirred for 1 h cold and then allowed to warm to room
temperature. Once the reaction appeared complete by TLC analysis,
the reaction mixture was poured into 10% aqueous NaHCO3 solu-
tion and extracted with DCM (2ꢂ). The organic layer washed again
with the NaHCO3 solution, brine, dried over Na2SO4, filtered and
then concentrated in vacuo. Crude oily material obtained was
purified by silica gel column chromatography with a 1:10–1:1 ethyl
acetate: DCM gradient solvent system to give solid upon standing.
d
143.65, 135.80,134.39, 128.88, 128.83, 128.76, 128.66, 119.32, 114.45,
67.00, 55.65. HRMS calcd. For C18H15NO4, 310.1079 found 310.1071.
Elemental calcd. for C18H15NO4: C, 69.89; H, 4.89; N, 4.53; found C,
69.67; H, 4.92; N, 4.46.
6.5.2. Benzyl 2-(2-(benzyloxy)phenyl)oxazole-4-carboxylate (63)
(S)-Benzyl
2-(2-(benzyloxy)phenyl)-4,5-dihydrooxazole-4-
carboxylate (3, 5.88 g, 15.2 mmol) was dissolved in 100 mL of dry
DCM and chilled in an ice bath (0 ꢀC bath temperature). Next, DBU
(6.8 mL, 45.5 mmol) was added followed by slow addition of bro-
motrichloromethane (4.5 mL, 45.5 mmol). The reaction was stirred
for 1 h cold and then allowed to warm to room temperature. Once
the reaction appeared complete by TLC analysis, the reaction
mixture was poured into 10% aqueous NaHCO3 solution and
extracted with DCM (2ꢂ). The combined organic layers were
washed again with the NaHCO3 solution, brine, dried over Na2SO4 ,
filtered and then concentrated in vacuo. Crude material obtained
was purified by silica gel column chromatography with a 1:10–1:1
ethyl acetate: DCM gradient solvent system to give 4.7 g (81%) of 63
6.5.1. Benzyl 2-(4-methoxyphenyl)oxazole-4-carboxylate (60)
4-Methoxybenzoyl chloride (4.11 g, 23.4 mmol) was dissolved in
120 mL of DCM and then L-serine benzyl ester hydrochloride
(5.86 g, 24.5 mmol) and diisopropylethylamine (10.2 mL,
58.4 mmol) were added while the reaction was chilled in an ice
bath (0 ꢀC bath temperature). The reaction mixture was warmed to
room temperature and then heated to reflux, 50 ꢀC (bath temper-
ature) for 14 h. The reaction mixture was cooled, washed with 10%

1714
G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
as a white solid. Rf ¼ 0.6 (DCM); mp 74–75 ꢀC; IR (neat) 1740 (C]O),
The organic phase was collected and dried over sodium sulfate
(Na2SO4), filtered and then concentrated in vacuo The crude
material obtained was purified using silica gel column chroma-
tography with 5%–50% ethyl acetate: DCM as the solvent system to
give (S)-benzyl 2-(2-chlorobenzamido)-3-hydroxypropanoate
(9.9 g, 99%) as a yellow oil which solidified upon standing. Rf
1453, 1319, 1145, 1121 cmꢁ1; 1H NMR (300 MHz, CDCl3)
d 8.31 (1H,
d, J ¼ 2.4 Hz), 8.06 (1H, dd, J ¼ 8.1, 1.7 Hz), 7.57–7.27 (11H, m), 7.11–
7.03 (2H, m), 5.41 (2H, s), 5.22 (2H, s). 13C NMR (126 MHz, CDCl3)
d
161.32, 156.80, 143.92, 136.55, 135.59, 133.89, 132.44, 131.10,
128.57, 128.50, 128.49, 128.36, 127.72, 126.77, 120.98, 116.17, 113.47,
70.48, 66.68. HRMS calcd. For C24H19NO4, 386.1392 found 386.1385.
Elemental calcd. for C24H19NO4: C, 74.79; H, 4.97; N, 3.63; found C,
74.59; H, 4.97; N, 3.66.
product ¼ 0.16 (DCM). 1H NMR (300 MHz, CDCl3)
d 7.72–7.64 (1H,
m), 7.46–7.18 (8H, m), 5.28 (2H, d, J ¼ 12.2 Hz), 4.97–4.86 (1H, m),
4.10 (2H, s).
