Searching for New Leads for Tuberculosis: Design, Synthesis, and Biological Evaluation of Novel 2-Quinolin-4-yloxyacetamides

Article abstract of DOI:10.1021/acs.jmedchem.6b00245

In this study, a new series of more than 60 quinoline derivatives has been synthesized and evaluated against Mycobacterium tuberculosis (H37Rv). Apart from the SAR exploration around the initial hits, the optimization process focused on the improvement of the physicochemical properties, cytotoxicity, and metabolic stability of the series. The best compounds obtained exhibited MIC values in the low micromolar range, excellent intracellular antimycobacterial activity, and an improved physicochemical profile without cytotoxic effects. Further investigation revealed that the amide bond was the source for the poor blood stability observed, while some of the compounds exhibited hERG affinity. Compound 83 which contains a benzoxazole ring instead of the amide group was found to be a good alternative, with good blood stability and no hERG affinity, providing new opportunities for the series. Overall, the obtained results suggest that further optimization of solubility and microsomal stability of the series could provide a strong lead for a new anti-TB drug development program.

Full text of DOI:10.1021/acs.jmedchem.6b00245

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Article
Searching for new leads for Tuberculosis: Design, synthesis
and biological evaluation of novel 2-quinolin-4-yloxyacetamides
Eleni Pitta, Maciej K. Rogacki, Olga Balabon, Sophie Huss, Fraser Cunningham, Eva Maria Lopez-
Roman, Jurgen Joossens, Koen Augustyns, Lluis Ballell, Robert H. Bates, and Pieter Van der Veken
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00245 • Publication Date (Web): 27 Jun 2016
Downloaded from http://pubs.acs.org on June 28, 2016
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Searching for new leads for Tuberculosis: Design,
synthesis and biological evaluation of novel 2ꢀ
quinolinꢀ4ꢀyloxyacetamides
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a,b
a,b
a,b
b
b
Eleni Pitta , Maciej K. Rogacki , Olga Balabon , Sophie Huss , Fraser Cunningham , Eva
b
a
a
b
b,*
Maria Lopez-Roman , Jurgen Joossens , Koen Augustyns , Lluis Ballell , Robert H. Bates ,
a,*
Pieter Van der Veken
a
Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp,
Universitieitsplein 1, Bꢀ2610 Wilrijk, Belgium
b
Diseases of the Developing World (DDW), Tres Cantos Medicines Development Campus
(TCMDC), GlaxoSmithKline, Severo Ochoa 2, 28760, Tres Cantos Madrid, Spain
Keywords: tuberculosis, mycobacteria, antiꢀmycobacterial, antiꢀinfective, HTS, quinoline,
quinoloxyacetamides
ABSTRACT
In this study, a new series of more than sixty quinoline derivatives has been synthesized and
evaluated against Mycobacterium tuberculosis (H37Rv). Apart from the SAR exploration around
the initial hits, the optimization process focused on the improvement of the physicochemical
properties, cytotoxicity and metabolic stability of the series. The best compounds obtained
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exhibited MIC values in the low micromolar range, excellent intracellular antimycobacterial
activity and an improved physicochemical profile without cytotoxic effects. Further investigation
revealed that the amide bond was the source for the poor blood stability observed while some of
the compounds exhibited hERG affinity. Compound 83 which contains a benzoxazole ring
instead of the amide group was found to be a good alternative, with good blood stability and no
hERG affinity, providing new opportunities for the series. Overall, the obtained results suggest
that further optimization of solubility and microsomal stability of the series could provide a
strong lead for a new antiꢀTB drug development program.
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INTRODUCTION
Tuberculosis (TB) is a worldwide pandemic caused by Mycobacterium tuberculosis (Mtb). The
threat it represents to global health is escalating because of the increased prevalence of multidrug
resistant TB (MDRꢀTB) and extensively drug resistant TB (XDRꢀTB) strains. The World Health
Organization has estimated that one third of the world’s population is infected with Mtb,
1
resulting in 1.5 million TB deaths in 2014.
The firstꢀline drugs for the treatment of drugꢀsusceptible TB are isoniazid, pyrazinamide,
2
rifampicin, ethambutol and streptomycin. The current treatment regimen for drugꢀsensitive TB
3
consists of a combination of 3ꢀ4 firstꢀline drugs that must be taken for 6 months or longer.
Infection relapse and emergence of drugꢀresistant strains have been reported in many cases as
4
often patients partly or completely drop the therapy due to its side effects and long duration. In
case of drugꢀresistant strains, treatment consists of secondꢀline drugs which are administered for
2
years or longer. Apart from the high cost, many secondꢀline drugs are toxic and have severe
5
side effects, posing a significant challenge to health systems.
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Consequently, the need for novel, more effective drugs is evident. Ideally, any new drug
should be able to shorten the duration of treatment, avoid significant drugꢀdrug interactions with
current regimens, be efficacious against MDRꢀTB and XDRꢀTB and preferably operate via a
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new mode of action.
One approach to address this drug discovery need is through high throughput phenotypic
screening of small molecule libraries directly against mycobacteria in order to identify a variety
7
of new active scaffolds. A high throughput screening (HTS) campaign performed by
GlaxoSmithKline (GSK) delivered several compound families that passed multiple drugꢀlike
6
property filters and were progressed for further profiling. The quinoloxyacetamides (QOA)
constitute one of these families and the two most active hit compounds (1 and 2, shown in Fig. 1)
were selected for further structureꢀactivity relationship (SAR) studies and optimization of their
properties. It is noteworthy that quinolines represent a common substructure of several known
antiꢀtubercular drugs, e.g., bedaquiline, mefloquine and fluoroquinolones such as moxifloxacin
8
,9,10,11,12
and gatifloxacin.
• Variation of substituents on aryl residue
• Introduction of heterocycles
•
•
•
Hybrid molecules
Introduction of substituents on
methylene group
Conformational constraint
•
Variation of substituents on the
quinoline core
Figure 1. Hit compounds 1, 2 and SAR design.
As shown in Table 1, the hit compounds 1 and 2 were found to possess significant antiꢀ
mycobacterial activity with minimum inhibitory concentration (MIC) values 1.9 µM and 1.4 µM
respectively, against M. tuberculosis (H37Rv). The cytotoxicity (HepG ) of the initial hits was
2
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also evaluated and hit 1 displayed a level of cytotoxicity (IC50 19.95 µM) while hit 2 did not
exhibit cytotoxic effects. Moreover, two key physicochemical parameters (solubility and
permeability) were investigated, revealing workable, but suboptimal properties of 1 and 2. Also,
the mouse and human microsomal stabilities were determined, and the results indicated that
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further optimization was required before progressing to an in vivo proofꢀofꢀconcept.
