3-(Oxazolo[4,5-b]pyridin-2-yl)anilides as a novel class of potent inhibitors for the kinetoplastid Trypanosoma brucei, the causative agent for human African trypanosomiasis

Article abstract of DOI:10.1016/j.ejmech.2013.05.007

A whole organism high-throughput screen of approximately 87,000 compounds against Trypanosoma brucei brucei led to the recent discovery of several novel compound classes with low micromolar activity against this organism and without appreciable cytotoxici

Full text of DOI:10.1016/j.ejmech.2013.05.007

European Journal of Medicinal Chemistry 66 (2013) 450e465
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
3-(Oxazolo[4,5-b]pyridin-2-yl)anilides as a novel class of potent
inhibitors for the kinetoplastid Trypanosoma brucei, the causative
agent for human African trypanosomiasis
Lori Ferrins a, Raphaël Rahmani a, Melissa L. Sykes b, Amy J. Jones b, Vicky M. Avery b,
,
,1
Eliott Teston c, Basmah Almohaywi d, JieXiang Yin d 1, Jason Smith d, Chris Hyland d
,
Karen L. White e, Eileen Ryan e, Michael Campbell e, Susan A. Charman a e, Marcel Kaiser f
,
,
,g
,c,h,
Jonathan B. Baell a
*
a Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia
b Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane Innovation Park, Don Young Road, Nathan, Queensland 4111, Australia
c The Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, Victoria 3052, Australia
d School of Chemistry, University of Tasmania, Private Bag 75, Hobart 7001, Australia
e Centre for Drug Candidate Optimisation, Monash University, Parkville, Victoria 3052, Australia
f Swiss Tropical and Public Health Institute, Socinstrasse 57, Basel 4051, Switzerland
g University of Basel, Petersplatz 1, Basel 4003, Switzerland
h Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A whole organism high-throughput screen of approximately 87,000 compounds against Trypanosoma
brucei brucei led to the recent discovery of several novel compound classes with low micromolar activity
against this organism and without appreciable cytotoxicity to mammalian cells. Herein we report a
structureeactivity relationship (SAR) investigation around one of these hit classes, the 3-(oxazolo[4,5-b]
pyridin-2-yl)anilides. Sharp SAR is revealed, with our most active compound (5) exhibiting an IC50 of
91 nM against the human pathogenic strain T.b. rhodesiense and being more than 700 times less toxic
towards the L6 mammalian cell line. Physicochemical properties are attractive for many compounds in
this series. For the most potent representatives, we show that solubility and metabolic stability are key
parameters to target during future optimisation.
Received 17 February 2013
Received in revised form
1 May 2013
Accepted 7 May 2013
Available online 16 May 2013
Keywords:
3-(Oxazolo[4,5-b]pyridin-2-yl)anilides
Human African trypanosomiasis
Sleeping sickness
Trypanosomacidal agent
Trypanosoma brucei
Ó 2013 Published by Elsevier Masson SAS.
Abbreviations: BBB, bloodebrain barrier; cPPB, chromatographic plasma protein binding; cytotox., cytotoxicity; DCM, dichloromethane; DMAP, N,N-dimethylamino-
pyridine; DMEM, Dulbecco’s modified Eagle’s medium; DMF, N,N-dimethylformamide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EH, hepatic extraction; FCS,
foetal calf serum; HAT, human African trypanosomiasis; HBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; STPHI, Swiss Tropical and Public Health Institute; TBAB, tetrabutylammonium bromide; L. donovani, Leishmania Donovani; P. falciparum,
Plasmodium falciparum; T.b. brucei, Trypanosoma brucei brucei; T.b. gambiense, Trypanosoma brucei gambiense; T.b. rhodesiense, Trypanosoma brucei rhodesiense; WHO, World
Health Organisation.
* Corresponding author. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia. Tel.: þ61 9903 9044.
E-mail address: Jonathan.Baell@monash.edu (J.B. Baell).
School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia.
1
0223-5234/$ e see front matter Ó 2013 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2013.05.007

L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
451
1. Introduction
Human African trypanosomiasis (HAT), more commonly known
as sleeping sickness, is a vector-borne parasitic disease caused by
infection of the host with either Trypanosoma brucei gambiense or
Trypanosoma brucei rhodesiense [1]. The World Health Organisation
(WHO) currently estimates that there are approximately 30,000
cases of HAT in Africa, which has a significant socioeconomic
impact [2]. HAT is largely confined to the sub-Saharan continent
where the vector, parasite and animal reservoirs co-exist [1]. The
disease consists of two stages; the first involves the invasion of the
haemolymphatic system by the parasite, and the second is associ-
ated with the transmission of the parasites across the bloodebrain
barrier (BBB) and into the central nervous system (CNS) [1].
Infection of the CNS leads to a number of symptoms including
mental impairment, severe headaches, fever, chronic encephalop-
athy and eventual death. This stage of the disease is particularly in
need of improved therapies [1].
There are a number of treatment options currently available for
HAT, though none of them are ideal. Pentamidine and suramin are
used to treat the first stage of HAT where T.b. gambiense and T.b.
rhodesiense are the causative agents, respectively. However, neither
of these drugs are able to cross the BBB making both ineffective
against the second stage of HAT [3e5]. In addition, both treatments
have significant side effects. Suramin has been linked to exfoliative
dermatitis and renal failure [4], whilst pentamidine use is associ-
ated with diabetes mellitus and nephrotoxicity [6]. Melarsoprol is
the primary treatment option available for second stage HAT being
effective against both subspecies of trypanosome [6,7]. However,
high failure rates have been reported even though resistance has
not been proven [7]. Alternatively, eflornithine can also be used to
treat second stage HAT [7]. It is a safer option but it is not effective
against T.b. rhodesiense and the high costs may make it prohibitive
[7]. Furthermore, administration requires four intravenous in-
fusions daily for 14 days which is impractical in rural African fa-
(a)
(b)
Fig. 1. (a) Structure and physicochemical profile of an initial screening hit; (b) numbering
used herein.
81 Å2 that could have potential for CNS penetration to treat stage
two HAT [14], and an attractively low cLogP of 2.6.
Furthermore, testing against
confirmed potent activity not only against T.b. rhodesiense (IC50
0.59 M), but also against Trypanosoma cruzi (IC50 0.23 M). In both
cases 1 was highly selective for these kinetoplastids compared with
the L6 mammalian cell line, with respective selectivity indices of 39
and 99 (Table 1). Activity was significantly weaker against the un-
a wider panel of parasites
m
m
related protozoan Plasmodium falciparum (IC50 7.8
causative agent of malaria [15].
mM), a major
As such, 1 was of interest as a starting point for drug develop-
ment resulting in the initiation of an SAR study. Herein we report
our discovery of a new class of compounds that are potent and
specific against T.b. brucei and T.b. rhodesiense, as well as T. cruzi, the
causative agent of Chagas’ disease [16].
2. Chemistry
After mining our database for analogues, it was determined that
directed synthesis was required to establish a focused SAR inves-
tigation. We have recently discussed the database mining results
elsewhere [11]. Investigations initially centred around the “north-
ern” heterocyclic group. As shown in Scheme 1, the synthesis was
relatively straightforward requiring a one-step cyclocondensation
reaction between 2-amino-3-hydroxypyridine and a substituted
3-aminobenzoic acid, mediated by PPA, followed by amide bond
formation between the amino group and the relevant heteroaryl
carboxylic acid using EDCI and DMAP. For selected compounds,
amide N-methylation (c) was undertaken. This yielded the target
compounds, which are listed in Table 2 along with their IC50 values
against T.b. brucei.
cilities [8]. Recently nifurtimox has been introduced as
a
combination therapy with eflornithine (NECT) [8]. The combination
therapy has the advantage of having a shorter and simplified
regimen which has made it the current first line treatment for
second stage HAT caused by T.b. gambiense [8]. Orally bioavailable
oxaborole 6-carboxamides and orally active benzoxaboroles were
identified and selected to enter pre-clinical studies in 2009 [9].
Also, fexinidazole has been progressed through phase 1 clinical
trials and is currently recruiting for a phase 2 clinical trial [10].
However, there is still a great need for new trypanosomacidal
compounds given the current chemotherapeutic options available,
particularly for the CNS-resident second stage of this disease.
We recently screened approximately 87,000 compounds against
T.b. brucei using an Alamar BlueÒ based 384-well viability assay
[11]. Being the same species but just a different sub-species that is
non-pathogenic to humans, T.b. brucei has a long history of useful
and relevant application as a surrogate for T.b. rhodesiense and T.b.
gambiense for early HAT drug discovery projects and comprises the
mainstay of most primary animal models for HAT [12]. Indeed, T.b.
brucei and T.b. rhodesiense differ in one gene e the serum resistance
gene that allows T.b. brucei to survive in human serum e and there
is recent strong evidence that T.b. rhodesiense is only a phenotypic
variant of T.b. brucei [13]. Eight structurally unique compound
3. Results and discussion
Introduction of a 3-methyl group (2) into the furan ring leads to
a slight loss of activity with an IC50 of 0.65
0.22 M for 1 (Table 2). An isoxazole is less tolerated and 3 is more
than 10-fold less active than 1 while the oxazole (4) is only slightly
better than this with an IC50 of 1.5 M. On the other hand, a 3-furyl
group (5) is well tolerated with an IC50 of 0.3 M. The thiazole (6) is
mM relative to an IC50 of
m
m
m
about 10-fold less active than 1 while the corresponding pyrazole
Table 1
Biological activitya profile of 1 against a number of parasites.
Parasite
IC50
(
mM)
SI
classes were identified with IC50 values of less than 10 mM against
T.b. brucei
T.b. rhodesiense
T. cruzi
0.22
0.59
0.23
7.8
>345b
39c
T.b. brucei, that were more than ten times selective for T.b. brucei
over the mammalian cell line HEK293, and had favourable physi-
cochemical properties. One compound of particular interest was an
oxazolopyridine derivative with an IC50 against T.b. brucei of
99c
P. falciparum
e
a
Values are the mean of 3 experiments, <ꢀ50%.
Selectivity relative to HEK293 cells.
Selectivity relative to L6 (rat skeletal myoblast) cells.
b
c
0.22
mM, shown in Fig. 1. This compound (1) had a moderately low
molecular weight of 339.5, a reasonably low polar surface area of

452
L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
a Reagents and conditions: (a) substituted 3-aminobenzoic acid, [PPA, 180 °C, 4 h]; (b) EDCI,
DMAP, R3-COOH, [DMF, rt, 12 h]; (c) MeI, TBAB (cat.), K2CO3, [Acetone, rt, 12 h].
Scheme 1. Synthetic overview of oxazolopyridine derivatives.
(7) and imidazole (8) led to a loss of activity. Fusing a phenyl ring to
the furan to give benzofuran (9) also gave rise to a loss of activity.
Interestingly, while weaker, the phenyl analogue 10 has an IC50 of
The aryl property of the furan appeared to be important because
appropriate connectivity in the saturated ether (16) led to a loss of
activity.
1.5
m
M. It was hypothesized that a suitably placed ethereal oxygen
In summary, the requirements for the amide group are strin-
gent, and 2- and 3-furans are highly favoured over other groups. As
we are developing SAR in a whole cell assay, it is possible that
negative SAR could be a reflection of poorer permeability rather
than lower affinity to intracellular targets. However, apart from the
pyrazole (7) and imidazole (8), it seems unlikely that permeability
of this set of compounds would differ significantly from each other.
Therefore we suggest steric hindrance in this region requires a
small aryl group with one side being hydrophobic and the other
containing a hydrogen-bond acceptor.
atom could return potent activity like the furan, but substitution of
a 2-methoxy (11) or 3-methoxy (12) on the phenyl ring only led to
loss of activity. Introduction of endocyclic nitrogen atoms at the 2-
and 4-positions in 13 and 14 respectively were not well tolerated
with an IC50 of 5.7 mM and 3.4 mM respectively while introduction of
the nitrogen at the 3-position in 15 led to a complete loss of activity.
Table 2
T.b. brucei inhibitory activitya of targets with varying amide groups.
Following this, substitutions around the central ring were
investigated. As shown in Table 3, removal of the 6-chloro (17) led to
a compound with significant potency and selectivity. The 2-position
appears to be unfavourable for substitution since methyl (22), fluoro
(23) or chloro (24) led to a loss in activity. An endocyclic nitrogen
(25) or chloro (29) at the 4-position led to a decrease in activity while
a methyl (27) or fluoro (28) at the same position led to a loss of
activity. Interestingly, a methoxy in the 4-position (26) retained
ID
R3
IC50
(
mM)
SIb
ID
R3
IC50
(m
M)
SIb
some activity with an IC50 of 1.1 mM. Substitutions at the 5-position
with an endocyclic nitrogen (30) or fluoro (31), both led to loss of
activity. Replacement of the 6-chloro with a with a 6-fluoro (37) was
potent with an IC50 of 0.29 mM and an SI of >288. Replacement with a
1
0.22
>345
9
>10
e
methyl group (32) was somewhat less tolerated.
Thus, the central ring is remarkably intolerant of substitution at
the 2-, 4- or 5-positions. However, the 6-chloro group can be
removed or replaced with a 6-fluoro to give compounds that are
essentially equally active with the 6-chloro parent compound but
lower in both molecular weight and hydrophobicity. In most cases
it appears unlikely that permeability would differ greatly amongst
this set of compounds and so we attribute this SAR e at least at the
2-, 4- and 5-positions e to steric interactions with the intracellular
target. It is possible that “ortho” substitution at the 2- and
4-positions could also conformationally influence the disposition of
the amide group relative to the fused heterocycle.
2
3
4
0.65
3.2
>130
e
10
11
12
1.5
54
e
>10
1.5
>45
>10
e
We were then interested in whether crossover SAR could be
observed in combining active heterocycles in Table 2 with the
active central ring substituents just identified. The reason for this
was two-fold. Firstly, furans are known to be potentially metabol-
ically liable [17] and we reasoned that even though alternative
heterocycles such as an oxazole may lead to less active compounds,
other favourable metabolic and physicochemical properties such as
solubility could counter such drawbacks during drug development.
Secondly, we reasoned that it was not necessarily the case that
these alternative heterocycles would stay correspondingly weaker
with substitution changes in the central ring. Shown in Table 3 are
the top central ring substitutions (17, 32, 37) which were combined
with the 2- and 3-furans, 3-methylfuran, oxazole and thiazole
heterocycles. An increase in trypanosomacidal activity for some of
the heterocyclic amides was observed when the 6-substituent was
5
6
7
0.30
1.8
160
39
13
14
5.7
15
3.4
e
>10
e
e
15
16
>10
e
e
8
>10
CH2OMe
>10
a
Values are the mean of 3 experiments, <ꢀ50%.
b
Selectivity relative to HEK293 cells for selected potent compounds.

