Identification of 5-Substituted 2-Acylaminothiazoles That Activate Tat-Mediated Transcription in HIV-1 Latency Models

Article abstract of DOI:10.1021/acs.jmedchem.9b00462

The persistent reservoir of cells latently infected with human immunodeficiency virus (HIV)-integrated proviral DNA necessitates lifelong suppressive antiretroviral therapy (ART). Epigenetic targeted compounds have shown promise as potential latency-reversing agents; however, these drugs have undesirable toxicity and lack specificity for HIV. We utilized a novel HEK293-derived FlpIn dual-reporter cell line, which quantifies specific HIV provirus reactivation (LTR promoter) relative to nonspecific host cell gene expression (CMV promoter), to identify the 5-substituted 2-acylaminothiazole hit class. Here, we describe the optimization of the hit class, defining the functionality necessary for HIV gene activation and for improving in vitro metabolism and solubility. The optimized compounds displayed enhanced HIV gene expression in HEK293 and Jurkat 10.6 latency cellular models and increased unspliced HIV RNA in resting CD4+ T cells isolated from HIV-infected individuals on ART, demonstrating the potential of the 2-acylaminothiazole class as latency-reversing agents.

Full text of DOI:10.1021/acs.jmedchem.9b00462

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Article
Identification of 5-substituted 2-acylaminothiazoles that
activate Tat mediated transcription in HIV-1 latency models
William Nguyen, Jonathan Jacobson, Kate E. Jarman, Helene Jousset Sabroux, Leigh
Harty, James McMahon, Sharon Lewin, Damian F. J. Purcell, and Brad E. Sleebs
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00462 • Publication Date (Web): 11 Apr 2019
Downloaded from http://pubs.acs.org on April 12, 2019
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Identification of 5-substituted 2-
acylaminothiazoles that activate Tat mediated
transcription in HIV-1 latency models
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†
,‡
€
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†,‡
William Nguyen, Jonathan Jacobson, Kate E. Jarman, Helene Jousset Sabroux,
€
ǁ
ǁ, ¥
€
Leigh Harty, James McMahon, Sharon R. Lewin, Damian, F. Purcell, Brad E.
Sleebs.†,‡,*
†
‡
The Walter and Eliza Hall Institute for Medical Research, Parkville, Victoria, 3052,
Australia.
Department of Medical Biology, University of Melbourne, Parkville, Victoria, 3052,
Australia.
€
Department of Microbiology and Immunology, Peter Doherty Institute of Infection and
Immunity, University of Melbourne, Parkville, Victoria, 3000, Australia.
ǁ
Department of Infectious Diseases, Alfred Health and Monash University, Melbourne,
Victoria, 3004 Australia.
¥
The Peter Doherty Institute for Infection and Immunity, The University of Melbourne
and Royal Melbourne Hospital, Parkville, Victoria 3000, Australia.
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*
Correspondence addressed to this author.
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KEYWORDS
HIV-1, AIDS, latency, thiazole.
ABSTRACT
The persistent reservoir of cells latently infected with Human Immunodeficiency Virus (HIV)
integrated proviral DNA necessitates lifelong suppressive antiretroviral therapy (ART).
Epigenetic targeted compounds have shown promise as potential latency reversing agents,
however these drugs have undesirable toxicity and lack specificity for HIV. We utilised a novel
HEK293 derived FlpIn dual-reporter cell line, that quantifies specific HIV provirus reactivation
(LTR promoter) relative to non-specific host cell gene expression (CMV promoter), to identify
the 5-substituted-2-acylaminothiazole hit class. Here, we describe the optimization of the hit
class, defining the functionality necessary for HIV gene activation and for improving in vitro
metabolism and solubility. The optimized compounds displayed enhanced HIV gene
expression in HEK293 and Jurkat 10.6 latency cellular models and increase unspliced HIV
RNA in resting CD4+ T cells isolated from HIV-infected individuals on ART, demonstrating
the potential of the 2-acylaminothiazole class as latency reversing agents.
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INTRODUCTION
Antiretroviral therapies (ART) for HIV-infected individuals has transformed HIV-1 infection
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from a fatal illness into a chronic manageable disease. ART dramatically improves health,
returns life expectancy to normal and significantly reduces the risk of HIV transmission.