[
a]
Table 1. Biological profile for the hit compounds 1 and 2.
Structure
Cmpd
[
b]
MIC (ꢁM)
Cytotoxicity IC (ꢁM)
1.9
1.4
[c]
19.95
180
26
>100.00
120
5
0
[d]
Permeability (nm/sec)
[
e]
Solubility (ꢁM)
Microsomal Fraction Stability
38
[
f]
Mouse
Human
Mouse
Human
ꢀ
1 ꢀ1
Clint (mL min g )
18.9
<5
1.3
>30
<3
5.4
16
t (min)
>30
1/2
a
upon reꢀtesting the obtained data were found to differ in some cases from the data published in
reference 6; MIC against Mycobacterium tuberculosis (H37Rv); HepG , human caucasian
hepatocyte carcinoma; artificial membrane permeability; in vitro profiling for kinetic aqueous
solubility (CLND, chemiluminescent nitrogen detection); in vitro microsomal fraction stability
b
c
2
d
e
f
(mouse and human) results: intrinsic clearance (Cl ) and halfꢀlife time (t ) are reported;
int
1/2
ꢀ1 ꢀ1
imidazolam was used as control with Clint = 27.5 ±0.4 and 6.4 mL min g in mouse and human,
respectively and t = <5 and 9 min in mouse and human, respectively.
1
/2
Based on the promising initial data, a library of more than sixty novel analogues was designed
and synthesized, relying on iterative logicꢀbased SAR exploration of the chemical space around
the hits. Along with identifying the structural parameters that govern the antiꢀmycobacterial
properties of these molecules, the primary goals were to optimize physicochemical properties
and to improve metabolic stability. For practical reasons, hit 1 was selected as a reference and
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divided into three substructures to help organize the SAR exploration: the quinoline, the linker
and the northern aryl part (Fig.1). This was done by preparing three compound subꢀseries in
which each of the substructures was modified separately, while keeping the rest of the molecule
identical to the reference compound. Practically, the process of compound optimization was
organized around iterative cycles of design, synthesis and evaluation. At each stage,
experimentally obtained antiꢀmycobacterial, physicoꢀchemical, and in vitro toxicity data were
used to refine the decision model used for synthetic planning.
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RESULTS AND DISCUSSION
Chemistry. More than 60 novel compounds were synthesized for this study. The target
compounds are clustered according to the modification type they contain, relative to reference
compound 1. Lastly, three compounds which contain amide bond replacements are presented.
Modification of the quinoline substitution pattern. An important goal of these compounds was
to investigate the contribution to antiꢀmycobacterial activity of the 6ꢀmethoxy and 2ꢀmethyl
quinoline substituents in 1. The selection of substituents was carried out in a highly exploratory
manner and comprises groups with widely differing impact on the sterics and electronics of the
quinoline system. The general synthetic strategy to obtain the target compounds (1, 8-24)
consisted of coupling the modified quinoline core 5a-r to 2ꢀbromoꢀNꢀ(3,5ꢀ
dimethylphenyl)acetamide 7 in the presence of potassium carbonate (K CO ) (Scheme 1).
2
3
Construction of the 2ꢀmethyl and 2ꢀtrifluoromethyl quinolinꢀ4ꢀols (5a-c, 5f, 5h-m, 5o-p) was
achieved by condensation of a number of commercially available anilines (3) with ethyl
acetoacetate (4a) or ethyl 4,4,4ꢀtrifluoroacetoacetate (4b) following a ConradꢀLimpach
1
3,14,15
protocol.
The moderate to low yields obtained with the used ConradꢀLimpach protocols
were considered acceptable for our purposes. However, further optimization of the quinolone
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synthesis would be required in case of upscaling.The Nꢀaryl haloacetamide building block 7 was
obtained in excellent yield by acylation of aniline 6 with bromoacetyl bromide in the presence of
1
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triethylamine (TEA).
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a
Scheme 1. Synthesis of compounds with quinoline substitution modifications
a
Reagents and conditions: (a) Dowtherm A, H SO , 240ꢀ250 ºC, 35ꢀ60 min; (b) 130 °C, 90
2
4
min, then Dowtherm A, 250 °C, 1 h; (c) acetic acid, toluene, reflux, 2 h, then Dowtherm A, 240
ºC, 1 h; (d) triethylamine, anhydrous DCM, rt, 2 h; (e) potassium carbonate, anhydrous DMF, rt,
3h ꢀ 4d.
Modification of the linker. SAR investigation of the linker region was focused on three main
approaches: (1) introduction of substituents on the acetyl’s methylene group, (2) conformational
constraint of the linker and (3) other modification types (Schemes 2, 3 and 4, respectively).
The synthetic approach to target compounds with the first modification type (27-30,
summarized in Scheme 2), was analogous to the general strategy described earlier (Scheme 1).
The reaction of 3,5ꢀdimethylaniline (6) with acyl halides 25a-d gave intermediates 26a-d. The
bromoalkylacyl bromides 25a-c were commercially available, while 2ꢀbromoꢀ2ꢀphenylacetyl
chloride (25d) was prepared from phenylacetyl chloride in the presence of Nꢀbromosuccinimide
1
7
and 2,2′ꢀAzobis(2ꢀmethylpropionitrile) (AIBN) according to a literature procedure. The final
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products 27-30 were subsequently obtained by alkylation of 2ꢀmethylꢀ6ꢀmethoxyꢀquinolinol (5a)
with these halides (26a-d) in the presence of sodium hydride (NaH).
a
Scheme 2. Synthesis of compounds with introduction of substituents on the linker
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a
Reaction conditions: (a) triethylamine, anhydrous DCM, rt, 2hꢀovernight; (b) sodium hydride,
(
potassium iodide), anhydrous DMF, rt, 6ꢀ48 h.