L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
Table 3 (continued )
453
Table 3
T.b. brucei inhibitory activitya of targets with substitutions around central ring sys-
ID
R2
R3
IC50
(m
M)
SIb
tem and with varying amides.
34
6-Me
>10
e
35
36
6-Me
6-Me
0.31
2.6
103
ID
R2
H
R3
IC50
(
m
M)
SIb
32
17
0.34
0.17
>245
37
38
39
6-F
6-F
6-F
0.29
0.10
2.0
290
840
43
18
19
H
H
115
29
2.9
20
21
H
H
0.60
1.3
89
63
40
6-F
6-F
0.12
0.76
680
110
41
22
23
24
25
26
27
28
2-Me
2-F
>10
e
a
Values are the mean of 3 experiments, <ꢀ50%.
b
c
Selectivity relative to HEK293 cells for selected potent compounds.
Endocyclic nitrogen.
>10
>10
>10
e
changed. For example, the combination of the 6-fluoro with the
3-methylfuran (38) and 3-furan (40) gave our most promising re-
sults to date with IC50 values of 0.10 mM and 0.12 mM respectively,
2-Cl
e
whilst the oxazole (39) and thiazole (41) were both less active.
These trends were broadly emulated when the 6-fluoro was
replaced with hydrogen (18e21) or 6-methyl (33e36), except that
the former grouping was on the whole very slightly less active and
the latter group marginally less active again.
While the focus of this work is on the central phenyl ring and
northern amide group, some limited exploration was undertaken of
the left hand side fused heterocycle, with interesting results.
Shown in Table 4 are the results of installing a 6-phenyl group.
When unsubstituted, the resulting compound (42) is potently
4-[N]c
4-OMe
4-Me
4-F
e
1.1
74
13
13
6.2
6.3
inhibitory with an IC50 of 0.12 mM. Substitution with a p-methoxy
(43) or p-chloro (44) results in a slight loss of activity. While 42 is
potent, it is not significantly more so than 17, that has no 6-phenyl
group, suggesting a null effect and lack of any interaction with
intracellular targets. This position might therefore be useful to
tailor other molecular properties such as solubility without loss of
activity.
29
30
31
32
33
4-Cl
>10
e
5-[N]c
5-F
>10
>10
e
Also listed in Table 4 are the results of a core change where the
oxazole is replaced by an imidazole and both compounds 44 and 45
retain significant activity with respective IC50 values of 0.78
mM and
e
1.0 M. It is possible that the slight loss of activity is contributed at
m
least in part by poorer permeability, suggesting further investiga-
tion of this core heterocycle change is warranted. Interestingly,
removal of the pyridine nitrogen leads to a complete loss of activity,
suggesting it is essential for activity and there may be a hydrogen
bond interaction with the receptor.
6-Me
6-Me
0.94
>88
145
0.57
Finally, investigation of the effect of N-alkylation of the amide e
NH was performed. In all cases the loss of the amide eNH led to a
(continued on next page)

454
L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
Table 4
T.b. brucei inhibitory activitya of analogues of the oxazolopyridine core.
ID
R1
IC50
(
m
M)
SIb
84
130
150
54
42
43
44
45
46
47
Ph
0.12
0.63
0.55
0.78
1.0
4-OMeePh
4-ClePh
e
e
e
39
e
>10
a
Values are the mean of 3 experiments, <ꢀ50%.
b
Selectivity relative to HEK293 cells for selected potent compounds.
complete loss of activity, as seen in Table 5. This suggests that there
may be an essential hydrogen bond interaction present between
the compound and its putative receptor.
CNS-resident second stage of HAT. Likewise, distribution co-
efficients were close to ideal and in the range 1.7e3.3 at pH 7.4.
However, aqueous solubility for some compounds was poor and as
In order to investigate activity against the human pathogenic
subspecies T.b. rhodesiense, selected compounds were tested against
this organism as well as a small panel of parasites. As shown in
Table 6, T.b. rhodesiense activity tracked well with T.b. brucei activity.
The transfer of SAR was not linear and the 3-furylamide (5) was
low as 1.6e3.1 mg/mL for pH values of both 2 and 6.5 for several
compounds. Plasma protein binding was moderate and would not
likely limit distribution of the compound. However, most com-
pounds were degraded rapidly by microsomes indicating that they
are susceptible to cytochrome P450-mediated metabolism. The
most stable compound was oxazolamide (39) and the next most
stable thiazolamide (41), lending strong support to the hypothesis
that the furanyl group is the metabolic hot spot.
potent against T.b. rhodesiense with an IC50 of 0.091 mM. Moreover,
both 1 and 5 were potently active against T.cruzi and 5 in particular
was active against Leishmania donovani, a causative agent of Leish-
maniasis [18]. Furthermore, 5 exhibited low micromolar inhibitory
activity towards L. donovani in infected macrophages,
a
4. Conclusion
pathogenically-relevant assay for which actives are extremely diffi-
cult to discover [18]. All compounds exhibited low micromolar ac-
tivity against P. falciparum, but were relatively non-cytotoxic towards
mammalian L6 cells. Indeed, the selectivity index for 5 was over 700
in this regard with respect to T.b. rhodesiense activity.
An assessment of drug-likeness was then made. Listed in Table 7
are various physicochemical and metabolic parameters for selected
compounds. In general, all compounds are of relatively low mo-
lecular weight and in the range 319e340. Their polar surface areas
are relatively low and within the range where acceptable CNS
penetration is plausible [14]. This is important in order to treat the
We have described the discovery of 3-(oxazolo[4,5-b]pyridin-2-
yl)anilides as a novel class of potent inhibitors for the kinetoplastid
Trypanosoma brucei. Compound 5 exhibits an IC50 of 0.091
mM
against the human infective form, T.b. rhodesiense. As this is a whole
cell readout, binding to intracellular targets must be extremely
strong and with a tight fit with the receptor(s). We have discovered
several key regions of this class with which we cannot interfere.
Importantly, we have identified positions where activity can be
modulated favourably. For example, replacement of the ring chloro
with fluoro leads to improved activity and the 3-furyl is more active
Table 5
Table 6
Investigation of amide N-alkylation on activity against T.b. bruceia for 3 selected
Biological activity profile of selected compounds against a parasite panel (IC50, m
M).a
compounds.
ID
T.b.
T.b.
T. cruzic
L. don.
L. don. inf.
P. falc.
Cytotox.
L6g
brucei
rhod.b
axe.d
mac.e
NF54f
1
5
6
10
0.22
0.30
1.8
0.59
0.091
1.1
0.23
0.26
2.3
1.8
0.34
4.8
e
7.8
4.9
7.7
6.9
23
65
235
51
12
>84
37
1.5
4.2
2.9
6.1
a
Values are the mean of 2 experiments, <ꢀ50%.
b
T.b. rhodesiense strain STIB 900, bloodstream form (trypomastigotes). Melarso-
prol (IC50 0.005 M) was used as a control.
T. cruzi Tulahaen C4 strain, amastigote stage. Benznidazole was used as a control,
EC50 1.46 M.
L. donovani MHOM-ET-67/L82 strain, amastigote stage. Miltefosine was used as
a control, EC50 0.49 M.
L. donovani in infected macrophages. Miltefosine was used as a control, EC50
1.67 M.
P. falciparum NF54 strain, erythrocytic stage. Chloroquine was used as a control,
EC50 0.006 M.
Rat skeletal myoblast cell
0.017 M.
R3
IC50 (mM)
m
ID
c
m
d
48
>10
m
e
m
f
49
>10
m
g
L-6 strain. Podophyllotoxin was used as a control, EC50
a
Values are the mean of 3 experiments, <ꢀ50%.
m

L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
455
e
Table 7
Key physicochemical parameters and in vitro metabolic stability of selected compounds.
2
a
ꢀ
ID
MW
PSA(A )
LogDb
Solubility (
pH 2
m
g/mL)c
pH 6.5
cPPBd (%)
Half-life (min)
In vitro CLint
L/min/mg protein)e
Microsome-predicted EH
(
m
pH 7.4
1
340
319
333
319
337
324
323
340
81.2
81.2
81.2
81.2
81.2
94.1
81.2
109.2
2.7
2.9
3.3
2.7
2.9
1.7
2.4
2.3
1.6e3.1
6.3e12.5
1.6e3.1
12.5e25.0
1.6e3.1
6.3e12.5
12.5e25.0
3.1e6.3
1.6e3.1
3.1e6.3
<1.6
6.3e12.5
1.6e3.1
6.3e12.5
6.3e12.5
3.1e6.3
93.9
94.2
95.8
93.2
93.5
71.3
88.8
89.2
7
10
8
10
9
124
11
36
258
181
228
176
202
14
0.91
0.88
0.90
0.87
0.89
0.35
0.86
0.65
18
33
35
38
39
40
41
154
48
a
Calculated using ACD/Labs software, version 9.
Measured chromatographically.
Kinetic solubility determined by nephelometry.
Human plasma protein binding estimated using a chromatographic method.
In vitro intrinsic clearance determined in human liver microsomes and predicted hepatic extraction ratio calculated from in vitro data.
b
c
d
e
ꢀ
than the 2-furyl. Significantly, some of our compounds are also
potent against T. cruzi, the causative agent of Chagas’ disease [16],
and low micromolar activity is also observed for 10 against
L. donovani in infected macrophages. Despite this, these compounds
are not at all appreciably toxic to mammalian cells. Assessment of
predictive ADMET reveals these compounds to generally be drug-
like and favourable for further investigation. Moving forward, key
parameters to target for optimisation are aqueous solubility and
metabolic stability and these will be interrogated during future
refinement of SAR around the fused pyridyl ring and in looking at
substituted furylamides.
Phenomenex Luna C8 100 A, 5
m
m (150 ꢃ 4.6 mm I.D.) column. A
binary solvent system was used (solvent A: 0.1% aqueous TFA, sol-
vent B: 0.1% TFA/19.9% H2O/80% ACN), with UV detection at 214 nm.
A gradient of 0e80% buffer B over 10 min for a 20 min run time was
used, with a flow rate of 1 mL/min.
1H and 13C NMR spectra were recorded at 400.13 and
100.62 MHz, respectively, on a Bruker Avance III Nanobay spec-
trometer with BACS 60 sample changer, using solvents from Cam-
bridge Isotope Laboratories. Chemical shifts (d, ppm) are reported
relative to the solvent peak (CDCl3: 7.26 [1H] or 77.16 [13C]; DMSO-
d6: 2.50 [1H] or 39.52 [13C]). Proton resonances are annotated as:
The mechanism of action of these compounds is currently not
known. However, a few structurally similar compounds have been
reported by workers at Sirtris as being activators of NADþ-
dependent deacetylase SIRT1, a sirtuin [19]. Such activity may be
relevant to trypanosomes [20,21], but it remains to be determined if
this is relevant to the compounds reported herein. For interested
researchers, screening samples of compounds reported herein may
be obtained by contacting the corresponding author.
chemical shift (d), multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad), coupling constant (J, Hz), and
number of protons.
Low resolution mass spectrometry was performed on an Agilent
6100 Series Single Quad LC/MS coupled with an Agilent 1200 Series
HPLC, G1311A quaternary pump, G1329A thermostatted autosam-
pler, and G1314B variable wave length detector (214 and 254 nm).
ꢀ
LC conditions: Phenomenex Luna C8(2) column (100 A, 5 mm,
50 ꢃ 4.6 mm), 30 ꢂC; sample (5 mL) was eluted using a binary
gradient (solvent A: 0.1% aq. HCO2H; solvent B: 0.1% HCO2H in
CH3CN; 5e100% B [10 min], 100% B [10 min]; 0.5 mL/min). MS
conditions: quadrupole ion source with multimode-ESI; drying gas
temperature, 300 ꢂC; vaporizer temperature, 200 ꢂC; capillary
voltage, 2000 V (positive mode) or 4000 V (negative mode); scan
range, 100e1000 m/z; step size, 0.1 s over 10 min.
5. Experimental protocol
Analytical TLC was performed on silica gel 60F254 pre-coated
aluminium sheets (0.25 mm, Merck) and visualized at 254 nm or
by chemical staining with a solution ceric sulfate/phosphomolybdic
acid followed by heating. Flash column chromatography was car-
ried out using Merck silica gel 60, 0.63e0.20 mm (70e230 mesh).
Microwave reactions were performed on a CEM discovery fitted
with an intellivent explorer unit. The temperature range of the
unit is ꢁ80 ꢂC to 300 ꢂC, a pressure range of 0e27 bar, power range
of 0e300 W and no pre stirring was required.
High resolution MS was performed on an Agilent 6224 TOF
LC/MS coupled to an Agilent 1290 Infinity LC. All data were acquired
and reference mass corrected via a dual-spray electrospray ion-
isation (ESI) source. Each scan or data point on the total ion chro-
matogram (TIC) is an average of 13,700 transients, producing a
spectrum every second. Mass spectra were created by averaging the
scans across each peak and subtracting the background from first
10 s of the TIC. Acquisition was performed using the Agilent Mass
Hunter Data Acquisition software ver. B.05.00 Build 5.0.5042.2 and
analysis was performed using Mass Hunter Qualitative Analysis ver.
B.05.00 Build 5.0.519.13. Acquisition parameters: mode, ESI; drying
gas flow, 11 L/min; nebuliser pressure, 45 psi; drying gas temper-
ature, 325 ꢂC; voltages: capillary, 4000 V; fragmentor, 160 V;
skimmer, 65 V; octapole RF, 750 V; scan range, 100e1500 m/z;
positive ion mode internal reference ions, m/z 121.050873 and
922.009798. LC conditions: Agilent Zorbax SB-C18 Rapid Resolution
HT (2.1 ꢃ 50 mm, 1.8 mm column), 30 ꢂC; sample (5 mL) was eluted
using a binary gradient (solvent A: 0.1% aq. HCO2H; solvent B: 0.1%
HCO2H in CH3CN; 5e100% B [3.5 min], 0.5 mL/min).
The purity of all compounds for biological testing was >95% in
all cases, except where specified otherwise.
aExperimental conditions: 1H and 13C Nuclear Magnetic Reso-
nance spectra were recorded at 300 MHz and 75 MHz respectively,
on a Bruker UltraShield 300 MHz spectrometer running Bruker
Topspin, version 1.3.
Low Resolution Mass Spectrometry analyses were performed on
a Finnigan LCQ Advantage Max coupled with a Surveyor PDA de-
tector and running XCalibur. Analysis was run on a Phenomenex
ꢀ
column (Gemini, 5
m
m, C18, 110 A, 50 ꢃ 2 mm).
Preparative High Performance Liquid Chromatography was
carried out on the Waters Alliance HT 2795 coupled with a Waters
2996 photodiode array detector and a Waters collector fraction III
running Empower Pro. It was fitted with a Phenomenex, Luna,
5
m
m, C18, 110 A, 150 ꢃ 10 mm column.
cExperimental conditions: 1H and 13C NMR spectra were recorded
at 300 MHz and 75 MHz respectively on a Varian Mercury 2000
spectrometer.
ꢀ
bExperimental conditions: Analytical reverse-phase HPLC was
carried out on a Waters Millennium 2690 system, fitted with a

