Differential characterization using readily accessible NMR experiments of novel N- and O-alkylated quinolin-4-ol, 1,5-naphthyridin-4-ol and quinazolin-4-ol derivatives with antimycobacterial activity

Article abstract of DOI:10.1016/j.ejmech.2016.10.014

During the construction of bioactive molecules, regioselective alkylation of heterocyclic, N/O ambident nucleophiles is a frequently encountered synthetic transformation. In this framework, specific attention is required to unambiguously determine the str

Full text of DOI:10.1016/j.ejmech.2016.10.014

Accepted Manuscript
Differential characterization using readily accessible NMR experiments of novel N-
and O-alkylated quinolin-4-ol, 1,5-naphthyridin-4-ol and quinazolin-4-ol derivatives
with antimycobacterial activity
Eleni Pitta, Olga Balabon, Maciej K. Rogacki, Jesús Gómez, Fraser Cunningham,
Jurgen Joosens, Koen Augustyns, Pieter van der Veken, Robert Bates
PII:
S0223-5234(16)30876-5
10.1016/j.ejmech.2016.10.014
EJMECH 8979
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2016
Revised Date: 13 September 2016
Accepted Date: 7 October 2016
Please cite this article as: E. Pitta, O. Balabon, M.K. Rogacki, J. Gómez, F. Cunningham, J. Joosens,
K. Augustyns, P. van der Veken, R. Bates, Differential characterization using readily accessible
NMR experiments of novel N- and O-alkylated quinolin-4-ol, 1,5-naphthyridin-4-ol and quinazolin-4-
ol derivatives with antimycobacterial activity, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.10.014.
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GRAPHICAL ABSTRACT

ACCEPTED MANUSCRIPT
Differential characterization using readily accessible NMR
experiments of novel N- and O-alkylated quinolin-4-ol, 1,5-
naphthyridin-4-ol and quinazolin-4-ol derivatives with
antimycobacterial activity
Eleni Pittaa, Olga Balabona, Maciej K. Rogackia, Jesús Gómezb, Fraser Cunninghamb, Jurgen
Joosensa, Koen Augustynsa, Pieter van der Vekena,*, Robert Batesb,*
aMedicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp,
Universitieitsplein 1, B-2610 Wilrijk, Belgium
aDiseases of the Developing World (DDW), Tres Cantos Medicines Development Campus
(TCMDC), GlaxoSmithKline, Severo Ochoa 2, 28760, Tres Cantos Madrid, Spain
*Corresponding Authors
For R.B.: phone, +34 650395529; e-mail, robert.h.bates@gsk.com.
For P.V.V: phone, +32 32652708; e-mail, pieter.vanderveken@uantwerpen.be.
Keywords: quinolinol, naphthyridinol, quinazolinol, tuberculosis, alkylation,
regioselectivity, NMR
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Abstract
During the construction of bioactive molecules, regioselective alkylation of heterocyclic,
N/O ambident nucleophiles is a frequently encountered synthetic transformation. In this
framework, specific attention is required to unambiguously determine the structures of
obtained reaction products. As part of our project on quinoloxyacetamide based
antimycobacterial agents, a series of N- or O- alkylated quinolin-4-ol, 1,5-naphthyridin-4-ol
and quinazolin-4-ol derivatives were prepared during the course of which we observed
unexpected selectivity issues. After finding that no consistent procedure existed in the
literature for assigning regioisomers of this type, we applied three readily accessible NMR
experiment types (13C-NMR, HSQC/HMBC and NOE) to resolve any uncertainties regarding
the obtained regioisomeric structures. Furthermore, the antimycobacterial activity of all
final compounds was evaluated with the best compound 23 showing potent antitubercular
activity (MIC = 1.25 µM) without cytotoxic effects.
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1. Introduction
Despite continuing advances in synthetic organic chemistry, regioselective alkylation of
heterocyclic N/O ambident nucleophiles remains a challenging transformation. Many
scientists have tried to identify reaction parameters and conditions (e.g., countercation,
solvent, temperature, electrophile) that influence the outcome in a predictive manner [1–
4]. However, in general the outcome can still vary among different heterocycles and even
between nearly identical scaffolds depending on their substitution pattern [5]. Likewise,
several theoretical approaches to offer insight in this matter have been proposed.
Systematic investigations by Kornblum and Tieckelmann were integrated in Pearson’s
concept of “Hard and Soft Acids and Bases” (HSAB) which became the most popular
approach to rationalize ambident reactivity [6,7]. The HSAB rationale was complemented
with the Klopman-Salem theory of charge and orbital control of organic reactions [8,9].
Although these concepts have been widely accepted, they have also been criticized by
Gompper and Wagner and by Mayer and co-workers [10,11]. More recently, Marcus
analysis has been proposed by Breugst and co-workers as a more successful alternative to
rationalize the behavior of ambident nucleophiles [12].
Several of these ambident heteroaromatic scaffolds such as quinolinols and quinazolinols
have notable value for drug discovery since they often display drug-like properties [13,14].
As evidence of these properties, they can be found in many drugs such as antibiotics,
antimalarials, antidiabetics, anti-inflammatories etc. [15–17].
We recently reported the synthesis and antimycobacterial activity of a number of 2-
substituted quinolines with a 4-oxyacetyl-derived substituent [18]. Based on the promising
results obtained, we decided to explore the Structure-Activity-Relationship (SAR) of this
novel class of antimycobacterials in greater detail. Herein, we describe a further expansion
of the initial set with compounds lacking the 2-substituent (compounds of type A, Figure 1)
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along with replacements of the quinoline system with related bicyclics such as
naphthyridine and quinazoline (compounds of types B and C, Figure 1). The rationale for
the scaffold replacement approach was to decrease the lipophilicity of the molecules
(chromlogD was used as an indicator of lipophilicity) and improve solubility by
introducing additional nitrogen atoms [19,20].
During the course of our investigation we were surprised to find that N-, O- selectivity of
our compounds was not always as expected and therefore each scaffold was carefully
examined using NMR techniques to fully assign the structure and rationalise the selectivity
of the core heterocycles.
Figure 1. Initial hit compound and design of new compounds.
2. Results and discussion
2.1. Chemistry
The general synthetic approach planned for all target compounds of this study consisted of
the initial preparation of the required heterocyclic cores equipped with a 4-
hydroxyfunction. Subsequently, the O-substituent was to be introduced using an alkylation
protocol that had proven earlier to regioselectively render the required O-alkylated
isomers in the 2-substituted quinolone series [18]. The observed O-regioselectivity was in
agreement with the literature reports of 2-(trifluoro)methyl quinolin-4-ols [21–23]. The
alkylation protocol consisted of stirring the heterocyclic scaffold with the required
haloacetyl derivative in N,N-dimethylformamide (DMF) at room temperature and using
potassium carbonate (K2CO3) as a base.
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For preparing the heterocyclic core of novel compounds from the quinoline series, the
Conrad-Limpach strategy used earlier for the 2-substituted series was considered
unsuitable because this methodology performs sluggishly for the production of 2-
unsubstituted derivatives [24]. The alternative condensation reaction that was used [25],
involved two steps as shown in Scheme 1. First, aniline 2 was added to Meldrum’s acid
derivative 3 in order to deliver enamine 4. Afterwards, thermal cyclization of 4 gave the
desired 2H-quinolin-4-ol 5b. The N-aryl haloacetamide 8 was obtained in excellent yield by
acylation of aniline 6 with bromoacetyl bromide 7 in the presence of triethylamine (Et3N).