Next, four conformationally constrained analogues were prepared (compounds 31, 32, 37 and
4
0, Scheme 3). While the synthetic preparation of the target compounds 31 and 32 could be
achieved using the general alkylation procedure, a more lengthy approach was required for
compound 37. Nitration and subsequent zinc/ammonium chloride (Zn/NH Cl) reduction of 2ꢀ
4
14
methylꢀ6ꢀmethoxyquinolinꢀ4ꢀol (5a) yielded 3ꢀaminoquinolinꢀ4ꢀol 34. The latter compound
was Nꢀacylated with intermediate 35, which was prepared from oxalyl chloride and 3,5ꢀ
dimethylaniline (6). Dehydration of the obtained intermediate 36 with phosphorus pentasulfide
(
P S ) led to the assembly of the annulated thiazole ring of thiazoloquinoline 37. The associated
4 10
formation of a thioamide in 37 during P S ꢀmediated dehydration was not considered
4
10
problematic, and the obtained compound was allowed to enter biological evaluation after
purification. Comparable attempts to prepare the oxazole analogue of 37 (compound 38) by
dehydration of 36 with phosphorus pentoxide (P O ), were not successful. Compound 40 was
4
10
obtained from the acylation reaction of intermediate 35 with the commercially available 2ꢀ
methylꢀ4ꢀaminoꢀ6ꢀmethyl quinoline 39.
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Scheme 3. Synthesis of compounds with conformational constraint of the linker
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potassium carbonate, anhydrous DMF, rt, 2ꢀ72 h; (b) nitric acid, propionic acid, 110 °C, 2 h; (c)
Zn, THF/sat. aq. NH Cl: 2/1, rt, 1 h; (d) 0 °C, 1 h; (e) anhydrous DCM:DMF (10:1), 0 °C, 1 h;
(f) phosphorus pentasulfide, anhydrous pyridine, reflux, overnight; (g) phosphorus pentoxide,
anhydrous pyridine, reflux, overnight; (h) sodium hydride, anhydrous DMF, rt, overnight.
4
Subsequently, three more analogues (42, 45, 47) were prepared with linkerꢀmodifications that
were not covered in the aforementioned sets (Scheme 4). The synthetic approach to 42 was
completely analogous to the general strategy mentioned earlier. While for the preparation of 45,
the hydroxy group of quinolinol 5a was converted to bromine by phosphorous tribromide (PBr3)
leading to intermediate 43. Separately, the reaction of 3,5ꢀdimethylaniline (6) and 2ꢀ
bromoethanol resulted in intermediate 44, which was subsequently coupled with 43 to yield the
18
target compound 45 using Cu(I)ꢀcatalysis (Ullmann reaction). For the synthesis of 47,
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intermediate 43 was coupled with glycine ethyl ester under nucleophilic aromatic substitution
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conditions to yield the carboxylic ester 46a. After basic hydrolysis of 46a in methanol,
carboxylate 46b was obtained in quantitive yield and then it was converted to the acyl chloride
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6c using thionyl chloride (SOCl ). Amide bond formation between intermediate 46c and 3,5ꢀ
2
dimethylaniline (6) was achieved using basic acylating conditions to afford final compound 47.
a
Scheme 4. Synthesis of compounds which contain other modifications types
a
Reaction conditions: (a) potassium carbonate, anhydrous DMF, rt, 26 h; (b) PBr , DMF, 50
3
°
C, 4 d; (c) 90 °C, 4 h; (d) CuI, TMEDA, cesium carbonate, anhydrous DMF, 95 °C, 2 d; (e)
.
glycine ethyl ester HCl, phenol, 120 °C, overnight; (f) sodium hydroxide, MeOH, reflux, 90 min;
g) thionyl chloride, anhydrous DCM, reflux, 2 d; (h) 3,5ꢀdimethylaniline (6), anhydrous DCM,
rt, 16 h.
(
Modification of the northern aryl fragment. Analogues in this section are divided into three
categories: (1) derivatives with modified phenyl substitution (49-66), (2) compounds with
heteroaryl groups (70-78) and (3) hybrid molecules in which known antiꢀtubercular drugs replace
the aniline of the hits (79-81) (Scheme 5). The synthesis of the first set of compounds (49-66)
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relied on the general alkylationꢀbased methodology. Thus, haloacetamide intermediates 48a-r
were first prepared by acylation of a number of commercially available anilines coupled with
bromoacetyl bromide, as depicted in Scheme 5 (Method A). Subsequently, Oꢀalkylation of the
quinolinol 5a with these halides resulted in the final products 49-66.
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The target compounds 70-81 were obtained by an alternative route (Method B) shown in
Scheme 5. According to this, quinolinol 5a was alkylated with ethyl 2ꢀbromoacetate followed by
hydrolysis of ester 67 under basic conditions and conversion of the corresponding carboxylic
acid 68 to acyl chloride 69 using thionyl chloride (SOCl ). Subsequently, reaction with a number
2
of commercially available heterocyclic amines afforded the final compounds (70-78).
Lastly, the same methodology (method B) was used to synthesize three “hybrid” compounds,
in which the quinoloxyacetamide core was covalently linked to known antiꢀTB drugs with
20
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a
Scheme 5. Synthesis of compounds with modifications of the northern aryl fragment
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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
a
Reaction conditions: (a) triethylamine, anhydrous DCM, rt, 2ꢀ48 h; (b) potassium carbonate,
anhydrous DMF, rt, 3ꢀ96 h; (c) potassium hydroxide, MeOH or EtOH, reflux, 1.5ꢀ3.5 h; (d)
thionyl chloride, anhydrous DCM, 20 ºC to 40 ºC, 24ꢀ48 h; (e) anhydrous DCM, rt to reflux, 18ꢀ
4
8 h.
Amide bond replacements. Lastly, three compounds (82-84) which could avoid amide bond
hydrolysis were prepared. Deprotonation of compound 1 using sodium hydride (NaH) and
subsequent methylation with methyl iodide (MeI) afforded the target compound 82. Preparation
of compounds 83 and 84 was similar to the general alkylation method described previously.
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9
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
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 6. Synthesis of compounds with amide bond replacements
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
a
Reaction conditions: (a) methyl iodide, sodium hydride, anhydrous THF, 0 ºC to 20 ºC,
overnight; (b) potassium carbonate, anhydrous DMF, rt, overnight; (c) sodium hydride,
anhydrous DMF, rt, overnight.
Biological evaluation. The hit compound 1 obtained from high throughput screening (HTS)
was resynthesized to confirm activity and evaluated together with 62 novel derivatives. All
synthesized compounds were evaluated for their ability to inhibit the growth of Mtb H Rv strain
37
and for cytotoxicity in HepG cells. In addition, three physicochemical properties were
2
measured: artificial membrane permeability, kinetic aqueous solubility (CLND,
21,22
chemiluminescent nitrogen detection) and ChromlogD.
