Synthesis, Structure-Activity Relationship, and Mechanistic Studies of Aminoquinazolinones Displaying Antimycobacterial Activity

Article abstract of DOI:10.1021/acsinfecdis.0c00252

Phenotypic whole-cell screening against Mycobacterium tuberculosis (Mtb) in glycerol-alanine-salts supplemented with Tween 80 and iron (GASTE-Fe) media led to the identification of a 2-aminoquinazolinone hit compound, sulfone 1 which was optimized for solubility by replacing the sulfone moiety with a sulfoxide 2. The synthesis and structure-activity relationship (SAR) studies identified several compounds with potent antimycobacterial activity, which were metabolically stable and noncytotoxic. Compound 2 displayed favorable in vitro properties and was therefore selected for in vivo pharmacokinetic (PK) studies where it was found to be extensively metabolized to the sulfone 1. Both derivatives exhibited promising PK parameters; however, when 2 was evaluated for in vivo efficacy in an acute TB infection mouse model, it was found to be inactive. In order to understand the in vitro and in vivo discrepancy, compound 2 was subsequently retested in vitro using different Mtb strains cultured in different media. This revealed that activity was only observed in media containing glycerol and led to the hypothesis that glycerol was not used as a primary carbon source by Mtb in the mouse lungs, as has previously been observed. Support for this hypothesis was provided by spontaneous-resistant mutant generation and whole genome sequencing studies, which revealed mutations mapping to glycerol metabolizing genes indicating that the 2-aminoquinazolinones kill Mtb in vitro via a glycerol-dependent mechanism of action.

Full text of DOI:10.1021/acsinfecdis.0c00252

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Article
Synthesis, Structure Activity Relationship and Mechanistic Studies
of Aminoquinazolinones Displaying Antimycobacterial Activity
Jessica N. Akester, Paul Njaria, Aloysius T Nchinda, Claire Le Manach, Alissa Myrick, Vinayak
Singh, Nina Lawrence, Mathew Njoroge, Dale Taylor, Atica Moosa, Anthony Smith, Elizabeth Brooks,
Anne J. Lenaerts, Gregory T. Robertson, Thomas R Ioerger, Rudolf Mueller, and Kelly Chibale
ACS Infect. Dis., Just Accepted Manuscript • Publication Date (Web): 29 May 2020
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Synthesis, Structure Activity Relationship and
Mechanistic Studies of Aminoquinazolinones
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Displaying Antimycobacterial Activity
Jessica N. Akestera, Paul Njariab, Aloysius Nchindaa, Claire le Manacha, Alissa Myricka,c,†
,
Vinayak Singha,c,h, Nina Lawrenced, Mathew Njoroged, Dale Taylord, Atica Moosae, Anthony
J. Smithf, Elizabeth J. Brooksf, Anne J. Lenaertsf, Gregory T. Robertsonf, Thomas R. Ioergerg,
Rudolf Muellera and Kelly Chibale a,b,c,h*
a Drug Discovery and Development Centre (H3D), Department of Chemistry,
University of Cape Town, Rondebosch 7701, South Africa
b Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
c Institute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa
d H3D, Division of Clinical Pharmacology, Department of Medicine, University of
Cape Town, Observatory, 7925, South Africa
e MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of
Pathology, University of Cape Town, Rondebosch 7701, South Africa
fMycobacteria Research Laboratories, Department of Microbiology, Immunology, and
Pathology, Colorado State University, 200 West Lake Street, Fort Collins, Colorado
80523, United States
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g Department of Computer Science, Texas A&M University, College Station, TX 77843-
3112, USA
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h South African Medical Research Council Drug Discovery and Development
Research Unit, Department of Chemistry, University of Cape Town, Rondebosch
7701, South Africa
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†Deceased. This paper is dedicated to Alissa Myrick.
Abstract
Phenotypic whole-cell screening against Mycobacterium tuberculosis (Mtb) in GASTE-Fe
media led to the identification of a 2-aminoquinazolinone hit compound, sulfone 1 which was
optimized for solubility by replacing the sulfone moiety with a sulfoxide 2. The synthesis and
structure activity relationship studies (SAR) identified several compounds with potent
antimycobacterial activity, which were metabolically stable and non-cytotoxic. Compound 2
displayed favourable in vitro properties and was therefore selected for in vivo pharmacokinetic
(PK) studies where it was found to be extensively metabolised to the sulfone 1. Both derivatives
exhibited promising PK parameters, however, when 2 was evaluated for in vivo efficacy in an
acute TB infection mouse model, it was found to be inactive. In order to understand the in vitro
and in vivo discrepancy, compound 2 was subsequently retested in vitro using different Mtb
strains cultured in different media. This revealed that activity was only observed in media
containing glycerol and led to the hypothesis that glycerol was not used as a primary carbon
source by Mtb in the mouse lungs, as has previously been observed. Support for this hypothesis
was provided by spontaneous resistant mutant generation and whole genome sequencing
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studies, which revealed mutations mapping to glycerol metabolising genes indicating that the
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2-aminoquinazolinones kill Mtb in vitro via a glycerol-dependent mechanism of action.
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KEYWORDS. Tuberculosis, Mycobacterium tuberculosis, Drug Discovery, 2-
aminoquinazolinones
Tuberculosis (TB) is an infectious disease, caused by the bacillus Mycobacterium
tuberculosis (Mtb), and is the leading cause of death worldwide from a single infectious agent.
According to the World Health Organisation (WHO), approximately 10 million people
acquired TB in 2018 and 1.2 million deaths occurred globally.1 Treatments for drug-susceptible
TB have been available for 60 years, with the current regimen comprising of an initial two-
month intensive phase with four frontline drugs (isoniazid, rifampicin, ethambutol and
pyrazinamide), followed by a four-month continuation phase using isoniazid and rifampicin.
However, resistant strains have emerged over the years possibly as a result of poor patient
compliance due to the extensive drug regimen, prolonged treatment time, socioeconomic
factors, toxic effects and the tendency to be asymptomatic before safe completion of the
prescribed course.2,3
In the past 15 years, several new classes of anti-TB drugs have emerged in the developmental
pipeline including three new agents. Bedaquiline and delamanid received accelerated
regulatory approval in 2012 and 2014, respectively, and are being progressed into phase III
clinical trials for the treatment of multi-drug resistant TB (MDR-TB).2–4 Most recently,
pretomanid was approved as part of a six month, three-drug regimen consisting of bedaquiline,
pretonamid and linezolid (BPaL) for the treatment of people with extensively drug resistant TB
(XDR-TB).5 Although there are a number of new chemical entities at various phases of clinical
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development,6 the high attrition rate requires a continuous feed of new chemical matter into
the drug discovery pipeline. The development of new TB treatments involves numerous
challenges as the TB research community needs to focus on many aspects, e.g. shorter and
simpler regimen, less side-effects, effectiveness against drug resistant TB, reduced drug-drug
interactions for human immunodeficiency virus (HIV)-coinfected patients and affordable cost
for patients in developing countries where the majority of TB cases exist.7,8
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2-Aminoquinazolin-4(3H)-ones are derived from the quinazolinone class of compounds, which
have been extensively explored in a wide range of therapeutic areas, whereas literature
references on 2-aminoquinazolinones are relatively scarce. Secondary and tertiary 2-amino
analogues have been found to have pharmacological relevance, with examples of antifungal,9
anti-inflammatory, anticancer10,11, antibacterial,12,13 antihypertensive,14 antidepressant15 and
antimycobacterial activity16,17. On the other hand, primary 2-aminoquinazolinone derivatives
have not yet been commercially developed but have shown a variety of biological activities as
antimalarial18, antiviral19,20 and anticancer21 agents. Although there is no known
antimycobacterial activity, they appear as a valuable novel scaffold to explore as a potential
anti-TB chemotype.