(S)-Benzyl 2-(2-chlorobenzamido)-3-hydroxypropanoate (5.9 g,
16.3 mmol) was dissolved in DCM (160 mL) and cooled to ꢁ78 ꢀC
(bath temp, dry ice-acetone). DAST (2.4 mL, 18.7 mmol) was added
slowly to the cold mixture under argon where it stirred for 30 min
K2CO3 (6.0 g, 43.9 mmol) was added and the reaction was allowed
to warm to room temperature. Once the reaction appeared
complete by TLC analysis, the reaction mixture was poured into 10%
aqueous NaHCO3 and extracted with DCM (2ꢂ). The organic layer
washed again with the NaHCO3 solution, brine, dried over Na2SO4 ,
filtered and then concentrated in vacuo. Crude material obtained
was purified by silica gel column chromatography with a 1:10 to 1:1
ethyl acetate: DCM gradient solvent system to give 4.9 g (87%) of
(S)-benzyl 2-(2-chlorophenyl)-4,5-dihydrooxazole-4-carboxylate,
16, as a white solid. Rf product ¼ 0.36 (DCM). 1H NMR (300 MHz,
6.5.3. Benzyl 2-phenyloxazole-4-carboxylate (65)
(S)-Benzyl 4,5-dihydro-2-phenyloxazole-4-carboxylate (15,
6.27 g, 22.3 mmol) was dissolved in 75 mL of dry DCM and chilled in
an ice bath (0 ꢀC bath temperature). DBU (10 mL, 66.9 mmol) was
added followed by slow addition of bromotrichloromethane
(6.6 mL, 66.9 mmol). The reaction was stirred for 1 h cold and then
allowed to warm to room temperature. Once the reaction appeared
complete by TLC analysis, the reaction mixture was poured into 10%
aqueous NaHCO3 solution and extracted with DCM (2ꢂ). The
organic layer washed again with the NaHCO3 solution, brine, dried
over Na2SO4 , filtered and then concentrated in vacuo. Crude
material obtained was purified by silica gel column chromatog-
raphy with a 1:10–1:1 ethyl acetate: DCM gradient solvent system
to give 5.64 g (91%) of 65 as a white solid. Rf ¼ 0.37 (DCM); mp 94–
CDCl3)
d
7.82 (1H, dd, J ¼ 7.6, 1.7 Hz), 7.49–7.27 (8H, m), 5.33–5.19
(2H, m), 5.04 (1H, dd, J ¼ 10.6, 7.7 Hz), 4.77–4.58 (2H, m).
95 ꢀC; IR (neat) 1741 (C]O), 1323, 1144, 1114 cmꢁ1
(300 MHz, CDCl3)
8.28 (1H, s), 8.12 (2H, dt, J ¼ 4.9, 4.7, 3.1 Hz),
7.52–7.31 (8H, m), 5.41 (2H, s). 13C NMR (126 MHz, CDCl3)
162.49,
;
1H NMR
(S)-Benzyl 2-(2-chlorophenyl)-4,5-dihydrooxazole-4-carbox-
ylate (16, 5.88 g, 15.2 mmol) was dissolved in 100 mL of dry DCM
and chilled in an ice bath (0 ꢀC bath temperature). DBU (6.8 mL,
45.5 mmol) was added followed by slow addition of bromotri-
chloromethane (4.5 mL, 45.5 mmol). The reaction was stirred for
1 h cold and then allowed to warm to room temperature. Once the
reaction appeared complete by TLC analysis, the reaction mixture
was poured into 10% aqueous NaHCO3 solution and extracted with
DCM (2ꢂ). The organic layer washed again with the NaHCO3
d
d
161.13, 143.84, 135.47, 134.32, 131.15, 128.96, 128.78, 128.57, 128.50,
128.46, 128.41, 126.86, 126.35, 66.79. HRMS calcd. For C17H13NO3,
280.0974 found 280.0973. Elemental calcd. for C17H13NO3: C, 73.11;
H, 4.69; N, 5.02; found C, 72.82; H, 4.67; N, 4.96.
