Design, synthesis, and evaluation of new thiadiazole-based direct inhibitors of enoyl acyl carrier protein reductase (InhA) for the treatment of tuberculosis

Article abstract of DOI:10.1021/jm501029r

Mycobacterial enoyl acyl carrier protein reductase (InhA) is a clinically validated target for the treatment of tuberculosis infections, a disease that still causes the death of at least a million people annually. A known class of potent, direct, and competitive InhA inhibitors based on a tetracyclic thiadiazole structure has been shown to have in vivo activity in murine models of tuberculosis infection. On the basis of this template, we have here explored the medicinal chemistry of truncated analogues that have only three aromatic rings. In particular, compounds 8b, 8d, 8f, 8l, and 8n show interesting features, including low nanomolar InhA IC₅₀, submicromolar antimycobacterial potency, and improved physicochemical profiles in comparison with the tetracyclic analogues. From this series, 8d is identified as having the best balance of potency and properties, whereby the resolved 8d S-enatiomer shows encouraging in vivo efficacy.

Full text of DOI:10.1021/jm501029r

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Article
Design, Synthesis and Evaluation of New Thiadiazole-
based Direct Inhibitors of Enoyl Acyl Carrier Protein
Reductase (InhA) for the Treatment of Tuberculosis
Roman Šink, Izidor Sosi#, Matej Živec, Raquel Fernandez-Menendez, Samo Turk, Stane Pajk, Daniel
Alvarez-Gomez, Eva Maria Lopez-Roman, Carolina Gonzales-Cortez, Joaquin Rullas-Triconado, Inigo
Angulo-Barturen, David Barros, Lluis Ballell Pages, Robert J. Young, Lourdes Encinas, and Stanislav Gobec
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501029r • Publication Date (Web): 17 Dec 2014
Downloaded from http://pubs.acs.org on December 23, 2014
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Design, Synthesis and Evaluation of New
Thiadiazoleꢀbased Direct Inhibitors of Enoyl Acyl
Carrier Protein Reductase (InhA) for the Treatment
of Tuberculosis
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†
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‡
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Roman Šink , Izidor Sosič , Matej Živec , Raquel FernandezꢀMenendez , Samo Turk , Stane Pajk ,
‡
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Daniel AlvarezꢀGomez , Eva Maria LopezꢀRoman , Carolina Gonzalez Cortes , Joaquin Rullasꢀ
‡
‡
‡
‡
§
Trincado , Inigo AnguloꢀBarturen , David Barros , Lluís BallellꢀPages , Robert J. Young ,
‡
*
†*
Lourdes Encinas , Stanislav Gobec
†
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenija
Diseases of the Developing World; Tres Cantos Medicines Development Campus
‡
GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
§
Medicines Research Centre, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY,
UK
KEYWORDS: (InhA inhibitors Tuberculosis·Isoniazid Thiadiazoles Antimycobacterial)
ABSTRACT
Mycobacterial enoyl acyl carrier protein reductase (InhA) is a clinically validated target
for the treatment of tuberculosis infections, a disease that still causes the death of at least a
million people annually. A known class of potent, direct and competitive InhA inhibitors based
on a tetracyclic thiadiazole structure has been shown to have inꢀvivo activity in murine models of
tuberculosis infection. Based on this template, we have here explored the medicinal chemistry of
truncated analogues that have only three aromatic rings. In particular, compounds 8b, 8d, 8f, 8l
and 8n show interesting features, including low nanomolar InhA IC , subꢀmicromolar
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antimycobacterial potency, and improved physicochemical profiles in comparison with the
tetracyclic analogues. From this series, 8d is identified as having the best balance of potency and
properties, whereby the resolved 8d Sꢀenatiomer shows encouraging inꢀvivo efficacy.
INTRODUCTION
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According to World Health Organisation figures, tuberculosis was the sole cause of death
1
of almost one million people in 2011, with a further 8.7 million people infected. Concurrently,
there were another half a million people infected with HIV who also died of tuberculosis that
1
2
year. This treatable disease is caused by infection by the Mycobacterium tuberculosis complex,
although this treatment can be hindered by the emergence of drug resistance. This is manifested
by the rapid spread of multidrugꢀresistant tuberculosis (MDRꢀTB) and extensively drugꢀresistant
tuberculosis (XDRꢀTB). Worldwide, 3.7% of new cases and 20% of previously treated cases
3
have been estimated to be due to infection by MDRꢀTB. The highest percentages of patients
4
infected with MDRꢀTB are in eastern Europe and central Asia.
MDRꢀTB is defined as an infection with a M. tuberculosis strain that is resistant to the
two most commonly used frontꢀline antimycobacterial drugs: isoniazid and rifampicin.
Furthermore, XDRꢀTB is caused by a strain that is additionally resistant to at least three of the
six classes of the secondꢀline antimycobacterials. These secondꢀline agents consist of
aminoglycosides, polypeptides, fluoroquinolones, thioamides, cyloserine and paraꢀ
3
aminosalicylic acid. Recently, the term ‘totally drugꢀresistant tuberculosis’ (TDRꢀTB) was
coined to describe infections by a strain that is resistant to all firstꢀline and secondꢀline
5
antitubercular drugs.
The firstꢀline and secondꢀline antimycobacterial drugs have been in clinical use for well
over 30 years now, and due to the widespread resistance and longꢀterm treatment, there is an
2
urgent need for the development of new drugs and new treatment regimes. New
antimycobacterial drugs should ideally treat MDRꢀTB, XDRꢀTB and TDRꢀTB with a new
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mechanism of action, while also shortening the treatment duration, having a good
pharmacokinetic profile for a lower dosing frequency, and have minimal side effects and drugꢀ
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drug interactions.
The urgency of the situation is perhaps embodied in the profile of bedaquiline, the first
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new antimycobacterial for a generation, which was approved at the end of 2012. Bedaquiline
acts by the novel mechanism of mycobacterial adenosine 5’ꢀtriphosphate (ATP)ꢀsynthase
inhibition, with good clinical efficacy against multiply resistant strains. Although it has certainly
saved lives, it was approved with a black box warning that highlighted the possibility of severe
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cardiovascular side effects. However, more recent increased efforts towards the design and
discovery of antimycobacterial agents have produced other promising new molecules and modes
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2
of action. Some of these are in preclinical development, while eight are in phase II clinical
2
2, 10
studies, and four are undergoing phase III clinical studies.
The bacterial fattyꢀacid biosynthesis, or FAS, pathway is an attractive target for the
1
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development of new antimycobacterial agents. Mammalian fattyꢀacid biosynthesis follows a
different pathway, FAS I, to that used by bacteria, FAS II, which offers an attractive means of
achieving selective actions with new antibacterial agents. Mycobacteria contain both fatty acid
synthases, FASI and FASII systems. Furthermore, there is a particular variation in the substrate
specificity of the FAS II enzyme cascade that is unique to mycobacteria, which synthesise
mycolic acids, as essential components of their cell envelope. Transformations within these
11b
pathways thus represent particularly attractive drug targets. The M. tuberculosis enoyl acyl
carrier protein reductase (InhA) is a key component of the M. tuberculosis FAS II pathway. It is
an NADHꢀdependent enzyme that facilitates the reduction of longꢀchain transꢀ2ꢀenoylꢀacyl
1
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carrier protein fatty acids (Figure 1). The Xꢀray structure of InhA has revealed that each
subunit consists of a single domain with a central core that has a Rossmann fold, which contains
13
the NADH binding site (Figure 1). Within the InhA structure, several αꢀhelices and βꢀstrands of
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the Rossmann fold extend beyond the NADH binding site, which creates a deep crevice for the
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lipophilic acyl substrate. InhA is the target for the frontline antimycobacterial drug isoniazid
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(Figure 1), a prodrug that is activated by the mycobacterial catalaseꢀperoxidase enzyme KatG,
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to form a reactive species that acylates the nicotinamide moiety of NADH. Investigations into
the mechanisms of resistance to isoniazid have revealed that KatG mutations are the primary
cause, as without the oxidation of isoniazid to the active species, the necessary acylation of the
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nicotinamide cannot occur. Therefore, compounds that can directly inhibit InhA without the
need for activation by KatG present a very promising opportunity to combat MDRꢀTB, XDRꢀTB
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5, 17
and TDRꢀTB.