However, although ART suppresses HIV to undetectable levels, discontinuation results in the
rapid rebound of the virus to pre-treatment levels.2
HIV persists in individuals on suppressive ART in resting CD4+ T cells that contain an
intact, replication-competent integrated latent HIV provirus which is unaffected by ART.3, 4
One strategy being developed to eliminate latently infected cells is referred to as the “shock
and kill” approach. This approach aims to activate proviral DNA templated RNA transcription
and in subsequent steps mRNA processing, protein translation and virion production so the
infected cells are identified and then destroyed by either by immune-mediated processes and/or
viral cytopathic effects.5, 6 The shock and kill approach used in conjunction with ART could
potentially deplete replication competent virus from an infected individual.
Many existing epigenetic modulators have been investigated as strategies to activate HIV
transcription and several have advanced to clinical trials yielding varied results. In clinical
trials, Vorinostat (Figure 1), a histone deacetylase (HDAC) inhibitor, showed enhanced levels
of HIV RNA expression in CD4+ T cells in blood from HIV-infected individuals on ART.7, 8
However, there were no statistically significant changes in plasma HIV RNA, concentration of
HIV DNA, integrated DNA and inducible virus in CD4+ T-cells. In addition, significant
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changes in host gene expression were also observed highlighting safety concerns with its long-
term use. Similar findings have been described with other HDAC inhibitors, including
Panobinostat9, 10 and Romidepsin11, 12 (Figure 1). JQ1 (Figure 1), a bromodomain and extra-
terminal motif (BET) protein inhibitor, can also reactivate HIV by enhancing transcription
processivity in both Jurkat latency (J.Lat) clones and primary CD4+ T-cell models.13, 14 JQ1
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also possesses a short half-life in humans and moreover is associated with mild cytotoxicity
making it an unsuitable clinical candidate for chronic regimens. Finally, these epigenetic
modulators lack specificity for HIV-1 transcriptional activation and are of modest potency with
respect to virus activation. Therefore, there is an unmet need to identify new latency reversing
agents (LRAs) with a novel mechanism of action.
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Figure 1. Structures of small molecules commonly used as latency reversing agents in the
“shock and kill” strategy.
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In latently infected cells, HIV-1 is predominantly integrated into the non-coding introns of
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transcriptionally active host genes. Transcription of pre-mRNA from the strong upstream
cellular promoter reads through the HIV provirus within these introns.13, 18 Alternative RNA
splicing can lead to the formation of chimeric cellular Tat (trans-activator of transcription)
mRNAs19-21 that translate basal levels of Tat protein using internal ribosome entry site (IRES)-
mediated translation.22 HIV transcription is then stimulated by Tat binding to the trans-
activation response element and recruits the positive transcription elongation factor-b (pTEFb)
enhancing Long Terminal Repeat (LTR) driven HIV transcription. Tat is the master regulator
for HIV gene expression and is key in promoting productive viral infection.23, 24 Therefore,
identifying novel small molecules that complement low level Tat expression is potentially a
viable strategy to increase HIV transcription in latently infected cells.
We recently developed a dual luciferase reporter cellular assay to identify small molecules
capable of activating HIV-1 gene expression (Figure S1).25, 26 The HEK293 derived reporter
cell line, named FlpIn.FM, mimics HIV-1 post integration latency and read-through
transcription by translating a low level of HIV-1 Tat – human growth hormone chimeric protein
using a IRES mechanism. The cell line expresses a HIV-1 Nef – click beetle red (CBR) fusion
protein driven by Tat from the long terminal repeat (LTR) HIV promoter, allowing for
detection of viral gene expression. The cell line also contains a click beetle green (CBG)
luciferase reporter driven from a cytomegalovirus (CMV) promoter allowing the detection of
non-specific host cell protein expression. The dual reporter FlpIn.FM cell line was
characterized for its high responsiveness to Tat and shown to be applicable for detecting latency
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reversing agents. In this study, Vorinostat and JQ1 (Figure 1) were shown to enhance the LTR
CBR response, but also increased CMV or host cell reporter to similar levels,25, 26 consistent
27
with literature. This study established the FlpIn.FM HEK293 cell line as an effective tool for
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identifying novel latency reversing agents that act via a Tat-dependent mechanism.