On the basis of structural similarity shared with a previously discovered antimalarial 2-
aminopyridine series,22 2-aminoquinazolinones were synthesized for evaluation against the
human malaria parasite Plasmodium falciparum. Subsequent phenotypic whole-cell cross-
screening against Mtb identified the 2-aminoquinazolinone 1 as an attractive hit compound to
follow up on despite its low solubility. During preliminary studies towards improving the
solubility of 1, the sulfone substituent was converted to the sulfoxide 2, which displayed
improved aqueous solubility whilst maintaining potency (Figure 1). As a result, medicinal
chemistry efforts were subsequently pursued in order to explore the antimycobacterial potential
of this 2-aminoquinazolinone series.
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CF3
CF3
O
N
O
N
MeOS
N
MeO2S
N
8
NH2
NH2
9
1
2
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Mtb MIC90 (H37RV): <0.244 M
Solubility (pH 7.4): <5 M
Mtb MIC90 (H37RV): 0.64 M
Solubility (pH 7.4): 80 M
Figure 1. Identification of 2-aminoquinazolinones with in vitro antimycobacterial activity.
Herein, we present the synthesis, structure-activity relationship, in vitro absorption,
distribution, metabolism, excretion, and toxicity (ADMET) properties, and in vivo
pharmacokinetic (PK) properties of this series. In vivo efficacy and subsequent mechanistic
studies are also presented.
Results and Discussion
Chemistry. Structural modifications were explored around the aminoquinazolinone core
with the aim of improving potency and aqueous solubility at pH 7.4. Four main points of
diversity were established at positions 2, 3, 6 and 7 of the scaffold as indicated in Figure 2. R1
and R2 groups were investigated to identify appropriately substituted ring structures with
various electronic, steric and hydrophobicity properties. The addition of heteroatoms or water
solubilising groups was envisaged to reduce lipophilicity, and therefore improve aqueous
solubility. Additionally, the strategy of increasing Sp3 character through saturation of aromatic
rings was employed to disrupt the crystal packing of molecules. The position of the substituent
on the left hand-side (LHS) from the 6- to the 7-position of the molecule was also explored to
determine which of the LHS positions was optimal for activity. Modifications were also made
to the 2-position to understand whether the 2-amino group was essential for potency.
O
5
R1
R2
R3
R4
N
6
3
N
7
2
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Figure 2. General structure of the quinazolinone core indicating modification sites.
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The synthesis of compounds 1, 2 and 7-36 was achieved following a relatively straightforward
five-step synthetic route adapted from Leivers et al, starting with the commercially available
2-aminoiodobenzoic acid (Scheme 1).19 Briefly, a cyclization reaction with the appropriately
substituted phenyl isothiocyanate afforded intermediate 3. Chlorination of the thioxo
intermediate to 4 was obtained using phosphorous oxychloride (POCl3) in the presence of
phosphorous pentachloride (PCl5). The 2-amino group was introduced via a 4-
methoxybenzylamine intermediate (5) followed by deprotection of the PMB group to afford 6.
Finally, a Suzuki cross-coupling reaction with the appropriate boronic acids yielded the desired
compounds 1, 2 and 7-36.
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R3
R3
O
O
COOH
NH2
i
ii
N
N
I
R3
+
I
I
SCN
N
H
S
N
Cl
4
3
a:
b:
c:
d:
e:
6-I, R3 = p-CF3
6-I, R3 = m-CF3
6-I, R3 = H
6-I, R3 = p-CH3
7-Br, R3 = p-CF3
a:
b:
c:
d:
e:
6-I, R3 = p-CF3
6-I, R3 = m-CF3
6-I, R3 = H
6-I, R3 = p-CH3
7-Br, R3 = p-CF3
iii
R3
R3
R3
O
N
N
O
O
N
R1
R2
iv
v
N
N
I
I
NH2
N
NH2
N
H
OMe
1,2, 7-36
6
5
a:
b:
c:
d:
e:
a:
b:
c:
d:
e:
6-I, R3 = p-CF3
6-I, R3 = m-CF3
6-I, R3 = H
6-I, R3 = p-CH3
7-Br, R3 = p-CF3
6-I, R3 = p-CF3
6-I, R3 = m-CF3
6-I, R3 = H
6-I, R3 = p-CH3
7-Br, R3 = p-CF3
Scheme 1. General Synthetic Route for the Preparation of Compounds E
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Reagents and conditions: (i) Dioxane, Et3N, reflux (110°C), 4 h; (ii) POCl3, PCl5, N2, 110 °C,
14 h; (iii) DMF, 4-methoxybenzylamine, DIPEA, 80 °C, 4 h; (iv) TFA, reflux, 48 h or MW
(110 °C) 20 min, (v) Dioxane/water, R1/R2B(OH)2, PdCl2(PPh3)2, K2CO3, 80 °C, 1-5 h.
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The procedure to obtain the analogue without the 2-amino substituent was adapted from
Güngör et al.23 (Scheme 2). This approach involved refluxing 2-amino-5-iodobenzoic acid with
4-(trifluoromethyl) aniline in the presence of triethoxymethane to afford the iodo intermediate
37. Subsequent Suzuki-coupling reaction with (3-(methylsulfonyl)phenyl)boronic acid yielded
the desired derivative devoid of the 2-amino group 38.
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CF3
CF3
O
N
O
N
i
CF3
I
COOH
+
ii
I
MeO2S
N
N
H2N
NH2
37
38
Scheme 2. Synthesis of quinazolinone analogue.
Reagents and conditions: (i) Triethoxymethane, dioxane, 100 °C, 16 h; (ii) Dioxane,
R1B(OH)2, PdCl2(ddpf)2-DCM, Cs2CO3, 90 °C, 5 h. 23
Modifications to the 2-amino substituent were achieved using a similar synthetic route to
Scheme 1, whereby the appropriate amine was installed by substituting the chloro group of
intermediate 4a under basic conditions. Thereafter, the final target compounds 40-43 were
obtained using the Suzuki-coupling conditions described previously (Scheme 3).
CF3
CF3
CF3
O
N
O
N
O
I
I
R1
ii
i
N
N
N
Cl
R4
N
R4
4a
40-43
39
Scheme 3. Synthetic route used to access core modifications.
Reagents and conditions: (i) Relevant amine, DMF/THF, base, 80 °C, 3h; (ii) Dioxane/water,
R1B(OH)2, PdCl2(PPh3)2, K2CO3, 80 °C, 2 h.
Lastly, compounds 47-58 were prepared using a modified synthetic route established by
Lecoutey and coworkers to introduce structural diversity at the R3 position (Scheme 4).24
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Initially, methyl 2-amino-5-bromobenzoate was reacted with ethoxycarbonyl isothiocyanate to
form the isolated thioureido intermediate 44. Thereafter, cyclisation and simultaneous amide
coupling with the appropriate amine using the coupling agent EDCI afforded intermediate 45,
which was deprotected to give 46. Finally, the methylsulfinylphenyl moiety was installed via
a Suzuki-coupling reaction to yield compounds 47-58.
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O
O
N
Br
O
O
Br
R3
O
Br
N
O
ii
i
NH
+
O
NCS
NH
NH2
S
NH
CO2Et
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45 a-i
44
CO2Et
iii
O
N
O
R3
Br
R3
iv
MeOS
N
N
NH2
N
NH2
46 a-i
47-58
Scheme 4. General route for the synthesis of LHS modifications.