456
L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
Infrared spectra were recorded on a Shimadzu FTIR 8400s
sodium bicarbonate (2 ꢃ 10 mL). The organic extract was dried with
sodium sulphate, filtered and evaporated to give the crude product
which was purified by flash chromatography, eluting 1:1 ethyl
acetate/hexane to give the desired compound.
spectrometer as thins films on NaCl plates or as solids using a
Perkin Elmer Spectrum 100 FT-IR spectrometer fitted with a dia-
mond window universal ATR sampling accessory.
5.1. General procedure A1: synthesis of the benzoxazole core
5.6. General procedure D1: N-methylation
To 34 mL of polyphosphoric acid (1.5 g/mmol of limiting re-
agent) was added 2-amino-3-hydroxypyridine (1.0 eq) and the
relevant benzoic acid (1.1 eq). The mixture was heated to 220 ꢂC
and stirred for 4 h, cooled and poured into 10% sodium bicarbonate
solution. The suspension was stirred until gas evolution ceased,
then filtered and washed with water. The aqueous medium was
extracted three times with ethyl acetate. The combined organic
layers were then washed with brine, dried with sodium sulphate
and the solvent was removed in vacuo. Purification by column
chromatography on silica gel, eluting cyclohexane/ethyl acetate
20e50% to give the desired benzoxazole core.
To a solution of N-(4-chloro-3-(oxazolo[4,5-b]pyridin-2-yl)
phenyl)benzamide (0.029 mmol) in DMF (1 mL) was added methyl
iodide (0.043 mmol) and caesium carbonate (0.086 mmol). The
mixture was stirred at room temperature under a nitrogen atmo-
sphere overnight. The reaction was diluted with ethyl acetate
before washing with saturated sodium carbonate. The aqueous
layer was extracted three times with ethyl acetate and the com-
bined organic layers were washed with brine and dried with so-
dium sulphate. The solvent was removed in vacuo and the crude
solid was purified on silica gel, eluting with n-heptane/ethyl acetate
1:2, to give the desired compound.
5.2. General procedure A2: synthesis of the benzoxazole core
5.7. General procedure E1: oxidative addition
To 34 mL of polyphosphoric acid (1.5 g/mmol of limiting re-
agent) was added 2-amino-3-hydroxypyridine (1.0 eq) and the
relevant benzoic acid (1.1 eq). The mixture was heated to 180 ꢂC and
stirred for 4 h, cooled and poured into 5 M sodium hydroxide. The
resulting precipitate was filtered and recrystallised from ethyl ac-
etate to give the desired benzoxazole core.
To a solution of oxazolo[4,5-b]pyridine (52) (1.0 eq) in dry DMF
(30 mL/mmol of limiting reagent) was added the relevant bromo-
furancarboxamide (2.0 eq), caesium carbonate (1.1 eq), tri-tert-
butylphosphonium hexaboron salt (0.1 eq), palladium diacetate
(0.05 eq) and copper bromide (0.2 eq). The mixture was stirred for
3 h at 150 ꢂC in a sealed tube. After filtration and expansion with
ethyl acetate, the solution was washed with saturated sodium bi-
carbonate. The combined organic layers were washed with brine and
dried with sodium sulphate. The solvent was removed in vacuo and
the crude material was purified on silica gel, eluting DCM. Further
purification by preparative TLC was carried out with an eluent of
n-heptanes/ethyl acetate 1:2 to give the desired compound.
5.3. General procedure B1: amide coupling
To a solution of the benzoxazole core (1.0 eq) in DCM (2 mL) was
added the carboxylic acid (1.2 eq), EDCI (1.2 eq) and DMAP (0.1 eq).
The solution was stirred under nitrogen at 45 ꢂC overnight.
Work-up 1 (W1): The solution was extended with DCM, a satu-
rated solution of sodium bicarbonate was added and the mixture
was extracted three times with DCM. The combined organic layers
were then washed with brine, dried with sodium sulphate and the
solvent was removed in vacuo. Purification by column chromatog-
raphy on silica gel, eluting ethyl acetate/cyclohexane 50:50 led to
the desired carboxamide.
5.8. Compound characterisation
5.8.1. 4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)-aniline (50)b
Title compound was prepared according to General procedure
A1 to give a yellow powder (20%). LRMS [M þ H]þ 246.1 m/z;
HRMS [M þ H]þ 246.0426 m/z, found 246.0431 m/z; 1H NMR
Work-up 2 (W2): Water (approximately 2 mL) was added to the
reaction mixture and it was transferred to the fridge overnight. The
resulting precipitate was filtered and gave the desired carboxamide.
(DMSO, 300 MHz)
d
8.59 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.27 (dd, J ¼ 8.2,
1.4 Hz, 1H), 7.51 (dd, J ¼ 8.2, 4.9 Hz, 1H), 7.41 (d, J ¼ 2.8 Hz, 1H), 7.32
(d, J ¼ 8.7 Hz,1H), 6.83 (dd, J ¼ 8.7, 2.8 Hz,1H), 5.70 (s, 2H); 13C NMR
(DMSO, 101 MHz)
d 164.0, 155.5, 148.6, 147.2, 142.2, 132.2, 125.2,
5.4. General procedure B2: amide coupling
121.5, 119.7, 119.0, 118.1, 116.5.
To
a
solution of the benzoxazole core (1.0 eq) in DMF
5.8.2. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (1)b
(10 mL/mmol of limiting reagent) was added the carboxylic acid
(1.2 eq), HBTU (1.2 eq) and triethylamine (5.0 eq). The solution
was stirred under nitrogen at 45 ꢂC overnight. The solution was
extended with ethyl acetate, a saturated solution of sodium bicar-
bonate was added and the mixture was extracted three times with
ethyl acetate. The combined organic layers were then washed with
brine, dried with sodium sulphate and the solvent was removed in
vacuo. Purification by column chromatography on silica gel, eluting
ethyl acetate/cyclohexane 50:50 led to the desired carboxamide.
Title compound was prepared from 50 according to General
procedure B1 (W1) as a brown solid (51%). LRMS [M þ H]þ 340.1
m/z; HRMS [M þ H]þ 340.0483 m/z, found 340.0483 m/z; 1H NMR
(DMSO, 400 MHz)
d
10.62 (s, 1H), 8.75 (d, J ¼ 2.6 Hz, 1H), 8.62 (dd,
J ¼ 4.8, 0.8 Hz, 1H), 8.32 (dd, J ¼ 8.2, 0.8 Hz, 1H), 8.07 (dd, J ¼ 8.8,
2.6 Hz, 1H), 8.03e7.93 (m, 1H), 7.72 (d, J ¼ 8.8 Hz, 1H), 7.54 (dd,
J ¼ 8.2, 4.9 Hz, 1H), 7.41 (d, J ¼ 3.5 Hz, 1H), 6.74 (dd, J ¼ 3.5, 1.7 Hz,
1H); 13C NMR (DMSO, 101 MHz)
d 162.6, 156.5, 155.0, 147.0, 146.3,
142.6,138.2,131.9,126.5,124.8,124.7,122.9,121.4,119.4,115.5,112.4,
Note: there is a quaternary carbon missing from the NMR which is
assumed to be under one of the other peaks.
5.5. General procedure C1: Suzuki coupling
A solution of N-(3-(6-bromooxazolo[4,5-b]pyridin-2-yl)phenyl)
furan-2-carboxamide (1.0 eq), boronic acid (3.3 eq) and Pd(PPh3)4
(0.1 eq) in toluene:ethanol:2 M aqueous sodium carbonate (10:1:1
15 mL total volume/0.1 mmol of limiting reagent) was refluxed for
10 h under an atmosphere of nitrogen. Ethyl acetate (20 mL) was
added and the organic phase washed with saturated solution
5.8.3. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (2)b
The title compound was prepared from 50 according to General
procedure B1 (W1) except purification was performed by column
chromatography, eluting 0e40% methanol in DCM. Further

L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
457
purification was performed using the HPLC, to give the title com-
pound as an off-white solid (21%). LRMS [M þ H]þ 354.1 m/z; HRMS
[M þ H]þ 354.0640 m/z, found 354.0637 m/z; 1H NMR (DMSO,
triethylamine (0.612 mmol). The solution was stirred under nitrogen
at room temperature overnight. A white solid precipitated out of the
solution. Filtration and trituration of the precipitate solid led to the
title compound as a white solid (20%). LRMS [M þ H]þ 340.1 m/z.
400 MHz)
d
10.51 (s, 1H), 8.84 (d, J ¼ 2.6 Hz, 1H), 8.61 (dd, J ¼ 4.8,
1.4 Hz,1H), 8.32 (dd, J ¼ 8.2, 1.4 Hz,1H), 8.03 (dd, J ¼ 8.9, 2.7 Hz,1H),
7.84 (d, J ¼ 1.7 Hz, 1H), 7.68 (d, J ¼ 8.8 Hz, 1H), 7.53 (dd, J ¼ 8.2,
4.8 Hz, 1H), 6.62 (d, J ¼ 1.6 Hz, 1H), 2.36 (s, 3H); 13C NMR (DMSO,
5.8.10. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)
benzofuran-2-carboxamide (9)a
101 MHz)
d
162.7, 157.6, 155.0, 146.9, 144.1, 142.5, 141.4, 138.3, 131.7,
To a solution of 50 (0.204 mmol) in DCM (2 mL) was added
benzofuran-2-carboxylic acid (0.244 mmol), EDCI (0.244 mmol)
and DMAP (0.0204 mmol). The solution was stirred under nitrogen
at 45 ꢂC overnight. A white solid precipitated out of the solution.
Filtration and trituration with DCM led to the clean product as a
purple solid (61%). LRMS [M þ H]þ 390.2 m/z; 1H NMR (CDCl3,
128.8, 126.3, 124.8, 124.7, 122.8, 121.4, 119.4, 115.9, 11.1.
5.8.4. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)isoxazole-
5-carboxamide (3)a
Title compound was prepared from 50 according to General
procedure B1 (W1) as an off-white solid (42%). LRMS [M þ H]þ
300 MHz)
d
8.69 (br s,1H), 8.65 (d, J ¼ 4.8 Hz,1H), 8.54 (d, J ¼ 2.6 Hz,
341.2 m/z; 1H NMR (DMSO, 300 MHz)
d
11.14 (br s, 1H), 8.83 (m, 1H),
1H), 8.11 (dd, J ¼ 8.8, 2.9 Hz, 1H), 7.97 (d, J ¼ 8.1 Hz, 1H), 7.71 (d,
J ¼ 7.9 Hz,1H), 7.66 (s, 1H), 7.57 (m, 2H), 7.39 (dd, J ¼ 8.1, 4.9 Hz, 1H),
7.33 (m, 2H).
8.75 (m, 1H), 8.61 (m, 1H), 8.32 (m, 1H), 8.07 (m, 1H), 7.70
(d, J ¼ 8.8 Hz, 1H), 7.53 (m, 1H), 7.30 (m, 1H).
5.8.5. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)oxazole-
5-carboxamide (4)a
5.8.11. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)
benzamide (10)b
Title compound was prepared from 50 according to General
Title compound was prepared from 50 according to General
procedure B1 (W1) as an off-white solid (69%). LRMS [M þ H]þ
350.1 m/z; HRMS [M þ H]þ 350.0691 m/z, found 350.0686 m/z; 1H
procedure B1 (W1) as a brown solid (9%). LRMS [M þ H]þ 341.1
m/z; 1H NMR (DMSO, 300 MHz)
d
10.86 (br s,1H), 8.68 (d, J ¼ 2.6 Hz,
1H), 8.67 (m, 1H), 8.61 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.32 (dd, J ¼ 8.2,
1.5 Hz, 1H), 8.03 (dd, J ¼ 8.9, 2.7 Hz, 1H), 8.02 (s, 1H), 7.73
(d, J ¼ 8.8 Hz, 1H), 7.53 (dd, J ¼ 8.2, 4.9 Hz, 1H).
NMR (DMSO, 400 MHz)
d
10.66 (s, 1H), 8.81 (d, J ¼ 2.6 Hz, 1H), 8.62
(dd, J ¼ 4.8, 1.4 Hz, 1H), 8.33 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.10 (dd, J ¼ 8.8,
2.6 Hz, 1H), 8.05e7.95 (m, 2H), 7.74 (d, J ¼ 8.8 Hz, 1H), 7.69e7.48 (m,
4H); 13C NMR (DMSO, 101 MHz)
d 165.9, 162.7, 155.0, 147.0, 142.6,
5.8.6. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-3-
carboxamide (5)b
138.7, 134.3, 132.0, 131.9, 128.5, 127.8, 126.4, 124.7, 124.7, 122.9,
121.4, 119.4.
Title compound was prepared from 50 according to General
procedure B1 (W1) as a yellow solid (35%). LRMS [M þ H]þ 340.1 m/
z; HRMS [M þ H]þ 340.0483 m/z, found 340.0483 m/z; 1H NMR
5.8.12. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-2-
methoxybenzamide (11)a
(DMSO, 400 MHz)
d
10.32 (s, 1H), 8.67 (d, J ¼ 2.6 Hz, 1H), 8.61 (dd,
Title compound was prepared from 50 according to General
J ¼ 4.8,1.4 Hz,1H), 8.43 (dd, J ¼ 1.5, 0.8 Hz,1H), 8.31 (dd, J ¼ 8.2,1.4 Hz,
1H), 8.06 (dd, J ¼ 8.8, 2.7 Hz, 1H), 7.83 (t, J ¼ 1.7 Hz, 1H), 7.71 (d,
J ¼ 8.8 Hz,1H), 7.53 (dd,J ¼ 8.2,4.8 Hz,1H), 7.03 (dd, J¼ 1.9, 0.8Hz,1H);
procedure B1 (W1) as an orange solid (58%). LRMS [M þ H]þ
380.2 m/z; 1H NMR (CDCl3, 300 MHz)
d 10.88 (br s, 1H), 8.80
(m, 2H), 8.77 (d, J ¼ 2.3 Hz, 1H), 8.60 (dd, J ¼ 4.7, 1.4 Hz, 1H), 8.32
(m, 1H), 8.07 (dd, J ¼ 8.3, 2.5 Hz, 1H), 7.89 (d, J ¼ 6.0 Hz, 2H), 7.75
(d, J ¼ 8.7 Hz, 1H), 7.52 (m, 1H), 4.11 (s, 3H).
13C NMR (DMSO, 101 MHz)
d 162.6, 160.8, 154.9, 147.0, 146.3, 144.5,
142.5, 138.4, 131.9, 126.3, 124.7, 124.4, 122.6, 122.6, 121.4, 119.4, 109.2.
5.8.7. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)thiazole-
4-carboxamide (6)b
5.8.13. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-3-
methoxybenzamide (12)a
Title compound was prepared from 50 according to General
procedure B1 (W1) as a yellow solid (83%). LRMS [M þ H]þ 357.1
m/z; HRMS [M þ H]þ 357.0208 m/z, found 357.0207 m/z; 1H NMR
Title compound was prepared from 50 according to General
procedure B1 (W1) as a dark brown solid (52%). LRMS [M þ H]þ
380.2 m/z; 1H NMR (CD3CN, 300 MHz)
d 9.02 (br s, 1H), 8.74 (d,
(DMSO, 400 MHz)
d
10.87 (s, 1H), 9.31 (d, J ¼ 2.0 Hz, 1H), 8.94 (d,
J ¼ 2.6 Hz,1H), 8.59 (d, J ¼ 4.9 Hz,1H), 8.10 (d, J ¼ 8.2 Hz,1H), 7.96 (m,
J ¼ 2.6 Hz, 1H), 8.63 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.58 (d, J ¼ 2.0 Hz, 1H),
8.34 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.14 (dd, J ¼ 8.9, 2.7 Hz, 1H), 7.74 (d,
J ¼ 8.8 Hz, 1H), 7.56 (dd, J ¼ 8.2, 4.8 Hz, 1H); 13C NMR (DMSO,
1H), 7.63 (m, 1H), 7.53 (m, 1H), 7.47 (m, 3H), 7.16 (m,1H), 3.88 (s, 3H).
5.8.14. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)
picolinamide (13)a
101 MHz) d 164.8,160.2,155.4,152.2, 152.2, 147.8, 143.4,138.9, 137.0,
132.0, 131.6, 130.3, 130.0, 128.2, 126.6, 124.6.
Title compound was prepared from 50 according to General
procedure B2 as a yellow solid (34%). LRMS [M þ H]þ 351.1 m/z; 1H
5.8.8. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-1H-
pyrazole-3-carboxamide (7)a
NMR (CD3OD, 300 MHz)
d
7.06 (m, 2H), 6.66 (dd, J ¼ 8.4, 1.5 Hz, 1H),
6.44 (m, 1H), 6.00 (m, 4H), 5.80 (m, 1H), 5.40 (m, 1H). Note: the
amide proton can be seen in the 1H NMR when run in DMSO at
11.11 ppm.
Title compound was prepared from 50 according to General
procedure B1 (W1) as an off-white solid (79%). LRMS [M þ H]þ
340.1 m/z; 1H NMR (DMSO, 300 MHz)
d 10.54 (br s, 1H), 8.85 (d,
J ¼ 1.4 Hz, 1H), 8.60 (d, J ¼ 4.8 Hz, 1H), 8.30 (m, 1H), 8.09 (m, 1H),
7.88 (s, 1H), 7.68 (dd, J ¼ 8.8, 0.9 Hz, 1H), 7.52 (m, 1H), 6.84 (s, 1H),
Note: the pyrazole eNH signal is presumed to be downfield of the
sweep width used.
5.8.15. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)
isonicotinamide (14)b
To a solution of 50 (0.204 mmol) in DCM (2 mL) and DMF
(0.2 mL) was added isonicotinic acid (0.244 mmol), EDCI
(0.244 mmol) and DMAP (0.0204 mmol). The solution was stirred
under nitrogen at 45 ꢂC. After 3 h a white solid precipitated out of
the solution. The mixture was filtered and triturated with DCM,
which led to the title compound as a white solid (35%). LRMS
[M þ H]þ 351.1 m/z; HRMS [M þ H]þ 351.0643 m/z, found 351.0637
5.8.9. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-1H-
imidazole-4-carboxamide (8)a
To a solution of 50 (0.204 mmol) in DMF (2 mL) was added
4-imidazole carboxylic acid (0.286 mmol), HBTU (0.286 mmol) and