Triclosan (a highly effective broadꢀspectrum antibacterial agent that targets
FabI, the nonmycobacterial equivalent of InhA; Figure 1) was demonstrated to act as a direct
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InhA inhibitor, and modifications to its structure have led to further potent inhibitors, although
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their progress is hampered by nonꢀideal pharmacokinetic properties.
Figure 1. Left: Threeꢀdimensional structure of InhA. Magenta, NADH; blue, fatty acid substrate.
Right: Reaction catalysed by InhA, and structures of isoniazid and triclosan.
A series of thiadiazoleꢀbased InhA inhibitors was initially discovered in a highꢀ
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throughput screening campaign by GlaxoSmithKline. These are exemplified by I (Scheme 1),
which was shown to be a direct, reversible inhibitor of InhA through binding in the active site,
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where it makes contacts with NADH. Preliminary structureꢀactivity relationship studies were
later performed by AstraZeneca, where it was also demonstrated that this binding is dependent
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on the oxidation state of NADH.
These aforeꢀmentioned compounds were all lipophilic tetracyclic structures with a high
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proportion of sp centres. Systematic variations on this thiadiazole template are explored in the
present study, wherein we describe the design, synthesis and biological evaluation of novel and
potent tricyclic InhA inhibitors that show improved physicochemical properties.
RESULTS AND DISCUSSION
Design
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The compounds on which the present study was based were identified as potent
nanomolar) inhibitors of InhA with antimycobacterial activity, with minimum inhibitory
(
concentrations (MICs) in the low micromolar range. The most promising compounds, e.g.,
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compound I, are based on the thiadiazole linkage as ring C. The goal of the present study was
to conserve rings C and D, and to truncate the leftꢀhand side of the molecule (see Scheme 1),
whilst retaining potent inhibition of InhA and good levels of antimycobacterial activity.
Inspection of the Xꢀray structure of I when coꢀcrystallised into InhA (Figure 2) supported
the feasibility of this approach. This revealed that one thiadiazole ring nitrogen and the NH
group connecting rings B and C of I form hydrogen bonds with the backbone NH and carbonyl
of Met98 of InhA, respectively. At the same time, the thiazole (ring D) nitrogen forms a
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hydrogen bond with the 2’ꢀhydroxyl of the ribose attached to the coꢀfactor nicotinamide. Ring
B appears to make subꢀoptimal contacts.
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Figure 2. Binding mode of I (cyan) in the active site of InhA (PDB entry: 4bqp).
Proof of this concept was obtained with our synthesis of compound 8a (see below), which
showed a reasonable level of activity and led to a fuller exploration of the structureꢀactivity
relationships around such tricyclic structures. However, no further liganded structures were
obtained to fully enable structureꢀbased design for the series of tricyclic analogues. In the
absence of such information, rings A and B were systematically replaced with substituted aminoꢀ
phenyls, aminoꢀpyridines, and fiveꢀmembered aminoꢀaromatic heterocycles and motifs (see
Scheme 1). The first goal was to explore the favoured substituents and the substitution patterns
that provided good InhA inhibitory activity whilst maintaining good physical properties.
Scheme 1. Replacement of the leftꢀhand side of lead compound I.
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Chemistry
The compounds were synthesised via the synthetic route described in Scheme 2. First, 4ꢀ
methylthiazol (1) was sequentially treated with
n
ꢀbutyl lithium and then ethyl pyruvate, to obtain
22
racemic ethyl 2ꢀhydroxyꢀ2ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)propanoate (3), which was reacted with
hydrazine hydrate to produce the crucial intermediate 2ꢀhydroxyꢀ2ꢀ(4ꢀmethylthiazolꢀ2ꢀ
yl)propanehydrazide (4). The reactions of hydrazide 4 with the
Nꢀ2ꢀpyridinylꢀ1Hꢀimidazoleꢀ1ꢀ
carbothioamide 6a or with aromatic isothiocynates 6bꢀ6w, prepared from the corresponding
aromatic amines 5aꢀ5w produced the hydrazineꢀ1ꢀcarbothioamide intermediates 7aꢀ7w. These
were then treated with 96% sulphuric acid to obtain the racemic final compounds 8aꢀw.
Cyclization of thiosemicarbazides 7aꢀ7w to 8aꢀ8w occurs most probably via cyclodehydration
23
mechanism,
which
was
studied
by
Siwek
et
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First,
mercapto(methylamino)methylene)formohydrazonic acid is formed, followed by dehydration
13
and formation of the desired 1,3,4ꢀthiadiazole. In the CꢀNMR spectra, two major signal shifts
can be observed when comparing the spectra of compounds 7aꢀ7w with 8aꢀ8w. For example,
thioamide signal at 174.36 ppm (compound 7l) is shifted to 151.84 ppm (compound 8l).
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Additionally, the signal for carbonyl carbon of 7l is shifted from 172.64 ppm to 167.89 ppm in
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a
Scheme 2. General synthetic route for compounds 8aꢀ8w.
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Reagents and conditions: (a)
n
ꢀBuLi, THF, ꢀ78 °C, 2 h; (b) H
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×H
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O, EtOH, reflux, 2 h;
(6a); Cl CS,
(c) 1,1′ꢀThiocarbonyldiimidazole (TCDI), Et N, THF, reflux,
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h
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CH
2
Cl
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/NaHCO3(aq), 0 °C, 2 h (6bꢀ6w); (d) THF, 75 °C, 2ꢀ18 h; (e) H
2 4
SO , 2 h.
The 6ꢀsubstituted 2ꢀaminopyridines 5h and 5i were synthesised via nucleophilic aromatic
substitution between 2ꢀaminoꢀ6ꢀbromopyridine (5d) and morpholine or piperidine (Scheme 3).
The 6ꢀcyclopropylꢀ2ꢀaminopyridine intermediate (5g) was prepared as presented in Scheme 4,
using the Suzuki crossꢀcoupling reaction conditions between 5d and cyclopropylboronic acid 10.
a
Scheme 3. Synthesis of 6ꢀsubstituted 2ꢀaminopyridines 5h and 5i.
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a
Reagents and conditions (a) morpholine, MW, 150 °C, 4 h; (b) piperidine, NMP, MW, 150 °C,
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h.
a
Scheme 4. Synthesis of the substituted 2ꢀaminopyridine 5g.
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a
3 4 2
Reagents and conditions (a) K PO , tricyclohexylphosphine, Pd(OAc) , 100 °C, 24 h.
Hydroxypyridine 8j was synthesised via the isopropyl ether protected 5j, followed by
concurrent thiadiazole formation and deprotection of the hydroxyl group in the last step (Scheme
5
).
a
Scheme 5. Synthesis of the final compound 8j.
a
Reagents and conditions (a) 2ꢀpropanol, NaOH, MW, 150 °C, 6 h; (b) Cl CS,
2
CH Cl /NaHCO , 0 °C, 2 h; (c) THF, 75 °C, 2ꢀ18 h; (d) H SO , 2 h.