Reagents and conditions: (i) Acetonitrile, 25°C, 10 min; (ii) DCM/DMF, EDCI, R3NH2,
25°C, 18 h; (iii) TFA, microwave, 110°C, 20 min; (iv) Dioxane/water (4:1), R1B(OH)2,
PdCl2(PPh3)2, K2CO3, 80 °C, 5 h.
In vitro antimycobacterial activity. Target compounds were initially evaluated for
their in vitro antimycobacterial activity against the drug susceptible Mtb H37Rv strain, under
replicating conditions using GASTE-Fe (Glycerol-Alanine-Salts supplemented with Tween 80
and Iron) culture media. The minimum inhibitory concentration (MIC90) was read 14 days after
incubation. Additionally, compounds were screened for in vitro cytotoxicity against a
mammalian cell-line, Chinese Hamster Ovarian (CHO). Initial SAR exploration was performed
on the LHS with variations on the 6-position whilst keeping the 2-aminoquinazolinone core
unchanged and the right-hand side (RHS) constant as a para-trifluoromethyl phenyl group
(Table 1).
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Table 1. SAR and in vitro ADMET data for LHS and core modifications.
CF3
O
6
7
8
9
3
6
R1
R2
N
2
7
N
R4
MIC90
GAST
Fe
Metabolic
Stability
H/R/Md
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Solubility
pH 7.4c
(µM)
Cytotoxicitya
µM (SI)
clogDb
R1
R2
R4
(µM)
(% Rem.)
1
2
7
8
H
H
H
H
NH2
NH2
NH2
NH2
<0.244
0.64
>100 (>400x)
>100 (>156x)
>100 (>24x)
0.021 (0.01x)
3.29
3.56
3.38
3.56
<5
80
53
66
97/100/92
88/-/-
S
O
O
S
O
4.22
65/86/90
85/98/97
S
O
O
O
2.35
S
O
H2N
9
H
H
H
NH2
NH2
NH2
<0.244
2.15
>100 (>400x)
0.64 (0.3x)
3.10
3.14
3.26
<5
46
<5
97/-/-
O
O
N
10
11
85/86/89
98/100/100
H
N
0.78
96.3 (123x)
O
O
N
H
N
12
13
14
H
H
H
NH2
NH2
NH2
9.22
0.82
1.03
ND
2.83
3.14
2.92
39
85/85/75
82/40/56
81/83/90
H
N
>50 (>61x)
>50 (>49x)
168
193
N
O
H
N
HO
O
H
N
15
16
17
18
H
H
H
H
NH2
NH2
NH2
NH2
1.45
4.93
83
8.77 (6x)
>50 (>10x)
ND
3.82
3.44
ND
<5
56
51/62/59
71/77/78
ND
N
O
O
O
N
O
N
O
S
N
ND
46
O
N
0.656
>50 (>76x)
3.67
8/23/22
O
N
N
19
H
NH2
1.57
>50 (32x)
3.27
51
97/86/94
O
HO
20
21
22
23
24
H
H
H
H
H
NH2
NH2
NH2
NH2
NH2
112
10
>100 (1x)
>100 (>10x)
>100 (>160x)
>100 (40x)
>100 (10x)
2.08
3.03
3.34
3.50
3.43
29
40
20
<5
40
99/97/98
94/-/-
O
N
N
N
0.625
2.5
68/-/-
N
87/-/-
N
10
72/-/-
O
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25
26
H
H
NH2
NH2
10
10
ND
ND
ND
ND
ND
ND
ND
O
>100 (10x)
7
8
9
27
H
NH2
2.5
>100 (40x)
3.76
20
37/-/-
N
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
28
29
H
H
NH2
NH2
0.75
>100 (130x)
ND
4.01
4.50
40
<5
88/-/-
73/-/-
F3C
0.675
Cl
O
S
30
H
NH2
1.39
>50 (>36x)
3.35
<5
99/99/99
O
O
S
31
32
33
38
40
41
42
43
H
H
H
NH2
NH2
NH2
H
1.32
0.80
1.22
2.96
1.4
>50 (>38x)
>50 (>63x)
ND
3.60
4.02
3.15
4.24
3.62
3.34
3.61
3.36
<5
<5
98/73/78
95/89/82
96/93/92
ND
F3C
O
H2N
58
H
H
H
H
H
ND
<5
S
O
O
O
O
>50 (>36x)
>50 (>0.4x)
ND
64
89/90/87
98/100/100
82/86/80
85/94/90
N
H
S
O
>125
2.89
<5
N
H
S
O
N
N
<5
S
O
>125
0.0039
1.562
ND
21.6
S
O
Rifampicin
Kanamycin
a50% inhibitory concentration (IC50) on Chinese Hamster Ovarian (CHO) cell lines. bclogD
calculated using StarDrop. cKinetic solubility assay at pH 7.4 using HPLC method. dH/R/M:
Human/Rat/Mouse. ND: Not determined. SI: Safety Index
The R1 phenyl group was first altered to explore the impact of various electronic effects and
water solubilising groups on activity. Changing the methyl sulfone in 1 to the more lipophilic
cyclopropyl sulfone (8) led to a loss of activity, as did the introduction of a methyl group at the
ortho-position of the biaryl moiety (7). Amide analogues (9, 10, 11) were generally equipotent
to the sulfone derivative. Potency was also maintained when an aliphatic chain was extended
off the amide functionality as in 13 and 14. Various morpholino-type carboxamides were
synthesized to improve aqueous solubility whereby the thiomorpholine analogue, 18, was
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9
found to be highly potent (0.656 µM). Amides in the meta position in combination with polar,
ionizable groups, such as pyridyl groups 12 and 17 resulted in a 10- and 15-fold loss in activity,
respectively. Transformation to the carboxylic acid group 20 was not tolerated (MIC90 = 112
µM).
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
Replacements of the phenyl group by heterocycles were also investigated. Whilst the
unsubstituted pyridine (22) was equipotent to the frontrunner compounds 1 and 2, the meta-
substituted counterparts (23 and 24) displayed a loss in potency (MIC90 > 2 µM). Likewise, the
pyrimidine analogue (21) was inactive. Exploration of electronic and lipophilic effects
identified that electron withdrawing, lipophilic substituents (e.g., CF3 and Cl groups as in 28
and 29, respectively) were highly beneficial for potency, whereas electron donating groups
(e.g., OMe, tBu, NMe2 as in 25, 26, 27, respectively) were not well-tolerated.
Subsequent SAR explorations focused on moving the LHS substituent from the 6- to the 7-
position as well as varying the 2-amino moiety (Table 1). All 7-position derivatives (using
sidechains in the 6-position, which led to active compounds in order to have matched pairs)
maintained potency (< 2 µM), thus allowing variation on this position and broadening the SAR
scope on the LHS. Thereafter, several analogues were made to explore the scope of the 2-amino
moiety whilst keeping either the phenyl sulfone or sulfoxide group at position 6. Removal of
the 2-amino group as in compound 38 led to a 10-fold loss in potency (MIC90 = 2.96 µM)
compared to the parent analogue 1. Secondary (40) and tertiary (42) 2-amino analogues
interestingly maintained some potency when the 6-position contained the sulfoxide substituent
(MIC90 = 1.4 and 2.89 µM, respectively), whereas the sulfone counterparts (41 and 43) were
inactive (MIC90 > 125 µM). The majority of compounds in this series were found to be non-
cytotoxic except for the cyclopropyl sulfone (8), tertiary amide (10) and the morpholino (16)
substituted analogues. Although cytotoxicity data always has to be evaluated critically when
they are higher than the solubility data.