458
L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
m/z; 1H NMR (DMSO, 400 MHz)
d
10.90 (s, 1H), 8.93e8.71 (m, 3H),
5.8.21. N-(3-(Oxazolo[4,5-b]pyridin-2-yl)phenyl)oxazole-5-
8.62 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.33 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.09 (dd,
J ¼ 8.8, 2.6 Hz, 1H), 7.91 (dd, J ¼ 4.5, 1.6 Hz, 2H), 7.76 (d, J ¼ 8.8 Hz,
1H), 7.55 (dd, J ¼ 8.2, 4.8 Hz, 1H); 13C NMR (DMSO, 101 MHz)
carboxamide (19)b
Title compound was prepared from 51 according to General
procedure B1 (W2) as a pale-orange solid (70%). LRMS [M þ H]þ
307.1 m/z; HRMS [M þ H]þ 307.0826 m/z, found 307.0837 m/z; 1H
d
164.8, 163.0, 155.4, 150.9 (2C), 147.5, 143.0, 142.8, 141.8, 138.6,
132.5, 127.5, 125.3, 123.5, 122.1 (2C), 121.9, 119.9.
NMR (DMSO, 400 MHz)
d
10.76 (s, 1H), 8.74 (dd, J ¼ 8.3, 6.6 Hz, 2H),
8.58 (dd, J ¼ 4.8, 1.3 Hz, 1H), 8.29 (dd, J ¼ 8.2, 1.3 Hz, 1H), 8.12e7.94
5.8.16. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)
nicotinamide (15)b
(m, 3H), 7.66 (t, J ¼ 8.0 Hz, 1H), 7.50 (dd, J ¼ 8.2, 4.9 Hz, 1H); 13C
NMR (DMSO, 101 MHz)
d 164.6, 155.4, 155.2, 154.1, 146.6, 144.9,
Title compound was prepared from 50 according to General
procedure B1 (W1) as a white solid (69%). LRMS [M þ H]þ 351.1
m/z; HRMS [M þ H]þ 351.0643 m/z, found 351.0640 m/z; 1H NMR
142.8, 139.1, 130.5, 130.0, 126.4, 124.2, 123.2, 120.9, 119.1, 119.1.
5.8.22. N-(3-(Oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-3-
carboxamide (20)a
Title compound was prepared from 51 according to General
procedure B1 (W1) as an off-white solid (79%). LRMS Mþ 305.0
m/z; HRMS Mþ 305.08004 m/z, found 305.08019 m/z; 1H NMR
(DMSO, 400 MHz)
d
10.87 (s, 1H), 9.17 (dd, J ¼ 2.3, 0.7 Hz, 1H), 8.80
(dd, J ¼ 4.5, 1.9 Hz, 2H), 8.64 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.40e8.30 (m,
2H), 8.10 (dd, J ¼ 8.8, 2.7 Hz, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H), 7.62 (ddd,
J ¼ 8.0, 4.8, 0.8 Hz, 1H), 7.56 (dd, J ¼ 8.2, 4.8 Hz, 1H); 13C NMR
(DMSO, 101 MHz)
d
164.5, 162.6, 154.9, 152.6, 142.6, 138.4, 135.7,
(CDCl3, 300 MHz)
d
8.54 (d, J ¼ 5.1 Hz, 1H), 8.28 (s, 1H), 8.45 (s, 1H),
135.6, 132.0, 130.0, 126.8, 124.8, 124.8, 123.6 (2C), 121.4 (2C), 119.4.
8.15 (d, J ¼ 0.9 Hz, 1H), 8.00 (m, 2H), 7.82 (dd, J ¼ 8.1, 3.6 Hz, 1H),
7.50e7.45 (m, 2H), 7.29 (t, J ¼ 4.8 Hz,1H), 6.83 (d, J ¼ 3.6 Hz,1H); 13
C
5.8.17. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-2-
methoxyacetamide (16)a
NMR (CDCl3, 75 MHz) d 165.4, 161.3, 156.4, 146.9, 145.8, 144.3, 143.4,
138.8, 130.1, 127.2, 124.5, 124.1, 123.0, 120.5, 119.7, 118.6, 108.7.
Title compound was prepared from 50 according to General
procedure B2 as a yellow solid (79%). LRMS [M þ H]þ 318.2 m/z; 1H
5.8.23. N-(3-(Oxazolo[4,5-b]pyridin-2-yl)phenyl)thiazole-4-
NMR (DMSO, 300 MHz)
d
10.21 (br s, 1H), 8.67 (d, J ¼ 2.5 Hz, 1H),
carboxamide (21)b
8.60 (dd, J ¼ 4.8, 1.3 Hz, 1H), 8.30 (d, J ¼ 8.2 Hz, 1H), 7.93 (dd, J ¼ 8.8,
2.6 Hz, 1H), 7.66 (d, J ¼ 8.8 Hz, 1H), 7.51 (dd, J ¼ 8.2, 4.8 Hz, 1H), 4.04
(s, 2H), 3.38 (s, 3H).
Title compound was prepared from 51 according to General
procedure B2 as a pale-yellow solid (20%). LRMS [M þ H]þ 323.1 m/
z; HRMS [M þ H]þ 323.0597 m/z, found 323.0613 m/z; 1H NMR
(DMSO, 400 MHz)
d
10.75 (s, 1H), 9.32 (d, J ¼ 2.0 Hz, 1H), 8.97 (t,
5.8.18. 3-(Oxazolo[4,5-b]pyridin-2-yl)aniline (51)b
J ¼ 1.8 Hz, 1H), 8.70e8.46 (m, 2H), 8.29 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.11
Title compound was prepared according to General procedure
(ddd, J ¼ 8.2, 2.1, 0.9 Hz, 1H), 8.05e7.93 (m, 1H), 7.64 (t, J ¼ 8.0 Hz,
A1 as a pale-yellow solid (35%). LRMS [M þ H]þ 212.2 m/z; 1H
1H), 7.50 (dd, J ¼ 8.2, 4.9 Hz,1H); 13C NMR (DMSO,101 MHz)
d 164.7,
NMR (DMSO, 400 MHz)
d
8.53 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.22 (dd,
159.5, 155.5, 155.2, 150.4, 146.6, 142.8, 139.5, 129.8, 126.3, 126.1,
124.5, 123.0, 120.8, 119.2, 119.1.
J ¼ 8.1, 1.4 Hz, 1H), 7.60e7.33 (m, 3H), 7.27 (t, J ¼ 7.8 Hz, 1H), 6.85
(ddd, J ¼ 8.0, 2.3, 1.0 Hz, 1H), 5.55 (s, 2H); 13C NMR (DMSO,
101 MHz)
d
165.6, 155.6, 149.5, 146.3, 142.6, 129.8, 126.3, 120.5,
5.8.24. Oxazolo[4,5-b]pyridine (52)b
118.8, 118.0, 114.9, 112.3.
To a solution of 2-amino-3-hydroxypyridine (90.1 mmol) in
triethylorthoformate (720.7 mmol) was added p-toluenesulphonic
acid (4.50 mmol) and the mixture was stirred under nitrogen at
160 ꢂC overnight. The triethylorthoformate was removed in vacuo
and the crude material was purified by column chromatography,
eluting with cyclohexane/ethyl acetate 5%e40% to give the title
compound as a beige solid (68%). LRMS [M þ H]þ 121.1 m/z;
5.8.19. N-(3-(Oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (17)b
To a solution of 51 (0.142 mmol) in DCM (2 mL) was added
2-furoic acid (0.199 mmol), EDCI (0.199 mmol) and DMAP
(0.0142 mmol). The solution was stirred under nitrogen at 45 ꢂC
overnight. The solution was stirred under nitrogen at 45 ꢂC over-
night. A white solid precipitated from the solution. Filtration and
trituration with DCM led to the title compound as a brown solid
(51%). LRMS [M þ H]þ 306.1 m/z; HRMS [M þ H]þ 306.0873 m/z,
1H NMR (DMSO, 400 MHz)
d
9.04 (s, 1H), 8.58 (dd, J ¼ 4.8, 1.4 Hz,
1H), 8.26 (dd, J ¼ 8.2, 1.4 Hz, 1H), 7.50 (dd, J ¼ 8.2, 4.8 Hz, 1H), 6.41
(s, 2H); 13C NMR (DMSO, 101 MHz)
120.1.
d 157.7, 154.5, 147.1, 142.0, 121.5,
found 306.0871 m/z; 1H NMR (DMSO, 400 MHz)
d 10.62 (s, 1H), 8.80
(t, J ¼ 1.8 Hz, 1H), 8.57 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.30 (dd, J ¼ 8.2,
1.4 Hz,1H), 8.08 (ddd, J ¼ 8.2, 2.1,1.0 Hz,1H), 8.02e7.89 (m, 2H), 7.61
(t, J ¼ 8.0 Hz,1H), 7.50 (ddd, J ¼ 6.6, 4.8, 4.0 Hz, 2H), 6.72 (dd, J ¼ 3.5,
5.8.25. 2-Methyl-3-(oxazolo[4,5-b]pyridin-2-yl)aniline (53)b
Title compound was prepared according to General procedure
A2 as a dark-red solid (48%). LRMS [M þ H]þ 226.2 m/z; HRMS
[M þ H]þ 226.0975 m/z, found 226.0975 m/z; 1H NMR (DMSO,
1.7 Hz, 1H); 13C NMR (DMSO, 101 MHz)
d 165.0, 156.6, 155.2, 147.2,
146.2 (2C), 143.1, 139.7, 129.9, 126.2, 124.3, 122.9, 121.0, 119.7, 119.1,
115.4, 112.3.
400 MHz)
d
8.54 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.21 (dd, J ¼ 8.1, 1.4 Hz, 1H),
7.45 (dd, J ¼ 8.1, 4.9 Hz, 1H), 7.28 (dd, J ¼ 7.7, 1.0 Hz, 1H), 7.12 (t,
J ¼ 7.8 Hz,1H), 6.92 (dd, J ¼ 7.9, 0.9 Hz,1H), 5.26 (s, 2H), 2.48 (s, 3H);
5.8.20. 3-Methyl-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (18)b
13C NMR (DMSO, 101 MHz)
d 166.1, 155.5, 148.1, 146.2, 142.1, 126.4,
125.7, 121.5, 120.6, 118.8, 117.9, 117.5, 14.3.
Title compound was prepared from 51 according to General
procedure B1 (W2) as an off-white solid (48%). LRMS [M þ H]þ
320.1 m/z; HRMS [M þ H]þ 320.1030 m/z, found 320.1040 m/z; 1H
5.8.26. N-(2-Methyl-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (22)b
NMR (DMSO, 400 MHz)
d
10.42 (s, 1H), 8.89 (t, J ¼ 1.8 Hz, 1H), 8.57
Title compound was prepared from 53 according to General
(dd, J ¼ 4.9, 1.4 Hz, 1H), 8.29 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.10e7.90 (m,
2H), 7.85 (d, J ¼ 1.5 Hz, 1H), 7.61 (t, J ¼ 8.0 Hz, 1H), 7.49 (dd, J ¼ 8.2,
4.9 Hz, 1H), 6.64 (d, J ¼ 1.4 Hz, 1H), 2.39 (s, 3H); 13C NMR (DMSO,
procedure B1 (W2) as a pale-yellow solid (68%). 1H NMR (DMSO,
400 MHz)
d
10.13 (s, 1H), 8.59 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.29 (dd,
J ¼ 8.2, 1.4 Hz, 1H), 8.10e8.02 (m, 1H), 7.97 (dd, J ¼ 1.7, 0.8 Hz, 1H),
7.62 (d, J ¼ 6.8 Hz, 1H), 7.55e7.44 (m, 2H), 7.35 (dd, J ¼ 3.5, 0.7 Hz,
1H), 6.74 (dd, J ¼ 3.5, 1.8 Hz, 1H), 2.63 (s, 3H).
101 MHz) d 164.9, 157.7, 155.5, 146.6, 143.9, 142.8, 141.6, 139.6, 129.7,
128.4, 126.2, 124.2, 122.7, 120.8, 119.1, 119.0, 115.8, 11.7.