We utilised the FlpIn.FM HEK293 cellular assay to screen a chemically diverse library of
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14,000 small molecules28, 29 to primarily identify compounds capable of enhancing HIV LTR
(CBR) activity. The CMV (CBG) reporter was used to monitor Tat non-dependent or non-
specific host cell gene expression. The high throughput screen identified the 2-
acylaminothiazole 1 (Figure 2) as a hit compound and possessed an EC of 24 µM for the HIV
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LTR reporter, while displaying no detectable activity for the CMV reporter at the highest
concentration tested (40 µM) (Table 1).
To demonstrate the hit compound 1 was genuinely activating gene expression and not
an assay artefact, two counterscreen approaches were implemented. The first
counterscreen utilized the FlpIn.RV HEK293 cell line in which the luciferase reporters
were reversed in comparison to the FlpIn.FM cell line (Figure S1). Using the FlpIn.RV cell
line, the hit compound 1 was shown to possess an EC of 29 M against the LTR (CBG)
5
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reporter while the EC for the CMV (CBR) reporter was >40 M (Figure S2), consistent
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with the activity of compound 1 using the FlpIn.FM cell line. The second counter screen
assay involved the addition of compound 1 to the FlpIn.FM cell line followed immediately
by the addition of and incubation with luciferase reagents (time point 0 hours in Figure
S3A). Under these conditions no bioluminescence signal was measured compared to
incubation with the compound for 48 hours (Figure S3B). Collectively the counterscreen
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data demonstrated hit compound 1 was not an assay interference artefact and was
genuinely activating LTR gene expression.
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Figure 2. Hit compound 1 identified from a high throughput screen of a diverse chemical
library of 114,000 compounds using FlpIn.FM dual reporter HEK293 cellular assay.
Herein, we describe the optimization and the characterization of the structure activity
relationship (SAR) of the 5-substituted 2-acylaminothiazole scaffold to generate a series of
compounds with enhanced HIV LTR activity in the FlpIn.FM cell line. Physicochemical
parameters were also assessed in parallel and assisted in the design and selection of analogues
for further evaluation in HIV-1 cellular models. Compounds with suitable properties and
activity in the FlpIn.FM HEK293 cellular assay were then evaluated in the Jurkat latently
infected cell line and subsequently in CD4+ T cells from HIV-infected individuals on
suppressive ART.
RESULTS AND DISCUSSION
The initial focus of optimising the 2-acylaminothiazole class was to establish the SAR,
providing a foundation to improve LTR reporter activity using the FlpIn.FM HEK293 cell
line. The hit 2-acylaminothiazole compound 1, possessed limited aqueous solubility (<1 μM
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at pH 6.5) and was rapidly degraded by both human and mouse liver microsomes (CL 175
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and 815 μL/min/mg protein, respectively) (Table 10). The thiazole moiety potentially
possesses intrinsic in vivo toxicity, because of cytochrome P450-mediated oxidative
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conversion of the 4,5-unsaturated bond to an epoxide acting as a trap for nucleophiles.30
The physicochemical properties, in vitro metabolism and the thiazole toxicophore are
factors taken into consideration during the optimization of the 2-acylaminothiazole series.
Chemistry
The general synthetic route (Scheme 1) undertaken to generate the 5-substituted 2-
acylaminothiazole analogues involved alkylation of a substituted phenol with a protected
halogenated butanoate. Subsequent deprotection of the ester and then an amide coupling
to a substituted 2-aminothiazole gave the 2-acylaminothiazole product.
Scheme 1. General pathway to synthesize 2-acylaminothiazole analogues. Reagents and
conditions: (a) substituted phenol, K CO or NaH, MeCN or DMF; (b) For R = Et: LiOH,
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3
MeOH, H O, 50°C; For R = tBu: HCl, dioxane, 20°C; (c) substituted 2-acylaminothiazole,
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EDC.HCl, DMAP, DCM.