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Table 2: SAR and in vitro ADME data for RHS modifications
5
6
7
8
O
6
3
N
R3
MeOS
2
7
N
NH2
9
MIC90
Met. Stab.
H/R/Md
(% Rem.)
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
Solubilityc
pH 7.4 (μM)
R3
GAST Fea
clogDb
(μM)
CF3
2
0.64
3.56
80
88/-/-
CF3
34
35
36
47
48
14.8
33.4
3.09
0.922
>125
4.84
67
3.55
ND
164
ND
172
<5
50/58/51
ND
2.92
3.19
ND
99/68/71
93/99/93
ND
N
CF3
F3C
ND
38
F
49
2.88
ND
82/93/96
ND
F
50
51
52
53
54
55
56
57
58
ND
ND
ND
128
16
O
O
S
>125
>125
6.75
9.28
43
ND
ND
OH
ND
ND
O
2.67
2.73
ND
100/103/99
92/98/91
ND
CN
ND
125
ND
104
CN
F
F
45.7
2.75
>31
3.36
ND
95/81/83
ND
CF3
3.62
52/61/69
CF3
a14 day readout against H37Rv Mtb-GFP strain in GASTE-Fe media. bclogD calculated using
StarDrop. cKinetic solubility assay at pH 7.4 using HPLC method. dH/R/M:
Human/Rat/Mouse. ND: Not determined.
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9
As far as the SAR exploration on the RHS is concerned, diverse groups were investigated to
replace the trifluoromethyl substituent, whilst keeping the 2-aminoquinazolinone core
unchanged and keeping the LHS group as a phenyl sulfoxide at position 6 constant, so that
matched pairs could be compared (Table 2). Initially, exploring the substitution pattern around
the ring highlighted that the CF3 substituent exhibited optimal potency when at the para-
position compared to ortho- (34) and meta- (48) positions. Solubility improvement strategies
by adding a pyridyl group (47) or introducing a saturated ring (57) maintained moderate
potency (MIC90 = 0.922 and 2.75 µM, respectively) whereas the addition of a benzylic
methylene linker (58) led to a loss in potency. Replacement of the trifluoromethyl substituent
by polar groups (SO2Me, OH, OMe, CN) was detrimental to activity whilst lipophilic
substituents (F, Me) allowed to retain moderate potency. The unsubstituted phenyl ring
analogue (35) was not tolerated (MIC90 = 33 µM). Overall, the RHS SAR revealed that directly
attached phenyl rings bearing lipophilic groups at the para position were optimal for activity.
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
Physicochemical and In Vitro Microsomal Metabolic Stability Profiling. As
mentioned previously, solubility was established as a critical parameter to optimise. Using the
kinetic solubility assay, we aimed to achieve values >100μM but compounds with moderate
solubility and potency were also progressed. To that effect, we looked at introducing
hydrophilic groups as well as reducing overall aromaticity and planarity of the molecules
(Table 1). For the latter, a methyl substituent was inserted at the ortho position to the LHS
sulfone, as exemplified by 7, in order to disrupt the crystal planarity of the biaryl scaffold. This
resulted in a 10-fold improvement in solubility (53.3 µM) with moderate potency (4.22 µM).
Generally, derivatives from the R1 SAR displayed poor to moderate solubility, except for 13
and 14 for which the addition of the aliphatic amide substituents drastically improved the
solubility (168 and 192 µM, respectively). The addition of heteroatoms as in 22 and 23 did not
lead to improvements in solubility, except when the heterocycle contained an additional meta-
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8
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substituent such as an amide (12) or methoxy (24) moiety (40 µM). Solubility for the
morpholine-, thiomorpholine- and piperazine-type derivatives (16, 18, 19) was moderately
improved (46 - 56 µM).
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
With regards to microsomal metabolic stability, the LHS modifications led to generally
metabolically stable derivatives across all species (human, rat, mouse). A few exceptions
though include the morpholino derivative (15), the thiomorpholine (18), the pyridine (22) and
the tertiary amine (27).
Modifications to the 2-position of the core scaffold maintained good metabolic stability but no
significant improvements were made in terms of solubility (Table 1). Removal of the 2-amino
group decreased the solubility (38 = < 5 µM) of the sulfoxide derivative potentially due to the
increase in lipophilicity (clogD = 4.25). Moving the LHS group from position 6 to position 7
on the scaffold resulted in poor solubility, except for the primary amide derivative (33), which
exhibited a 10-fold improvement compared to 9.
RHS modifications were found to be the most favourable to improve aqueous solubility with
five analogues (34, 58, 36, 53 and 56) exhibiting moderate to high solubility (> 100 µM) (Table
2). Introducing a pyridyl nitrogen (47) did not improve solubility as seen previously on the
LHS, this could be due to the electron withdrawing nature of the trifluoromethyl substituent
which lowers the pKa and reduces the ionizability of the nitrogen. Moving the trifluoromethyl
substituent from the para to the meta-position was shown to be beneficial for solubility (164
µM), however, a reduction in metabolic stability was observed. Similarly, the incorporation of
a benzyl linker improved solubility albeit detrimental to metabolic stability. Variations of the
trifluoromethyl substituent with hydrophilic, electron donating groups such as the methyl (36)
and methoxy (53) moieties, resulted in improved solubility (172 and 128 µM, respectively)
whereas electron withdrawing groups as in the mono-fluoro (49) and cyano (54) derivatives
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were relatively insoluble (37.8 and 16.3 µM). As expected, increasing Sp3 character through
5
6
saturation of the aromatic ring led to improved solubility (56 = 125 µM).
7
8
9
Despite multiple analogues being synthesized and exhibiting favourable in vitro properties, the
two frontrunner compounds remained the best overall compounds in terms of combined
potency, solubility, metabolic stability and cytotoxicity properties. Therefore compounds 2 and
its active metabolite, 1, were selected for further in vivo PK studies.
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
In vivo Pharmacokinetic Studies. In vivo PK studies were performed on compound 1
(sulfone) and compound 2 (sulfoxide) (Table 3). All studies and procedures were conducted
with prior approval of the animal ethics committee of the University of Cape Town (approval
numbers 013/028 and 014/028) in accordance with the South African National Standard (SANS
10386:008) for the Care and Use of Animals for Scientific Purposes, and guidelines from the
Department of Health. The sulfone 1 was poorly absorbed achieving a bioavailability of 18%.
Compound 2 had much higher oral bioavailability and notably, was converted in vivo to the
sulfone (compound 1). This resulted in a higher clearance for the sulfoxide 2 (21.8 vs 1.3
ml/min/kg). Higher exposure of the sulfone was achieved when the sulfoxide was dosed and
underwent metabolic conversion, than when the sulfone was dosed directly (AUC 18430 vs
7249 min.µmol.L-1) as indicated in Figure 3.
Table 3. Pharmacokinetic parameters for 1 and 2 after intravenous and oral administration.