6.5.4. Benzyl 2-(4-fluoro-3-nitrophenyl)oxazole-4-carboxylate (66)
(S)-4-Fluoro-3-nitrobenzyl 2-(benzamido)-3-hydroxypropanoate
(29, 5.10 g, 14.8 mmol) was dissolved in 200 mL of dry DCM and
chilled in an ice bath (0 ꢀC bath temperature). DBU (6.6 mL,
44.4 mmol) was added followed by slow addition of bromotri-
chloromethane (4.4 mL, 44.4 mmol). The reaction was stirred for 1 h
cold and then allowed to warm to room temperature. Once the
reaction appeared complete by TLC analysis, the reaction mixturewas
poured into 10% aqueous NaHCO3 solution and extracted with DCM
(2ꢂ). The combined organic layers were washed again with NaHCO3,
brine dried over Na2SO4 , filtered and then concentrated in vacuo.
Crude material obtained was purified by silica gel column chroma-
tography with a1:10–1:1 ethyl acetate: DCM gradient solvent system
to give 3.1 g (60%) of 66 as a light yellow solid upon standing. Rf ¼ 0.29
(DCM); mp 120–121 ꢀC; IR (neat) 1732 (C]O), 1534 (C]O), 1349,
solution, brine, and dried over Na2SO4
, filtered and then
concentrated in vacuo. Crude material obtained was purified by
silica gel column chromatography with a 1:10–1:1 ethyl acetate:
DCM gradient solvent system to give 4.7 g (81%) of 68 as a white
solid. Rf ¼ 0.59 (DCM); mp 62–63 ꢀC IR (neat) 1741 (C]O), 1314,
1145, 1118 cmꢁ1
; d 8.36 (1H, d,
1H NMR (500 MHz, CDCl3)
J ¼ 1.2 Hz), 8.09–7.99 (1H, m), 7.56–7.21 (8H, m), 5.41 (2H, s). 13C
NMR (126 MHz, CDCl3)
d 160.94, 160.67, 144.32, 135.44, 134.14,
132.88, 131.86, 131.64, 131.07, 128.59, 128.53, 128.44, 126.85,
125.51, 66.85. For C17H12ClNO3, 314.0584 found 314.0571.
Elemental calcd. for C17H12ClNO3: C, 59.65; H, 3.24; F, 5.55; N,
8.18; found C, 65.08; H, 3.86; N, 4.46; found C, 65.07; H, 3.93; N,
4.42.
1322, 1151 cmꢁ1; 1H NMR (300 MHz, CDCl3)
2.2 Hz), 8.40(1H, ddd, J ¼ 8.8, 4.2, 2.3 Hz), 8.33 (1H, d, J ¼ 2.6 Hz), 7.50–
7.31 (6H, m), 5.41 (2H, s). 13C NMR (126 MHz, CDCl3)
160.62,159.33,
d
8.79 (1H, dd, J ¼ 7.0,
6.6. General procedure for preparation of threonine amides (9, open
d
L
-threonine form)
157.89, 155.74, 144.60, 135.17, 134.85, 133.59, 128.67, 128.62, 124.70,
124.68, 123.53, 123.50, 119.57, 119.40, 67.15. For C17H11FN2O5,
343.0730 found 343.0751.Elemental calcd. for C17H11FN2O5: C, 59.65;
H, 3.24; F, 5.55; N, 8.18; found C, 59.83; H, 3.29; N, 8.00.
Benzoic acid (414 mg, 3.3 mmol, 1 equiv) and L-threonine benzyl
ester hydrochloride (874 mg, 3.5 mmol, 1.05 equiv) were dissolved
in 7 mL of acetonitrile (ACN) and treated with N-(3-dimethylami-
nopropyl)-N0-ethylcarbodiimide hydrochloride (EDC-HCl,
1 g,
6.5.5. Benzyl 2-(2-chlorophenyl)oxazole-4-carboxylate (68)
2-Chlorobenzoyl chloride (5.39 g, 29.9 mmol) was dissolved in
150 mL of DCM and then L-serine benzyl ester hydrochloride
(7.49 g, 31.4 mmol) and diisopropylethylamine (13 mL, 74.7 mmol)
were added while the reaction was chilled in an ice bath (0 ꢀC bath
temperature). The reaction mixture was warmed to room temper-
ature and then heated to reflux, 50 ꢀC (bath temperature) for 14 h.