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Structure and activity relationship
To establish the SAR, we systematically investigated moieties on the 2-acylamino
scaffold independently and the effect on LTR reporter activity in the FlpIn.FM HEK293
cell line. We first investigated substitution on the phenoxy ring with different functionality
to determine whether the steric and electronic nature of groups would affect LTR activity
(Table 1). It was shown that substitution of the 2- and 4- positions on the phenoxy ring
were generally not tolerated, except for the 2-methoxy substitution (12), that possessed
enhanced LTR activity (EC 1.2 µM), and the 2-methyl substitution (2) that was equally as
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active (EC 19 µM) compared to 1. The 2-methoxy substitution 12 also gave an increase in
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CMV reporter activity (EC 6.2 µM) and therefore further iterations to the 2-position of
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the phenoxy ring were not further pursued. In contrast, 3-methyl (3), 3-chloro (6), 3-
trifluoromethyl (8) and 3-nitrile (10) substituents enhanced activity, all possessing LTR
EC values of approximately 1 to 2 µM. The 3-chloro and 3-trifluoromethyl analogues (6
5
0
and 8) did not display CMV reporter activity at the highest concentration tested (EC >40
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0
µM), but the 3-methyl and 3-nitrile analogues (3 and 10) exhibited CMV reporter activity
EC of 2.1 and 9.9 µM respectively). Electron donating substituents, such as hydroxyl (15),
(
5
0
amino (17) and aminoacyl (19) were not tolerated in the 3-position (LTR EC s all >40 µM).
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A methyl carboxylate group in the 3-position (23) was also detrimental to activity (LTR
EC s all >40 µM) suggesting larger polar substituents were not tolerated. The 3-methylene
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hydroxy group (21) was accepted (LTR EC 6.4 µM), but also exhibited the CMV reporter
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activity (EC 7.1 µM). Thus, the activity data on the phenoxy ring substitution indicated
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0
3
-chloro and 3-trifluormethyl possessed the largest improvement in LTR activity while not
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affecting the CMV reporter (EC values >40 µM), but the 3-methyl and 3-methylene
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hydroxy substitution conveyed equipotent LTR and CMV activity. This selectivity data
suggested that electron withdrawing groups in the 3-position were the most effective at
enhancing LTR activity while only minimally effecting the CMV reporter. The 3-chloro
and 3-trifluormethyl modifications (6 and 8) slightly increased in cLogP, translating into a
minor increase in LipE (2.1 and 1.5) while the nitrile analogue (10) exhibited a 2-fold
improvement in LipE (2.7) compared to the hit compound 1 (LipE 1.4). Metabolically the
3-trifluoromethyl substitution (8) prevents O-dealkylation of the phenoxy group, while the
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-chloro (6) and 3-nitrile (10) substitution did not block O-dealkylation (Table S2).
Table 1. The activity of aryl substitution in the FlpIn.FM dual reporter cell assay.
Cmpd
LTR
EC50
CMV
EC50
cLog
P
PSA
(Å )
LipE
R2
R3
R4
b
2
b
c
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(
SD) M (SD) M
a
a
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H
CH3
H
H
Cl
H
H
H
H
H
H
H
CH3
H
H
Cl
H
CF3
H
CN
H
H
H
H
H
CH3
H
H
Cl
H
CF3
H
CN
H
H
OCH3
H
OH
H
24 (3.6)
19 (1.6)
1.7 (0.2) 2.1 (0.7)
>40
>40
3.2
3.8
3.8
3.8
3.8
3.8
3.8
4.1
4.1
3.1
3.1
3.1
3.1
3.1
2.9
2.9
2.4
51
51
51
51
51
51
51
51
51
75
75
60
60
60
71
71
77
1.4
0.9
2.0
-
-
2.1
-
1.5
-
2.7
-
2.8
-
-
>40
>40
>40
>40
>40
>40
>40
>40
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.3 (0.2)
>40
2.6 (0.7)
>40
1.5 (0.3) 9.9 (4.4)
>40 >40
1.2 (0.3) 6.2 (1.1)
10
1
1
1
1
2
3
H
OCH3
H
OCH3
H
OH
H
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
14
H
H
H
1
1
5
6
-
-
17
H
NH2
-
1
8
H
H
H
H
H
NHAc
H
NH2
H
>40
>40
>40
>40
>40
>40
2.4
2.5
2.5
2.5
77
80
80
71
-
-
19
2
0
NHAc
H
-
21
CH OH
6.4 (1.4) 7.1 (2.4)
2.7
2
2
2
2
3
H
H
H
H
CH OH
>40
>40
>40
>40
>40
>40
2.5
3.2
3.2
71
78
78
-
-
-
2
CO CH
H
2
3
24
H
CO CH
2
3
a
EC data represents means and SDs for three or more independent experiments measuring
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
5
0
b
23
c
Calculated using the LTR EC value.