L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
459
5.8.27. 3-Amino-2-fluorobenzoic acid (54)b
400 MHz)
d
8.85 (d, J ¼ 5 Hz, 1H), 8.75 (dd, J ¼ 5, 1 Hz, 1H), 7.83
0.05 M Solution of 2-fluoro-3-nitrobenzoic acid (0.27 mmol)
was made up in 50:50 ethyl acetate/ethanol (54 mL). This was
loaded on to the ThalesNano H-CubeÒ with a 10% Pd/C CatCartÒ.
The reaction was monitored by LRMS until no more starting ma-
terial was present and the solvent was removed under reduced
pressure to give the amine as a pale-yellow solid (95%). There was a
small amount of starting material by 1H NMR though it was not
possible to separate the two compounds and so the material was
used directly in the next reaction. LRMS [M þ H]þ 156.1 m/z; 1H
(m, 1H), 7.65 ( dd, J ¼ 7, 5 Hz, 1H), 7.58 (d, J ¼ 7 Hz, 1H), 7.38
(dd, J ¼ 5, 2 Hz, 1H), 6.25 (s, 2H).
5.8.34. N-(4-(Oxazolo[4,5-b]pyridin-2-yl)pyridin-2-yl)furan-2-
carboxamide (25)b
Title compound was prepared from 58 according to General
procedure B2 as a pale-brown solid (85%). LRMS [M þ H]þ 307.1 m/
z; HRMS [M þ H]þ 307.0826 m/z, found 307.0826 m/z; 1H NMR
(DMSO, 400 MHz)
d
9.23 (s, 1H), 8.59 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.31 (s,
NMR (DMSO, 400 MHz)
d
7.03e6.83 (m, 3H), 5.32 (s, 2H).
1H), 8.28 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.16 (d, J ¼ 5.2 Hz, 1H), 7.52 (dd,
J ¼ 8.2, 4.8 Hz, 1H), 7.29e7.23 (m, 1H), 7.20 (dd, J ¼ 5.2, 1.5 Hz, 1H),
5.8.28. 2-Fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)aniline (55)b
Title compound was prepared using 53 according to General
procedure A2 as a pale-brown solid (63%). LRMS [M þ H]þ 230.1
m/z; HRMS [M þ H]þ 230.0724 m/z, found 230.0723 m/z; 1H NMR
6.42 (s, 2H); 13C NMR (DMSO, 101 MHz)
d 170.9, 162.6, 159.0, 157.4,
148.2, 147.5, 147.0, 146.0, 143.8, 142.6, 114.7, 114.4, 113.6, 112.8, 107.3,
102.1.
(DMSO, 400 MHz)
d
8.58 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.26 (dd, J ¼ 8.2,
5.8.35. 2-Methoxy-5-(oxazolo[4,5-b]pyridin-2-yl)aniline (59)b
Title compound was prepared according to General procedure
A2 as a pale-brown solid (85%). LRMS [M þ H]þ 242.2 m/z; 1H
1.4 Hz,1H), 7.49 (dd, J ¼ 8.2, 4.9 Hz,1H), 7.35 (ddd, J ¼ 7.8, 6.1, 1.8 Hz,
1H), 7.15e7.01 (m, 2H), 5.57 (s, 2H); 13C NMR (DMSO, 101 MHz)
d
162.3 (JCeF ¼ 4 Hz), 155.2, 148.5 (JCeF ¼ 252 Hz), 146.7, 142.4, 138.0
NMR (DMSO, 400 MHz)
d
8.48 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.15 (dd,
(JCeF ¼ 12 Hz), 124.9 (JCeF ¼ 4 Hz), 120.9, 119.95 (JCeF ¼ 5 Hz), 119.1,
116.2, 113.9 (JCeF ¼ 8 Hz).
J ¼ 8.1, 1.4 Hz, 1H), 7.54 (d, J ¼ 2.2 Hz, 1H), 7.48 (dd, J ¼ 8.3, 2.2 Hz,
1H), 7.39 (dd, J ¼ 8.1, 4.9 Hz, 1H), 7.02 (d, J ¼ 8.4 Hz, 1H), 5.17 (s, 2H),
3.88 (s, 3H); 13C NMR (DMSO, 101 MHz)
d 165.8, 156.0, 150.0, 146.0,
5.8.29. N-(2-Fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (23)b
142.5, 138.5, 120.0, 118.4, 118.2, 116.7, 111.9, 110.6, 55.6.
Title compound was prepared from 55 according to General
5.8.36. N-(2-Methoxy-5-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-
2-carboxamide (26)b
procedure B1 (W2) as a pale-brown solid (44%). 1H NMR (DMSO,
400 MHz)
d
10.27 (s, 1H), 8.61 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.32 (dd,
Title compound was prepared from 59 according to General
J ¼ 8.2, 1.4 Hz, 1H), 8.19e8.08 (m, 1H), 8.00 (dd, J ¼ 1.6, 0.7 Hz, 1H),
7.96e7.87 (m, 1H), 7.58e7.44 (m, 2H), 7.41 (dd, J ¼ 3.5, 0.7 Hz, 1H),
6.75 (dd, J ¼ 3.5, 1.8 Hz, 1H).
procedure B1 (W2) as a pale-brown solid (70%). LRMS [M þ H]þ
336.1 m/z; HRMS [M þ H]þ 336.0979 m/z, found 336.0976 m/z; 1
H
NMR (DMSO, 400 MHz)
d
9.29 (s, 1H), 8.89 (d, J ¼ 2.1 Hz, 1H), 8.52
(dd, J ¼ 4.9, 1.4 Hz, 1H), 8.22 (dd, J ¼ 8.1, 1.4 Hz, 1H), 8.08 (dd, J ¼ 8.6,
2.2 Hz, 1H), 7.99 (dd, J ¼ 1.7, 0.7 Hz, 1H), 7.44 (dd, J ¼ 8.1, 4.9 Hz, 1H),
7.38 (dd, J ¼ 4.7, 4.0 Hz, 2H), 6.75 (dd, J ¼ 3.5, 1.7 Hz, 1H), 3.34 (s,
5.8.30. 2-(2-Chloro-3-nitrophenyl)oxazolo[4,5-b]pyridine (56)c
Title compound was prepared according to General procedure
A2 as a pale-yellow solid (60%) and used directly in the next reac-
3H); 13C NMR (DMSO, 101 MHz)
d 164.8, 155.8, 155.7, 153.4, 147.0,
tion without further purification. 1H NMR (CDCl3, 300 MHz)
d
7.24
146.3, 146.0, 142.7, 127.1, 125.3, 120.9, 120.3, 118.7, 118.0, 115.4, 112.5,
112.0, 56.5.
(dd, J ¼ 8.4, 5.1 Hz,1H), 7.61 (t, J ¼ 8.1 Hz, 1H), 7.94 (ddd, J ¼ 21.9, 8.1,
1.5 Hz, 2H), 8.43 (dd, J ¼ 7.8, 1.5 Hz, 1H), 8.68 (J ¼ 4.8, 1.5 Hz, 1H).
5.8.37. 2-Methyl-5-(oxazolo[4,5-b]pyridin-2-yl)aniline (60)b
Title compound was prepared according to General procedure
A1 as a pale-brown solid (34%). LRMS [M þ H]þ 226.2 m/z; HRMS
[M þ H]þ 226.0975 m/z, found 226.0979 m/z; 1H NMR (DMSO,
5.8.31. 2-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)aniline (57)c
56 (1.45 mmol) was reduced by heating with iron powder
(4.35 mmol) and concentrated hydrochloric acid (20.3 mmol) in
refluxing ethanol (5 mL). The reaction mixture was diluted and
basified with a saturated sodium bicarbonate solution. The aqueous
phase was extracted with ethyl acetate (3 ꢃ 30 mL) and the extract
was dried with sodium sulphate, filtered and evaporated to give the
title compound as a brown solid (66%). LRMS Mþ 245.0 m/z; HRMS
Mþ 245.03559 m/z, found 245.03559 m/z; 1H NMR (CDCl3,
400 MHz)
d
8.50 (d, J ¼ 4.8 Hz, 1H), 7.95 (d, J ¼ 7.7 Hz, 1H), 7.78 (dd,
J ¼ 7.7, 4.8 Hz, 1H), 7.75 (d, J ¼ 8.4 Hz, 1H), 7.50 (s, 1H), 7.28 (d,
J ¼ 0.3 Hz, 1H), 5.45 (s, 2H), 2.30 (s, 3H); 13C NMR (DMSO, 101 MHz)
d
167.1, 155.3, 147.4, 145.0,142.9, 126.5, 116.2, 126.5, 122.9 (2C), 117.6,
114.1, 18.1.
300 MHz)
d
8.60 (s, 1H), 7.89 (d, J ¼ 8.1 Hz, 1H), 7.70 (d, J ¼ 7.5 Hz,
5.8.38. N-(2-Methyl-5-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (27)b
1H), 7.35e7.19 (m, 3H), 6.97 (d, J ¼ 6.9 Hz, 1H).
Title compound was prepared from 60 according to General
procedure B2 as a pale-brown solid (54%). HPLC >85% purity at
254 nm. LRMS [M þ H]þ 320.3 m/z; 1H NMR (DMSO, 400 MHz)
5.8.32. N-(2-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (24)c
Title compound was prepared from 57 according to General
procedure B1 (W1) as a pale-brown solid (54%). LRMS Mþ 339 m/z;
HRMS Mþ 339.04107 m/z, found 339.04141 m/z; 1H NMR (CDCl3,
d
10.12 (s, 1H), 8.61 (dd, J ¼ 6.5, 1.8 Hz, 1H), 8.50 (dd, J ¼ 1.7, 0.8 Hz,
1H), 7.95 (dd, J ¼ 1.7, 0.9 Hz,1H), 7.65e7.75 (m, 2H), 7.46 (dd, J ¼ 8.5,
6.5 Hz,1H), 7.40 (dd, J ¼ 8.1, 0.8 Hz,1H), 7.29 (dd, J ¼ 3.4, 0.9 Hz, 1H),
6.72 (dd, J ¼ 3.4, 1.7 Hz, 1H), 2.27 (3H); 13C NMR (DMSO, 101 MHz)
300 MHz)
d
9.02 (s, 1H), 8.78 (dd, J ¼ 8.4,1.5 Hz, 1H), 8.64 (dd, J ¼ 4.8,
1.5 Hz, 1H), 7.96 (J ¼ 14.4, 8.1, 1.5 Hz, 2H), 7.60 (d, J ¼ 2.7 Hz, 1H), 7.49
(t, J¼ 8.4Hz,1H), 7.37 (dd,J¼ 8.1, 4.8 Hz,1H), 7.31(d, J¼ 4.5 Hz,1H), 6.6
(dd, J ¼ 3.6, 1.8 Hz, 1H).
d 167.0, 161.9, 154.9, 148.2 (C2), 147.1, 142.6, 131.4, 133.1, 130.0, 123.6,
119.6 (C2), 119.6, 119.3, 115.3, 111.7, 17.3.
5.8.39. N-(2-Fluoro-5-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (28)b
Title compound was prepared according to General procedure
B2 as a pale-brown solid (72%). LRMS [M þ H]þ 324.1 m/z; HRMS
[M þ H]þ 324.0779 m/z, found 324.0779 m/z; 1H NMR (DMSO,
5.8.33. 4-(Oxazolo[4,5-b]pyridin-2-yl)pyridin-2-amine (58)b
Title compound was prepared according to General procedure
A1 as a pale-brown solid (36%). LRMS [M þ H]þ 213.2 m/z; HRMS
[M þ H]þ 213.0771 m/z, found 213.0774 m/z; 1H NMR (DMSO,