1
2
1 as metabolite of 2
Oral
20 of compound 2
Parameter
Dose (mg/kg)
iv
5
Oral
20
iv
5
Oral
20
Cmax (µM)
-
5.98
3
-
3.54
0.5
-
10.4
Tmax (h)
-
-
3
Vd (L/kg)
0.81
7.3
1.3
8589
-
-
11.1
6.2
21.8
523
-
Apparent t1/2 (h)
CLtotal (mL.min-1.kg-1)
AUC0-∞ (min.μmol.L-1)
Bioavailability (%)
-
-
10.4
-
-
-
18430
-
7249
18.4
2152
93
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5
6
7
CF3
CF3
O
N
O
N
Metabolism
MeO2S
N
MeOS
N
NH2
NH2
1
2
100
100
10
1
8
9
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
10
1
1 iv 5 mpk
1 po 20 mpk
2 iv 5 mpk
2 po 20 mpk
0.1
0.01
0.1
1 as metabolite of 2
(po 20 mpk)
0.01
0
6
12
18
24
0
6
12
Time (h)
18
24
Time (h)
Figure 3. Concentration-time profiles for compound 2 (LHS graph) and its active metabolite
1 (RHS graph) at 20 mg/kg oral doses (po); and 5 mg/kg intravenous (IV) dose.
In vivo Tolerability and Efficacy Studies. The high exposure of the sulfone 1 achieved
by dosing the sulfoxide 2 suggested that the sulfoxide could be used for in vivo efficacy
experiments. Compound 2 was therefore dosed orally at 200 mg/kg to determine the exposure
of the compound at the efficacious dose, and to confirm that the exposure would be maintained
long enough for efficacy. The free concentration of compound 2, and its sulfone metabolite 1
are represented in Figure 4 below.
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7
8
2
1
po 200 mg/kg
(metabolite of
4
3
2
1
0
2)
9
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
MIC90 of 2
MIC90 of 1
0
8
16
24
32
40
48
Time (h)
Figure 4. Free concentration-time profile of sulfoxide 2 and its sulfone metabolite 2 at 200
mg/kg dosed orally.
The free exposure was maintained above the MIC90 of compound 1 for more than 24 hrs and
this dose was therefore projected to give in vivo efficacy.
To test compound 2 for in vivo efficacy we employed the BALB/c acute TB mouse infection
model (see Supporting Information, Section 4.2). In this model, mice are infected by low dose
aerosol resulting in a mean of 2.86 log10 CFU Mtb Erdman-Lux in lungs 1 day following
aerosol. The lung burdens increased to 4.16 log10 CFU at the start of treatment on day 7. Mice
were then dosed once per day with 2 for 12 consecutive days by oral gavage in 0.2 mL volume
at 100 and 200 mg/kg. All mice completed the non-lethal acute TB efficacy trial as scheduled
and there was no evidence of compound tolerability issues. Three days following the last dose,
the mean lung burdens were 6.51 log10 CFU for untreated mice compared to 5.72, 4.15, 7.84,
7.75 log10 CFU for mice treated with rifampin at 10 mg/kg, ethambutol at 100 mg/kg, or 2 at
100 or 200 mg/kg, respectively. Thus, compound 2 was found to lack efficacy in the BALB/c
acute infection model, showing higher overall lung burdens relative to the untreated controls.
In contrast, rifampin at 10 mg/kg and ethambutol at 100 mg/kg reduced lung burdens as
expected by 0.8 and 2.4 logs, respectively.
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7
8
9
Based on the in vivo PK and tolerability studies, the lack of in vivo efficacy is unlikely to be
accounted for by low exposure and/or toxicity issues. In addition, plasma concentrations in
infected mice at 1 hr and 24 hr were comparable to those in healthy mice at the same time
points (Supporting Information, Table S1). This suggests that PK was similar between healthy
and infected animals. Further investigation into the discrepancy between in vitro and in vivo
results was therefore undertaken to understand the in vitro/in vivo disconnect.
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
Mechanistic Studies. Compound 2 was tested in biology triage assays to assess the
activity against promiscuous targets. It did not show hypersensitivity against a cytochrome-bd
oxidase knockout mutant strain (Δcyd GAST Fe MIC90 = 2.5 µM), which is hypersusceptible
to compounds that inhibit the QcrB-containing cytochrome bc1 complex, thereby eliminating
QcrB as a potential target of 2.25,26 Additionally, 2 did not show a positive signal in two
standard bioluminescence reporter assays: PiniB-LUX27 – detects modulation in iniB
expression if compounds target Mtb cell-wall biosynthesis, and PrecA-LUX27 – detects
modulation in recA expression, an indicator of genotoxic compounds. Based on this initial
triage process, the 2-aminoquinazolinone scaffold was identified as a hit series with a potential
novel mechanism of action (MOA).
However, to further understand the discrepancy between in vitro and in vivo results, compound
2 was subsequently retested in vitro using different Mtb strains cultured in different media. As
shown in Table 4, 2 was active against both H37Rv and Erdman Mtb strains when grown in
media containing glycerol as the carbon source (7H9, glycerol, ADC, and Tween-80). In
contrast to these observations, 2 lost activity against both strains when cultured in glycerol-free
media (7H12, casitone, palmitic acid, BSA, catalase and Tween-80).
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5
Table 4. In vitro antimycobacterial activity of 2 in different assay conditions.
MIC90 (mg/L):
6
7
8
2
Rifampin
Isoniazid
Strain
Media
Erdman
7H9 + glycerol + ADC + Tween-80
1
0.03
0.016
9
H37Rv
Erdman
H37Rv
7H9 + glycerol + ADC + Tween-80
0.5
>16
>16
0.016
<0.008
<0.008
0.016
0.016
0.016
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
7H12 + casitone + palmitic acid + BSA + catalase
+ Tween-80
7H12 + casitone + palmitic acid + BSA + catalase
+ Tween-80
Additional in vitro assays using glucose as the carbon source were performed on 15 other
aminoquinazolinone analogues. In all cases, no activity was observed in the absence of glycerol
in the culture medium, thus suggesting that the glycerol metabolism played a key role in
effectively inhibiting Mtb growth. We therefore hypothesized that the in vitro/in vivo
disconnect was due to the fact that glycerol was not used as a primary carbon source by Mtb in
the mouse lungs, as has previously been observed.28–30
In order to test this hypothesis, MOA studies using spontaneous-resistant mutant generation
combined with potential target identification using whole genome sequencing were performed
with 2. The spontaneous-resistant mutants were raised on Middlebrook 7H10 containing
Glycerol and OCDC (Oleic acid, casitone, dextrose, and catalse) using Mtb H37Rv MA strain.
The mutants arose with a mutation frequency of 5 x 10-7. Random isolated colonies were
picked, grown in compound free medium, and tested for MIC at a compound concentration
range of 0.16 - 160 µM. This led to the isolation of six Mtb spontaneous mutants (MIC90 of
compound 2, >160 µM) with full genome sequencing revealing single nucleotide
polymorphisms (SNPs) mapping exclusively to glycerol metabolizing genes, specifically glpK
and glpD2, as described in Table 5. GlpK (Rv3696) encodes for glycerol kinase, which is the
first committing enzyme for glycerol metabolism in most bacteria. The second metabolite in
the glycerol dissimilation pathway is dihydroxyacetone-phosphate (DHAP), which is formed
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from glycerol-3-phosphate (G3P) by the enzyme glycerol-3-phosphate dehydrogenase encoded
5
6
by the glpD1 and glpD2 genes.28
7
8
9
Previous reports observed that frameshift mutations and SNPs in glpK were linked to resistance
to Mtb growth inhibitors.28–30 These studies showed that compounds with glycerol-specific
activity promote the accumulation of G3P resulting in the rapid depletion of adenosine
triphosphate (ATP) utilized for phosphorylation of glycerol, ultimately leading to self-
poisoning of Mtb.28,29 The activity of these compounds was therefore found to be associated
with the metabolism of glycerol contained in 7H9 media.28–30 No such reports have, however,
been observed for glpD2 mutants related to in vitro drug screening.