5
0
We then explored the effect of disubstitution of the phenoxy ring on LTR activity,
primarily concentrating on maintaining one of the electron withdrawing substituents in
the 3-position while altering the functionality at either the 4- or 5-position (Table 2).
Analogues that possessed either a 3-chloro or 3-trifluoromethyl group with a 4-chloro
substituent (26 and 27) displayed slightly decreased LTR activity (EC 9.2 and 3.6 µM)
50
compared to their 3-monosubstituted parent compounds 6 and 8 (EC 1.3 and 2.6 µM). The
5
0
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
-chloro-4-methyl substituted analogue (25) displayed an EC of 4.7 µM indicating the
5
0
installation of a 4-methyl group was not beneficial to activity. Consistent with activity
values for monosubstituted analogues, addition of 4-chloro and 4-nitrile substituents on 3-
methyl phenoxy ring, the 3-methyl-4-chloro or 3-methyl-4-nitrile analogues (28 and 29)
were detrimental to LTR activity (EC50 13 and >40 µM). 3,5-Disubstitution was also
detrimental to the LTR activity of analogues 30-31 and 33 (EC >10 µM) compared to 3-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
mono substituted compounds (EC s <3 µM). Overall 3,4-distubsutituon was tolerated but
5
0
did not enhance LTR activity and the addition of extra hydrophobic functionality in the 4-
position also decreased LipE values. Therefore, this suggested mono-substitution with
electron withdrawing groups in the 3-position of the phenoxy ring were the most effective
at increasing LTR activity while not increasing the CMV reporter activity.
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2
3
4
5
6
7
8
9
Table 2. Activity of analogues with aryl disubstitution on Flp.FM dual reporter cell
activity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cmpd
LTR
EC50
CMV
EC50
PSA
(Å )
R3
R4
R5
cLogP b
LipE c
2
b
(SD) M (SD)
a
a
M
1
25
H
Cl
Cl
H
CH3
Cl
CF3
Cl
CN
H
H
H
H
H
H
H
H
F
Cl
24 (3.6)
4.7 (1.0)
9.2 (1.5)
3.6 (0.8)
13 (2.9)
>40
40 (2.8)
>40
3.3 (1.1)
11 (3.0)
2.3 (0.3)
>40
>40
>40
>40
38
>40
>40
>40
>40
>40
>40
3.2
4.4
4.5
4.7
4.4
3.7
3.5
4.5
4.4
3.0
4.3
51
51
51
51
51
75
51
51
51
99
51
1.4
0.9
0.5
0.7
0.5
-
0.9
-
1.1
2.0
1.3
2
2
6
7
Cl
28
CH3
CH3
F
Cl
Cl
2
3
3
3
9
0
1
2
H
H
H
CH3
CN
CH3
33
CN
CH3
3
4
a
EC data represents means and SDs for three or more independent experiments measuring
5
0
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
b
23
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
c
Calculated using the LTR EC value.