Unexpectedly, alkylation of quinolinols 5a-c with alkyl halide 8 using the previously
mentioned alkylation protocol exclusively rendered the N-alkylated products 9a-c
(unambiguous structural identification was carried out as described in the next section).
Scheme 1. Synthesis of the N-alkylated quinolin-4-ones 9a-c. Reagents and conditions: (a) CH(OEt)3, EtOH,
reflux, 2 h; (b) Dowtherm A, 220 °C, 10 min; (c) Et3N, anhydrous DCM, rt, 2 h; (d) K2CO3, anhydrous DMF, rt, 3 h
A likely explanation for this inverted regioselectivity could be that the 2-methyl substituent
exerts sufficient steric hindrance around the quinoline ring nitrogen to render the oxygen
more reactive. In agreement with our findings, Wang and co-workers in 1996 [26], Oyama
et al. in 2015 [27] and Pissinate et al. [28] reported exclusively N-alkylated products in
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case of 2H-quinolin-4-ols under the same experimental conditions. On the other hand, one
can find publications [29,30] where O-alkylated products were reported when applying the
same protocol, although after careful examination we found that they did not include full,
unambiguous characterization data.
A different synthetic approach was designed to obtain the originally desired O-alkylated
analogues by employing a SNAr reaction of the desired alcohols with 4-chloroquinazoline.
The synthesis (shown in Scheme 2) began with preparing 2-hydroxyacetamide 11 by
heating the corresponding aniline 6 and glycolic acid 10 at 130 °C without solvent
according to a procedure described by Hung et al. [31]. Subsequently, 2-hydroxyacetamide
11 was coupled with 4-chloroquinolines 12a-b, in an Ullman-type reaction, catalysed by
copper iodide/tetramethyl ethylenediamine (CuI/TMEDA), according to Zhou et al. [32].
Cesium carbonate (Cs2CO3) was used as a base and dimethyl sulfoxide (DMSO) as a solvent.
Heating the reaction mixture overnight at 95 °C afforded final products 13a-b.
Scheme 2. Synthesis of the O-alkylated quinolin-4-ols 13a-b. Reagents and conditions: (a) 130 °C, 5.5 h; (b)
CuI, TMEDA, Cs2CO3, anhydrous DMF, 95 °C, overnight
To obtain 6-methoxy-2-methyl-1,5-naphthyridin-4-ol 16, a classical Conrad-Limpach
protocol [33,34] could be employed as shown is Scheme 3. Thus, 6-methoxypyridin-3-
amine 14 and ethyl acetoacetate 15 were heated at 130 °C for 3 h, followed by addition of
Dowtherm A and heating at 240-250 °C for 1 h. Subsequently, intermediate 16 was
subjected to the selected alkylation protocol with alkyl bromide 8 to obtain the desired O-
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alkylated analogue 17 (Scheme 3). As previously hypothesized, the presence of a
substituent ortho to the ring-nitrogen likely accounts for the observed regioselectivity.
Scheme 3. Synthesis of the O-alkylated 1,5-naphthyrid-4-ol 17. Reagents and conditions: (a) 130 °C, 3h, then
Dowtherm A, 250 °C, 1 h; (b) K2CO3, anhydrous DMF, rt, 18 h
For the ring synthesis of 4-hydroxy quinazolines 20a-c, a Niementowski protocol [35] was
employed as shown in Scheme 4. The appropriate anthranilic acids 18a-b and formamide
19a or acetamide 19b were heated at 150-165 °C to afford intermediates 20a-c.
Afterwards, their alkylation with alkyl bromide 8 under the standard conditions (K2CO3,
anhydrous DMF, r.t.) provided the N(3)-alkylated products 21a-c in all cases (2-substituted
and 2-unsubstituted). Clearly in this case, steric factors can no longer reasonably explain
the observed regioselectivity.
Scheme 4. Synthesis of the N(3)-alkylated quinazolin-4-ones 21a-c. Reagents and conditions: (a) 150-165 °C,
7-54 h; (b) 8, K2CO3, anhydrous DMF, rt, 3h
As shown in Figure 2, 3,4-dihydroquinazoline-4-ones have three potential alkylation sites,
i.e., the N(1), N(3) and OH groups.
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Figure 2. 3,4-dihydroquinazolin-4-one and possible alkylation positions.
It is known that 4-quinazolinones react normally with alkyl halides at N(3) of the
quinazoline ring and occasionally at the oxygen atom [5]. Therefore, the fact that we
obtained N(3)-alkylated products 21a-c should not be surprising. As highlighted by Spulak
et al. [36], there is likely an artificially high number of O-alkylated products in the
literature due to mischaracterization or insufficient data which fully, unambiguously
characterise the obtained products [37–39].
As shown in Scheme 5, a different synthetic approach was selected to obtain the desired O-
alkylated quinazoline 23. First, quinazolin-4-ol 20c was prepared using a modified
Niementowski protocol by means of microwave irradiation [35]. Oxychloro-exchange was
achieved by treatment with phosphoryl chloride (POCl3) under reflux for 60 h resulting in
4-chloroquinazoline 22. Subsequently, alcohol 11 was treated with sodium hydride (NaH)
in tetrahydrofuran (THF) for 1 h and 4-chloroquinazoline 22 was added to deliver the
desired O-alkylated compound 23 [40].
Scheme 5. Synthesis of the O-alkylated quinazolin-4-ol 23. Reagents and conditions: (a) microwave
irradiation, 160 °C, 12 h; (b) POCl3, reflux, 60 h; (c) 11, NaH, anhydrous THF, rt, 3.5 h
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2.2. Structure elucidation
The routine analytical tools that are used for addressing structural identity and purity of
compounds in most cases are incapable of unambiguously distinguishing O- and N-
1
alkylated analogues. Both regioisomers often produce similar H NMR spectra and have
identical masses. IR spectroscopy has been demonstrated to be helpful to determine the
alkylation position in structurally simple molecules [41]. In case of N-alkylation, a
carbonyl-type group is preserved and its characteristic stretching bands are potentially
easy to recognize in a relatively isolated spectral region [42]. However, this is not always
true for more complex molecules, especially if other carbonyl groups are present, leading
to possible overlap of the key IR signals. Herein, we describe a number of standard 1D and
2D NMR techniques which allowed for the unambiguous characterization of these type of
heterocyclic systems. All 1D and 2D NMR experiments described here are pre-programmed
on standard, contemporary NMR spectrometers.
13C NMR chemical shifts: The sensitivity of 13C chemical shifts to the carbon’s environment
has been commonly used for structure determination and differentiation between
compounds, as reflected in many examples from the literature. We exploited the fact that
the carbon atom of the methylene linker (-CH2-) directly bonded to an oxygen versus a
nitrogen atom will possess a notably different 13C chemical shift as an initial indication of
the obtained regioisomer. The desired O-analogue will produce a significant and
predictable downfield shift of the methylene signal in comparison to the N-analogue. The
examples provided in Table 1 and 2 demonstrate that typical 13C chemical shifts for O-
analogues fall into the 50-80 ppm range, while in the case of N-analogues, they appear at
higher field (typically 40-60 ppm).