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: Description of mutations obtained from spontaneous resistant mutant generation
MIC90
of 2
No.
Gene
Mutation
Descriptor
> 160 µM
1
2
3
4
5
6
glpK
glpK
SNP
Frameshift mutation
Frameshift mutation
SNP
T96M
C insertion at a.a. 189
C insertion at a.a. 189
F122V
> 160 µM
> 160 µM
> 160 µM
> 160 µM
> 160 µM
glpK
glpD2
glpD2
glpD2
SNP
F122L
SNP
F122L
All considered together, the results from the MOA studies suggest that the aminoquinazolinone
series potentially exert its inhibitory effect by dysregulating the glycerol dissimilation
pathway,31 leading to the toxic accumulation of sugar phosphates such as G3P and DHAP in
Mtb.28 When glycerol is not used as a carbon-source by Mtb in vivo, compounds with such
MOA do not show efficacy in the mouse model.
These results highlight the importance of validating the relevance of in vitro assay conditions
to reproduce the environment encountered by Mtb in the infected host.28,30 The culture media
currently used for sufficient Mtb growth were empirically devised several decades ago for
optimal propagation of the bacterium in vitro and were not optimised for drug screening efforts.
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5
6
7
8
9
Consequently, these media were not developed to replicate the conditions encountered by Mtb
in vivo, thus limiting the predictive value of the in vitro assays. There is no clear consensus
thus far as to which in vitro methods best reflect the in vivo biology in order to effectively
identify novel candidates that will inhibit Mtb growth in vivo.28,29,32
10
11
12
13
14
15
16
17
18
19
20
21
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Conclusion
This study identified a new class of aminoquinazolinone compounds active against Mtb
through phenotypic whole-cell assays. Although these compounds exhibited excellent in vitro
potency and favourable pharmacological properties, they were found to be inactive in an
BALB/c mouse acute TB infection model. The discrepancy between in vitro activity and in
vivo efficacy was elucidated through retesting in vitro using different Mtb strains cultured in
different media, which revealed that activity was only observed in media containing glycerol.
This was supported by spontaneous resistant mutant generation and whole genome sequencing
studies, which revealed mutations mapping to glycerol metabolising genes, suggesting a major
difference in carbon metabolism between bacteria growing in standard TB culture medium
containing glycerol compared to those found in TB-infected lungs.
Methods
General methods. All reagents and solvents were obtained from commercial sources and
used as received. All dry solvents were obtained from an SP-1 standalone solvent purification
system from LC Technology Solutions Inc. Reactions were monitored by thin layer
chromatography (TLC) using Merck TLC silica gel 60 F254 aluminium-backed precoated plates
and were visualized by ultraviolet light at 254 nm. Purification of compounds was carried out
by either column chromatography on silica gel 60 (Fluka), particle size 0.063−0.2 mm (70−230
mesh ASTM), as the stationary phase, or by Waters’ HPLC using X-bridge C18 5µm column
(4.6 x 150 mm); organic phase: 10 mM Ammonium acetate (pH 3.7) in HPLC grade methanol,
aqueous phase: 10 mM Ammonium acetate (pH 3.7) in HPLC grade water; flow rate = 15.00
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mL/min; detector: photodiode array (PDA). All target compounds and intermediates were
characterised by 1H NMR, 13C NMR and MS. NMR spectra were recorded on either a Varian
Mercury-300 (1H 300.1 MHz, 13C 75.5 MHz) or Bruker-400 (1H 400.2 MHz, 13C 100.6 MHz)
instrument using CDCl3, MeOH-d4 and DMSO-d6 as solvents. The 1H NMR data are reported
as follows: chemical shift in parts per million (δ) downfield of tetramethylsilane (TMS),
multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd =doublets of
doublets, dt = triplets of doublets and m = multiplet), coupling constant (Hz), and integrated
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value. The C NMR spectra were measured with complete proton decoupling. LC–MS/MS
analysis was performed using an Agilent® 1260 Infinity Binary Pump, Agilent® 1260 Infinity
Diode Array Detector (DAD), Agilent® 1290 Infinity Column Compartment, Agilent® 1260
Infinity Standard Autosampler, and a Agilent® 6120 Quadrupole (Single) MS, with APCI/ESI
multimode ionisation source. The purities were determined by Agilent® LCMS/MS or Waters’
HPLC using X-bridge C18 5µm column (4.6 x 150 mm); organic phase: 10 mM Ammonium
acetate (pH 3.7) in HPLC grade methanol, aqueous phase: 10 mM Ammonium acetate (pH 3.7)
in HPLC grade water; flow rate = 1.20 mL/min; detector: photodiode array (PDA). The purities
of all compounds were found to be > 95 %.
6-Iodo-2-thioxo-3-(4-(trifluoromethyl)phenyl)-2,3-dihydroquinazolin-4(1H)-one (3a).
A
solution of 2-amino-5-iodobenzoic acid (5g, 19 mmol, 1 equiv) and 4-(Trifluoromethyl)phenyl
isothiocyanate (4g, 20mmol, 1.05 equiv) in anhydrous 1,4-dioxane (75mL) was treated with
triethylamine (4mL, 28.5 mmol, 1.5 equiv). The solution was heated under refluxed for 4 hours,
cooled to room temperature, and the solids that formed were filtered. The solids were
resuspended in diethyl ether (Et2O) and filtered again to afford a white solid (7.84 g, 92%); 1H
NMR (300 MHz, DMSO-d6) δ 13.18 (s, 1H), 8.20 (d, J = 1.9 Hz, 1H), 8.10 (dd, J = 8.6, 2.0
Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.6 Hz, 1H); 13C NMR
(101 MHz, DMSO-d6) δ 176.14, 159.02, 144.21, 143.32, 139.64, 135.69, 130.72 (2C), 126.60
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(2C), 126.56, 118.77, 118.54, 88.18, 66.84. LC-MS (ESI): found m/z = 448.9 [M+H]+, (calcd
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6
for C15H8F3IN2OS: 447.0); HPLC purity 99%.
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2-Chloro-6-iodo-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (4a). PCl (6.37g, 30.63
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mmol, 1.75 equiv) was added in one portion to a mixture of intermediate 3 (7.84, 17.5 mmol,
1 equiv) in POCl3 (40mL, 25 equiv) at room temperature. The mixture was stirred for 15
minutes at room temperature and was subsequently heated to 110°C for 14 hours under
nitrogen. The reaction was cooled to room temperature, concentrated in vacuo and then diluted
with ethyl acetate to be added portionwise to stirred ice – saturated sodium bicarbonate and
stirred until all solids dissolved. The organic layer was then separated, washed with brine, dried
over magnesium sulphate (Mg2SO4) and concentrated under reduced pressure. The resulting
residue was triturated in Et2O and filtered to obtain an off-white solid (6.26 g, 79%); 1H NMR
(300 MHz, DMSO-d6) δ 8.39 (d, J = 1.8 Hz, 1H), 8.22 (dd, J = 8.5, 2.1 Hz, 1H), 7.99 (d, J =
8.3 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.7 Hz, 1H); 13C NMR (101 MHz, DMSO-
d6) δ 150.20, 143.82, 139.91, 135.86, 135.42, 130.71 (2C), 130.44, 129.22, 127.13 (2C),
126.41, 118.23, 116.99, 85.69. LC-MS (ESI): found m/z = 450.9 [M+H]+, (calcd for
C15H7ClF3IN2O: 449.9); HPLC purity 90%.
6-Iodo-2-((4-methoxybenzyl)amino)-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (5a).