2
2
3(aq)
2
4
InhAꢀinhibitory and antimycobacterial activities of compounds 8aꢀ8w
Compounds 8aꢀ8w were first assayed for their inhibitory activity against InhA, which
showed that several were potent inhibitors of InhA, with tens of nanomolar levels of activity.
Compound 8a with an unsubstituted 2ꢀaminopyridine as ring B showed inhibitory activity with
an IC of 386 nM. The methyl (8b; IC , 260 nM) and methoxy (8c; IC , 264 nM) groups were
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tolerated in the 6ꢀposition, while introducing 6ꢀbromine led to the most potent InhA inhibitor in
this series (8d; IC50, 13 nM) with a value comparable to the fourꢀmembered ring compound I.
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Fluorine at position 6 on the pyridine ring (8e; IC , 551 nM) showed a similar level of activity
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to the proton and methyl substitutions. The incorporation of bulkier groups at position 6 of the 2ꢀ
aminopyridine, such as CF and cyclopropyl, provided activities comparable to that of the
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bromine substitution, with IC50 values of 18 nM (8f) and 77 nM (8g). The introduction of larger
aminoꢀlinked saturated rings in this position was not well tolerated (i.e., morpholine (8h) or
piperidine (8i)). Of note, the polar 6ꢀhydroxyl substituent of 8j rendered this analogue inactive.
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The preference for lipophilic substituents such as CF and Br was very apparent from this set of
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compounds.
Structureꢀactivity relationship investigations of position 5 of the 2ꢀaminopyridine ring
showed that halogens were tolerated. The addition of fluorine (8k) showed almost no difference
in activity compared with the unsubstituted pyridine 8a, while the introduction of bromine at
position 5 in compounds 8l (IC , 197 nM) and 8m (IC , 179 nM) did not produce the marked
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increase in potency that was achieved in the 6ꢀposition. Surprisingly, if bromine was introduced
at position 5 of the 2ꢀaminopyridine together with trifluoromethyl group at position 6 (8n; IC50
46 nM), there was no significant enhancement of potency over the 6ꢀmono substituted analogue
f (IC , 18 nM). Taking into account the variability of the biochemical assay used, IC values
,
8
5
0
50
within ~3ꢀ4ꢀfold differences can be considered as similar (observed by variation of results for
positive control). These results illustrate a preference for lipophilic substituents in both the 5 and
6 positions of the 2ꢀamionopyridine ring. This is consistent with the active site being a largely
hydrophobic pocket where the alkyl chain of the fatty acid is accommodated during the InhA
catalytic cycle. Further variations of ring B in this series revealed that pyrimidine (8o), isomeric
pyridines (8pꢀ8s), substituted phenyl (8t), and 5ꢀmembered heteroaromatics (8uꢀ8w) were not
well tolerated. However, two compounds showed moderate inhibition. One was a 3ꢀ
aminopyridine derivative 8p, which was the sole pyridineꢀcontaining inhibitor that does not
contain the 2ꢀaminopyridine moiety. When 2ꢀaminopyridine was replaced with 1ꢀmethylꢀ1Hꢀ
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pyrazoleꢀ3ꢀamine (8u), moderate inhibitory activity was seen. This activity was expected, as this
compound contains an aromatic nitrogen at position 2 with respect to the amino group, which
connects the 1ꢀmethylꢀ1Hꢀpyrazole with the thiadiazole ring, and it is a simplified version of
compound I. The inactivity of the 2ꢀaminopyrimidine 8o indicated a probable requirement for a
modestly basic nitrogen, or that two polar motifs were not tolerated within the ring.
The exact kinetic mode of inhibition was not studied. However, given the fact that
Compound I showed uncompetitive inhibition modality towards NADH, we can postulate that
the active compounds in the presented series show similar inhibition mechanism.
Table 1. Drugꢀlike properties and pharmacodynamic activities of the final compounds 8aꢀ8w
and compound I.
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32
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34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Number
Structure
Chrom Property
LE
LLE
InhA
MIC Mtb
log
Forecast
Index
IC50
(µM)
a
D
pH7.4
(nM)
I
3.95
7.95
0.40 6.24
4
0.2
8
a
2.92
3.46
3.80
5.92
6.46
6.80
0.42 6.05
0.41 5.72
0.39 5.43
386
260
264
8
4
8b
8c
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2
2
2
2
2
2
2
2
2
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3
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5
5
5
5
5
5
5
6
8d
3.93
6.93
0.49 6.63
13
2
0
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2
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6
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8
9
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8
9
0
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8
9
0
8e
8f
3.60
4.01
6.60
7.01
0.39 5.72
0.42 6.44
551
18
16
3
8g
4.44
3.38
7.44
6.38
0.41 5.81
77
32
8h
ND ND >1000
>32
8i
5.16
8.16
0.33 4.77
299
16
8j
1.89
3.26
4.89
6.26
ND ND >1000
>31.25
8k
0.40 5.90
363
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8l
4.46
5.07
5.29
7.46
8.07
8.29
0.42 5.44
0.40 4.99
0.39 5.15
197
179
46
4
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8m
8n
8o
2.33
2.64
5.33
5.64
ND ND >1000
>32
32
8p
0.37 4.98
741
8q
8r
8s
2.58
2.45
1.78
5.58
5.45
4.78
ND ND >1000
ND ND >1000
ND ND >1000
>32
>32
>32
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8t
4.93
7.93
ND ND >1000
>32
8u
8v
2.16
1.60
1.52
5.16
4.60
0.39 6.14
894
62.5
>32
0
1
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8
9
0
1
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9
0
1
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9
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1
2
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7
8
9
0
ND ND >1000
ND ND >1000
8w
4.152
>125
a
InhA IC50 values are an average of two measurements without standard errors due to robustness of the assay, which does not
allow the exact determination of the inhibitory activity. IC50 values below 100 nM can be interpreted as similar. InhA IC50 values
within ~3ꢀ4ꢀfold differences can be considered as similar. For illustration, see the concentration response curves in the
Supporting Information (Pages S28ꢀS34); Mtb, M. Tuberculosis; ND, not determined
Physicochemical measurements were obtained to further explore the profiles of these
molecules, and in particular, the hydrophobicity was measured using the chromatography
24
technique, to generate Chrom log
D
pH7.4 values. These showed that these compounds have a
hydrophobicity that is in a reasonable range for drugꢀlike molecules, which goes with them
having only three aromatic rings. This gives these compounds some advantages over the
2
4ꢀ25
tetracyclic series, in terms of their Property Forecast Index (PFI).
The PFI is defined as the
sum of Chrom log pH7.4 plus the number of aromatic rings, and this value has been shown to be
D
a reasonable predictor of the likelihood that a particular molecule will show activity in a number
of the soꢀcalled ‘developability’ measures, such as solubility, permeation, cytochrome P450s,
intrinsic clearance, and human etherꢀaꢀgoꢀgoꢀrelated gene (hERG) binding and promiscuity. As
all of the synthesised compounds here contain three aromatic rings in the structure, the PFI for
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these compounds is Chrom logDpH7.4 + 3. A value of around 7 or below is regarded as a good
guide for lowꢀrisk progression of compounds that are likely to be soluble. Amongst the most
potent compounds 8b, 8d and 8n have the targeted PFI of 6.46, 6.93 and 7.01, respectively; for
comparison, the PFI for compound I is 7.95. In addition to PFI, ligand efficiency (LE) and ligand
lipophilicity efficiency (LLE) were calculated for active compounds. LE and LLE are both
helpful metrics for assessing the potency and lipophilicity of drugꢀlike molecules during lead
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optimization. As an indication of drugꢀlike properties, compound 8d showed the best ligand
26b
efficiency value of 0.49 kcal/mol/atom (the commonly used threshold value is 0.3 kcal/mol) ,
and LLE of 6.63. Altogether, this represents an improvement when compared to compound I (LE
= 0.40, LLE = 6.24).