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Table 1. Comparison of 13C-NMR chemical shifts in DMSO-d6 of the methylene group (-CH2-) for the N-
alkylated quinolin-4-ones 9a-c, O-alkylated quinolin-4-ols 13a-b and 1,5-naphthyridin-4-ol 17
N-alkylated
Structure
O-alkylated
Structure
13C shifts (ppm)
Cmpd
9a
13C shifts (ppm) Cmpd
54.6
13a
13b
67.2
67.1
9b
9c
54.7
54.5
17
67.3
Table 2. Comparison of 13C-NMR chemical shifts in DMSO-d6 of the methylene group (-CH2-) for the N-
alkylated quinazolin-4-ones 21a-c and O-alkylated quinazolin-4-ol 23
N-alkylated
Structure
O-alkylated
Structure
13C shifts (ppm)
Cmpd
21a
13C shifts (ppm) Cmpd
48.8
21b
21c
48.8
47.0
23
64.5
A faster alternative to 13C NMR acquisition is a highly sensitive 2D NMR Heteronuclear
Single Quantum Correlation (HSQC) experiment, which detects correlations between nuclei
of two different types separated by one bond [43,44]. HSQC crosspeaks correlate protons
of the methylene linker (-CH2-) with their attached carbon, facilitating the rapid
determination of the 13C chemical shift of interest.
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This methodology does not render definitive proof on atom connectivity. However, in a
series of structurally related compounds, the complete assignment of some representatives
allows a trend in the 13C chemical shifts to be established which can be utilised for
preliminary assignment of target compounds.
1
HSQC & HMBC: H-13C HMBC (Heteronuclear Multi-Bond Connectivity) correlates coupled
spins across multiple bonds, optimized in a way to detect proton-carbon correlation over
two or three bonds [45,46]. Single-bond HSQC and multiple-bond HMBC experiments are
complementary and their combination allows full resonance assignment and unambiguous
structure determination.
As an example, relevant HMBC correlations of quinolin-4-ols 9a, 13a and quinazolin-4-ols
21c, 23 are displayed in Figures 3 and 4 respectively, to demonstrate their appropriateness
for confident differentiation between N- and O- analogues. All available NMR data and full
resonance assignments are provided in the Supplementary information.
For 9a, consistency with an N-linked connectivity was clear due to the presence of H1’/C2
and H1’/C8a crosspeaks, meaning that H1’ should be within two or three bond distances
from C2 and C8a of the quinolin-4-ol core. This pattern of correlations was impossible for the
O-alkylated analogue. Instead, the acetyl protons H1’ in compound 13a had a distinctive
correlation with C4 of the core, indicating the formation of O-alkylated regioisomer (Figure
3).
Figure 3. Relevant HMBC connectivities in 9a and 13a for N- vs O- differentiation.
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The HMBC spectrum of compound 21c demonstrated crosspeaks H1’/C2 and H1’/C4,
which were consistent with alkylation on the nitrogen atom at position 3. It is worth noting
that the correlation of H1’ with C4 of the carbonyl group and absence of H11/C8a
crosspeak excluded the possibility of alkylation on nitrogen atom at position 1. In contrast,
in the regioisomer 23 only one crosspeak was noted, which was proven to represent
H1’/C4 correlation, consistent with the O-substituted analogue (Figure 4).
Figure 4. HMBC connectivity in 21c and 23 for N- vs O- differentiation.
NOE: The Nuclear Overhauser Effect is a transfer of nuclear spin polarization through
space, not through chemical bonds. It provides valuable information on the intramolecular
distances as the interaction is strongly dependent on the distance between a pair of nuclei.
In our studies, 1D NOE experiments were performed by irradiating the methylene linker to
produce an intensive signal, while neighbouring nuclei were identified by weaker signals of
the opposite phase (see Supplementary information). The peak to be irradiated should
possess a relatively isolated signal in 1H NMR in order to produce a precise NOE. 2D NOESY
experiments can be used as an alternative to 1D NOE, as it is a pre-programmed NMR
experiment which gives connectivity through space for the whole molecule, not only for a
selected signal.
In many cases an intrinsic limitation of the NOE approach arises from necessity of a
previous full proton and carbon assignments (often by 2D NMR) for correlation of the
observed peaks with the compound structure. Nevertheless, when the groups
1
neighbouring to the bond in question are easily assigned by H NMR, their chemical shifts
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can be used directly as references in 1D NOE. In such case, the NOE approach becomes the
fastest unambiguous method to assess the compound structure.
We have used this method to confirm our findings from the previous methods in case of
compounds 9a and 13a. The presence of H2, H8 and H3’ peaks in the NOE spectrum of the
quinolin-4-ol derivative 9a upon irradiation of H1’, indicated that H1’ is in proximity of
those protons. This was consistent with the N-alkylated analogue. In contrast, the NOE data
of compound 13a reported H3 and H3’ peaks upon irradiation of methylene protons H1’,
consistent with the O-alkylated analogue (Figure 5).
Figure 5. NOE correlations upon irradiation of H1’ in 9a and 13a for N- vs O- differentiation.
The quinazolin-4-ols 21c and 23 are an excellent demonstration of the NOE method
1
potential. The 2-methyl group (2-CH3) was readily assigned from H NMR spectra. In case
of quinazolin-4-ol derivative 21c irradiation of methylene protons H1’ resulted in two
peaks: H3’ (NH of the linker) and 2-CH3, which gave a direct indication of the linker
proximity to the methyl group. This was consistent with alkylation on the nitrogen atom in
position 3. It is worth noting that no H8 peak was observed, which would be expected in
case of alkylation on nitrogen atom in position 1. On the other hand, in 23 only a weak H3’
signal (corresponding to the acetamide NH) was registered upon irradiation of methylene
proton H1’, which was in a good agreement with the O-alkylated analogue (Figure 6).
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Figure 6. NOE correlations upon irradiation of H1’ in 21c and 23 for N- vs O- differentiation.
2.3. Biological evaluation
All final compounds were first evaluated for their antimycobacterial activity against
Mycobacterium tuberculosis H37Rv strain. In parallel, cytotoxicity in HepG2 cells and
physicochemical measurements were obtained to further explore the profiles of these
molecules. In particular, artificial membrane permeability (AMP), kinetic aqueous
solubility (CLND, chemiluminescent nitrogen detection) and hydrophobicity was measured
chromatographically to generate ChromlogD (pH 7.4) values. The obtained results are
shown in Table 3.
Table 3. Antimycobacterial activity, toxicity and physicochemical properties of final compounds.
M.tb MIC
(μΜ)[a]
1.9
Cytotoxicity
Permeability
(nm/sec)[c]
180
Solubility
C
hromlogD[e]
5.64
Cmpd
[b]
pIC50
CLND (μΜ) [d]
1
13a
13b
17
4.7
<4
4.3
<4
<4
4.2
<4
4
26
59
51
19
3
>80
460
5.03
10
280
5.30
3.8
510
5.57
23
1.25
>250
>250
>250
>250
>250
250
370
5.59
9a
450
34
30
86
20
8
3.54
9b
190
3.72
9c
260
4.39
21a
21b
21c
INH[g]
4.1
<4
<4
600
4.29
n.d. [f]
580
4.50
74
4.77
3.6
aMIC against Mycobacterium tuberculosis (H37Rv), data provided are the average of 2 experimental
replicates; bHepG2, human caucasian hepatocyte carcinoma; cartificial membrane permeability; din vitro
e
profiling for kinetic aqueous solubility (CLND, chemiluminescent nitrogen detection); chromlogD values at
pH = 7.4; fn.d. = not determined; gINH = isoniazid.