Table 2 presents the results for the reference compound 1 and the first set of compounds
possessing variations to the substitution pattern of the quinoline system (8-24). The main goal
was to investigate the contribution to antiꢀmycobacterial activity of the 6ꢀmethoxy and 2ꢀmethyl
quinoline substituents. Therefore, the 6ꢀmethoxy substituent was removed or replaced with
halides, methylthio, alkyl or alkoxy substituents (8, 9, 11-16, Table 2). In addition, a regioꢀ
isomer of compound 1 with the methoxy group shifted from position 6 to 7 was prepared (10,
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Table 2). Since the 2ꢀmethyl substituent of hits 1 and 2 was suspected to be a metabolically labile
site, a few analogues possessing a trifluoromethyl group were synthesized (19ꢀ23, Table 2).
Additionally, the 2ꢀmethyl group was replaced by a propyl group in an attempt to explore the
available space (24).
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
Table 2. Biological profile of the compounds with a modified quinoline part.
MIC
(µM)
Cytotoxicity Permeability Solubility Chrom
Compd
[a]
[b]
[c]
[d]
[e]
IC (µM)
(nm/sec)
(µM)
logD
5
0
R1
R2
1
6ꢀOCH
3
3
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
ꢀCH
3
3
3
3
3
3
3
3
3
3
3
3
1.9
19.95
180
370
26
55
3
5.64
5.39
6.42
5.59
5.79
6.38
5.95
6.67
6.79
5.98
4.84
7.03
7.02
7.96
7.55
8
ꢀH
24
>100.00
>100.00
50.12
[
[
f]
f]
9
6ꢀSCH
7ꢀOCH
6ꢀF
3
>125
>250
15.6
40
n.d.
n.d.
310
n.d.
520
[f]
10
11
12
13
n.d.
63.10
28
8
[
f]
6ꢀCl
>100.00
15.85
6ꢀCH
6ꢀCF
3.9
83
10
3
14
15
16
17
18
19
20
21
3
>250
>250
>250
>250
>250
>250
>250
>250
>100.00
>100.00
>100.00
10.00
<30
<30
<30
605
<10
<10
<3
[
[
[
f]
f]
f]
6ꢀOCF
6ꢀOEt
n.d.
n.d.
n.d.
<1
3
6,7ꢀdiOCH
3
6ꢀOCH
6ꢀOCH
Ph
>100.00
>100.00
>100.00
>100.00
2
ꢀCF
ꢀCF
ꢀCF
15
3
3
6ꢀOCF
6ꢀOEt
34
3
3
<3
<1
3
6
,7ꢀmethylenꢀ
22
ꢀCF
3
>250
>100.00
<30
<1
6.73
dioxy
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8
9
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
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
4
8ꢀCF
3
ꢀCF
3
>250
>250
>100.00
12.59
<10
<1
12
7.73
6.44
[f]
2
6ꢀOCH
3
ꢀPr
n.d.
a
b
MIC against Mycobacterium tuberculosis (H37Rv); HepG , human caucasian hepatocyte
2
c
d
carcinoma; artificial membrane permeability; in vitro profiling for kinetic aqueous solubility
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
e
f
(
CLND, chemiluminescent nitrogen detection); chromlogD values at pH = 7.4; n.d. = not
determined.
As shown in Table 2, SAR analysis of the quinoline part of the molecule showed that only a
methyl can be considered an acceptable alternative for the original 6ꢀmethoxy group (13, MIC =
3
.9 ꢁM). This compound showed slightly better solubility (CLND) and high permeability,
although it displayed similar cytotoxicity to 1. The presence of fluorine or no substituent (ꢀH) at
the same position led to decreased activity (11 and 8; MIC = 15.6 and 24 ꢁM, respectively).
Chlorine resulted in a further drop of the potency (12, MIC = 40 ꢁM). All the other substituents
in the series were inactive. Steric limitations might be involved here, although other factors most
likely contribute, as reflected by the absence of activity for the 6ꢀtrifluoromethyl containing
2
3
analogue 15. Regarding the methyl group at position 2, its replacement with a trifluoromethyl
or a propyl group led to inactive compounds (19 and 24). For all the compounds (19-24)
possessing a trifluoromethyl group in position 2, the extremely poor solubility and higher
chromlogD values could play a role in the loss of potency.
Table 3 presents the results for the second set of compounds possessing modifications on the
linker. The first area of exploration was around the acetyl methylene group. Monoꢀsubstitution at
this prochiral position is the most straightforward way of introducing a chiral center in these
compounds without affecting the quinoloxyacetamide basic framework. In addition, this site was
also suspected of being a contributor to the fast (oxidative) metabolism observed for the hit
compounds. In this initial proofꢀofꢀconcept compound set, the substituent choice was limited to
small aliphatic groups (methyl and ethyl) and a phenyl ring (27-30).
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Next, four conformationally constrained analogues were prepared (31, 32, 37 and 40). In
compounds 31, 32 and 37 conformational locking was accomplished by an additional ring
closure, while in the case of analogue 40 the oxalyl diamide fragment was expected to rigidify
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
24, 25
the linker region into a constrained conformation.
Subsequently, three more analogues (42, 45, 47) were prepared with linkerꢀmodifications that
were not covered in the aforementioned sets. Compound 42 contains a benzylamide function as a
means to avoid potential toxicity and metabolic stability concerns that are associated with anilide
groups. Compound 45 possesses a fully reduced linker, while in compound 47 the ether bridge
between the linker region and the quinoline residue is replaced by an amine function.
Table 3. Biological profile of the compounds with linker modifications.
MIC
Cytotoxicity Permeability Solubility Chrom
IC (µM)
Cmpd Structure
[a]
[b]
[c]
[d]
[e]
(µM)
(nm/sec)
(µM)
logD
5.76
6.37
6.28
7.13
5
0
2
2
2
3
7
8
9
0
R
R
R
R
4
4
4
4
= CH
3
125
39.81
320
61
[
f]
= diCH
= Et
>250
>250
>125
>100.00
79.43
n.d.
390
20
3
123
2
= Ph
>100.00
<30
31
32
31.62
470
21
5.48
32
36
125
100.00
550
215
10
4.99
3.76
[
f]
>125
>100.00
n.d.
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1
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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
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[f]
3
4
7
0
>125
>125
>100.00
>100.00
<30
35
10
n.d.
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
[e]
n.d.