The reaction mixture was cooled, washed with 10% aqueous
NaHCO3 solution (2ꢂ), water, 0.5 N citric acid (2ꢂ) and then brine.
6.6 mmol, 2 equiv) and N,N-dimethylaminopyridine (DMAP, 1.2 g,
9.9 mmol, 3 equiv). The resulting solution was stirred for 12 h at
room temperature. The reaction mixture was diluted with EtOAc,
washed with 0.5 N citric acid (2ꢂ), water, 10% aqueous NaHCO3
solution (2ꢂ). The organic phase was collected, dried over sodium
sulfate (Na2SO4), filtered and then concentrated in vacuo. Crude
material obtained was purified by silica gel column chromatog-
raphy with a 1:10–1:1 ethyl acetate: dichloromethane (DCM)
gradient solvent system.

G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
1715
6.6.1. Method 2
monkey kidney cells (VERO) was determined using a colorimetric
assay as previously described [32].
Benzoyl chloride (449 mg, 0.4 mL, 3.1 mmol, 0.95 equiv equiv)
was dissolved in 6 mL of DCM and then L-threonine benzyl ester
hydrochloride (711 mg, 3.3 mmol, 1 equiv) and triethylamine (1 g,
1.4 mL, 9.9 mmol, 3 equiv) were added. The reaction mixture was
warmed to room temperature and then heated to reflux, 50 ꢀC
(bath temperature) for 14 h. The reaction mixture was cooled,
washed with 10% aqueous NaHCO3 solution (2ꢂ), water, 0.5 N citric
acid (2ꢂ) and then brine. The organic phase was collected and dried
over Na2SO4, filtered and then concentrated in vacuo The crude
material obtained was purified by silica gel column chromatog-
raphy with a 1:10–1:1 ethyl acetate: DCM gradient solvent system.
7.1. Simulated gastric fluid stability
Compounds were incubated in simulated gastric fluid [35]
(consisting of 3.2 mg/mL pepsin in 0.03 M sodium chloride at pH
1.2) at 37 ꢀC at a concentration of 50
mM. Aliquots (500
mL) were
taken at 0 h and 2 h, 500
mL of 10% sodium bicarbonate containing
internal standard was added, followed by centrifugation for 5 min
at 12,000 rpm. Aliquots of the supernatants were analyzed by
reversed-phase HPLC with UV detection at 254 nm. The mobile
phase consisted of elution on a YMC 5
m
-basic 4.6 mm i.d. ꢂ 15 cm
6.7. General procedure for preparation of threonine-derived
oxazoline products (compounds 10, 101–105)
column at 1 mL/min with 5-min isocratic elution with 10% aceto-
nitrile/90% water, 20-min linear gradient from 10% acetonitrile to
90% acetonitrile, and 10-min isocratic elution with 90% acetonitrile/
10% water. The peak area ratio relative to internal starndard was
measured and compared to that at time 0 and reported as percent
remaining.
L-Threonine amide 9 (330 mg, 1.1 mmol, 1 equiv) was dissolved
in DCM (5 mL) and cooled to ꢁ78 ꢀC (bath temp, dry ice-acetone).
DAST (0.16 mL, 1.3 mmol, 1.2 equiv) was added slowly to the cold
mixture under argon and the reaction was stirred for 30 min. Then
K2CO3 (393 mg, 2.8 mmol, 2.7 equiv) was added and the reaction
was allowed to warm to room temperature. Once the reaction
appeared complete by TLC analysis, the reaction mixture was
poured into 10% aqueous NaHCO3 solution and extracted with DCM
(2ꢂ). The combined organic layers were washed with the NaHCO3
solution, brine, dried over Na2SO4 , filtered and then concentrated
in vacuo. Crude material obtained was purified by silica gel column
chromatography with a 1:10–1:1 ethyl acetate: DCM as the
gradient solvent system.