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The obtained data demonstrated that removal of both substituents from the quinoline
core led to the inactive compound 13a, while removal of the methyl group at position 2
preserved some antimycobacterial activity (13b, MIC = 10 μΜ). The most interesting
compounds, possessing a naphthyridine or quinazoline core (17 and 23), showed excellent
antimycobacterial activity with MIC values of 3.8 and 1.25 μΜ, respectively, comparable or
more potent than isoniazid, the reference compound 1. Additionally, these compounds did
not exhibit cytotoxicity against HepG2 cells, an improvement over the reference compound
1. Unfortunately, chromlogD values for compounds 17 and 23 remained at the same level
as 1, while their solubility continued to be very poor (<30 μΜ). All N-alkylated compounds
(9a-c and 21a-c) completely lost their activity against Mycobacterium tuberculosis,
suggesting that alkylation at the O- is essential for the activity of this series.
3. Conclusion
In continuation of our antimycobacterial research on quinoloxyacetamides, we explored
the alkylation of heterocyclic N/O-ambident nucleophilic scaffolds (quinolin-4-ol,
naphthyridin-4-ol and quinazolin-4-ol) under the same experimental conditions (K2CO3,
DMF, rt). Alkylation of 2-unsubstituted quinolinols led solely to N-alkylated compounds
9a-c, in contrary with the 2-methyl quinolin-4-ols/naphthyridin-4-ol where O-alkylated
compounds were obtained. All quinazolin-4-ols gave exclusively N(3)-alkylated products
21a-c whether they were 2-substituted or not. The desired alkoxy- analogues were
unambiguously prepared via the conversion of the 4-hydroxy quinazolines/quinolines into
the corresponding 4-chloro compounds followed by nucleophilic aromatic substitution by
alkoxides. Given the lack of commonly applied methods for the unambiguous assignment of
N/O-ambident alkylation reaction products in the literature, three NMR methods (13C-NMR
chemical shifts, 2D HSQC/HMBC and 1D NOE) were applied for structure determination.
Reporting correct structures is of particular importance in medicinal chemistry, and the
above mentioned NMR techniques should be utilized for full structural assignment of
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compounds when tackling similar regioselectivity issues. Furthermore, antimycobacterial
activity was evaluated for all final compounds, indicating that O-alkylation is essential for
activity, with the most active compound 23 exhibiting excellent potency against M.
tuberculosis H37Rv (MIC = 1.25 μM) without cytotoxic effects. Further research is
necessary in order to address lipophilicity and solubility issues of the series.
4. Experimental
4.1. Chemistry
General Information. Unless otherwise stated, laboratory reagent grade solvents were
used. Reagents were purchased from Sigma-Aldrich, Acros Organics, TCI or Enamine and
were used without further purification unless otherwise described. 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
Characterization of all compounds was done using H NMR and 13C-NMR spectroscopy
1
and mass spectrometry. H NMR (400 MHz) and 13C-NMR (100 MHz) spectra were
recorded on a Bruker Avance III Nanobay Ultrashield 400 or a Bruker 400 DPX
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. CDCl3, CD3OD or DMSO-
d6 were used as the standard NMR 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
(Reichert-Jung Optische Werke) microscope was used.
Purity and mass were verified using a UPLC-MS system and purities of all final products
were found to be >95%. UPLC-MS involved the following: Waters Acquity UPLC system
coupled to a Waters TQD ESI mass spectrometer and Waters TUV detector. A Waters
Acquity UPLC BEH C18 1.7 μm, 2.1 mm × 50 mm column was used. Solvent A consisted of
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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. For method B, a Waters Acquity UPLC system was coupled to a Waters SQ
detector and an Acquity UPLC BEH C18 1.7 μm, 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 NH4 25mM + 10% ACN at pH 6.6 /ACN, 0.0-0.2 min 99.9:
0.1, 0.2-1.0 min 10:90, 1.0-1.8 min 10:90, 1.9-2.0 min 99.9:0.1 at temperature 40 °C. The UV
detection was an averaged signal from wavelength of 210 nm to 400 nm. The quasi-
molecular ions [M+H]+ or [M-H]- 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.
Where necessary, flash purification was performed on a Biotage ISOLERA One flash
system equipped with an internal variable dual-wavelength diode array detector. For
normal phase purifications, pre-packed Biotage SNAP and Merck cartridges (10-50 g) were
used, and reverse-phase purifications were done making use of KP-C18 containing
cartridges. Dry sample loading was done by self-packing samplet cartridges using silica or
Celite 545, respectively, for normal and reversed phase purifications. HPLC purifications
were performed using XBridge columns 19-30 and as eluent acetonitrile in water plus
0.1% ammonium bicarbonate (NH4HCO3).
The following section comprises the synthetic procedures and analytical data for all
intermediates and final compounds reported in this publication. Complementary data
regarding NMR spectra can be found in the Supporting Information. Synthetic procedures
17

ACCEPTED MANUSCRIPT
that were used in the preparation of several products are summarized here as “General
Methods”.
General Method A. The appropriate 4-hydroxyquinoline/quinazoline/naphthyridine (1
eq.), 2-bromo-N-(3,5-dimethylphenyl)acetamide (1-1.1 eq.) and potassium carbonate (3
eq.) were dissolved in anhydrous DMF (0.09-0.11 M) and stirred for 3-18 h under nitrogen
atmosphere at room temperature. Then, the reaction mixture was poured into water and
the resulting precipitate was filtered, washed with water and dried. If necessary, it was
purified by silica gel column chromatography using n-heptane/ethyl acetate as eluent or by
recrystallization using ethyl acetate as solvent.
5-((4-Methoxyphenylamino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (4). A solution of
para-anisidine (1 g, 8.12 mmol), 2,2-dimethyl-1,3-dioxane-4,6-dione (1.381 g, 9.58 mmol)
and triethoxymethane (1.6 ml, 9.58 mmol) in ethanol (10 mL) was heated under reflux for
2 h and then allowed to cool down to room temperature. The resulting precipitate was
filtered, washed with cold absolute ethanol and dried under reduced pressure afforded the
title compound. Yield 90% (white solid, 2.028 g, 7.31 mmol); TLC, Rf 0.6 (ethyl acetate/n-
1
heptane, 50/50); H NMR (400 MHz, CDCl3) ppm 11.23 (d, J=14.6 Hz, 1H), 8.55 (d, J=14.3
Hz, 1H), 7.16 - 7.23 (m, 2H), 6.93 - 6.98 (m, 2H), 3.84 (s, 3H), 1.76 (s, 6H); UPLC-MS (ESI)
(A), RT 1.63 min, m/z 278.4 [M+H]+ (>95%).