460
L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
400 MHz)
d
9.98 (s, 1H), 8.66 (m, 2H), 7.85e7.95 (m, 2H), 7.84 (d,
5.8.46. N-(3-Fluoro-5-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (31)b
J ¼ 8.2 Hz, 1H), 7.57 (d, J ¼ 8.2 Hz, 1H), 7.53 (dd, J ¼ 7.7, 5.3 Hz, 1H),
7.29 (d, J ¼ 3.4 Hz,1H), 6.71 (dd, J ¼ 3.4,1.8 Hz,1H); 13C NMR (DMSO,
Title compound was prepared from 64 according to General
procedure B2 as a pale brown solid (61%). LRMS [M þ H]þ 324.2 m/z;
HRMS [M þ H]þ 324.0779 m/z, found 324.0771 m/z; 1H NMR (DMSO,
101 MHz) d 167.0, 162.7, 158.3,154.9,148.2, 148.0, 143.8, 142.6, 124.7,
121.9, 121.1, 119.6, 119.9, 118.2, 116.2, 115.3, 111.7.
400 MHz)
d
9.65 (s, 1H), 8.69 (d, J ¼ 5.3 Hz, 1H), 8.09 (s, 1H), 8.00 (d,
5.8.40. 2-(4-Chloro-3-nitrophenyl)oxazolo[4,5-b]pyridine (61)c
J ¼ 1.9 Hz,1H), 7.85e7.95 (m, 3H), 7.57 (dd, J ¼ 8.1, 5.3 Hz,1H), 7.31 (d,
Title compound prepared according to General procedure A1 as a
J ¼ 3.4 Hz, 1H), 6.74 (dd, J ¼ 3.4, 1.7 Hz, 1H); 13C NMR (DMSO,
grey solid (70%). 1H NMR (CDCl3, 300 MHz)
d
9.16 (d, J ¼ 2.7 Hz, 1H),
101 MHz) d 110.9, 112.3, 115.3 (2C), 116.4, 119.0, 120.1, 127.9, 139.6,
8.70 (d, J ¼ 4.8 Hz, 1H), 8.34 (dd, J ¼ 8.8, 2.7 Hz, 1H), 8.02 (dd, J ¼ 8.2,
143.0, 143.5, 148.2 (2C), 161.0, 162.5, 163.9, 167.0.
1.5 Hz, 1H), 7.80 (d, J ¼ 8.8 Hz, 1H), 7.44 (dd, J ¼ 8.2, 4.8 Hz, 1H).
5.8.47. 4-Methyl-3-(oxazolo[4,5-b]pyridin-2-yl)aniline (65)b
Title compound was prepared according to General procedure
A1 as a pale-yellow solid (30%). LRMS [M þ H]þ 226.2 m/z; 1H
5.8.41. 2-Chloro-5-(oxazolo[4,5-b]pyridin-2-yl)aniline (62)c
61 (1.45 mmol) was reduced by heating with iron powder
(4.35 mmol) and concentrated hydrochloric acid (20.3 mmol) in
refluxing ethanol (5 mL). The reaction mixture was diluted and
basified with a saturated sodium bicarbonate solution. The aqueous
phase was extracted with ethyl acetate (3 ꢃ 30 mL) and the extract
was dried with sodium sulphate, filtered and evaporated to give the
title compound as a pale yellow solid (66%). LRMS Mþ 245.0 m/z;
HRMS Mþ 245.03559 m/z, found 245.03559 m/z; 1H NMR (CDCl3,
NMR (DMSO, 400 MHz)
d
8.54 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.22 (dd,
J ¼ 8.1, 1.4 Hz, 1H), 7.52e7.41 (m, 2H), 7.11 (d, J ¼ 8.2 Hz, 1H), 6.77
(dd, J ¼ 8.2, 2.5 Hz, 1H), 5.30 (s, 2H), 2.59 (s, 3H); 13C NMR (DMSO,
101 MHz)
d 165.70, 155.61, 147.01, 146.23, 141.96, 132.56, 125.45,
124.77, 120.51, 118.66, 117.96, 114.29, 20.85.
5.8.48. N-(4-Methyl-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (32)b
300 MHz)
d
8.59 (d, J ¼ 4.8 Hz, 1H), 7.85 (d, J ¼ 8.1 Hz, 1H), 7.74 (d,
J ¼ 2.1 Hz, 1H), 7.62 (dd, J ¼ 8.1, 1.8 Hz, 1H), 7.41 (d, J ¼ 8.1 Hz, 1H),
7.30 (dd, J ¼ 8.1, 5.1 Hz, 1H), Note: The amine was observed as a
broad peak and so was not assigned; 13C NMR (CDCl3, 75 MHz)
Title compound was prepared from 65 according to General
procedure B2 as an off-white solid (7% yield). LRMS [M þ H]þ 320.2
m/z; HRMS [M þ H]þ 320.1030 m/z, found 320.1027 m/z; 1H NMR
d
165.4, 156.5, 146.9, 143.7, 143.4, 130.3, 125.9, 123.7, 120.4, 118.5,
(DMSO, 400 MHz)
d
10.44 (s, 1H), 8.70 (d, J ¼ 2.3 Hz, 1H), 8.57 (dd,
118.4, 114.9.
J ¼ 4.9, 1.4 Hz, 1H), 8.28 (dd, J ¼ 8.1, 1.4 Hz, 1H), 8.03e7.86 (m, 2H),
7.56e7.42 (m, 2H), 7.39 (dd, J ¼ 3.5, 0.6 Hz, 1H), 6.73 (dd, J ¼ 3.5,
5.8.42. N-(2-Chloro-5-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (29)c
Title compound was prepared from 62 according to General
procedure B1 (W1) as a pale-brown solid (47%). LRMS Mþ 339.0
m/z; HRMS Mþ 339.04107 m/z, found 339.04141 m/z; 1H NMR
1.7 Hz, 1H), 2.75 (s, 3H); 13C NMR (DMSO, 101 MHz)
d 164.8, 156.3,
155.5, 147.3, 146.5, 146.0, 142.1, 137.0, 134.10, 132.4, 124.9, 123.7,
121.0, 120.9, 119.0, 115.0, 112.2, 21.4.
5.8.49. 3-Methyl-N-(4-methyl-3-(oxazolo[4,5-b]pyridin-2-yl)
phenyl)furan-2-carboxamide (33)b
(CDCl3, 300 MHz)
d
9.45 (d, J ¼ 1.8 Hz, 1H), 8.79 (s, 1H), 8.57 (dd,
J ¼ 4.8, 1.2 Hz, 1H), 8.09 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.88 (dd, J ¼ 8.1,
Title compound was prepared from 65 according to General
procedure B1 (W1) as a pale-orange solid (56%). LRMS [M þ H]þ
334.2 m/z; HRMS [M þ H]þ 334.1186 m/z, found 334.1195 m/z;
1.5 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 2H), 7.33 (d, J ¼ 4.2 Hz, 2H), 6.60
(J ¼ 3.6, 1.8 Hz, 1H); 13C NMR (DMSO, 75 MHz)
d 164.7, 156.3, 156.1,
147.1, 146.8, 145.1, 143.4, 135.1, 127.0, 126.3, 124.7, 120.6, 120.2, 118.7,
116.5, 113.1, 112.6.
1H NMR (DMSO, 400 MHz)
d
10.33 (s, 1H), 8.80 (d, J ¼ 2.3 Hz, 1H),
8.58 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.30 (dd, J ¼ 8.2, 1.4 Hz, 1H), 7.90
(dd, J ¼ 8.3, 2.3 Hz, 1H), 7.83 (d, J ¼ 1.7 Hz, 1H), 7.60e7.37 (m, 2H),
6.62 (d, J ¼ 1.7 Hz, 1H), 2.75 (s, 3H), 2.38 (s, 3H); 13C NMR
5.8.43. N-(5-Bromopyridin-3-yl)furan-2-carboxamide (63)b
Title compound was prepared according to General procedure
B1 (W1) to give the title compound as an orange solid (9% yield).
(DMSO, 101 MHz)
d 164.9, 157.5, 155.5, 146.5, 143.8, 142.1, 141.7,
137.1, 133.9, 132.3, 128.1, 124.7, 123.8, 121.0, 120.8, 118.9, 115.7,
21.3, 11.1.
LRMS [M þ H, Br79 þ
]
d
267.0 m/z, [M þ H, Br81 þ
]
269.0 m/z; 1H NMR
10.59 (s, 1H), 8.90 (d, J ¼ 2.1 Hz, 1H), 8.55e8.35
(DMSO, 400 MHz)
(m, 2H), 8.00 (dd, J ¼ 1.7, 0.8 Hz, 1H), 7.39 (dd, J ¼ 3.5, 0.8 Hz, 1H),
5.8.50. N-(4-Methyl-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)oxazole-
5-carboxamide (34)b
6.74 (dd, J ¼ 3.5, 1.7 Hz, 1H); 13C NMR (DMSO, 101 MHz)
d 156.6,
146.7, 146.4, 144.83, 140.2, 136.4, 129.2, 119.5, 115.9, 112.5.
Title compound was prepared from 65 according to General
procedure B1 (W1) as an off-white solid (67%). LRMS [M þ H]þ
321.1 m/z; HRMS [M ꢁ H]ꢁ 319.0837 m/z, found 319.0834 m/z; 1H
5.8.44. N-(5-(Oxazolo[4,5-b]pyridin-2-yl)pyridin-3-yl)furan-2-
carboxamide (30)a
NMR (DMSO, 400 MHz) d 10.68 (s, 1H), 8.77e8.62 (m, 2H), 8.58 (dd,
Title compound was prepared from 63 according to General
J ¼ 4.8, 1.4 Hz, 1H), 8.28 (dd, J ¼ 8.1, 1.4 Hz, 1H), 8.04 (s, 1H), 7.93 (dd,
procedure E1 as a brown solid (17% yield). LRMS [M þ H]þ 307.2
J ¼ 8.3, 2.3 Hz, 1H), 7.61e7.40 (m, 2H), 2.76 (s, 3H); 13C NMR (DMSO,
m/z; 1H NMR (CDCl3, 300 MHz) :
d
9.80 (m, 2H), 9.74 (s, 1H), 9.29 (s,
101 MHz) d 164.7, 155.4, 155.0,154.0,146.5,145.0, 142.1, 136.6, 134.5,
1H), 8.69 (d, J ¼ 3.7 Hz, 1H), 7.99 (d, J ¼ 8.7 Hz, 1H), 7.66 (s, 1H), 7.62
132.5, 130.2, 124.9, 123.7, 121.1, 120.9, 118.9, 21.4.
(s, 1H), 7.43 (dd, J ¼ 8.1, 4.6 Hz, 1H), 6.63 (m, 1H).
5.8.51. N-(4-Methyl-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-3-
carboxamide (35)b
5.8.45. 3-Fluoro-5-(oxazolo[4,5-b]pyridin-2-yl)aniline (64)b
Title compound was prepared according to General procedure
A2 as a pale-brown solid (61%). HPLC >90% purity at 254 nm;
LRMS [M þ H]þ 230.1 m/z; HRMS [M þ H]þ 230.0724 m/z, found
Title compound was prepared from 65 according to General
procedure B1 (W1) as an off-white solid (79%). LRMS [M þ H]þ
320.2 m/z; HRMS [M ꢁ H]ꢁ 318.0884 m/z, found 318.0876 m/z; 1H
230.0725 m/z; 1H NMR (DMSO, 400 MHz)
d
8.64 (d, J ¼ 5.1 Hz, 1H),
NMR (DMSO, 400 MHz) d 10.18 (s, 1H), 8.77e8.49 (m, 2H), 8.43 (d,
7.72 (m, 2H), 7.52 (dd, J ¼ 7.6, 5.2 Hz, 1H), 7.50 (br s, 1H), 7.44 (s, 1H),
5.62 (s, 2H); 13C NMR (DMSO, 101 MHz)
106.9, 111.2, 118.3, 121.0
(2C), 129.0, 140.1, 148.0, 150.1, 155.2, 164.4, 167.0.
J ¼ 0.6 Hz, 1H), 8.28 (dd, J ¼ 8.1, 1.4 Hz, 1H), 7.94 (dd, J ¼ 8.3, 2.3 Hz,
d
1H), 7.83 (t, J ¼ 1.7 Hz, 1H), 7.58e7.36 (m, 2H), 7.04 (dd, J ¼ 1.8,
0.7 Hz, 1H), 2.76 (s, 3H); 13C NMR (DMSO, 101 MHz)
d 164.8, 160.5,