In parallel with the assessment of InhA inhibition, the translation of this effect into
antimycobacterial activity was also investigated, in terms of their MICs (Table 1). These data
were generally consistent with the InhA inhibitory activities, as a more potent InhA activity
tended to translate into better MICs against M. tuberculosis. The most potent InhA inhibitor, 8d
(IC50, 13 nM), was also the best antimycobacterial compound (MIC, 2 µM) in this series of
truncated analogues. Compound 8d is slightly less potent than compound I. Of note, 8d also has
one of the better physicochemical profiles.
A summary of the main features in the straightforward structureꢀactivity relationships for
these inhibitors is as follows:
•
•
•
2ꢀaminopyridine and 1
which is essential for InhA inhibitory activity;
3
hydrophobic substituents, H, OCH , CH , CF , Br, cyclopropyl and piperidinꢀ1yl are
Hꢀpyrazoleꢀ3ꢀamine have a nitrogen in the equivalent position,
3
3
allowed at position 6 on the 2ꢀamionopyridine ring;
hydrophobic substituents H, Br and F at position 5 on the 2ꢀaminopyridine ring
contribute to better inhibitory activity against InhA;
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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4
5
5
5
5
5
5
5
5
5
5
6
•
•
a biꢀsubstitution pattern at positions 5 and 6 (combinations of hydrophobic substituents
OCH
3 3 3
, CH , CF , Br) does not drastically improve the inhibitory activity against InhA;
hydroxyl and morpholinyl substituents are not allowed at position 6 on the pyridine ring.
Due to the robustness of the in vitro InhA assay that does not allow a more significant degree of
differentiation between more potent compounds, we decided to prioritize compounds based on
the wholeꢀcell activity and the inꢀvitro profiling.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In-vitro profiling of compounds 8b, 8d, 8f, 8l and 8n in comparison with compound I
To better understand the properties of these compounds beyond their early activity, basic
inꢀvitro profiling of the compounds with MICs <5
µM was completed. Artificial membrane
permeability, kinetic solubility, cytochrome P450s (CYP) inhibition, and hERG binding and
cytotoxicity were evaluated. The rapid generation of these data contributed to enhance the
absorption, distribution, metabolism, excretion and toxicology knowledge, and to minimise the
number of compounds to take forward into animal models. Generally, compounds with high
ꢀ6
27
permeability have a permeability coefficient higher than 1.0×10 cm/s and some compounds in
24
this series were highly permeable (Table 2) in the artificial membrane permeability assay, as
24
expected, given the range of PFI values (values between 5 and 8 were shown to be optimal).
The most potent InhA inhibitors 8b (PFI, 6.46), 8d (PFI, 6.93), 8g (PFI, 7.01), 8f (PFI, 7.01) and
8n (PFI, 8.29) also have high permeability. Based on the measured chemiluminescent nitrogen
24
detection (CLND) solubility criteria, all of these compounds showed modest solubility.
Additionally, potential drugꢀdrug interactions were evaluated according to the guidelines for
28
29
drug interactions studies of the US Food and Drug Administration and to literature data.
Therefore, we assayed the compounds on the two important cytochrome P450 enzymes,
CYP3A4VR and CYP3A4VG (Table 2). Here, most of the inhibitors showed inhibitory values in
a range that are indicative of a minimal risk of drugꢀdrug interactions, only compounds I and 8n
have shown values under 10 µM. No activity was observed in the hERG dofetilide binding assay
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(pIC <4.3) and a similar low risk was indicated in the cytotoxicity evaluation using HepG2,
5
0
where no activity was observed below 50 ꢁM for compound 8d. Hence, 8d is the most potent
compound, and it also shows the best balance between physicochemical properties and inꢀvitro
absorption, distribution, metabolism, excretion and toxicology profile.
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Table 2. Safety profile, metabolic profile and pharmacokinetic parameters of the final
synthesised compounds and compound I.
Compound
number
hERG IC50
(µM)
Solubility
(µM)
Permeability
(cm/s)
CYP 3A4VR
(µM)
CYP 3A4VG
(µM)
Cytotoxicity
Tox 50 (µM)
I
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
318ꢀ458
121
245
82
ND
10.49
>50.12
19.95
32.36
19.05
8.71
6.71
40.74
15.85
11.22
13.18
5.62
ND
>100
>50
ꢀ
5
5
8b
5.1×10
2.1×10
3.5×10
4.0×10
1.9×10
ꢀ
8
d
ꢀ5
8
f
>100
79.4
31.6
ꢀ
5
5
8
l
93
ꢀ
8n
43
Profile of compound 8d Sꢀenantiomer
To determine potential differences in the activities, the two enantiomers of compound 8d
were separated using chiral HPLC resolution. The absolute configuration of both enantiomers
was determined by ab initio vibrational circular dichroism (VCD). The resolution demonstrated
that the 8d
S
ꢀenantiomer ((
S
)ꢀ8d) is the eutomer, while the
Rꢀconfiguration of 8d showed an
InhA IC50 >1.0 µM. To complete its profile, (
S
)ꢀ8d was tested for its activity against intracellular
bacteria, where it showed a MIC of 0.48 µM. This significant intracellular activity (in THPꢀ1
cells) was accompanied by a clean safety profile and an acceptable profile for CYP3A4VR and
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CYP3A4VG inhibition. The measured intrinsic clearance of (
S)ꢀ8d was 5.4 ml/min/kg, and its
elimination halfꢀtime (t ) was 13.3 min in the mouse microsomal fraction, which suggested that
1/2
(S
)ꢀ8d would be removed rapidly from the body. In a human microsomal fraction, (
S
)ꢀ8d showed
)ꢀ8d was
lower clearance, at 1.3 ml/min/kg, and a t1/2 >30 min. On the basis of these results, (
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chosen to be evaluated in the inꢀvivo 4 day mouse model of M. tuberculosis infection. This inꢀ
vivo assay is a rapid and economic tool that provides useful feedback with regard to the
therapeutic efficacy of a new product, as only 70 mg and 2 mice per compound are needed. The
efficacy of (S)ꢀ8d in this murine disease model was demonstrated (Table 3), whereby a 0.7 log10
colony forming units (logCFU) drop was observed, compared to the untreated group (untreated
mice, 7.1 ± 0.07 logCFU; treated mice, 6.4 ± 0.06 logCFU). The positive control, isoniazid,
reduced the CFU by 3.1 logs from the control. The further development of the active (S)ꢀ8d was
thus precluded by this modest inꢀvivo potency.
If we compare the activities of this thiadiazole series of direct InhA inhibitors with other
published compounds, we can observe that other literature InhA inhibitors also inhibit the
enzyme in nanomolar concentrations, however they lack the inꢀvivo activity after oral
30
administration (or it is not published). The reason for this is most probably the poor inꢀvivo
pharmacokinetic properties or compound availability at the same location where the lung
infected cells are. The inꢀvivo efficacy of the most potent tricyclic thiadiazole, presented in this
manuscript, although moderate, proved the concept that InhA can be inhibited by smaller
molecules, thus enabling further investigations into this concept. The premise that direct InhA
inhibitors should be active against isoniazidꢀresistant strains, and absence of toxicity alerts, grant
us the confidence for the development potential of this class of compounds. The opportunities for
possible systematic modifications to the chemical class will enable a rational optimisation
approach that should ultimately lead to improved inꢀvivo response.