6-Methoxyquinolin-4-ol (5b). 5-((4-methoxyphenylamino)methylene)-2,2-dimethyl-1,3-
dioxane-4,6-dione (1 g, 3.61 mmol) was added portion wise to Dowtherm A (10 mL) at
220 C. After bubbling stopped, the mixture was heated for additional 10 min and then
allowed to cool down to room temperature. The mixture was poured into n-heptane (50
mL), the brown solid was collected by filtration and washed with n-heptane (20 mL x 3). It
was purified by silica gel column chromatography using n-heptane/ethyl acetate and then
ethyl acetate/methanol as eluents. Yield 64% (off-white solid, 403 mg, 2.30 mmol); TLC, Rf
18

ACCEPTED MANUSCRIPT
0.4 (ethyl acetate/methanol, 80/20); UPLC-MS (ESI) (A), RT 0.56 min, m/z 176.4 [M+H]+
(>95%).
N-(3,5-dimethylphenyl)-2-(4-oxoquinolin-1(4H)-yl)acetamide (9a). The title compound was
prepared according to the general method A, using 4-hydroxy-1-azanaphthalene (300 mg,
2.07 mmol), 2-bromo-N-(3,5-dimethylphenyl)acetamide (550 mg, 2.27 mmol) and
potassium carbonate (857 mg, 6.20 mmol) in anhydrous DMF (24 mL) and the reaction
time was 3 h. Yield 84% (white solid, 553 mg, 1.74 mmol); TLC, Rf 0.4 (ethyl
1
acetate/methanol, 90/10); H NMR (400 MHz, DMSO-d6) δ ppm 10.38 (s, 1H), 8.19 (dd,
J=8.0, 1.4 Hz, 1H), 7.98 (d, J=7.6 Hz, 1H), 7.70 (ddd, J=8.6, 7.1, 1.5 Hz, 1H), 7.48 (d, J=8.6 Hz,
1H), 7.37 (t, J=7.5 Hz, 1H), 7.21 (s, 2H), 6.72 (s, 1H), 6.10 (d, J=7.8 Hz, 1H), 5.11 (s, 2H), 2.22
(s, 6H); 13C NMR (101 MHz, DMSO-d6) δ ppm 176.5, 165.2, 145.8, 140.5, 138.3, 137.9,
132.1, 126.4, 125.7, 125.2, 123.3, 116.9, 116.0, 108.7, 54.6, 21.0; UPLC-MS (ESI) (A), RT
1.64 min, m/z 307.5 [M+H]+ (>95%).
N-(3,5-dimethylphenyl)-2-(6-methoxy-4-oxoquinolin-1(4H)-yl)acetamide (9b). The title
compound was prepared according to the general method A, using 6-methoxyquinolin-4-ol
(400 mg, 2,28 mmol), 2-bromo-N-(3,5-dimethylphenyl)acetamide (608 mg, 2.51 mmol)
and potassium carbonate (947 mg, 6,85 mmol) in anhydrous DMF (25 mL) and the
reaction time was 3 h. Yield 51% (white solid, 393 mg, 1.17 mmol; TLC, Rf 0.35 (ethyl
acetate/methanol, 90/10); 1H NMR (400 MHz, DMSO-d6) δ ppm 10.35 (s, 1H), 7.92 (d, J=7.6
Hz, 1H), 7.61 (d, J=3.0 Hz, 1H), 7.45 (d, J=9.3 Hz, 1H), 7.34 (dd, J=9.1, 3.0 Hz, 1H), 7.20 (s,
2H), 6.72 (s, 1H), 6.05 (d, J=7.6 Hz, 1H), 5.10 (s, 2H), 3.84 (s, 3H), 2.22 (s, 6H); 13C NMR
(101 MHz, DMSO-d6) δ ppm 175.9, 165.2, 155.5, 144.8, 138.3, 137.9, 135.1, 127.6, 125.2,
121.8, 117.9, 116.9, 107.6, 105.5, 55.4, 54.7, 21.0; UPLC-MS (ESI) (A), RT 1.69 min, m/z
337.6 [M+H]+ (>95%).
19

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2-(7-Chloro-4-oxoquinolin-1(4H)-yl)-N-(3,5-dimethylphenyl)acetamide (9c). The title
compound was prepared according to the general method A, using 7-chloro-4-
hydroxyquinoline (500 mg, 2.78 mmol), 2-bromo-N-(3,5-dimethylphenyl)acetamide (674
mg, 2.78 mmol) and potassium carbonate (1.154 g, 8.35 mmol) in anhydrous DMF (31 mL)
and the reaction time was 3 h. Yield 5% (white solid, 45 mg, 0.13 mmol); TLC, Rf 0.63 (ethyl
acetate/methanol, 90/10); 1H NMR (400 MHz, DMSO-d6) δ ppm 10.33 (s, 1H), 8.17 (d, J=8.6
Hz, 1H), 7.98 (d, J=7.8 Hz, 1H), 7.64 (s, 1H), 7.41 (dd, J=8.6, 1.0 Hz, 1H), 7.21 (s, 2H), 6.73 (s,
1H), 6.12 (d, J=7.8 Hz, 1H), 5.12 (s, 2H), 2.22 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ ppm
175.9, 165.0, 146.2, 141.5, 138.3, 137.9, 137.0, 127.9, 125.2, 125.0, 123.7, 116.9, 115.9,
109.4, 54.5, 21.0; UPLC-MS (ESI) (A), RT 1.81 min, m/z 341.5, 343.5 (3:1) [M+H]+ (>95%).
N-(3,5-dimethylphenyl)-2-hydroxyacetamide (11). A mixture of 3,5-dimethylaniline (1 g,
8.25 mmol) and glycolic acid (628 mg, 8.25 mmol) were stirred at 130 °C for 5.5 h. The
mixture was then cooled to room temperature and dissolved in ethyl acetate (50 mL). The
organic phase was washed with ammonium chloride (NH4Cl, 1N, 50 mL), aqueous
saturated solution of sodium bicarbonate (NaHCO3, 50 ml) and brine (NaCl, 50 mL). The
organic phase was dried under magnesium sulfate (MgSO4) and the solvent was removed
1
under reduced pressure. Yield 81% (brown solid, 1.2 g, 6.70 mmol); H NMR (400 MHz,
DMSO-d6) ppm 9.42 (s, 1H), 7.30 (s, 2H), 6.70 (s, 1H), 5.62 (t, J=5.9 Hz, 1H), 3.96 (d, J=5.8
Hz, 2H), 2.23 (s, 6H); 13C NMR (101 MHz, DMSO-d6) ppm 170.6, 138.3, 137.6, 124.9, 117.3,
61.8, 21.1; UPLC-MS (ESI) (B) RT 0.99 min, m/z 180 [M+H]+ (>95%).
N-(3,5-dimethylphenyl)-2-(quinolin-4-yloxy)acetamide (13a). 4-Chloroquinoline (100 mg,
0.61 mmol), N-(3,5-dimethylphenyl)-2-hydroxyacetamide (110 mg, 0.61 mmol), N,N,N',N'-
tetramethylethylenediamine (7.1 mg, 0.06 mmol), copper(I) iodide (11.6 mg, 0.06 mmol)
and cesium carbonate (398 mg, 1.22 mmol) dissolved in anhydrous N,N-
dimethylformamide (DMF) (5 mL) were placed in a high pressure tube under nitrogen
20

ACCEPTED MANUSCRIPT
atmosphere. The reaction mixture was stirred at 95 °C overnight, cooled down and filtered
using a nylon syringe filter. The filtrate was evaporated and the residue was dissolved in
ethyl acetate (50 mL) and washed with brine (30 mL x 3). The organic layer was dried over
magnesium sulfate (MgSO4) and evaporated under reduced pressure. The residue was
purified by HPLC using acetonitrile/water plus 0.1% ammonium bicarbonate as eluent.