4-Methoxybenzylamine (2.36mL, 18 mmol, 1.3 equiv) and N,N-diisopropylethylamine
(DIPEA) (4.84mL, 27.8 mmol, 2 equiv) were dissolved in DMF (45mL) and the appropriate
intermediate 4a (6.26g, 13.9 mmol, 1.0 equiv) was added portionwise. The reaction was stirred
for 4 hours at 80°C and allowed to cool to room temperature. The solvent was concentrated
under reduced pressure and diluted with EtOAc. The organic phase was washed with 5%
lithium chloride (3 x 20mL), brine (20mL), dried over Mg2SO4 and concentrated in vacuo to
yield a brown solid (6.36 g, 83%); 1H NMR (300 MHz, DMSO-d6) δ 8.15 (d, J = 2.0 Hz, 1H),
7.97 (d, J = 8.3 Hz, 2H), 7.87 (dd, J = 8.6, 2.2 Hz, 1H), 7.67 (d, J = 8.1 Hz, 2H), 7.24 (d, J =
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8.7 Hz, 2H), 7.11 (d, J = 8.7 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.68 (t, J = 5.9 Hz, 1H), 4.44
(d, J = 5.9 Hz, 2H), 3.71 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 161.00, 158.55, 150.59,
149.58, 143.08, 139.05, 134.95, 132.01, 130.97 (2C), 130.70, 130.14 (q, J = 32.0 Hz, 1C, C-
CF3), 128.88 (2C), 124.7 (q, J = 272.5 Hz, 1C, CF3), 127.75 (2C), 127.60, 119.49, 114.00 (2C),
84.74, 44.18. LC-MS (ESI): found m/z = 552.0 [M+H]+, (calcd for C23H17F3IN3O2: 551.0);
HPLC purity 96%.
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2-Amino-6-iodo-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (6a). Intermediate 5a
(6.35, 11.5 mmol, 1.0 equiv) was dissolved in TFA (56mL, 64 equiv) and refluxed at 80°C for
48 hours. The reaction was cooled to room temperature and the solvent was concentrated. The
residue was resuspended in DCM and added portionwise to a stirred ice – saturated sodium
bicarbonate solution. The organic phase was separated, the aqueous phase was extracted with
DCM and the combined organic phases were diluted with Et2O to a 1:1 ratio DCM/Et2O. The
solids were filtered, washed with Et2O and dried under vacuum to afford an off-white solid
(4.34 g, 87%); 1H NMR (300 MHz, DMSO-d6) δ 8.14 (d, J = 2.0 Hz, 1H), 7.93 (d, J = 8.3 Hz,
2H), 7.87 (dd, J = 8.7, 2.2 Hz, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 8.7 Hz, 1H), 6.60 (s,
2H); 13C NMR (101 MHz, DMSO-d6) δ 161.06, 152.21, 150.20, 143.04, 139.51, 135.01,
130.59 (2C), 130.03, 127.59, 127.55 (2C), 126.93, 119.24, 84.35. LC-MS (ESI): found m/z =
432.0 [M+H]+, (calcd for C15H9 F3IN3O: 431.0); HPLC purity 98%.
General protocol for the synthesis of compounds 1, 2 and 7 - 36
To a solution of the relevant intermediate 6a-e (1 equiv) and the appropriate arylboronic acid
or ester (1.5 equiv) in anhydrous 1,4-dioxane (2mL) and flushed with nitrogen.
Bis(triphenylphosphine) palladium(II) dichloride, PdCl2(PPh3)2, (0.1 equiv) was added to the
solution, cesium carbonate or potassium carbonate (3 equiv) was dissolved in water (0.5mL)
and subsequently added to the reaction mixture. The solution was heated at 80°C until TLC
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monitoring showed completion (1-5 hours), allowed to cool to room temperature and diluted
with EtOAc to be filtered through celite. The organic phase was washed with water (2 x 20mL)
and brine (20mL), dried over Mg2SO4 and concentrated under reduced pressure. The resulting
crude residue was purified by silica-gel flash chromatography eluting a gradient of either 0 –
100% EtOAc in hexane or 0 – 10% MeOH in DCM. Combined pure fractions were
concentrated in vacuo, triturated with Et2O, filtered and dried under vacuum to give the relevant
target compounds.
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2-Amino-6-(3-(methylsulfonyl)phenyl)-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one
(1). Yield 77%. 1H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J = 2.1 Hz, 1H), 8.18 (t, J= 1.7 Hz,
1H), 8.11 (ddd, J = 7.8, 1.8, 1.1 Hz, 1H), 8.09 (dd, J = 8.6, 2.4 Hz, 1H), 7.94 (d, J = 8.3 Hz,
2H), 7.88 (ddd, J = 7.8, 1.8, 1.0 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.37
(d, J = 8.4 Hz, 1H), 6.57 (br s, 2H), 3.29 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 162.24,
152.18, 150.88, 142.21, 140.96, 139.69, 133.66, 131.86, 131.66, 130.64 (2C), 130.17 (q, J =
32.0 Hz, 1C, C-CF3), 127.61, 127.58 (2C), 125.83, 125.41, 124.96, 124.87, 124.54 (q, J = 272.5
Hz, 1C, CF3), 117.40, 43.95. LC-MS (ESI): found m/z = 460.1 [M+H]+, (calcd for
C22H16F3N3O3S: 459.09); HPLC purity 98%.
2-amino-6-(3-(methylsulfinyl)phenyl)-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (2).
Yield 57%. 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 2.2 Hz, 1H), 8.03 (dd, J = 8.6, 2.2
Hz, 1H), 7.98 (t, J = 2.2 Hz, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.86 (dt, J = 6.6, 2.2 Hz, 1H), 7.70–
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7.62 (m, 4H), 7.37 (d, J = 8.6 Hz, 1H), 6.55 (br s, 2H), 2.81 (s, 3H); C NMR (101 MHz,
DMSO-d6) δ 162.28, 152.08, 150.68, 147.89, 140.82, 139.73, 133.64, 132.55, 130.66 (2C),
130.45, 130.14 (q, J = 32.0 Hz, 1C, C-CF3), 128.86, 127.61, 127.57, 125.34, 124.75, 124.7 (q,
J = 272.48 Hz, 1C, CF3), 122.64, 121.49, 117.37, 43.74. LC-MS (ESI): found m/z = 444.1
[M+H]+, (calcd for C22H16F3N3O2S: 443.1); HPLC purity 99%.
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6-iodo-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (37). To a solution of 2-amino-5-
iodobenzoic acid (330mg, 1.25 mmol, 1 equiv) and 4-(trifluoromethyl)aniline (200mg, 1.25
mmol, 1 equiv) in Dioxane (30 ml) was added triethoxymethane (2g, 12.5 mmol, 10 equiv) and
the resulting reaction mixture heated in a sealed tube at a temperature of 100˚C. After 16 hours,
a precipitate is formed. The reaction mixture was cooled to room temperature at 25˚C, the
precipitate was filtered, washed with Et2O (10ml) and dried to afford a light yellow solid
(150mg, 0.36 mmol, 29%). LC-MS (ESI): found m/z = 417.0 [M+H]+, (calcd for C15H8F3IN2O:
416.0); HPLC purity 80%.