Table 3. Characteristics and pharmacokinetics of the 8d Sꢀenantiomer.
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Characteristic/ pharmacokinetics
Molecular weight
Value
398.3
ClogP
1.26
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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50
51
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60
a
Chrom log D pH = 7.4/PFI
4.11/7.11
96.1
HSA binding mouse (%)
ꢀ5
Art. membrane permeability (cm/s)
3.80×10
b
Solubility CLND (ꢁM)
130
Inhibitory activity
InhA IC (ꢁM)
0.043
1
50
M. tuberculosis MIC (ꢁM)
M. tuberculosis intracell MIC (ꢁM)
CYP 3A4 VR IC50 (ꢁM)
0.48
25.1
15.8
CYP 3A4 VG IC50 (ꢁM)
c
Acute drug efficacy study in mice
LOG_CFU_REDUCTION
0.7
Microsomal clearance (in vitro
Mouse t1/2 (min)
)
13.3
5.4
Mouse Cl (ml/min×g)
Human t1/2 (min)
>30
1.3
Human Cl (ml/min×g)
a
ClogP, Fragment based prediction of logarithm of partition coefficient; Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A.
b
P. Drug Discovery Today 2011, 16, 822−830 CLND solubility values that are within 85% of maximum possible concentration
c
(as determined from DMSO stock concentration). (p <0.05) Means were compared by Oneway ANOVA analysis (Dunnet post
test).
CONCLUSIONS
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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4
4
4
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5
5
5
5
5
5
5
5
5
6
We have designed, synthesised and biologically evaluated a series of 23 tricyclic
inhibitors of InhA that are based on the thiadiazole scaffold. These inhibitors were designed
starting from the tetracyclic thiadiazole derivatives, with the aim to improve their
physicochemical properties and drugꢀlikeness while retaining their InhA inhibitory potency and
antimycobacterial activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Potent nanomolar inhibitors (8d, 8f, 8n), in the same range of compound I, were
synthesised that showed excellent antimycobacterial activity, and inꢀvitro profiling studies
revealed that they have good artificial membrane permeability, do not interact with hERG, and
have an acceptable profile of cytochrome inhibition. The best compound in the tricyclic series
was the (
antimycobacterial activity in THPꢀ1 cells, and good intrinsic clearance in a human microsomal
fraction. Finally, although ( )ꢀ8d was less active in the inꢀvivo mouse model of tuberculosis
S)ꢀ8d (IC50, 43 nM, M. tuberculosis MIC, 1 µM), which showed good intracellular
S
infection than isoniazid, its activity confirms the development potential of these smallꢀmolecule
inhibitors of InhA.
GENERAL PROCEDURES
Reagents and solvents were obtained from commercial sources (Fluka, SigmaꢀAldrich, Acros
Organics, Alfa Aesar, TCI, CombiꢀBlocks). Solvents were distilled before use, while the other
chemicals were used as received. Liquid compounds used in the reactions with
nꢀbutyl lithium
(n
ꢀBuLi) were placed on the activated molecular sieves (4 Å) under an argon atmosphere. All of
the reactions were carried out under a normal atmosphere with magnetic stirring, unless stated
otherwise. Microwaveꢀassisted reactions were performed using a focused microwave reactor
(Discover, CEM Corporation, Matthews, NC, USA). Analytical TLC was performed on Merck
silica gel (60F254) preꢀcoated plates (0.25 mm). The compounds were visualised under UV light
(254 nm) and/or stained with the relevant reagent. Flash column chromatography was performed
on silica gel with pore size 60 Å, 230ꢀ400 mesh particle size, and 40ꢀ63 ꢁm particle size, with
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the indicated solvents. The yields refer to the purified products, and they were not optimised. All
1
of the products were obtained as amorphous solids, and melting points were not measured. H
NMR spectra were recorded on a Bruker Avance III NMR spectrometer and in a Bruker DPX
Avance 400 MHz instrument equipped with a QNP probe. Measurements were made at a
temperature of 295 K, and are reported in ppm using tetramethylsilane or solvent as an internal
10
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standard (DMSOꢀd6 at 2.50 ppm; CDCl at 7.26 ppm). The coupling constants (J) are given in
3
Hz, and the splitting patterns are designated as: s, singlet; bs, broad singlet; d, doublet; dd,
13
double doublet; t, triplet; and m, multiplet. C NMR spectra were recorded on a Bruker Avance
III NMR spectrometer at 295 K, and are reported in ppm using solvent as an internal standard
3
(DMSOꢀd6 at 39.5 ppm; CDCl at 77.0 ppm). Mass spectra data and highꢀresolution mass
measurements were performed on a VGꢀAnalytical Autospec Q mass spectrometer. IR spectra
were recorded on an FTꢀIR spectrometer Thermo Nicolet Nexus 470 ESP FTꢀIR. Analytical
1
purity was ≥95% unless stated otherwise, as determined by HꢀNMR and HPLC analyses. The
purities of the final compounds were checked using a Waters ZQ2000 coupled with LC Waters
2795 and Waters 2996 PDA detector. All mass spectra were performed using electrospray
ionisation. Optical rotations were measured on a JASCO Pꢀ2000 polarimeter at room
temperature using a 2.5x50 mm cell of 0.5 mL capacity.
EXPERIMENTAL SECTION
Synthesis of ethyl 2ꢀhydroxyꢀ2ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)propanoate (3)
22
In the synthesis of ethyl 2ꢀhydroxyꢀ2ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)propanoate 3,
methylthiazole 1 (1 eq) was dissolved in freshly distilled THF, cooled to –78 °C, and then treated
with 1.1 eq of ꢀBuLi. After addition of ꢀBuLi, the reaction mixture was left stirring at –78 °C
4ꢀ
n
n
for 30 min, before ethyl 2ꢀoxopropanoate was carefully added. Afterwards, the reaction mixture
was stirred at –78 °C for 1 h, and then left for an additional 1 h at room temperature. Then, the
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reaction mixture was quenched with a saturated aqueous solution of NH Cl, the THF was
4
evaporated, and the aqueous residue extracted with EtOAc. After evaporation of the EtOAc, the
crude product was loaded onto a silica column and purified using an
mobile phase. The reaction yield was on average about 40% to 45%.
nꢀhexane/EtOAc (1:5, v/v)
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Synthesis of 2ꢀhydroxyꢀ2ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)propanehydrazide (4)
ꢀHydroxyꢀ2ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)propanehydrazide 4 was synthesised by treating ethyl
ꢀhydroxyꢀ2ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)propanoate 3 with hydrazine hydrate (9 eq) in EtOH at 82 °C
2
2
for 3 h. After the completion of the reaction, the EtOH and the residual hydrazine hydrate were
evaporated. The crude product was then loaded onto a silica column and compound 4 was eluted
with a mobile phase of CH Cl and MeOH (15:1, v/v).
2
2
General synthetic procedures
General procedure 1:
Aryl isothiocyanates 6bꢀ6w were synthesised by treating aromatic amines 5bꢀ5u with
2 2 3
thiophosgene (1.1 eq) in a mixture of CH Cl and saturated aqueous solution of NaHCO (1:1,
v/v) at 0 °C for 2 h. Then, the layers were separated and the organic layer was evaporated to
produce compounds 6bꢀ6w. Compounds 6bꢀ6w differed substantially in stability, and therefore
the majority were used in the next step without further purification. Stable representative
compounds 6e, 6k, 6r and 6t were thoroughly analysed.