Yield 21% (off-white solid, 40 mg, 0.13 mmol); m.p. 162-163 °C; TLC, Rf 0.40 (ethyl
acetate); 1H NMR (400 MHz, DMSO-d6) δ ppm 10.12 (s, 1H), 8.73 (d, J=5.1 Hz, 1H), 8.30 (d,
J=7.6 Hz, 1H), 7.97 (d, J=8.3 Hz, 1H), 7.77 (ddd, J=8.3, 6.9, 1.4 Hz, 1H), 7.61 (ddd, J=8.3, 6.9,
1.4 Hz, 1H), 7.25 (s, 2H), 6.96 (d, J=5.3 Hz, 1H), 6.74 (s, 1H), 5.02 (s, 2H), 2.24 (s, 6H); 13C
NMR (101 MHz, DMSO-d6) δ ppm 165.3, 160.2, 151.4, 148.8, 138.1, 137.7, 129.8, 128.5,
125.7, 125.3, 121.9, 120.6, 117.4, 101.8, 67.2, 21.0; UPLC-MS (ESI) (B), RT 1.18 min, m/z
307 [M+H]+ (>95%); HRMS (ES) m/z calculated for C19H19N2O2 [M + H]+: 307.1441; found:
307.1440.
N-(3,5-dimethylphenyl)-2-((6-methoxyquinolin-4-yl)oxy)acetamide
(13b).
4-chloro-6-
methoxyquinoline (100 mg, 0.52 mmol), N,N,N',N'-tetramethylethylenediamine (6 mg, 0.05
mmol), copper(I) iodide (9.8 mg, 0.05 mmol) and cesium carbonate (337 mg, 1.03 mmol)
were placed in anhydrous N,N-dimethylformamide (DMF) (5 mL) in a high pressure tube
under nitrogen atmosphere. The reaction mixture was stirred at 95 °C overnight. The
reaction mixture was allowed to cool down and it was filtered through a nylon syringe
filter. The filtrate was evaporated and the residue was dissolved in ethyl acetate (50 mL)
and washed with brine (50 mL x 3). The organic layer was dried over sodium sulfate
(Na2SO4) and evaporated under reduced pressure. The mixture was purified by silica gel
column chromatography using cyclohexane/ethyl acetate as eluent and followed by HPLC
using acetonitrile/water plus 0.1% ammonium bicarbonate as eluent. Yield 4% (off-white
solid, 7.2 mg, 0.02 mmol); m.p. 156-157 °C; TLC: Rf 0.48 (ethyl acetate); 1H NMR (400 MHz,
21

ACCEPTED MANUSCRIPT
DMSO-d6) δ ppm 10.13 (s, 1H), 8.58 (d, J=5.1 Hz, 1H), 7.88 (d, J=9.1 Hz, 1H), 7.55 (d, J=3.0
Hz, 1H), 7.41 (dd, J=9.1, 2.8 Hz, 1H), 7.25 (s, 2H), 6.91 (d, J=5.3 Hz, 1H), 6.73 (s, 1H), 5.02 (s,
2H), 3.92 (s, 3H), 2.24 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ ppm 165.4, 159.3, 156.8,
148.7, 144.7, 138.2, 137.8, 130.2, 125.2, 121.9, 121.3, 117.3, 101.9, 99.9, 67.1, 55.5, 21.1;
UPLC-MS (ESI) (B), RT 1.18 min, m/z 337 [M+H]+ (>95%); HRMS (ESI) m/z calculated for
C20H21N2O3 [M+H]+: 337.1547; found: 337.1540.
6-Methoxy-2-methyl-1,5-naphthyridin-4-ol (16). In a round bottom flask, 5-amino-2-
methoxypyridine (1 g, 8.06 mmol) and ethyl acetoacetate (1 mL, 8.06 mmol) were stirred
at 130 °C for 3h (to form the corresponding imine). The reaction vessel was equipped with
a short distillation apparatus. Dowtherm A (10 mL) was added to the reaction mixture and
heated at 250 °C for 1 h. The reaction mixture was cooled down and poured into n-heptane
(50 mL). The formed sticky precipitate was washed with n-heptane (20 mL x 3) and ethyl
acetate (20 mL x 3) and the solid was filtered off. It was purified by silica gel column
chromatography using ethyl acetate/methanol as eluent. Yield 19% (grey solid, 290 mg,
1
1.53 mmol); TLC, Rf 0.20 (ethyl acetate/methanol/triethylamine, 90/10/0.01); H NMR
(400 MHz, DMSO-d6) δ 11.63 (s, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.05 (s,
1H), 3.91 (s, 3H), 2.32 (s, 3H). UPLC-MS (A) (ESI) RT 0.29 min, m/z 191.3 [M+H] + (>95%).
N-(3,5-dimethylphenyl)-2-((6-methoxy-2-methyl-1,5-naphthyridin-4-yl)oxy)acetamide (17).
The title compound was prepared according to the general method A, using 6-methoxy-2-
methyl-1,5-naphthyridin-4-ol (265 mg, 1.39 mmol), 2-bromo-N-(3,5-dimethylphenyl)
acetamide (337 mg, 1.39 mmol) and potassium carbonate (577 mg, 4.18 mmol) in
anhydrous DMF (13 mL) and the reaction time was 18 h. Yield 72% (white solid, 354 mg,
1.01 mmol); TLC, Rf 0.43 (ethyl acetate); 1H NMR (400 MHz, DMSO-d6) δ ppm 10.06 (br. s.,
1H), 8.13 (d, J=9.1 Hz, 1H), 7.24 (s, 2H), 7.21 (d, J=9.1 Hz, 1H), 7.06 (s, 1H), 6.73 (s, 1H),
5.03 (s, 2H), 4.01 (s, 3H), 2.55 (s, 3H), 2.23 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ ppm
22

ACCEPTED MANUSCRIPT
165.6, 160.4, 158.6, 157.2, 141.5, 139.6, 138.2, 137.8, 132.2, 125.2, 117.1, 116.1, 106.7,
67.3, 53.4, 24.8, 21.1; UPLC-MS (ESI) (A), RT 1.54 min, m/z 352.4 [M+H]+ (>95%).