L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
461
155.5, 146.5, 146.0, 144.3, 142.1, 137.3, 133.9, 132.4, 124.8, 123.5,
122.8, 120.9, 120.8, 118.9, 109.2, 21.3.
J ¼ 8.2, 1.4 Hz, 1H), 8.08 (ddd, J ¼ 9.0, 4.2, 2.8 Hz, 1H), 7.83 (t,
J ¼ 1.7 Hz, 1H), 7.59e7.46 (m, 2H), 7.04 (dd, J ¼ 1.9, 0.8 Hz, 1H).
5.8.52. N-(4-Methyl-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)
thiazole-4-carboxamide (36)b
5.8.58. N-(4-Fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)thiazole-
4-carboxamide (41)b
Title compound was prepared from 65 according to General
procedure B1 (W1) as a pale-yellow solid (56%). LRMS [M þ H]þ
337.1 m/z; HRMS [M þ H]þ 337.0754 m/z, found 337.0768 m/z; 1H
Title compound was prepared from 66 according to General
procedure B1 (W2) as an off-white solid (77%). LRMS [M þ H]þ
341.1 m/z; HRMS [M þ H]þ 341.0503 m/z, found 341.0512 m/z; 1H
NMR (DMSO, 400 MHz)
d
10.65 (s, 1H), 9.30 (d, J ¼ 2.0 Hz, 1H), 8.89
NMR (DMSO, 400 MHz)
d
10.80 (s, 1H), 9.31 (d, J ¼ 2.0 Hz, 1H), 8.96
(d, J ¼ 2.3 Hz, 1H), 8.66e8.44 (m, 2H), 8.29 (dd, J ¼ 8.1, 1.4 Hz, 1H),
(dd, J ¼ 6.5, 2.7 Hz, 1H), 8.69e8.47 (m, 2H), 8.32 (dd, J ¼ 8.2, 1.4 Hz,
7.98 (dd, J ¼ 8.3, 2.3 Hz, 1H), 7.62e7.33 (m, 2H), 2.76 (s, 3H); 13C
1H), 8.13 (ddd, J ¼ 9.0, 4.2, 2.9 Hz, 1H), 7.64e7.42 (m, 2H); 13C NMR
NMR (DMSO, 101 MHz)
d
164.8, 159.3, 155.5, 155.1, 150.5, 146.5,
(DMSO, 101 MHz) d 161.4, 161.4, 159.4, 155.2, 150.3, 146.9, 142.5,
142.1, 137.0, 134.3, 132.3, 125.8, 124.8, 124.0, 121.2, 120.9, 119.0, 21.4.
135.6, 126.6, 126.5, 126.1, 121.7, 121.2, 119.3, 117.7, 117.4.
5.8.53. 4-Fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)aniline (66)b
Title compound was prepared according to General procedure
A1 as a yellow solid (34%). LRMS [M þ H]þ 230.2 m/z; 1H NMR
5.8.59. 3-(6-Bromooxazolo[4,5-b]pyridin-2-yl)aniline (67)c
Title compound was prepared from 2-amino-5-bromo-3-
hydroxypyridine [22] according to General procedure A1 as a
grey solid (53%). LRMS [M þ H]þ 289.0 m/z; 1H NMR (CDCl3,
(DMSO, 400 MHz)
d
8.57 (dd, J ¼ 4.9, 1.4 Hz, 1H), 8.27 (dd, J ¼ 8.2,
1.4 Hz, 1H), 7.46 (ddd, J ¼ 8.9, 7.1, 3.9 Hz, 2H), 7.16 (dd, J ¼ 10.9,
300 MHz)
d
8.59 (d, J ¼ 2.1 Hz, 1H), 7.98 (d, J ¼ 2.1 Hz, 2H), 7.29 (t,
8.9 Hz, 1H), 6.86 (ddd, J ¼ 8.9, 4.1, 3.0 Hz, 1H), 5.42 (s, 2H); 13C NMR
2H), 7.6 (m, 2H), 6.89 (dd, J ¼ 8.1, 1.5 Hz, 1H); 13C NMR (CDCl3,
(DMSO, 101 MHz)
146.67,145.8 (JCeF ¼ 2 Hz),142.45,120.88,119.5 (JCeF ¼ 8 Hz),119.09,
117.6 (JCeF ¼ 22 Hz), 113.7 (JCeF ¼ 11 Hz), 113.5.
d
162.4 (JCeF ¼ 4 Hz), 155.1, 152.2 (JCeF ¼ 247 Hz),
101 MHz) d 165.6, 154.6, 147.9, 147.6, 143.0, 130.0, 126.6, 121.2, 119.4,
117.6, 115.6, 113.8.
5.8.60. N-(3-(6-Bromooxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (68)c
5.8.54. N-(4-fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (37)a
Title compound was prepared from 67 according to General
procedure B1 (W1) as a pale-yellow solid (60%). LRMS [M þ Na,
Title compound was prepared from 66 according to General
procedure B1 (W1) as an off-white solid (4%). LRMS [M þ H]þ
Br79 þ
]
406 m/z, [M þ Na, Br81 þ
]
408 m/z; HRMS Mþ 382.99055 m/z,
324.1 m/z; 1H NMR (DMSO, 400 MHz)
d
10.55 (s, 1H), 8.77 (dd,
found 382.99071 m/z; 1H NMR (CDCl3, 300 MHz)
d 8.64 (d,
J ¼ 6.5, 2.7 Hz, 1H), 8.59 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.39e8.23 (m, 1H),
8.07 (ddd, J ¼ 9.0, 4.2, 2.8 Hz, 1H), 7.97 (ddd, J ¼ 7.4, 1.7, 0.7 Hz, 1H),
7.51 (dt, J ¼ 9.5, 3.4 Hz, 2H), 7.38 (ddd, J ¼ 11.3, 3.5, 0.7 Hz, 1H),
J ¼ 2.1 Hz,1H), 8.50 (d, J ¼ 1.8 Hz, 1H), 8.30 (s, 1H), 8.03 (m, 3H), 7.55
(t, 2H), 7.3 (d, J ¼ 4.2 Hz, 1H), 6.60 (dd, J ¼ 3.6, 1.8 Hz, 1H); 13C NMR
(CDCl3, 75 MHz)
d 165.9, 156.4, 155.2, 148.2, 147.6, 144.8, 143.5,
6.79e6.63 (m, 1H); 13C NMR (DMSO, 101 MHz)
d
161.4, 156.4, 156.3
138.5, 130.3, 127.0, 124.4, 124.2, 121.4, 119.3, 116.1, 113.0, Note: there
is one carbon missing which is assumed to overlap with one of the
other signals.
(JCeF ¼ 249 Hz), 147.1, 146.9, 146.1, 142.6, 135.6, 126.2 (JCeF ¼ 8 Hz),
121.4 (JCeF ¼ 26 Hz) (þ1C identified by HSQC), 119.3, 117.7 (JCe
¼ 22 Hz), 115.3, 114.0 (JCeF ¼ 11 Hz), 112.3, Note: 1 quaternary
F
carbon is missing and is assumed to be overlapping with the other
signals.
5.8.61. N-(3-(6-Phenyloxazolo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (42)c
Title compound was prepared from 68 according to General
procedure C1 as a pale-yellow solid (90%). LRMS [M þ H]þ 382.0
m/z; HRMS Mþ 381.11134 m/z, found 381.11234 m/z; 1H NMR
(CDCl3, 300 MHz): 8.56 (s, 1H), 8.54 (s, 1H), 8.31 (s, 1H), 8.10 (d,
J ¼ 8.1 Hz,1H), 8.04 (m, 2H), 7.65 (m, 2H), 7.44e7.56 (m, 5H), 7.30 (d,
J ¼ 7.2 Hz,1H),6.6 (dd, J ¼ 3.6,1.8 Hz,1H); 13C NMR (CDCl3, 75 MHz):
165.7, 156.4, 155.7, 147.6, 146.3, 144.8, 138.4, 137.9, 130.3, 129.9,
129.5, 128.5, 127.8, 127.4, 124.3, 124.0, 119.3, 116.8, 116.1, 115.6, 113.0.
5.8.55. N-(4-Fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-3-
methylfuran-2-carboxamide (38)b
Title compound was prepared from 66 according to General
procedure B1 (W2) as a pale-orange solid (46%). LRMS [M þ H]þ
338.1 m/z; HRMS [M þ H]þ 338.0935 m/z, found 338.0952 m/z; 1H
NMR (DMSO, 400 MHz)
d
10.45 (s, 1H), 8.88 (dd, J ¼ 6.5, 2.7 Hz, 1H),
8.60 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.32 (dd, J ¼ 8.2, 1.4 Hz, 1H), 8.04 (ddd,
J ¼ 9.0, 4.3, 2.8 Hz, 1H), 7.84 (d, J ¼ 1.5 Hz, 1H), 7.59e7.42 (m, 2H),
6.63 (d, J ¼ 1.5 Hz, 1H), 2.37 (s, 3H).
5.8.62. N-(3-(6-(4-Methoxyphenyl)oxazolo[4,5-b]pyridin-2-yl)
phenyl)furan-2-carboxamide (43)c
5.8.56. N-(4-Fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)oxazole-
5-carboxamide (39)b
Title compound was prepared from 68 according to General
procedure C1 as a pale-yellow solid (33%). LRMS [M þ H]þ 412.0
m/z; HRMS [M þ H]þ 412.1292 m/z, found 412.1292 m/z; 1H NMR
Title compound was prepared from 66 according to General
procedure B1 (W2) as a pale-yellow solid (93%). LRMS [M þ H]þ
325.1 m/z; HRMS [M þ H]þ 325.0731 m/z, found 325.0747 m/z; 1H
(CDCl3, 300 MHz)
d
8.78 (d, J ¼ 2.0 Hz, 1H), 8.53 (m, 1H), 8.28 (s, 1H),
8.12 (m, 1H), 7.98 (d, J ¼ 2.0 Hz, 1H), 7.56 (m, 5H), 7.30 (dd, J ¼ 3.6,
0.6 Hz, 1H), 7.05 (d, J ¼ 8.7 Hz, 2H), 6.60 (dd, J ¼ 3.3,1.8 Hz, 1H), 3.89
NMR (DMSO, 400 MHz) d 10.79 (s, 1H), 8.84e8.67 (m, 2H), 8.61 (dd,
J ¼ 4.8, 1.2 Hz, 1H), 8.32 (dd, J ¼ 8.2, 1.2 Hz, 1H), 8.15e7.96 (m, 2H),
(s, 3H); 13C NMR (CDCl3, 75 MHz)
d 165.4, 160.1, 156.4, 155.2, 147.6,
7.64e7.43 (m, 2H).
146.0, 144.7, 143.8, 138.4, 134.5, 130.24, 128.9, 127.6, 124.3, 123.8,
119.1, 116.3, 116.1, 114.9, 113.0, 55.7, Note: there is one carbon
missing which is assumed to overlap with another signal.
5.8.57. N-(4-Fluoro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)furan-3-
carboxamide (40)b
Title compound was prepared from 66 according to General
procedure B1 (W2) as an off-white solid (80%). LRMS [M þ H]þ
324.1 m/z; HRMS [M ꢁ H]ꢁ 322.0633 m/z, found 322.0627 m/z; 1H
5.8.63. N-(3-(6-(4-Chlorophenyl)oxazolo[4,5-b]pyridin-2-yl)
phenyl)furan-2-carboxamide (44)c
Title compound was prepared from 68 according to General
NMR (DMSO, 400 MHz)
d
10.27 (s, 1H), 8.70 (dd, J ¼ 6.5, 2.7 Hz, 1H),
procedure C1 as a pale-yellow solid (69%). 1H NMR (CDCl3,
8.60 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.43 (dd, J ¼ 1.5, 0.8 Hz, 1H), 8.32 (dd,
300 MHz)
d
8.79 (bs, 1H), 8.56 (s, 1H), 8.26 (s, 1H), 8.14 (d, J ¼ 7.8 Hz,