General procedure 2:
Hydrazineꢀ1ꢀcarbothioamide intermediates 7bꢀ7w were synthesised by reacting
compounds 6bꢀ6w with equimolar quantity of 4 in THF (except 7k in EtOH at 50 ºC) and
heating the reaction mixture to 70 °C from 3 h to 18 h. After the completion of the reaction, the
THF was evaporated, the crude material was reꢀcrystallised from EtOH, or in the case of less
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pure compounds, loaded onto a silica column and eluted with a mobile phase of CH Cl and
2
2
MeOH (15:1, v/v).
General procedure 3:
The final compounds 8aꢀ8w were prepared by treating compounds 7aꢀ7w with
concentrated H SO at room temperature for 1ꢀ2 h. Afterwards, the reaction mixture was cooled
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4
to 0 °C and carefully neutralised with 25% aqueous solution of NH , until the pH was >10. The
3
precipitated compound was filteredꢀoff and washed thoroughly with cold water. Final
compounds 8s and 8t were reꢀcrystallised from EtOH, whereas compounds 8o and 8q were
2 2
purified by column chromatography using a mobile phase of CH Cl and MeOH (20:1, v/v),
whereas compounds 8a and 8j were purified by column chromatography using mobile phase of
Hexane and EtOAc (1:2, v/v) and EtOAc/MeOH (40:1, v/v) respectively.
Declaration of Purity
All assayed final compounds (8aꢀ8w) were more than 95% pure by HPLC.
Experimental properties of compounds 8aꢀ8w
Compounds 8a to 8w were synthesised via general procedure 3.
1ꢀ(4ꢀMethylthiazolꢀ2ꢀyl)ꢀ1ꢀ(5ꢀ(pyridinꢀ2ꢀylamino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ethanꢀ1ꢀol (8a)
Yield: 16%
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.65 (1H, s), 8.28ꢀ8.29 (1H, m), 7.71ꢀ7.75 (1H,
m), 7.34 (1H, s), 7.20ꢀ7.21 (1H, m), 7.12ꢀ7.14 (1H, m), 7.04ꢀ7.06 (1H, m), 2.30ꢀ2.31 (3H, m),
+
2
.02 (3H, br s). [ESꢀMS] (ESI+): m/z calcd for C13H13N5OS2 [M+H] , 320.06; found, 320.00.
1ꢀ(5ꢀ((6ꢀMethylpyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(8b) Offꢀwhite solid. Yield: 82%
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.43 (bs, 1H), 7.61 (1H, dd,
J
1
= 7.6 Hz,
J
2
=0.8
=0.8
Hz), 7.34 (1H, s), 7.21 (1H, d, J= 1.2 Hz), 6.86.ꢀ6.80 (2H, m), 2.42 (3H, s), 2.31 (3H, d, J
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4
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5
5
5
5
5
5
5
5
6
1
3
Hz), 2.02 (3H, s), C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ (175.43, 167.45, 161.16,
1
55.30, 151.91, 149.93, 138.65, 115.57, 114.86, 107.80, 74.33, 28.52, 23.40, 16.89)
1
ꢀ(5ꢀ((6ꢀMethoxypyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀ
ol (8c) Pale yellow solid. Yield: 83%.
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 7.63 (1H, t,
J
=8 Hz), 7.35 (1H, s), 7.22 (1H, d,
J
2
1
2
=1.2 Hz), 6.60 ( 1H, d, J=7.6 Hz), 6.36 (1H, d, J=8.0 Hz), 3.92 (3H, s), 2.31 (3H, d, J= 0.8 Hz),
1
3
.019 (3H, s); C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ (175.38, 167.38, 162.68, 161.25,
51.94, 149.61, 141.42, 114.92, 102.44, 101.25, 74.24, 54.42, 28.45, 16.87), IR (ATR) υ= 3198,
981, 1623, 1583, 1545, 1463, 1400, 1340, 1286, 1221, 1148, 1091, 1045, 994, 784, 670, 614,
ꢀ1
+
593, 575 cm , HRMS (ESI+): m/z calcd for C14
16 5 2
H N OS [M+H] , 350.0745; found, 350.0747.
1
ꢀ(5ꢀ((6ꢀBromopyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(
8d) Yellow solid. Yield: 80%
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.18 (1H, bs), 7.67 (1H, dd,
J
1
= 8.2 Hz,
= 7.7 Hz, = 0.5 Hz),
= 1.0 Hz), 2.03 (3H, s); C NMR (100.613
MHz, DMSOꢀd6, 295 K) δ 175.12, 168.06, 160.83, 151.94, 151.09, 141.14, 137.66, 119.72,
2
J = 7.7
Hz), 7.35 (1H, s), 7.44 (1H, bs), 7.22 (1H, d,
J
= 1.0 Hz), 7.19 (1H, dd,
J
1
J
2
1
3
7.06 (1H, dd, = 8.2 Hz, = 0.5 Hz), 2.31 (3H, d, J
J
1
J
2
1
14.92, 109.05, 74.29, 28.44, 16.85; IR (ATR) υ= 3121, 2925, 2762, 1620, 1583, 1532, 1453,
ꢀ1
1
427, 1395, 1340, 1161, 1134, 1093, 1060, 1039, 774, 722, 678, 655 cm ; HRMS (ESIꢀ): m/z
ꢀ
calcd for C13
H
11
N
5
OS
2
Br [MꢀH] , 395.9588; found, 395.9592.
1
ꢀ(5ꢀ((6ꢀFluoropyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(
8e). Pale ochre solid. Yield: 81%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 10.16 (1H, s), 7.90 (1H, dd, J1=8.0 Hz, J2=8.4
Hz), 7.47 (1H, s), 7.23 (d,
Hz, =1.6 Hz), 2,32 (3H, d,
K) δ 175.15, 168.15, 162.61 and 160.25 (1C,
J
= 0.8 Hz), 6.93 (1H, dd,
1 2 1
J =6.4 Hz, J =1.6 Hz), 6.69 (1H, dd, J =6.0
13
J
2
J
= 0.8 Hz), 2.04 (3H, s); C NMR (100.613 MHz, DMSOꢀd6, 295
1
JF, C = 236 Hz), 161.09, 152.02, 150,257 and
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150.11 (1C, JF, C = 13.7 Hz), 143.60 and 143.52 (1C, JF, C = 8.2 Hz), 114.97, 107.93 and 107.89
4
2
(
1C, JF, C = 3.6 Hz), 99.99 and 99.64 (1C, JF, C = 34.5 Hz), 74.27, 28.326, 16.89; IR (ATR) υ=
3
195, 3133, 2980, 2924, 1624, 1583, 1545, 1466, 1444, 1426, 1401, 1340, 1286, 1222, 1191,
ꢀ1
1
148, 1091, 1047, 994, 986, 785, 731, 671, 613, 593 cm , HRMS (ESI+): m/z calcd for
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11
12
13
14
15
16
17
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58
59
60
+
C
13
H
12FN
5
OS
2
[M+H] , 338.0546; found, 338.0540.
1ꢀ(4ꢀMethylthiazolꢀ2ꢀyl)ꢀ1ꢀ(5ꢀ((6ꢀ(trifluoromethyl)pyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
yl)ethanꢀ1ꢀol (8f) Pale yellow solid. Yield: 75%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 9.10 (1H, s), 7.99 (1H, dd, =0.8 Hz),
J
1
=8.0 Hz,
J
2
7
.450ꢀ7.431 (2H, m), 7.32 (1H, d, =8.4 Hz), 7.22 (1H, d, =0.8 Hz), 2.31 (3H, d, J=1.2 Hz),
J
J
1
3
2.03 (3H, s), C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ (175.16, 170.98, 168.38, 160.96,
51.95, 151.18, 143.63, 140.17, 115.11, 114.98, 113.21, 113.19, 74.32, 28.51, 16.85), IR (ATR)
υ= 3205, 3070, 3031, 1701, 1667, 1592, 1528, 1476, 1376, 1265, 1191, 1140, 1044, 1023, 963,
1
ꢀ1
+
9
08, 780, 696, 611, 556 cm , HRMS (ESI+): m/z calcd for C14
13 3 5 2
H F N OS [M+H] , 388.0514;
found, 388.0510.