Quinazolin-4-ol (20a). A 50 mL round bottom flask was charged with 2-aminobenzoicacid
(1 g, 7.29 mmol) and formamide (4 mL, 101 mmol) and heated for 7 h at 150 °C. Then, the
reaction mixture was cooled down to room temperature and a white precipitate was
formed, filtered, washed with water to remove the excess of formamide and dried. Yield
56% (white solid, 595 mg, 4.07 mmol); TLC, Rf 0.48 (ethyl acetate), Rf 0.83 (Ethyl
1
acetate/Methanol, 90/10); H NMR (400 MHz, MeOD-d4) ppm 8.22 (dd, J=8.2, 1.4 Hz, 1H),
8.09 (s, 1H), 7.83 (ddd, J=8.3, 7.2, 1.4 Hz, 1H), 7.70 (d, J=8.3 Hz, 1H), 7.55 (ddd, J=8.0, 7.1,
1.3 Hz, 1H); UPLC-MS (ESI) (A), RT 0.46 min, m/z 147.3 [M+H]+ (>95%).
6-Methoxyquinazolin-4-ol (20b). A 50 mL round bottom flask was charged with 2-amino-5-
methoxybenzoicacid (500 mg, 2.99 mmol) and formamide (3 mL, 75 mmol) and heated at
150 °C for 7 h. Then, the reaction mixture was cooled down to room temperature and the
precipitate was filtered, washed with water to remove the excess of formamide and dried.
Yield 65% (light brown solid; 343 mg, 1.95 mmol); TLC, Rf 0.66 (ethyl acetate/methanol,
90/10); 1H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 0.4H), 7.98 (s, 1H), 7.62 (d, J = 8.9 Hz, 1H),
7.50 (d, J = 3.0 Hz, 1H), 7.41 (dd, J = 8.9, 3.0 Hz, 1H), 4.04 (s, 0.6H), 3.86 (s, 3H); 13C NMR
(101 MHz, DMSO-d6) δ 157.8, 143.2, 143.1, 143.0, 129.0, 123.8, 123.5, 105.9, 55.6; UPLC-
MS (ESI) (A), RT 1.11 min, m/z 177.4 [M+H]+ (>95%).
6-Methoxy-2-methylquinazolin-4-ol (20c). Method A: A 50 mL round bottom flask was
charged with 2-amino-5-methoxybenzoicacid (500 mg, 2.99 mmol) and acetamide (2.297
g, 38.9 mmol) and heated at 150 °C for 29 h and at 165 °C for additional 25 h. Afterwards,
the reaction mixture was cooled down to room temperature and water was added. A dark
brown precipitate was formed which was filtered, washed with water and purified by
reverse phase column chromatography using methanol/water as eluent. Method B: A
23

ACCEPTED MANUSCRIPT
microwave vial (20 mL) was charged with 2-amino-5-methoxybenzoic acid (0.7 g, 4.19
mmol) and acetamide (4.95 g, 84 mmol). The reaction mixture was then irradiated for 12 h
at 165 °C. The obtained solid was washed with water (50 ml x 3). It was purified by an
amine column using ethyl acetate/ethanol as eluent. Yield 14/35% (A/B), (off-white solid,
77/280 mg (A/B), 0.41/1.47 mmol (A/B)); 1H NMR (400 MHz, DMSO-d6) δ ppm 12.15 (br.
s., 1H), 7.52 (d, J=8.8 Hz, 1H), 7.46 (d, J=2.8 Hz, 1H), 7.37 (dd, J=8.8, 3.0 Hz, 1H), 3.85 (s,
3H), 2.32 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ ppm 161.5, 157.1, 151.8, 143.4, 128.2,
123.7, 121.3, 105.7, 55.5, 21.2; UPLC-MS (ESI) (B), RT 1.11 min, m/z 191.1 [M+H]+ (>95%).
N-(3,5-dimethylphenyl)-2-(4-oxoquinazolin-3(4H)-yl)acetamide (21a). The title compound
was prepared according to the general method A, using quinazolin-4-ol (500 mg, 3.42
mmol), 2-bromo-N-(3,5-dimethylphenyl)acetamide (828 mg, 3.42 mmol) and potassium
carbonate (1.419 g, 10.26 mmol) in anhydrous DMF (38 mL) and the reaction time was 3
h. Yield 95% (white solid, 1.004 g, 3.27 mmol); TLC, Rf 0.65 (ethyl acetate/methanol,
90/10); 1H NMR (400 MHz, DMSO-d6) δ ppm 10.31 (s, 1H), 8.36 (s, 1H), 8.15 (dd, J=8.0, 1.1
Hz, 1H), 7.83 - 7.89 (m, 1H), 7.69 - 7.74 (m, 1H), 7.53 - 7.61 (m, 1H), 7.21 (s, 2H), 6.71 (s,
1H), 4.85 (s, 2H), 2.22 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ ppm 165.3, 160.3, 148.6,
148.1, 138.5, 137.8, 134.5, 127.2, 127.1, 126.0, 125.1, 121.4, 116.8, 48.8, 21.1; UPLC-MS
(ESI) (A), RT 1.75 min, m/z 308.5 [M+H]+ (>95%).
N-(3,5-dimethylphenyl)-2-(6-methoxy-4-oxoquinazolin-3(4H)-yl)acetamide (21b). The title
compound was prepared according to the general method A, using 6-methoxyquinazolin-4-
ol (300 mg, 1.70 mmol), 2-bromo-N-(3,5-dimethylphenyl) acetamide (412 mg, 1.70 mmol)
and potassium carbonate (706 mg, 5.11 mmol) in anhydrous DMF (19 mL) and the
reaction time was 3 h. Yield 93 % (light grey solid, 533 mg, 1.58 mmol); TLC, Rf 0.72 (ethyl
1
acetate); H NMR (400 MHz, DMSO-d6) δ ppm 10.29 (s, 1H), 8.25 (s, 1H), 7.67 (d, J=9.1 Hz,
1H), 7.51 (d, J=2.8 Hz, 1H), 7.46 (dd, J=8.8, 2.8 Hz, 1H), 7.21 (s, 2H), 6.71 (s, 1H), 4.84 (s,
24

ACCEPTED MANUSCRIPT
2H), 3.87 (s, 3H), 2.22 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ ppm 165.3, 160.1, 158.0,
146.4, 142.6, 138.5, 137.8, 128.9, 125.1, 123.9, 122.3, 116.8, 105.9, 55.7, 48.8, 21.1; UPLC-
MS (ESI) (A), RT 1.80 min, m/z 338.5 [M+H]+ (>95%).
N-(3,5-dimethylphenyl)-2-(6-methoxy-2-methyl-4-oxoquinazolin-3(4H)-yl)acetamide (21c).
The title compound was prepared according to the general method A, using 6-methoxy-2-
methylquinazolin-4-ol (77 mg, 0.41 mmol), 2-bromo-N-(3,5-dimethylphenyl) acetamide
(98 mg, 0,41 mmol) and potassium carbonate (168 mg, 1.22 mmol) in anhydrous DMF (4.5
mL) and the reaction time was 3 h. Yield: 84% (white solid, 119 mg, 0.34 mmol); TLC, Rf
1
0.49 (ethyl acetate); H NMR (400 MHz, DMSO-d6) δ ppm 10.30 (s, 1H), 7.57 (d, J=8.8 Hz,
1H), 7.45 (d, J=2.8 Hz, 1H), 7.41 (dd, J=8.6, 2.9 Hz, 1H), 7.20 (s, 2H), 6.71 (s, 1H), 4.93 (s,
2H), 3.85 (s, 3H), 2.22 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ ppm 165.4, 161.0, 157.4,
153.0, 141.7, 138.5, 137.8, 128.3, 125.1, 124.1, 120.4, 116.9, 106.0, 55.6, 47.0, 22.7, 21.1;
UPLC-MS (ESI) (A), RT 1.81 min, m/z 352.5 [M+H]+ (>95%).