<3
6.47
5.25
4
2
31
>100.00
92
4
4
5
7
32
47
31.62
6.31
190
310
33
6.93
3.59
204
a
b
MIC against Mycobacterium tuberculosis (H37Rv); HepG , human caucasian hepatocyte
carcinoma; artificial membrane permeability; in vitro profiling for kinetic aqueous solubility
(CLND, chemiluminescent nitrogen detection); chromlogD values at pH = 7.4; n.d. = not
determined.
2
c
d
e
f
Antimycobacterial screening of the compounds with an alphaꢀsubstituted acetyl group (27-30)
demonstrated that modifications of this type are detrimental to activity. Similarly, in the second
group of compounds the activity was lost or significantly reduced in all cases. In the same way,
compounds belonging to the third group (42, 45 and 47) showed some moderate potency giving
MIC values of 31, 32 and 47 ꢁM respectively.
To investigate the role of the hydrophobic phenyl ring in the northern part of the molecule,
thirty compounds (49-66, 70-78 and 79-81) possessing modifications on the northern aryl part
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were prepared and data from their biological and physicochemical evalution is presented in
Table 4.
Analogues in this section are divided into three categories: (1) derivatives with modified
phenyl substitution (49-66), (2) compounds with heteroaryl groups (70-78) and (3) hybrid
molecules in which 3 known antiꢀTB drugs replace the aniline of the hits compounds (79-81).
For the first category, the initial goal was to introduce a diverse set of commercially available
anilines. These molecules allow a more thorough investigation of the role of the aryl substituents
on the antimycobacterial properties of QOAꢀbased molecules. Furthermore, the metabolic
liability of the specific aryl residues present in 1 and 2 had also been proposed as a potential
reason for the observed microsomal instability. Therefore, additional value for this series could
come from its potential to deliver antimycobacterial products in which positions prone to CYPꢀ
mediated oxidation have been blocked or modified.
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
Similar to the substitutions discussed above, replacement of the phenyl ring with a heteroaryl
(70ꢀ78) offered the possibility of improving the microsomal stability, at least partially, by
reducing the overall lipophilicity of the molecule. Commercially available heteroaromatic
amines with an identical or similar dimethylꢀsubstitution pattern as present in reference 1 were
included in the series to allow direct comparisons.
Regarding the hybrid compounds, we hypothesized that the full hybrid constructs could still
possess antimycobacterial properties. In addition, if metabolic cleavage of the amide would take
place, either inside bacteria or mediated by host metabolism, a second antimycobacterial
compound could be released.
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8
9
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
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Biological profile of the compounds with modifications on the northern aryl.
MIC
Cytotoxicity Permeability Solubility Chrom
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
Cmpd
[a]
[b]
[c]
[d]
[e]
(µM)
IC (µM) (nm/sec)
(µM)
logD
5
0
R3
4
5
5
5
2
5
5
5
5
5
5
5
6
9
0
1
2
phenyl
2
39.81
370
355
810
630
120
470
420
320
n.d.
210
n.d.
n.d.
n.d.
n.d.
570
n.d.
620
230
n.d.
480
330
63
4.28
4.83
5.05
4.55
4.68
4.49
4.22
4.99
5.65
5.50
5.95
5.13
6.40
6.22
5.09
5.80
3.95
4.49
5.15
3.49
2.46
3ꢀCH
ꢀphenyl
2
39.81
193
45.5
112
38
3
2,5ꢀdiCH
2,6ꢀdiCH
ꢀphenyl
ꢀphenyl
8
>100.00
>100.00
>100.00
>100.00
>100.00
7.94
3
125
1.4
6.4
0.6
3
2ꢀOCH
3ꢀOCH
4ꢀOCH
ꢀphenyl
ꢀphenyl
ꢀphenyl
3
3
3
3
4
5
6
7
8
9
0
35
108
17
3,5ꢀdiCH
2ꢀOCH , 5ꢀCH
3,5ꢀdiCH
3ꢀCH , 4ꢀBrꢀphenyl
, 4ꢀOCH
ꢀphenyl 1
3
3
[
f]
ꢀphenyl
2.5
2
>100.00
>100.00
15.85
9.5
31.5
<1
3
3
, 4ꢀFꢀphenyl
3
[
[
[
[
f]
f]
f]
f]
3.9
3
3
3,5ꢀdiFꢀphenyl
2,4ꢀdiClꢀphenyl
>100.00
>100.00
79.43
13
2
20.5
17
61
62
63
64
65
66
3ꢀCF
3
, 4ꢀClꢀphenyl
47
>125
>125
62
12
3
3ꢀOMe, 4ꢀClꢀphenyl
2,5ꢀdiOMe, 4ꢀClꢀphenyl
3,4,5ꢀtriOMeꢀphenyl
4ꢀFꢀphenyl
>100.00
>100.00
>100.00
63.10
13
[
[
f]
f]
3
34
104
9
4ꢀClꢀphenyl
>100.00
>100.00
>100.00
7
7
0
1
pyridinꢀ2ꢀyl
15.65
62.5
29
pyridinꢀ3ꢀyl
32
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7
7
7
7
7
2
3
4
5
6
7
pyridinꢀ4ꢀyl
2,6ꢀdiCH ꢀpyridinꢀ4ꢀyl
pyrimidinꢀ4ꢀyl
2,6ꢀdiCH
thiazolꢀ2ꢀyl
4,5ꢀdiCH ꢀthiazolꢀ2ꢀyl
>250
>250
>250
>100.00
12.59
560
n.d.
525
12
91.5
12
2.50
3.01
2.53
3.24
3.20
4.24
[f]
3
>100.00
>100.00
>100.00
>100.00
27.5
5
ꢀpyrimidinꢀ4ꢀyl >250
3
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
>250
62
1120
440
54
31.5
3
7
7
8
9
187
>100.00
>100.00
625
<10
195.5
≥166
3.30
0.79
>125
8
8
0
1
125
62
>100.00
>100.00
15
33
≥381
≥375
2.20
1.48
a
b
MIC against Mycobacterium tuberculosis (H37Rv); HepG , human caucasian hepatocyte
2
c
d
carcinoma; artificial membrane permeability; in vitro profiling for kinetic aqueous solubility
e
f
(CLND, chemiluminescent nitrogen detection); chromlogD values at pH = 7.4; n.d. = not
determined.