1ꢀ(5ꢀ((6ꢀCyclopropylpyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀ
yl)ethanꢀ1ꢀol (8g)
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.16 (1H, bs), 7.55 (1H, dd, J1=7.5 Hz, J2=8.1
Hz), 7.32 (1H, s), 7.23 (1H, q, J=1.0 Hz), 6.91 (1H, d, J=7.5 Hz), 6.76 (1H, d, J=8.1 Hz), 2.32
13
(3H, d, J=1.0 Hz), 2.07ꢀ2.03 (1H, m), 2.02 (1H,s), 1.10ꢀ1.02 (2H, m), 1.00ꢀ0.91 (2H, m); ( C
NMR (100.613 MHz, DMSOꢀd6, 295 K) δ (175.51, 167.23, 161.15, 160.69, 151.97, 150.42,
1
38.27, 114.94, 114.44, 107.58, 74.28, 28.49, 16.93, 9.53, 9.32), HRMS (ESI+): m/z calcd for
C H N OS [M+H]+, 360.0951; found, 360.0953.
16
18
5
2
1ꢀ(4ꢀMethylthiazolꢀ2ꢀyl)ꢀ1ꢀ(5ꢀ((6ꢀmorpholinopyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
yl)ethanꢀ1ꢀol (8h) Offꢀwhite solid. Yield: 61%
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6
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.34 (1H, bs), 7.49 (1H, dd, J1= 8.0 Hz, J2= 8.0
Hz), 7.35 (1H, s), 7.28 (1H, bs), 7.25 (1H, d,
J
= 1.0 Hz), 6.37ꢀ6.33 (2H, m), 3.72ꢀ3.67 (4H, m),
13
3
2
6
.51ꢀ3.46 (4H, m), 2.31 (3H, d,
J
= 1.0 Hz), 2.02 (3H, s); C NMR (100.613 MHz, DMSOꢀd6
,
95 K) δ 175.60, 166.55, 161.06, 158.14, 151.99, 149.44, 139.66, 114.88, 99.71, 99.12, 73.99,
5.73, 45.71, 27.86, 16.85; IR (ATR) υ= 3327, 3202, 3104, 2960, 2926, 2867, 1625, 1585, 1552,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
1469, 1447, 1409, 1362, 1265, 1212, 1172, 1049, 1007, 896, 780, 655 cm ; HRMS (ESI+): m/z
+
calcd for C H N O S [M+H] , 405.1167; found, 405.1155.
17
21
6
2 2
1
ꢀ(4ꢀMethylthiazolꢀ2ꢀyl)ꢀ1ꢀ(5ꢀ((6ꢀ(piperidinꢀ1ꢀyl)pyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
yl)ethanꢀ1ꢀol (8i) Orange solid. Yield: 33%
1
1 2
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.25 (1H, bs), 7.67 (1H, dd, J = 7.9 Hz, J = 7.9
Hz), 7.35 (1H, s), 7.27 (1H, bs), 7.23 (1H, d,
J
= 1.0 Hz), 6.31 (1H, dd, J1= 7.9 Hz, J2= 0.5 Hz),
6
1
1
.24 (1H, dd, J1= 7.9 Hz, J2= 0.5 Hz), 3.54ꢀ3.49 (4H, m), 2.30 (3H, d, = 1.0 Hz), 2.02 (3H, s),
J
13
.65ꢀ1.58 (2H, m), 1.55ꢀ1.47 (4H, m); C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ 175.62,
66.50, 161.23, 157.77, 151.96, 149.43, 139.47, 114.81, 99.05, 98.20, 74.04, 46.33, 27.92,
24.88, 24.21, 16.84; IR (ATR) υ= 3189, 3095, 2987, 2927, 2849, 1622, 1576, 1548, 1447, 1417,
ꢀ1
+
1
4
6
224, 1170, 1140, 1039, 775 cm ; HRMS (ESI+): m/z calcd for C H N OS [M+H] ,
18 23 6 2
03.1375; found, 403.1366.
ꢀ((5ꢀ(1ꢀHydroxyꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)amino)pyridinꢀ2ꢀol
(8j) Offꢀwhite solid. Yield (after two consecutive steps): 27%
1
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.36 (1H, bs), 10.68 (1H, bs), 7.53 (1H, dd, J =
7
.8 Hz, J2= 7.8 Hz), 7.35 (1H, s), 7.20 (1H, d,
J= 1.0 Hz), 6.49 (1H, dd, J1= 7.8 Hz, J2= 0.4 Hz),
1
3
6
.18 (1H, dd, J1= 7.8 Hz, J2= 0.4 Hz), 2.30 (3H, d,
J
= 1.0 Hz), 2.02 (3H, s); C NMR (100.613
MHz, DMSOꢀd6, 295 K) δ 175.44, 167.54, 162.05, 161.60, 151.91, 149.75, 140.93, 114.76,
101.45, 100.43, 74.27, 28.48, 16.87; IR (ATR) υ= 2929, 2795, 1641, 1544, 1478, 1445, 1414,
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ꢀ
1
1374, 1333, 1278, 1226, 1151, 1129, 1110, 785 cm ; HRMS (ESI+): m/z calcd for
+
C H N O S [M+H] , 336.0589; found, 336.0586.
13
14
5
2 2
1
ꢀ(5ꢀ((5ꢀFluoropyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(8k) Solid. Yield: 26%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
6
1
H NMR (400.130 MHz, DMSOꢀd , 295 K) δ 11.65 (1H, s), 8.31 (1H, d, J = 3.03 Hz), 7.74 (1H,
dt, J1= 9.41 Hz, J2= 3.03 Hz ), 7.35 (1H, s), 7.20ꢀ7.21 (1H, m), 7.09 (1H, dd, J1= 9.26 Hz, J2
=
+
3
3
1
.54 Hz ), 2.30 (3H, s), 2.02 (3H, s a). [ESꢀMS] (ESI+): m/z calcd for [M+H] , 338.05; found,
38.00.
ꢀ(5ꢀ((5ꢀBromopyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(8l) Brownish solid. Yield: 76%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.56 (1H, s), 8.43 (1H, dd, J1=2.0 Hz, J2=0.2
Hz), 7.94 (1H, dd, J1=6.0 Hz, J2=2.8 Hz), 7.41 (1H, s), 7.22 (1H, d,
J
=1.2 Hz, 7.05 (1H, dd,
13
J =8.4 Hz, J =0.4 Hz), 2,31 (3H, d, J= 0.8 Hz), 2.03 (3H, s); C NMR (100.613 MHz, DMSOꢀ
1 2
d6, 295 K) δ (175.25, 167.89, 160.93, 151.98, 149.70, 146.96, 140.86, 114.86, 113.01, 110.82,
74.23, 28.26, 16.86), IR (ATR) υ= 3218, 3075, 2916, 1819, 1612, 1582, 1537, 1473, 1420, 1370,
ꢀ1
1
324, 1220, 1130, 1087, 1058, 1036, 895, 820, 154, 611, 588, 577 cm HRMS (ESI+): m/z calcd
+
for C H BrN OS [M+H] , 397.9745; found, 397.9734.