7.2. Metabolic stability
Compounds (20
mM) were mixed with NADPH (0.5 mM) in
potassium phosphate buffer (50 mM, pH 7.4) in a total volume of
0.5 mL and preincubated at 37 ꢀC. The reaction was started by
adding pooled male Sprague–Dawley liver microsomes (0.2 mg
protein). After 15-min incubation, the reaction was quenched with
0.5 mL acetonitrile containing internal standard and centrifuged to
remove protein. A second incubation was terminated at time 0. An
aliquot of the supernatant was analyzed by reversed-phase HPLC
with UV detection at 254 nm. The chromatographic conditions
were the same as for the gastric fluid stability. The peak area ratio
relative to internal standard was measured and compared relative
to that at time 0 and reported as percent remaining.
6.8. General procedure for preparation of threonine-derived
oxazoles (compounds 11, 106–111)
Threonine derived oxazoline 10 (195 mg, 0.66 mmol, 1 equiv)
was dissolved in 2 mL of dry DCM and chilled in an ice bath (0 ꢀC
bath temperature). DBU (0.3 mL, 2 mmol, 3 equiv) was added fol-
lowed by slow addition of bromotrichloromethane (0.2 mL,
2 mmol, 3 equiv). The reaction was stirred for 1 h cold and then
allowed to warm to room temperature. Once the reaction appeared
complete by TLC analysis, the reaction mixture was poured into 10%
aqueous NaHCO3 solution and extracted with DCM (2ꢂ). The
combined organic layers were washed again with the NaHCO3
solution, brine, dried over Na2SO4, filtered and then concentrated in
vacuo. Crude material obtained was purified by silica gel column
chromatography with a 1:10–1:1 ethyl acetate: DCM gradient
solvent system.
7.3. HPLC chromatography
The HPLC system consisted of a Perkin–Elmer series 200
quaternary pump (Perkin–Elmer Corp., Norwalk, CT), a Perkin–
Elmer UV detector series 200, and a Perkin–Elmer auto sampler
series 200. Samples were analyzed on a YMC 5
i.d. ꢂ 15 cm column (YMC Inc., Wilmington, NC) connected to
a YMC 5
m basic 4.0 mm i.d. ꢂ 2 cm guard cartridge.
mm basic 4.6 mm
m
Acknowledgment
7. Anti-tuberculosis and cytotoxicity assays
This work was supported by Grant PHS 398/2590 from the
National Institutes of Health. We would like to thank the University
of Notre Dame especially the MS facility (Bill Boggess, Nonka Sev-
ova) and Prof. Jennifer DuBois for profound scientific discussion.
The excellent technical assistance of Baojie Wan and Yuehong
Wang with anti-TB assays at UIC and Uta Wohfield with microbial
assays at the HKI is greatly appreciated. Adriel Villegas-Estrada was
a Fellow of the Chemistry–Biochemistry–Biology Interface (CBBI)
Program at the University of Notre Dame, supported by training
grant T32GM075762 from the National Institutes of Health.
Activity against replicating M. tuberculosis H37Rv (ATCC 27294,
American Type Culture Collection, Rockville, MD) was determined
using a fluorescence readout in the Microplate Alamar Blue Assay
(MABA) [32] following incubation for one week with test
compounds in glycerol-alanine-salts medium (GAS) as well as in
medium without added iron but with Tween 80 (GAST) [33]. The
MIC was defined as the minimum concentration inhibiting fluo-
rescence by 90% relative to bacteria-only controls. Activity against
non-replicating M. tuberculosis was determined using the Low
Oxygen Recovery Assay (LORA) [34] by exposing low oxygen-
adapted M. tuberculosis containing
a luciferase gene to test
compounds under anaerobic conditions for 10 days. After a 28 h
recovery in air, the luciferase signal was measured. MIC was defined
as the lowest concentration inhibiting recovery of luciferase signal
by ꢃ90% relative to bacteria-only controls. Toxicity to African green
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2009.12.072.

1716
G.C. Moraski et al. / European Journal of Medicinal Chemistry 45 (2010) 1703–1716
[21] Assay data from level two testing at the Tuberculosis Antimicrobial Acquisi-
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[19] 7.7
the GAST (low iron Tween 80 containing media) run in Scott Franzblau’s lab at
the University of Illinois at Chicago. The MIC has ranged from 2.2 M to
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mM is the average value from multiple tests (15 in total) of ND-005859 in
m
m