Modification of the substitution pattern on the phenyl ring resulted in a number of active
compounds with MIC values in the low micromolar range. Elimination of one or both methyl
groups (50, 49) did not affect the activity (MIC = 2 μM), indicating that the methyl groups are
not critical for potency. Moreover, solubity and chromlogD values were improved, although
cytotoxicity remained at similar levels as 1. It is worth noting that shifting one methyl group
from position 3 to 2 led to decreased activity (51, MIC = 8 µM), while shifting both methyls
from 3,5 to 2,6 positions led to the practically inactive compound 52 (MIC = 125 µM).
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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
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Exploration of the position of the methoxy group of the hit compound 2 indicated that the
para-position (compound 54, MIC = 0.6 µM) is more favourable than the ortho- (hit compound
2
, 1.2 µM) or meta- substitution (compound 53, MIC = 6.4 µM). Compound 54, apart from
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
excellent activity, also possessed an improved profile in comparison with the reference
compound 1. More specifically, it did not exhibit cytotoxic effects (IC >100 µM), had good
5
0
permeability (420 nm/sec), improved kinetic aqueous solubility (CLND) (108 µM) and
chromlogD values (4.22). Compound 55, where we preserved both methyl groups at positions 3
and 5 (as the reference compound 1) and a methoxy group at position 4 (as compound 54),
showed very good activity (MIC = 1 µM) but high cytotoxicity (IC50 7.94 µM) and low
solubility (17 µM). Several compounds possessing monoꢀ, diꢀ or triꢀ substituted phenyl rings
(56-60, 66) exhibited very good potencies, although they were less active than the reference
compounds. Most of them did not show cytotoxic effects (56, 57, 59, 60, 66), but, chromlogD
values were generally high (>5) and solubility low (>32).
Introduction of more hydrophilic rings such as pyridine, pyrimidine, thiazole, isoxazolidinone
or dimethylisoxazole did not provide the desired solubility improvement although the
chromlogD values (2.46ꢀ4.24) were lower than the phenyl derivatives, and the antitubercular
activity was lost for most of them. Comparison among compounds 70-72 revealed that 2ꢀ
pyridine (70) is the most favourable, however still less active than the hit compounds (1 and 2).
The last subset of compounds which includes three hybrid molecules (79-81), unfortunately, led
to loss of activity too.
Additionally, the intracellular activity for the hit compounds 1 and 2 and the most active, nonꢀ
cytotoxic synthesized compounds (54, 56, 57, 59, 60, 66) was evaluated. This assay determines
the effect of the compounds on mycobacteria growing inside phagocytes/macrophages. Activity
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in this assay is considered highly desirable as many of the bacteria during an active Mtbꢀ
infection are found intracellularly in phagocytotic cell types. The obtained results are shown in
Table 5.
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
Table 5. Intracellular IC and IC values for selected compounds.
5
0
90
Cmpd
Intracellular
IC50 IC90
R3
[a]
[a]
(
µM)
(µM)
1
3,5ꢀdiCH
3
ꢀphenyl
0.05
0.50
0.03
0.16
0.08
0.40
0.79
0.16
0.2
2
2ꢀOCH
4ꢀOCH
2ꢀOCH
3
3
3
ꢀphenyl
ꢀphenyl
1.58
0.25
0.63
0.25
2.51
>50
54
56
57
59
60
, 5ꢀCH
, 4ꢀFꢀphenyl
ꢀphenyl
3
3,5ꢀdiCH
3
3,5ꢀdiFꢀphenyl
2,4ꢀdiClꢀphenyl
4ꢀClꢀphenyl
66
0.50
a
IC and IC against infected Human THPꢀ1 macrophages with Mycobacterium tuberculosis
5
0
90
(H37Rv)
It is worth noting that 6 out of 8 tested compounds showed excellent intracellular IC values,
9
0
ranging from 0.25 to 2.51 µM. More specifically, compound 54, which had also shown the
highest MIC value, and compound 57 exhibited the highest intracellular potencies with IC90
values of 0.25 µM. They were followed by compounds 56 and 66 with IC values 0.50 and 0.63
9
0
µM, respectively. Less active but still very potent compounds were the hit compound 2 and
compound 59 (IC 1.58 and 2.51 µM, respectively). Lastly, compound 60, although possessing a
9
0
good MIC value, did not reach 90% inhibition at 50 µM (IC >50 µM) in the intracellular assay.
9
0
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2
2
2
2
2
2
2
2
2
2
3
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3
3
3
4
4
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4
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4
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5
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5
5
5
5
6
This could be due to poor permeability of the compounds through the cell membrane or due to
bacterial efflux pumps activated by the macrophage or inactivation of the compounds by host
2
6
cell derived metabolites such as reactive species or acidic pH.
0
1
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1
2
3
4
5
6
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9
0
Metabolic stability studies. In view of the fact that microsomal instabillity was identified as a
possible liability of the hits, six compounds were selected and the stability in mouse and human
microsomal fractions was evaluated before continuing with further synthetic efforts. The
selection of compounds was driven by structural criteria in an attempt to identify the metabolic
liabilities of the series. Three possible metabolic sites were explored: the methoxy group, the
amide bond and the phenyl ring of the reference compound 1. Therefore, compound 13 which
possesses a methyl group instead of methoxy group was selected in order to evaluate its stability.
Similarly, compounds 28 and 45, which have a sterically hindered amide and an amine,
respectively, were also chosen. Lastly, compounds 50, 75 and 57 were selected because they
contain substituents in different positions of the phenyl ring.
The six selected compounds together with the reference compound 1, were evaluated for their
stability in mouse and human microsomal fractions, and the obtained data are presented in Table
6. All the tested compounds proved to be highly unstable, especially when incubated with mouse
microsomal fractions. Comparison of the obtained data with the control without coꢀfactor data
indicated that cytochrome Pꢀ450 metabolism was not determinant for all the tested compounds
apart from compound 45, signifying that the amide bond is the most susceptible group of the
series and that the esterase hydrolysis might be involved.
In order to confirm that esterases are responsible for the rapid metabolism of the compounds,
the selected compounds were incubated in fresh whole CD1 mouse blood. A parallel run, after
pretreatment of the blood with panꢀesterase inhibitor sodium fluoride (NaF), was performed and
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the obtained results are depicted in Table 6. As expected and in accordance with the no coꢀfactor
control in the microsomal stability experiment, nearly all the compounds possessing an amide
bond were highly unstable. On the other hand, compounds 45 and 28, which possess an amine or
a sterically hindered amide, were stable. In addition, the instability is mitigated after NaF
pretreatment suggesting that it is mainly due to the hydrolysis of the amide. Microsomal stability
in human fractions was better, which is in agreement with the hypothesis of esterase hydrolysis,
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28
29
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31
32
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34
35
36
37
38
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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
2
7
since esterases in rodents are more active.