5
0
Thiazoles are known toxicophores and are susceptible to cytochrome P450 mediated
3
1
oxidative metabolism of the 4,5-double bond to produce an epoxide. The epoxide formed
can either act as a trap for nucleophiles or can be further oxidized to form reactive di-
carbonyl and thioamide metabolites.32 Meloxicam, a marketed nonsteroidal anti-
inflammatory analgesic, possesses a 5-methyl 2-aminothiazole. Metabolism studies show
that a 5-methyl substitution present on the thiazole in Meloxicam can delay or circumvent
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
this oxidative pathway. However, the metabolic outcome cannot always be predicted
with new chemical entities. Compound 1 is rapidly degraded in the presence of human and
mouse liver microsomes (Table 10). Identification of metabolite masses in this study
revealed several points of metabolism on the 2-acylaminothiazole scaffold (Table S2). Two
+16 mass metabolites were identification that were predicted to correspond to either the
oxidation of the 5-methyl substituent, oxidation of the phenol ring or epoxidation of the
thiazole ring. To de-risk the potential inherent metabolic and toxicophoric liability of the
thiazole we investigated two strategies. Firstly, determine functional group tolerance in the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
- and 5-position on the thiazole ring that may inadvertently mitigate oxidative metabolism
of the 4,5-unsaturated bond. The second approach was to replace the thiazole with suitable
isosteric 5- and 6-membered heterocyclic systems.
We first examined the effects of substitution in the 4- and 5-position of the thiazole ring
on LTR activity while keeping the scaffold of compound 1 constant (Table 3). In this set of
analogues, it was shown deleting the 5-methyl substituent (35) resulted in a loss of LTR
activity. 4-Methyl and 4,5-dimethyl substitution of the thiazole (47 and 50) also resulted
in loss activity, indicating the 4- and 5- positions of the thiazole system were sensitive to
change and the 5-substitution was critical for activity. Analogues with 4-trifluromethyl
and 4-nitrile substitution (48 and 49) were also generated and demonstrated functionality
other than a methyl group in the 5-position of the thiazole was not accepted.
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3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. The activity of 4- and 5-thiazole substitution in the Flp.FM dual reporter cell
assay.
Cmpd
LTR
EC50
CMV
EC50 (SD)
M a
PSA
cLog
P
LipE
R1
R2
2
(Å )
b
c
(
SD) M
b
a
1
35
CH3
H
Cl
Br
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CF3
CN
CH3
24 (3.6)
>40
>40
>40
>40
3.2
2.6
3.4
3.5
3.6
3.7
4.0
4.3
4.2
2.6
2.4
1.6
2.0
2.7
3.9
2.8
3.4
51
51
51
51
51
51
51
51
51
75
89
94
72
51
51
75
51
1.4
-
3
3
3
6
7
8
5.0 (1.0)
3.7 (0.8)
2.9 (0.9)
4.7 (1.2)
6.4 (1.2)
>40
>40
11 (0.9)
>40
1.9
1.9
1.9
1.6
1.2
-
-
2.4
-
-
-
-
-
>40
8.4 (3.8)
7.2 (2.0)
32 (15)
>40
39
CH CH
2
3
4
4
4
0
1
2
iPr
iBu
Ph
>40
>40
>40
>40
>40
>40
>40
>40
43
CN
4
4
4
4
5
6
COOH
CONH2
CONMe2
H
>40
>40
>40
>40
>40
>40
47
4
4
5
8
9
0
H
H
CH3
-
-
>40
a
EC data represents means and SDs for three or more independent experiments measuring
5
0
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
b
23
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
c
Calculated using the LTR EC value. 1 is included for comparison.
5
0
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Tolerance of functionality and steric volume in the 5-position of the thiazole were then
explored (Table 3). Replacing the 5-methyl group (1) with electron withdrawing chloro or
bromo halogens (36 and 37) gave a 5-fold enhancement in LTR activity (EC 5.0 and 3.7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
µM) without effecting the CMV reporter (EC >40 µM). 5-Trifluoromethyl substitution of
5
0
the thiazole (38) improved LTR activity 8-fold (EC 2.9 µM) but introduced CMV reporter
5
0
activity (EC 8.4 µM). Replacing the 5-methyl group (1) with larger substitution such as
5
0
ethyl and isopropyl groups (39 and 40) improved LTR activity (EC 4.7 and 6.4 µM), but
5
0
also increased CMV reporter activity (EC 7.2 and 32 µM). Analogues with substituents
5
0
larger than an isopropyl such as a 5-isobutyl and 5-phenyl substituents (41 and 42) were
inactive. A 5-nitrile substituent (43) was tolerated (11 µM), but larger polar groups such as
a carboxylic acid, carboxamide and dimethyl carboxamide at the 5-position (44, 45 and 46)
were detrimental to LTR activity (EC s >40 µM). The SAR of the 5-position of the thiazole
5
0
suggested hydrophobic and nitrile substituents with smaller steric size than an isopropyl
group were ideally occupying a pocket of the cellular target responsible for LTR activity,
however compounds that possess a 5-substitutent larger steric volume than a methyl or
bromo, were also potentially occupying a pocket of the cellular target conferring CMV
reporter activity. Compared to analogues substituted with hydrophobic functionality,
analogue 43 with a nitrile group in the 5-position possessed lower cLogP (2.6) and enhanced
LipE (2.4) and therefore was the most effective according to these parameters.