13
12
5
2
1
ꢀ(5ꢀ((5ꢀBromoꢀ6ꢀmethylpyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀ
yl)ethanꢀ1ꢀol (8m) Pale yellow solid. Yield: 86%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 11.71 (1H, s), 7.89 (1H, d,
J
=8.8 Hz), 7.22 (1H, d,
=1.2 Hz), 2.02 (3H,
s), C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ (175.31, 167.81, 161.05, 153.47, 151.94,
49.10, 141.86, 114.92, 110.92, 110.34, 74.32, 28.47, 23.85, 16.89), IR (ATR) υ= 3166, 2990,
J
= 1.2 Hz), 6.85 (1H, dd, J1= 8.4 Hz J2= 0.4 Hz), 2.52 (3H, s), 2.31 (3H, d, J
1
3
1
ꢀ1
2921, 1604, 1578, 1533, 1420, 1365, 1329, 1227, 1092, 1033, 955, 911, 821, 734, 613, 579 cm ,
+
HRMS (ESI+): m/z calcd for C14
H
15BrN
5
OS
2
[M+H] , 411.9901; found, 411.9893.
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1ꢀ(5ꢀ((5ꢀBromoꢀ6ꢀ(trifluoromethyl)pyridinꢀ2ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀ
methylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol (8n) Yellow solid. Yield: 75%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 8.20 (1H, d,
= 4.0 Hz, = 1.2 Hz), 2.32 (3H, d, =1.2 Hz), 2.03 (3H, s), C NMR (100.613 MHz,
DMSOꢀd6, 295 K) δ 175.42, 168.60, 160.94, 151.98, 149.37, 145.06, 141.14 and 140.80 (1C,
J= 8.8 Hz), 7.42 (1H, s,), 7.23 (2H,
13
dd,
J
1
J
2
J
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
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8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
JF, C = 33.7 Hz), 122.66 and 119.92 (1C, JF, C = 273.9 Hz), 116.63, 115.00, 107.55, 74.30,
28.44, 16.86; IR (ATR) υ= 3089, 2980, 2924, 1616, 1589, 1540, 1459, 1425, 1405, 1356, 1312,
ꢀ1
1
231, 1195, 1135, 1095, 1041, 835, 743, 715, 639, 617, 579 cm , HRMS (ESI+): m/z calcd for
+
C
14
H12BrF
3
N
5
OS
2
[M+H] , 465.9619; found, 465.9608.
1ꢀ(4ꢀMethylthiazolꢀ2ꢀyl)ꢀ1ꢀ(5ꢀ(pyrimidinꢀ2ꢀylamino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(8o)
Brown solid. Yield: 65%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 8.64 (2H, d,
=1.2 Hz), 7.08 (1H, t, =4.8 Hz), 3.31 (1H, s), 2.30 (3H, d,
100.613 MHz, DMSOꢀd6, 295 K) δ (175.29, 168.81, 161.50, 158.32, 156.37, 152.04, 143.55,
114.92, 114.63, 74.27, 28.29, 16.89), IR (ATR) υ= 3175, 3042, 2923, 1618, 1578, 1548, 1439,
J=4.8 Hz), 7.41 (1H, s), 7.22 (1H, d,
13
J
J
J= 0.8 Hz), 2.03 (3H, s), C NMR
(
ꢀ1
1
1393, 1314, 1074, 1052, 1002, 802, 742, 686, 638, 607, 554 cm , HRMS (ESI+): m/z calcd for
+
C H N OS [M+H] , 321.0581; found, 321.0592.
12
13
6
2
1ꢀ(5ꢀ((2ꢀMethoxypyridinꢀ4ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀ
ol (8p) Pale yellow solid. Yield: 87%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 10.86 (1H, s), 8.02 (1H, d, J=6.0 Hz), 7.54 (1H,
s), 7.24 (1H, d,
J
=1.2 Hz), 7.22 (1H, d,
1H, s), 2.31 (3H, d, =0.8 Hz), 2.01 (3H, s), C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ
175.377, 167.379, 162.677, 161.247, 151.942, 149.612, 141.145, 114.922, 102.440, 101.246
J=1.2 Hz), 7.00 (1H, dd, J1= 3.6 Hz, J2= 2.0 Hz), 3.85
1
3
(
(
J
74.243, 54.422, 28.451, 16.865), IR (ATR) υ = 3093, 2922, 2805, 1602, 1488, 1401, 1326, 1221,
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Journal of Medicinal Chemistry
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ꢀ
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1192, 1165, 1035, 966, 914, 843 ,733, 611, 560 cm , HRMS (ESI+): m/z calcd for
+
C H N O S [M+H] , 350.0745; found, 350.0741.
14
16
5
2 2
1
ꢀ(5ꢀ((5ꢀMethylpyridinꢀ3ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(8q) Brown solid. Yield: 55%.
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H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 8.50 (1H, d,
J
=2.4 Hz ), 8.04 (1H, d,
J=1.2 Hz),
7.98ꢀ7.97 (1H, m), 7.47 (1H, s), 7.23 (1H, d,
J
=1.2 Hz ), 2.31 (3H, d,
J=1.2 Hz), 2.40 (3H, s),
13
2
1
3
.00 (3H, s), C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ (174.82 166.34, 164.970, 152.06,
43.00, 136.91, 136.46, 133.11, 124.23, 115.03, 74.20, 28.19, 18.01, 16.88), IR (ATR) υ = 3060,
000, 2918, 1607, 1591, 1490, 1433, 1297, 1246, 1203, 1160, 1140, 1088, 1042, 861, 823, 748,
ꢀ1
+
16 5 2
700, 613, 570 cm , HRMS (ESI+): m/z calcd for C14H N OS [M+H] , 334.0796; found,
3
34.0789.
1
ꢀ(5ꢀ((6ꢀMethylpyridinꢀ3ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(8r) Brown solid. Yield: 63%.
1
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 8.60 (1H, d, J=2.8 Hz ), 7.98 (1H, dd, J = 5.60
13
Hz,
2
J = 2.8 Hz), 7.23ꢀ7.21 (2H, m), 7.41 (3H, s), 2.31 (3H, d, J=1.2 Hz), 2.00 (3H, s), C NMR
(100.613 MHz, DMSOꢀd6, 295 K) δ (174.82, 166.34, 164.97, 152.07, 143.01, 136.911, 136.45,
1
33.13, 124.23, 115.04, 74.20, 28.18, 18.01, 16.88), IR (ATR) υ= 3196, 3029, 1595, 1552, 1493,
ꢀ1
1
410, 1386, 1292, 1243, 1078, 1037, 310, 861, 828, 741, 610, 580 cm , HRMS (ESI+): m/z
+
calcd for C14
H
16
N
5
OS
2
[M+H] , 334.0796; found, 334.0797.
1
ꢀ(5ꢀ((2ꢀMethylpyridinꢀ3ꢀyl)amino)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ1ꢀ(4ꢀmethylthiazolꢀ2ꢀyl)ethanꢀ1ꢀol
(8s) Brown solid. Yield: 70%.
1
H NMR (400.130 MHz, DMSOꢀd6, 295 K) δ 8.33 (1H, dd, J1=7.2 Hz, J2=0.8Hz), 8.14 (1H, dd,
J
1
= 3.20 Hz,
J
2
=1.6 Hz), 7.41 (1H, s), 7.25ꢀ7.22 (2H, m), 2.47 (3H, s), 2.31 (3H, d,
J
=0.8 Hz),
13
1.99 (3H, s), C NMR (100.613 MHz, DMSOꢀd6, 295 K) δ (174.97, 166.25, 152.05, 148.50,
1
43.09, 135.16, 127.01, 121.67, 121.57, 115.01, 74.18, 28.14, 21.26 16.89), IR (ATR) υ= 3186,
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