Table 6. Stability of selected compounds in mouse/human microsomal fractions and blood.
[
a]
Microsomal fraction stability
[
b]
Blood stability
Mouse
Clint
Human
Clint
Cmpd
t1/2
t1/2
t1/2
ꢀ1
ꢀ1
ꢀ1 ꢀ1
NaF
(mL min g ) (min)
(mL min g ) (min) (min)
Partially
stabilized
Partially
stabilized
1
18.9
<5
<5
1.3
1.6
>30
>30
<5
<5
13
28
21.9
27.3
86.5
<5
<5
2.7
6.3
19
96
Stable
Stable
4
5
5
0
>30
>120
Partially
stabilized
Partially
stabilized
Partially
stabilized
18.1
50.2
19.3
<5
<5
<5
2.5
2.3
1.1
23
<5
<5
<5
51
24
5
a
7
>30
in vitro microsomal fraction stability (mouse and human) results: intrinsic clearance (Cl ) and
int
halfꢀlife time (t ) are reported; imidazolam was used as control with Cl 27.5 ±0.4 and 6.4 mL
1
/2
int =
ꢀ1 ꢀ1
min g in mouse and human, respectively and t = <5 and 9 min in mouse and human,
respectively; blood stability results: halfꢀlife time (t ) and effect in presence of NaF are
1
/2
b
1/2
reported.
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In the light of this evidence, further medicinal chemistry effort led to methylation of the amide
(
82) and replacement of the Nꢀphenyl amide with benzoxazole (83) or benzimidazole (84) rings
in an effort to overcome the rapid clearance observed in mice microsomal fractions. The ring
closure or the methylated amide bond could potentially stabilize the metabolic liability of the
series, in theory rendering an improved profile over the previous scaffold. Table 7 presents the
obtained data for compounds 82, 83 and 84. Lastly, Nꢀmethylation of the reference compound 1
was explored as a classic approach to reduce amide hydrolysis.
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
Table 7. Biological profile of compounds 82, 83 and 84.
Structure
Cmpd
82
83
84
[
a]
MIC (µM)
109.3
5.5
16
[b]
[c]
[c]
Intracellular IC (µM)
n.d.
5
n.d.
9
0
[
d]
Cytotoxicity IC (µM)
50.12
300
>100.00
86
>100.00
5
0
[
e]
[c]
Permeability (nm/sec)
n.d.
[f]
Solubility (µM)
373
1
13
[
g]
ChromlogD
Microsomal fraction stability
5.47
6.12
3.59
[
h]
ꢀ
1
ꢀ1
[g]
[c]
Cl [mL min g ]
n.d.
46.8 (m), 3.5 (h)
<5 (m), 15.7 (h)
n.d.
int
t (min)
1
/2
[
i]
Blood stability
(min)
[g]
[c]
n.d.
>240
b
n.d.
t
1
/2
a
MIC against Mycobacterium tuberculosis (H37Rv); IC against infected Human THPꢀ1
macrophages with Mycobacterium tuberculosis (H37Rv); n.d. = not determined; HepG , human
caucasian hepatocyte carcinoma; artificial membrane permeability; in vitro profiling for kinetic
aqueous solubility (CLND, chemiluminescent nitrogen detection); chromlogD values at pH =
7.4; in vitro microsomal fraction stability results; clearance (Cl ) and halfꢀlife time (t ) is
reported; imidazolam was used as control with Clint = 27.5 ±0.4 and 6.4 mL min g in mouse and
human, respectively and t = <5 and 9 min in mouse and human, respectively (h) = human, (m)
9
0
c
d
2
e
f
g
h
int
1/2
ꢀ1 ꢀ1
1
/2
i
=
mouse; blood stability results: halfꢀlife time (t ) is reported.
1/2
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Compound 83, which possesses a benzoxazole ring instead of anilide, showed significant antiꢀ
tubercular activity with an MIC value of 5.5 µM and intracellular IC90 of 5 ꢁM. Subsequent
testing for blood and microsomal stability revealed the expected improvement in blood stability
due to the removal of the labile amide bond. However, solubility of compound 83 was very poor
and in vitro microsomal clearance was high, suggesting that further medicinal chemistry effort is
required in order to overcome these problems.
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Lastly, a preliminary evaluation of the cardiosafety of the series was performed by measuring
the human etherꢀaꢀgoꢀgoꢀrelated gene (hERG) inhibition of selected compounds. Obtained
results are presented in Table 8.
Table 8. Results of hERG binding of selected compounds.
hERG
Cmpd R1
R3
(pIC50)
<4.3
5
1
6ꢀCH
3
3
3
3
3
3
3
Oꢀ
Oꢀ
ꢀ
3,5ꢀdiCH ꢀphenyl
3
2
6ꢀCH
6ꢀCH
6ꢀCH
6ꢀCH
6ꢀCH
6ꢀCH
2ꢀOCH
3
ꢀphenyl
ꢀphenyl
ꢀphenyl
13
54
56
57
66
3,5ꢀdiCH
3
<4.3
5.2
Oꢀ
Oꢀ
Oꢀ
Oꢀ
4ꢀOCH
2ꢀOCH
3
, 5ꢀCH
ꢀphenyl 5.3
3
3
3,5ꢀdiCH
, 4ꢀFꢀphenyl
<4.3
5.3
3
4ꢀClꢀphenyl
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1
1
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
3
<4.3
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
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The data in Table 8 indicate that four out of eight tested compounds displayed a hERG
inhibitory potency that was higher than the threshold value used for safety assessment (pIC =
5
0
4
.3). Interestingly, three of these (2, 54 and 56) possess a methoxyaniline substituent. The rest of
the evaluated compounds did not display potent hERG inhibition (pIC50 <4.3). Compound 83,
which was found to be of particular interest due to its blood stability, did not show interaction
with hERG.