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5
6
7
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9
In vitro metabolism studies were undertaken with analogue 43 in the presence of human
and mouse liver microsomes to determine if the 5-nitrile thiazole substitution circumvents
metabolic epoxidation of the thiazole ring. The metabolism results show there was no
difference in the human intrinsic clearance with 43 compared to the 5-methyl ortholog (1),
but there was an improvement in mouse intrinsic clearance (Table 10). Mass spectrometry
analysis of the metabolites in the liver microsome study showed there was a reduction in
the number of oxidative metabolism events with 43 compared to the 5-methyl ortholog (1)
(Table S2). It was proposed this difference was due to hydroxylation of the 5-methyl group
of 1 and therefore epoxidation of the 4,5-thiazole double bond was not observed under the
in vitro liver microsome conditions. It was concluded, inclusion of the 5-nitrile substituent
blocked 5-methyl hydroxylation and amide bond hydrolysis was responsible for the
improvement in mouse intrinsic clearance of analogue 43 compared to the 5-methyl
ortholog 1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Another approach to avoid thiazole related metabolism and improve physicochemical
properties was to explore suitable isosteric replacements of the thiazole with either 5-
methyl or 5-trifluoromethyl substitution (Table 4). 5-Membered heterocycles including
oxazoles (51 and 52), 1,3,4-thiadiazoles (53, 54) and 1,3,4-oxadiazoles (55, 56) were
examined, but none of these analogues displayed LTR reporter activity (Table 4). N-
Methylated pyrazoles (57 and 58) and 5-methyl isooxazole (59) were also inactive (Table
4
), highlighting the addition of an extra hetero nitrogen in the 4-postion and the exchange
of the thiazole sulfur for an oxygen or carbon atom in 5-membered heterocyclic systems
were not tolerated.
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Activity of 5-membered thiazole isosteres in the FlpIn.FM dual reporter cell
assay.
O
CMV
PSA
LTR EC
cLogP
O
50
a
(Å ) LipE c
2
Cmpd
1
EC50 (SD)
N
b
(SD) M
H
M a
b

N
24 (3.6)
>40
3.2
51
1.4
S
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2
3
4
5
6
7
8
9
N
51
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
2.1
2.7
1.8
3.0
1.0
2.2
2.3
1.7
2.4
64
64
64
64
77
77
56
56
64
-
-
-
-
-
-
-
-
-
O
N
5
5
5
2
3
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F3C
O
N N
S
N N
S
F3C
N N
O
55
N N
O
5
5
6
7
F3C
N N
N
58
N
5
9
O
N
a
EC data represents means and SDs for three or more independent experiments measuring
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
Calculated using the LTR EC value. 1 is included for comparison.