4-Chloro-6-methoxy-2-methylquinazoline (22). 6-methoxy-2-methylquinazolin-4-ol (233
mg, 1.23 mmol) was suspended in phosphorus oxychloride (POCl3) (10 mL, 108 mmol) and
the reaction mixture was stirred under reflux for 2.5 days during which the suspension
turned into a reddish brown solution. The mixture was allowed to cool down to room
temperature and phosphorus oxychloride was evaporated under reduced pressure. Then,
the residue was partitioned between aqueous solution of sodium bicarbonate (NaHCO3,
5%, 100 mL) and ethyl acetate (100 mL). The organic layers was washed with aqueous
solution of sodium bicarbonate (NaHCO3, 5%, 100 mL) and water (100 mL). Then, the
organic layer was dried over sodium sulfate (Na2SO4) and evaporated under reduced
pressure. The residue was purified by silica gel column chromatography using
cyclohexane/ethyl acetate as eluent. Yield 56% (white solid, 142 mg, 0681 mmol);1H NMR
(400 MHz, DMSO-d6) δ ppm 7.92 (d, J=9.1 Hz, 1H), 7.70 (dd, J=9.1, 2.8 Hz, 1H), 7.42 (d, J=2.8
25

ACCEPTED MANUSCRIPT
Hz, 1H), 3.96 (s, 3H), 2.71 (s, 3H); UPLC-MS (ESI) (B); RT 3.88 min, m/z 209 [M+H]+
(>95%).
N-(3,5-dimethylphenyl)-2-((6-methoxy-2-methylquinazolin-4-yl)oxy)acetamide (23). N-(3,5-
dimethylphenyl)-2-hydroxyacetamide (60.1 mg, 0.34 mmol) and sodium hydride (NaH),
60% dispersion in mineral oil (26.8 mg, 0.67 mmol) were suspended in anhydrous
tetrahydrofuran (THF) (4 mL) and left stirring at room temperature for 1 h, before 4-
chloro-6-methoxy-2-methylquinazoline (70 mg, 0.34 mmol) was added to the mixture.
Then, the reaction mixture was stirred at room temperature for 2.5 h. The product was
purified by silica gel column chromatography using cyclohexane/ethyl acetate as eluent,
followed by HPLC using acetonitrile/water plus 0.1% ammonium bicarbonate as eluent.
Yield 14% (white solid, 17 mg, 0.05 mmol); m.p. 180-181 °C; TLC, Rf 0.40 (cyclohexane
/ethyl acetate, 50/50), 0.39 (DCM/methanol: 90/10); 1H NMR (400 MHz, DMSO-d6) δ ppm
10.10 (s, 1H), 7.78 (d, J=9.1 Hz, 1H), 7.56 (dd, J=9.1, 2.8 Hz, 1H), 7.48 (d, J=2.8 Hz, 1H), 7.22
(s, 2H), 6.71 (s, 1H), 5.19 (s, 2H), 3.92 (s, 3H), 2.54 (s, 3H), 2.22 (s, 6H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 165.5, 164.6, 160.1, 157.2, 146.7, 138.4, 137.7, 128.4, 125.7, 125.0, 117.1,
114.3, 101.4, 64.5, 55.7, 25.7 (d, J=1.5 Hz), 21.0; UPLC-MS (ESI) (B), RT 1.22 min, m/z 352
[M+H]+ (>95%); HRMS (ES) m/z calcd for C20H22N3O3 [M+H]: 352.1656; found: 352.1649.
4.2. Biological assays
Strain and growth conditions. M. tuberculosis H37Rv (ATC25618) wild-type was grown
in Middlebrook 7H9-ADC broth (Difco) supplemented with 0.05% Tween 80 and on 7H10-
OADC or 7H11-OADC agar (Difco) at 37 °C. Isoniazid and hygromycin were purchased from
Sigma-Aldrich. Where required, hygromycin (50 µg/mL) was added to the culture medium.
MIC determination. The measurement of the Minimum Inhibitory Concentration (MIC)
against M. tuberculosis H37Rv for each tested compound was performed in 96-well flat-
bottom, polystyrene microtiter plates in a final volume of 200 μL. Ten two-fold drug
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dilutions in neat DMSO were performed. Middlebrook 7H9 (Difco) was used as medium.
Isoniazid (INH) (Sigma Aldrich) was used as a positive control with two-fold dilutions of
INH starting at 4 μg/mL placed at row 11 of the plate layout and rifampicin (Sigma Aldrich)
was used as no-growth control at concentration of 1 μM, placed at G-12 and H-12 wells.
The inoculum (200 μL) was added to the entire plate. All plates were placed in a sealed box
to prevent drying out of the peripheral wells and incubated at 37 °C without shaking for six
days. A Resazurin solution was prepared by dissolving one tablet of resazurin (Resazurin
Tablets for Milk Testing; Ref 330884Y' VWR International Ltd) in 30 mL of sterile PBS
(phosphate buffered saline). Of this solution, 25 μL were added to each well. Fluorescence
was measured (Spectramax M5 Molecular Devices, Excitation 530nm, Emission 590 nm)
after 48 hours to determine the MIC value. MIC data was the average of two experimental
replicates.
HepG2 cytotoxicity assay. HepG2 cells were cultured using Eagle’s MEM supplemented
with 10% heat-inactivated FBS, 1% NEAA and 1% penicillin/streptomycin. Prior to
addition of the cell suspension, 250 nL of test compounds per well were pre-dispensed in
TC-treated black clear-bottomed 384 well plates (Greiner, cat. #781091) with an Echo 555
instrument. After that, 25 μL of HepG2 (ATCC HB-8065) cells (~3000 cells/well) grown to
confluency in Eagle’s MEM supplemented with 10% heat-inactivated FBS, 1% NEAA and
1% Pencillin/Streptomycin were added to each well with the reagent dispenser. Plates
were allowed to incubate at 37 °C with 20% O2 and 5% CO2 for 48 h.
After the incubation period (48h), the plates were equilibrated to room temperature
before proceeding to develop the luminescent signal. ATP levels measured with CellTiter
Glo kit (Promega) were used as cell viability read-out. 25 μL of CellTiter Glo substrate
dissolved in the buffer was added to each well. Plates were incubated at room temperature
for 10 minutes for stabilization of luminescence signal and read on View Lux with
excitation and emission filters of 613 and 655 nm, respectively.
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The human biological samples were sourced ethically and their research use was in
accord with the terms of the informed consents.
Artificial membrane permeability, kinetic aqueous solubility (CLND) and
hydrophobicity (chromlogD at pH7.4) assays. Those assays were performed analogously to
previously described by Ballell et al. [47].
Supporting Information
Additional data (1H-NMR, 13C-NMR, HSQC, HMBC, NOE and COSY spectra) for the final
compounds.
Acknowledgements
This research was funded by a Marie Curie Intra-European Grant within the 7th
European Community Framework Programme (FP7-PEOPLE-2012-ITN) under
the European Industrial Doctorates Scheme of the Marie Curie Initial Training
Networks actions with grant agreement No 316773 and it is part of the OpenMedChem
project. The authors gratefully acknowledge the many scientists from GSK DDW, involved
in the research.
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