462
L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
1H), 8.01 (m, 2H), 7.60e7.48 (m, 6H), 7.31 (dd, J ¼ 3.3, 0.6 Hz, 1H),
6.6 (dd, J ¼ 5.1, 1.5 Hz, 1H).
chromatography, eluting cyclohexane/ethyl acetate 50:50. The
fraction containing the compound was further purified using a
silica coated glass plate eluting with cyclohexane/ethyl acetate
50:50 leading to the title compound as a white solid (64%). LRMS
5.8.64. 3-(1H-Imidazo[4,5-b]pyridin-2-yl)aniline (69) [23]c
Title compound was prepared from 2,3-diaminopyridine ac-
cording to General procedure A2 as a tan solid (80%) and was used
[M þ H]þ 354.2 m/z; 1H NMR (DMSO, 300 MHz)
d
8.61 (dd, J ¼ 4.7,
1.4 Hz, 1H), 8.29 (d, J ¼ 8.2 Hz, 1H), 8.15 (d, J ¼ 2.5 Hz, 1H), 7.76 (d,
J ¼ 8.6 Hz, 1H), 7.6 (m, 3H), 6.20 (s, 1H), 5.33 (m, 1H), 3.37 (s, 3H).
without further purification. 1H NMR (DMSO, 300 MHz)
d 8.29 (bs,
1H), 7.95 (bs, 1H), 7.46 (s, 1H), 7.33 (m, 1H), 7.19 (m, 2H), 6.71 (d,
J ¼ 7.6 Hz,1H), 5.05 (bs, 2H), Note: imidazole eNH was not observed
and is assumed to be downfield of 12 ppm; 13C NMR (DMSO,
5.8.70. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-N-
methylbenzamide (49)a
75 MHz)
116.9, 114.9, 112.7.
d
154.0, 149.8, 144.2, 136.2, 130.8, 130.1, 126.6, 119.7, 118.6,
Title compound was prepared from 10 according to General
procedure D1 as a brown solid (46%). LRMS [M þ H]þ 364.2 m/z;
1H NMR (DMSO, 300 MHz)
(d, J ¼ 8.3 Hz, 1H), 8.08 (d, J ¼ 2.6 Hz, 1H), 7.67 (m, 2H), 7.60
(d, J ¼ 8.6 Hz, 1H), 7.51 (m, 1H), 7.42 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.31
(m, 3H), 3.42 (s, 3H).
d
8.61 (d, J ¼ 4.9 Hz, 1H), 8.27
5.8.65. N-(3-(1H-Imidazo[4,5-b]pyridin-2-yl)phenyl)furan-2-
carboxamide (45)c
Title compound was prepared from 69 according to General
procedure B1(W1) as a tan solid (48%). LRMS Mþ 305.0 m/z;
HRMS Mþ 304.09603 m/z, found 304.09490 m/z; 1H NMR (CDCl3/
5.9. Physicochemistry
DMSO, 300 MHz)
d 9.33 (s, 1H), 8.18 (s, 1H), 8.10 (bs, 1H) 7.77 (m,
3H), 7.37 (m, 1H), 7.25 (m, 1H), 7.07 (d, J ¼ 3.5 Hz, 1H), 6.95 (dd,
5.9.1. Solubility Estimates using Nephelometry
J ¼ 7.8, 4.8 Hz, 1H), 6.34 (m, 1H); 13C NMR (CDCl3/DMSO, 75 MHz)
Stock solutions of compound prepared in DMSO were spiked
into either pH 6.5 phosphate buffer or 0.01 M HCl (approximately
pH 2.0) with the final DMSO concentration being 1% (v/v). Samples
were then analysed by nephelometry to determine the solubility
range as described previously [24].
d
156.8, 153.1, 147.8, 144.8, 144.1, 138.7, 130.5, 129.6, 122.9, 122.6,
118.9, 118.2, 115.2, 112.3, Note: there are three carbons missing
which are assumed to overlap with the other signals.
5.8.66. N-(3-(1H-Imidazo[4,5-b]pyridin-2-yl)phenyl)furan-3-
carboxamide (46)c
5.9.2. Chromatographic logD measurement
Title compound was prepared from 69 according to General
procedure B1 (W1) (44%). LRMS [M þ H]þ 305 m/z; HRMS
[M þ H]þ 305.1033 m/z, found 305.1031 m/z; 1H NMR (CDCl3,
Partition coefficients (logD) were estimated by comparing the
chromatographic retention properties of each compound at pH 3
and pH 7.4 to the retention characteristics of a series of standard
compounds with known partition coefficients. Data were collected
using a Waters 2795 HPLC instrument with a Waters 2487 dual
300 MHz)
d
10.15 (s, 1H), 8.63 (s, 1H), 8.36 (m, 2H), 8.05 (dd, J ¼ 8.1,
1.2 Hz, 1H), 7.9 (dd, J ¼ 8.1, 1.8 Hz, 1H), 7.80 (d, J ¼ 1.5 Hz, 1H), 7.54 (t,
J ¼ 7.8 Hz, 1H), 7.27 (dd, J ¼ 8.1, 5.1 Hz, 2H), 7.04 (dd, J ¼ 1.8, 0.9 Hz,
channel UV detector with
a
Synergy Hydro-RP
4
mm
1H); 13C NMR (DMSO, 75 MHz)
d
161.3, 146.8, 145.0, 144.4, 140.2,
(30 mm ꢃ 2 mm) column. The mobile phase consisted of aqueous
buffer (50 mM ammonium acetate, pH 7.4 or 50 mM ammonium
formate, pH 3.0) and acetonitrile, with an acetonitrile gradient of
0e100% over 10 min. Compound elution was monitored at 220 and
254 nm. This method is a gradient HPLC based derivation of the
method developed by Lombardo [25].
130.7, 130.1, 123.5, 123.0, 122.4, 119.5, 118.9, 109.9, Note: there are
four carbons missing which are assumed to overlap with other
signals.
5.8.67. 3-(Benzo[d]oxazol-2-yl)-4-chloroaniline (70)b
Title compound was prepared according to General procedure
A2 as a pale-pink solid (74%). LRMS [M þ H]þ 245.1 m/z; HRMS
[M þ H]þ 245.0476 m/z, found 245.0474 m/z; 1H NMR (DMSO,
5.9.3. Chromatographic protein binding estimation
Plasma protein binding values were estimated using a chro-
matographic method whereby the retention characteristics on a
human albumin column (Chromtech Chiral-HSA 50 mm ꢃ 3.0 mm,
400 MHz)
d
7.93e7.71 (m, 2H), 7.55e7.38 (m, 2H), 7.35 (d, J ¼ 2.8 Hz,
1H), 7.29 (d, J ¼ 8.7 Hz,1H), 6.79 (dd, J ¼ 8.7, 2.8 Hz,1H), 5.63 (s, 2H);
13C NMR (DMSO, 101 MHz)
d
160.9, 149.9, 148.1, 141.1, 131.5, 125.8,
5 mm, SigmaeAldrich) were compared to the retention characteris-
125.3, 124.9, 120.0, 118.0, 117.5, 115.9, 110.9.
tics of a series of compounds with known human protein binding
values. The method is a modification of a method published previ-
ously [26]. A Waters 2795 HPLC system equipped with a Waters
2487 dual channel UV detector (monitored at 220 and 254 nm) was
used with a mobile phase comprised of aqueous buffer (25 mM
ammonium acetate buffer, pH 7.4) and 30% isopropyl alcohol in the
same buffer. The isopropanol concentration gradient varied over
10 min and the column was reconditioned prior to the next injection.
5.8.68. N-(3-(Benzo[d]oxazol-2-yl)-4-chlorophenyl)furan-2-
carboxamide (47)b
Title compound was prepared according to General procedure
B1 (W1) except purification was performed on a silica coated
glass plate, eluting with 5% methanol in DCM to give the title
compound as a white solid (10%). LRMS [M þ H]þ 339.1 m/z; 1H
NMR (DMSO, 400 MHz)
d
10.58 (s, 1H), 8.69 (d, J ¼ 2.6 Hz, 1H), 8.04
(dd, J ¼ 8.8, 2.7 Hz, 1H), 7.98 (dd, J ¼ 1.7, 0.8 Hz, 1H), 7.92e7.87 (m,
1H), 7.87e7.81 (m, 1H), 7.69 (d, J ¼ 8.8 Hz, 1H), 7.48 (dqd, J ¼ 14.7,
7.4, 1.4 Hz, 2H), 7.41 (dd, J ¼ 3.5, 0.8 Hz, 1H), 6.73 (dd, J ¼ 3.5, 1.7 Hz,
5.9.4. In vitro metabolism in human liver microsomes
A 10 mg/mL stock solution was prepared in DMSO which was
diluted in 50% acetonitrile/water to a spiking concentration of
1H); 13C NMR (DMSO, 101 MHz)
d
160.0, 156.4, 150.0, 147.0, 146.2,
500
1 m
mM. This was diluted 1 in 500 to give a final concentration of
141.1, 138.1, 131.7, 126.2, 126.1, 125.3, 125.1, 124.1, 122.7, 120.3, 115.4,
112.3, 111.1.
M of compound which was incubated at 37 ꢂC in human liver
microsomes (XenoTech LLC, Lenexa, Kansas City) suspended in
0.1 M phosphate buffer (pH 7.4) at a final protein concentration of
0.4 mg/mL. An NADPH-regenerating system (1 mg/mL NADP, 1 mg/
mL glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehydro-
genase) and MgCl2 (0.67 mg/mL) was added to initiate the meta-
bolic reactions, which were subsequently quenched with ice-cold
5.8.69. N-(4-Chloro-3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-N-
methylfuran-3-carboxamide (48)a
Title compound was prepared from 5 according to General
procedure D1 except purification was performed by column

L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
463
acetonitrile at various time points over 60 min. Samples were
5.10.3. L. donovani intracellular amastigotes assay (infected
subjected to centrifugation and the amount of parent compound
remaining in the supernatant was quantified by LC-MS using a
Waters Micromass ZQ coupled to a Waters Alliance 2975 HPLC. The
first order rate constant for substrate depletion was determined by
fitting the data to an exponential decay function and these values
macrophages)
Mouse peritoneal macrophages (4 ꢃ 104 in 100
mL RPMI 1640
medium with 10% heat-inactivated FBS) were seeded into wells of
Lab-tek 16-chamber slides. After 24 h 1.2 ꢃ 105 amastigote L.
donovani in 100 mL were added. The amastigotes were taken from
were used to calculate the in vitro intrinsic clearance value (CLint
)
an axenic amastigote culture grown at pH 5.4. 4 h later the medium
containing free amastigote forms was removed and replaced by
fresh medium. Next day the medium was replaced by medium
containing different compound dilutions. Parasite growth in the
presence of the drug was compared to control wells. After 96 h of
incubation the medium was removed and the slides fixed with
methanol for 10 min followed by a staining with a 10% Giemsa
solution. Infected and non-infected macrophages were counted for
the control cultures and the ones exposed to the serial drug di-
lutions. The infection rates were determined. The results were
expressed as % reduction in parasite burden compared to control
wells, and the IC50 calculated by linear regression analysis [32].
and the predicted in vivo intrinsic clearance value (CLint vivo) as
described previously [27]. The predicted in vivo hepatic extraction
ratio (EH) was calculated using the following relationship: EH ¼ CLint
vivo/(Q þ CLint vivo) where Q is human liver blood flow (20.7 mL/
min/kg) [28].
5.10. Biological assays
All in vitro assays were carried out at least twice independently
in singleton. The IC50 values are the means of two independent
assays and vary less than ꢀ50%.
5.10.1. P. falciparum assay
5.10.4. T. cruzi assay
In vitro activity against erythrocytic stages of P. falciparum was
determined using a 3H-hypoxanthine incorporation assay [29,30],
using the drug sensitive NF54 strain [31] and the standard drug
chloroquine (Sigma C6628). Compounds were dissolved in DMSO at
10 mg/mL and added to parasite cultures incubated in RPMI 1640
medium without hypoxanthine, supplemented with HEPES (5.94 g/
L), NaHCO3 (2.1 g/L), neomycin (100 U/mL), AlbumaxR (5 g/L) and
washed human red cells Aþ at 2.5% haematocrit (0.3% para-
sitaemia). Serial drug dilutions of eleven 3-fold dilution steps
Rat skeletal myoblasts (L-6 cells) were seeded in 96-well
microtitre plates at 2000 cells/well in 100
with 10% FBS and 2 mM -glutamine. After 24 h the medium was
removed and replaced by 100 L per well containing 5000 trypo-
mastigote forms of T. cruzi Tulahuen strain C2C4 containing the
galactosidase (Lac Z) gene [35]. After 48 h the medium was
removed from the wells and replaced by 100 L fresh medium with
or without a serial drug dilution of eleven 3-fold dilution steps
covering a range from 100 to 0.002 g/mL. After 96 h of incubation
mL RPMI 1640 medium
L
m
b
-
m
m
covering a range from 100 to 0.002
m
g/mL were prepared. The 96-
the plates were inspected under an inverted microscope to assure
growth of the controls and sterility. Then the substrate CPRG/
Nonidet (50 mL) was added to all wells. A colour reaction developed
within 2e6 h and could be read photometrically at 540 nm. Data
were analysed with the graphic programme Softmax Pro (Molec-
ular Devices), which calculated IC50 values by linear regression [32]
from the sigmoidal dose inhibition curves. Benznidazole is used as
control.
well plates were incubated in a humidified atmosphere at 37 C; 4%
CO2, 3% O2, 93% N2. After 48 h 50
was added to each well of the plate. The plates were incubated for a
further 24 h under the same conditions. The plates were then
m
L of 3H-hypoxanthine (¼0.5
mCi)
harvested with
a BetaplateÔ cell harvester (Wallac, Zurich,
Switzerland), and the red blood cells transferred onto a glass fibre
filter then washed with distilled water. The dried filters were
inserted into a plastic foil with 10 mL of scintillation fluid, and
counted in a BetaplateÔ liquid scintillation counter (Wallac, Zurich,
Switzerland). IC50 values were calculated from sigmoidal inhibition
curves by linear regression [32] using Microsoft Excel. Chloroquine
and artemisinin are used as control.
5.10.5. T. brucei rhodesiense assay
This stock was isolated in 1982 from a human patient in
Tanzania and after several mouse passages cloned and adapted to
axenic culture conditions [36]. Minimum Essential Medium (50 mL)
supplemented with 25 mM HEPES, 1 g/L additional glucose, 1%
MEM non-essential amino acids (100ꢃ), 0.2 mM 2-
mercaptoethanol, 1 mM Na-pyruvate and 15% heat inactivated
horse serum was added to each well of a 96-well microtiter plate.
Serial drug dilutions of eleven 3-fold dilution steps covering a range
5.10.2. L. donovani axenic amastigotes assay
Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown
in axenic culture at 37 ꢂC in SM medium [33] at pH 5.4 supplemented
with 10% heat-inactivated foetal bovine serum under an atmosphere
of 5% CO2 in air. 100
m
L of culture medium with 105 amastigotes from
from 100 to 0.002
m
g/mL were prepared. Then 4ꢃ103 bloodstream
axenic culture with or without a serial drug dilution were seeded in
96-well microtitre plates. Serial drug dilutions of eleven 3-fold
dilution steps covering a range from 100 to 0.002
forms of T.b. rhodesiense STIB 900 in 50 mL was added to each well
and the plate incubated at 37 ꢂC under a 5% CO2 atmosphere for
70 h. 10 L Alamar Blue (resazurin, 12.5 mg in 100 mL double-
mg/mL were pre-
m
pared. After 70 h of incubation the plates were inspected under an
inverted microscope to assure growth of the controls and sterile
conditions. 10 mL of Alamar Blue (12.5 mg resazurin dissolved in
distilled water) was then added to each well and incubation
continued for a further 2e4 h [37]. Then the plates were read with a
Spectramax Gemini XS microplate fluorometer (Molecular Devices
Cooperation, Sunnyvale, CA, USA) using an excitation wave length
of 536 nm and an emission wave length of 588 nm. The IC50 values
were calculated by linear regression [32] from the sigmoidal dose
inhibition curves using Softmax Pro software (Molecular Devices
Cooperation, Sunnyvale, CA, USA). Melarsoprol is used as control.
100 mL distilled water) [34] were then added to each well and the
plates incubated for another 2 h. Then the plates were read with a
Spectramax Gemini XS microplate fluorometer (Molecular Devices
Cooperation, Sunnyvale, CA, USA) using an excitation wave length of
536 nm and an emission wave length of 588 nm. Data were analysed
using the software Softmax Pro (Molecular Devices Cooperation,
Sunnyvale, CA, USA). Decrease of fluorescence (¼inhibition) was
expressed as percentage of the fluorescence of control cultures and
plotted against the drug concentrations. From the sigmoidal inhibi-
tion curves the IC50 values were calculated by linear regression [32].
Miltefosine is used as control.
5.10.6. Rat skeletal myoblast cytotoxicity assay
Assays were performed in 96-well microtiter plates, each well
containing 100 mL of RPMI 1640 medium supplemented with 1% L-
glutamine (200 mM) and 10% foetal bovine serum, and 4000 L-6
cells (a primary cell line derived from rat skeletal myoblasts)

464
L. Ferrins et al. / European Journal of Medicinal Chemistry 66 (2013) 450e465
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0.0004 L of
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mL of this dilution was subsequently added to assay
m
m
70% Alamar BlueÒ (Invitrogen) prepared in HMI-9 media þ10% FCS
was added to assay plates and plates incubated for a further 2 h at
37 ꢂC/5% CO2 followed by 22 h at room temperature. Assay plates
were read at 535 nm excitation/590 nm emission on an EnvisionÒ
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in DMEM media. 5
assay plates to give final drug concentrations ranging from 83.34 to
0.0004 L of
M. Plates were incubated for 48 h at 37 ꢂC/5% CO2. 10
mL of this dilution was subsequently added to
m
m
70% Alamar BlueÒ (Invitrogen) prepared in DMEM media þ10% FCS
was added to assay plates and plates incubated for a further 5 h at
37 ꢂC/5% CO2 followed by 19 h at room temperature. Assay plates
were read at 535 nm excitation/590 nm emission on an En visionÒ
multiplate reader (PerkinElmer, Massachusetts, USA). Data was
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This research was supported by the NHMRC (IRIISS grant
number 361646; NHMRC Senior Research Fellowship 1020411
(J.B.B.), NHMRC Project Grant 1025581); Victorian Stage Govern-
ment OIS grant; Dr. Jason Dang for his assistance in obtaining some
of our analytical data; Dr. Sebastian Marcuccio (Advanced Molec-
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