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6-(3-(methylsulfonyl)phenyl)-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (38). 6-iodo-
3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one 37 (150mg, 0.3 6mmol, 1 equiv), (3-
(methylsulfonyl)phenyl)boronic acid (78mg, 0.39 mmol, 1.1 equiv) and PdCl2(dppf) (30 mg,
0.041 mmol, 0.11 equiv) were dissolved in Dioxane (3 mL). Thereafter, Cs2CO3 (230mg, 0.706
mmol, 2 equiv) and water (0.3 mL) were added. The mixture was heated in a pressure tube to
85˚C for 2 hours. The cooled mixture was diluted with 50 ml NaHCO3 solution and extracted
with EtOAc (2 x 50 ml). The organic phases were dried over Na2SO4 and evaporated. Flash
chromatography eluting a gradient of 30 – 50% EtOAc in DCM and evaporation of the product
1
fractions yielded a white solid (80mg, 50%). H NMR (300 MHz, CDCl3) δ (ppm) 8.63 (1H,
d, J = 3.0 Hz); 8.30-8.28 (1H, m); 8.18 (1H, s); 8.21 (1H, dd, J = 3.0 and 9.0 Hz); 8.04 -7.99
(2H, m); 7.93 (1H, d, J = 9.0 Hz); 7.88 (2H, d, J = 9.0 Hz); 7.77-7.71 (1H, m); 7.64 (2H, d, J
= 9.0 Hz); 3.16 (3H, s). LC-MS (ESI): found m/z = 445.1 [M+H]+, (calcd C22H15F3N2O3S,
444.08); HPLC purity 100%.
6-iodo-2-(methylamino)-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (39a). 2-chloro-
6-iodo-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (intermediate 4a) (200 mg, 0.44
mmol, 1.0 equiv) was dissolved in THF (2mL) and a 33% solution of methylamine in ethanol
was added and the mixture was heated in a pressure tube to 60˚C for 3 hours. Once cooled,
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10ml water was added and this mixture was extracted with ethyl acetate (2 x 50 ml). The
organic layers were dried over Na2SO4 and evaporated to afford an oily residue (240 mg,
121%). 1H NMR (300 MHz, CDCl3) δ (ppm) 8.44 (1H, d, J = 3.0 Hz); 7.93 (1H, dd, J = 3.0 &
9.0 Hz), 7.92 (2H, d, J = 9.0 Hz); 7.49 (2H, J = 9.0 Hz); 7.46 (1H, d, J = 9.0 Hz); 3.13-3.07
(3H, m). LC-MS (ESI): found m/z = 446.0 [M+H]+, (calcd C16H11F3IN3O, 444.99).
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General procedure for the synthesis of compounds 40 and 41
Intermediate 39 (120mg, 0.22 mmol, 1.0 equiv), the relevant boronic acid (56mg, 0.28 mmol,
1.3 equiv) and PdCl2(dppf) (20mg, 0.027 mmol, 0.12 equiv) were dissolved in Dioxane (2
mL). Thereafter, cesium carbonate (230mg, 0.71 mmol, 3 equiv) and water (0.3mL) were
added. The mixture was heated in a pressure tube to 80˚C for 3.5 hours. Once complete, the
mixture was cooled, diluted with water and extracted with EtOAc (2 x 50mL). The organic
layers were dried over Na2SO4 and evaporated. Flash chromatography eluting a gradient of 20
– 40% THF in DCM afforded the target compounds 40 and 41.
2-(methylamino)-6-(3-(methylsulfinyl)phenyl)-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-
one (40). Yield 75%. 1H NMR (300 MHz, CDCl3) δ (ppm) 8.22 (1H, d, J = 3.0 Hz); 8.06 (1H,
dd, J = 3.0 & 9.0 Hz); 8.00-7.94 (3H, m); 7.90-7.86 (1H, m); 7.70-7.68 (4H, m); 7.47 (1H, d, J
= 9.0 Hz); 6.08-6.02 ( 1H, m); 2.81 (6H, “s”). LC-MS (ESI): found m/z = 458.1 [M+H]+, (calcd
C23H18F3N3O2S, 457.11); HPLC purity 99%.
2-(methylamino)-6-(3-(methylsulfonyl)phenyl)-3-(4-(trifluoromethyl)phenyl)quinazolin-
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4(3H)-one (41). Yield 55%. H NMR (300 MHz, CDCl3) δ (ppm) 8.40 (1H, d, J = 3.0 hz);
8.24-8.22 (1H, m); 7.98-7.90 (5H, m); 7.72-7.65 (2H, m); 7.53 (2H, d, J = 9.0 Hz); 4.13 (1H,
sb); 3.13 (3H, s); 3.08 (3H, d, J = 6.0 Hz). LC-MS (ESI): found m/z = 474.1 [M+H]+, (calcd
C23H18F3N3O3S: 473.10); HPLC purity 98%.
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2-(dimethylamino)-6-iodo-3-(4-(trifluoromethyl) phenyl)quinazolin-4(3H)-one (39b). 2-
chloro-6-iodo-3-(4-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (4a) (220mg, 0.488 mmol,
1 equiv) was dissolved in DMF (5 mL), dimethylamine hydrochloride (350mg, 4.8 mmol, 10
equiv) and 1 ml triethylamine were heated in a pressure tube to 80˚C for 3 hours. To the cooled
mixture was added 50 ml water, and this mixture was extracted with ethyl acetate (2 x 50 ml).
The organic layers were dried over Na2SO4 and evaporated to afford a yellow solid (270mg,
120%). 1H NMR (300 MHz, CDCl3) δ (ppm) 8.36 (1H, d, J = 3.0 Hz); 7.83 (1H, dd, J = 3.0 &
9.0 Hz), 7.69 (2H, d, J = 9.0 Hz); 7.42 (2H, J = 9.0 Hz); 7.20 (1H, d, J = 9.0 Hz); 2.61 (6H, s).
LC-MS (ESI): found m/z = 460.0 [M+H]+, (calcd C17H13F3IN3O: 459.0); HPLC purity 98%.
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General procedure for the synthesis of 42 and 43
Intermediate 39b (135mg, 0.29 mmol, 1 equiv), the corresponding boronic acid (56mg, 0.28
mmol, 1 equiv) and PdCl2(dppf) (20mg, 0.027 mmol, 0.1 equiv) were dissolved in Dioxane (2
mL). Thereafter, cesium carbonate (250mg, 0.77 mmol, 3.0 equiv) and water (0.3mL) were
added. The mixture was heated in a pressure tube to 80˚C for 3.5 hours. Once complete, the
mixture was cooled, diluted with water and extracted with EtOAc (2 x 50mL). The organic
layers were dried over Na2SO4 and evaporated. Flash chromatography eluting a gradient of 40
– 80% EtOac in hexane afforded the desired final compounds 42 and 43.
2-(dimethylamino)-6-(3-(methylsulfinyl)phenyl)-3-(4-(trifluoromethyl)phenyl)quinazolin-
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4(3H)-one (42). Yield 80%. H NMR (300 MHz, CDCl3) δ (ppm) 8.40 (1H, d, J = 3.0 Hz);
8.00 (1H, dd, J = 3.0 & 9.0 Hz); 7.97-7.93 (1H, m); 7.84-7.62 (7H, m); 7.56 (1H, d, J = 9.0
Hz); 2.81 (3H, s); 2.78 ( 6H, s). LC-MS (ESI): found m/z = 472.1 [M+H]+, (calcd
C24H20F3N3O2S, 471.12); HPLC purity 98%.
2-(dimethylamino)-6-(3-(methylsulfonyl)phenyl)-3-(4-(trifluoromethyl)phenyl)quinazolin-
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4(3H)-one (43). Yield 88%. H NMR (300 MHz, CDCl3) δ (ppm) 8.41 (1H, d, J = 3.0 hz);
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