CONCLUSION
In the presented work, the synthesis of more than sixty novel quinoloxyacetamide derivatives
and their biological evaluation against Mycobacterium tuberculosis (H37Rv) is reported. Apart
from the SAR exploration around the initial hits, the optimization processes focused on the
improvement of the physicochemical properties, cytotoxicity and metabolic stability of the hit
compounds. Several compounds showed potent antiꢀtubercular activities with MICs in the low
micromolar range with the best compound (54) exhibiting a MIC value of 0.6 ꢁM and no
measurable cytotoxicity. This compound and other potent, nonꢀcytotoxic analogues also showed
excellent intracellular IC90 values ranging from 0.25 to 2.51 µM. Furthermore, the metabolic
stability of the series was investigated and it was shown that the amide bond is the most labile
group. Thus, synthetic efforts were focused on amide replacement and compound 83 which
possessed a benzoxazole was identified as a good alternative to improve blood stability (halfꢀlife
time >240 min). Evaluation of hERG binding indicated that mainly compounds possessing a
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methoxyaniline substituent, showed appreciable hERG inhibition, while the optimized
compound 83 displayed no activity in the assay. Further medicinal chemistry effort is ongoing to
increase solubility and microsomal stability of the series in order to provide a strong lead for a
new antiꢀtubercular drug discovery program.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
36
37
38
39
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
General Information. Unless otherwise stated, laboratory reagent grade solvents were used.
Reagents were purchased from SigmaꢀAldrich, Acros Organics, TCI or Enamine and were used
without further purification unless otherwise mentioned. Reactions were monitored by TLC on
silica gel with detection by UV light (254 nm). TLC analysis was performed using Polygram®
precoated silica gel TLC sheets SIL G/UV254
.
1
13
Characterization of all compounds was done using H NMR and CꢀNMR spectroscopy and
1
13
mass spectrometry. H NMR (400 MHz) and CꢀNMR (100 MHz) spectra were recorded on a
Bruker Avance III Nanobay Ultrashield 400 or a Bruker DPX 400 spectrometer. The chemical
shift (δ) values are expressed in parts per million (ppm) and coupling constants are in Hertz (Hz).
Minor rotamers of the amide bond, which were less than 10% of the major rotamer, are not
reported in the NMR data. CDCl
3
, CD
3
OD or DMSOꢀd were used as the standard NMR
6
solvents. Legend: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet,
dd = doublet of doublets, ddd = doublets of doublets of doublets, br = broad signal. For the
measurement of melting points, a Technoterm 7300 (ReichertꢀJung Optische Werke) microscope
was used.
Purity and mass were verified using a UPLCꢀMS system and purities of all final products were
found to be >95%. UPLCꢀMS involved the following: Waters Acquity UPLC system coupled to
a Waters TQD ESI mass spectrometer and Waters TUV detector. A Waters Acquity UPLC BEH
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C18 1.7 ꢁm, 2.1 mm × 50 mm column was used. Solvent A consisted of water with 0.1% formic
acid. Solvent B consisted of acetonitrile with 0.1% formic acid. Method A involved the
following: flow 0.4 mL/min, 0.15 min isocratic elution (95% A, 5% B), followed by gradient
elution during 1.85 min (from 95% A, 5% B to 95% B, 5% A), then 0.25min (0.350 mL/min)
isocratic elution (95% B, 5% A). The wavelength for UV detection was 254 nm. Method B: flow
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
0.4 mL/min, 0.25 min isocratic elution (95% A, 5% B), followed by gradient elution during 4.75
min ( 95% B, 5% A, then isocratic 0.25 min of isocratic elution (95% B, 5% A) followed by 0.75
min isocratic elution (95% A, 5% B). The wavelength for UV detection was 214nm. For method
C, a Waters Acquity UPLC system was coupled to a Waters SQ detector and an Acquity UPLC
BEH C18 1.7 ꢁm, 3x50 mm column was used. The concentration of the measured samples was
0.1 mg/ml and flow 0.8 mL/min. The method involved the following: Acetate NH 25mM + 10%
4
ACN at pH 6.6 /ACN, 0.0ꢀ0.2 min 99.9: 0.1, 0.2ꢀ1.0 min 10:90, 1.0ꢀ1.8 min 10:90, 1.9ꢀ2.0 min
99.9:0.1 at temperature 40ºC. The UV detection was an averaged signal from wavelength of 210
+
ꢀ
nm to 400 nm. The quasiꢀmolecular ions [M+H] or [MꢀH] were detected. Retention time (RT)
was indicated for the described method.
For the High Resolution Mass Spectrometry (HRMS) measurements: Positive ion mass spectra
were acquired using a QSTAR Elite (AB Sciex Instruments) mass spectrometer, equipped with a
turbospray source, over a mass range of 250–700.
When necessary, flash purification was performed on a Biotage ISOLERA One flash system
equipped with an internal variable dualꢀwavelength diode array detector (200ꢀ400 nm). For
normal phase purifications SNAP cartridges (10ꢀ100 g, flow rate of 10ꢀ100 mL/min) were used,
and reverseꢀphase purifications were done making use of KPꢀC18 containing cartridges. Dry
sample loading was done by selfꢀpacking samplet cartridges using silica or Celite 545,
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respectively, for normal and reversed phase purifications. Gradients used varied for each
purification. However, typical gradients used for normal phase were gradient of 0−100% ethyl
acetate in nꢀheptane or 0ꢀ15% methanol in ethyl acetate. For reverse phase a gradient of 5%
MeCN in water to 50% MeCN in water was used.
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11
12
13
14
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16
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18
19
20
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22
23
24
25
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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
The following section comprises the synthetic procedures and analytical data for all final
compounds and some representative intermediates reported in this publication. Complimentary
data for the rest of intermediates can be found in the Supporting Information. Synthetic
procedures that were used in the preparation of several products are summarized here as
“General Procedures”.
For the synthesis of the quinolinꢀ4ꢀols, three related methods were used (General procedures
A, B and C).
General procedure A. Formation of 2-methyl-quinolin-4-ols (5a, 5l) and 2-
trifluoromethyl-quinolin-4-ols (5m, 5o, 5p). According to Brouet et al., the appropriate
substituted aniline (1 eq.) and ethyl acetoacetate or ethyl 4,4,4ꢀtrifluoroacetoacetate (1.25ꢀ2.5
eq.) were dissolved in Dowtherm A (molarity: 0.5 M) and concentrated sulfuric acid (1ꢀ3 drops)
1
was added to the stirred mixture. The reaction vessel was equipped with a short distillation
apparatus. The reaction mixture was heated gradually to 240ꢀ250 °C for 35ꢀ60 min and the
produced water/ethanol was removed by distillation as the reaction progressed. Subsequently, the
reaction mixture was cooled down to room temperature and poured into nꢀheptane to give a
precipitate. The precipitate was collected by filtration and washed with nꢀheptane and EtOAc. If
necessary, the product was further purified by recrystallization or silica gel flash
chromatography.
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