5
0
b
23
c
5
0
5-Methyl pyridine was also attempted as a suitable 6-membered heterocyclic isostere of
the 5-methyl thiazole system (Table 5). The 5-methyl pyridine analogue (60) exhibited
comparable LTR reporter activity (EC 20 µM) and CMV reporter activity (EC 39 µM) to
5
0
50
the thiazole ortholog 1. This data demonstrated the pyridyl system was a potential
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
replacement for the thiazole, and therefore several 5-subtituted pyridyl analogues were
generated and their LTR and CMV activities (Table 5) compared to the activity of the 5-
substituted thiazole orthologs (Table 3). The chloro and bromo substitution in the 5-
position of the pyridine (62 and 63) resulted in a 3-fold improvement in LTR activity (EC50
of 6.7 and 8.0 µM) compared to the 5-methyl pyridine analogue 60 (EC 20 µM). The 3-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
fold shift in LTR activity was slightly less than the 5-fold shift between the halogenated
thiazole analogues 36 and 37, and the 5-methyl thiazole analogue 1. The 5-trifluoromethyl
pyridine analogue 61 displayed equipotent LTR and CMV activity (EC 14 and 32 µM,
5
0
respectively) compared to the methyl pyridine analogue 60 (EC50 20 and 39 µM
respectively), but decreased LTR and CMV activity when compared to the 5-
trifluoromethyl thiazole ortholog 38 (EC50 2.9 and 8.4 µM). The 5-isopropyl pyridine
analogue (64) exhibited no LTR activity (EC >40 µM), in contrast to the LTR activity (EC
5
0
50
6.4 µM) exhibited by the 5-isopropyl thiazole ortholog 40. Moving the pyridinyl endocyclic
nitrogen to the 2-position (66) or the methyl substitution to the 4-position (65) also
eliminated LTR activity. Collectively, this activity data demonstrated that although the 5-
substituted pyridyl system was a suitable isostere for the 5-functionalized thiazole, the
pyridyl system generally displayed decreased LTR reporter activity and a smaller selectivity
window over the CMV reporter compared to the thiazole analogues. Furthermore, the
pyridyl system did not enhance physicochemical properties or LipE and therefore was not
further pursued.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 5. Activity of 6-membered thiazole isosteres in the FlpIn.FM dual reporter cell
assay.
LTR
CMV
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
PSA
EC50
EC50
(SD)
a
cLog
P
O
(Å ) LipE c
2
Cmpd
60
N
b
(SD) M
H
b
a

M
20 (0.6) 39 (0.5)
13 (1.0) 32 (5.7)
3.1
3.5
3.2
3.4
3.9
3.1
2.2
51
51
51
51
51
51
51
1.6
1.4
2.0
1.7
-
N
F3C
Cl
6
6
6
1
2
3
N
6.7 (1.4)
8.0 (1.7)
>40
>40
>40
>40
>40
>40
N
Br
N
N
64
6
6
5
6
>40
-
N
>40
-
N
a
EC data represents means and SDs for three or more independent experiments measuring
5
0
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
b
23
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
c
Calculated using the LTR EC value.
5
0
We next determined whether the length of the three-carbon chain, between the
phenoxy and the carboxamide, was optimum for LTR activity (Table 6). Shortening the
chain to one- or two-carbons (67 and 68) or extending the chain to four-carbons (69)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 122
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
eliminated LTR activity, suggesting the three-carbon chain between the phenoxy and the
carboxamide is necessary for LTR activity. We then investigated the importance of the
oxygen atom on the phenoxy group by replacing it with carbon and nitrogen atoms. It was
shown that exchange of the phenoxy oxygen with a carbon atom (70) resulted in a 2-fold
improvement in LTR activity (EC 13 µM) compared to its phenoxy counterpart 1 (EC
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
2
4 µM), but also introduced CMV reporter activity (EC 34 µM) (Table 6). Replacing the
5
0
phenoxy group with aniline (71) preserved the LTR activity (EC 22 µM) but also increased
5
0
the CMV reporter activity (EC 27 µM). Substituting the oxygen of the phenoxy group
5
0
with a N-methyl group (72) increased both the LTR and CMV reporter activity (EC 6.3
5
0
and 14 µM respectively) compared to the phenoxy comparator compound 1. This data
highlighted that a carbon atom was a suitable replacement for the oxygen, and although
not tested, 70 would be metabolically devoid of the O-dealkylation observed with the
phenoxy analogue 1 in the presence of liver microsomes (Table S2). However, compound
7
0 possesses significantly higher cLogP and decreased LipE compared to the
complementary phenoxy analogue 1, and therefore incorporation of the carbon-linked
phenyl would only be considered in future work if metabolic O-dealkylation was not
overcome.
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Page 23 of 122
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. The activity of acyl carbon chain in the Flp.FM dual reporter cell assay.
LTR
EC50
CMV
EC50
(SD)
cLogP
PSA
(Å )
LipE
Cmpd
1
b
2
b
c
(
SD) M
a
a
M
O
24 (3.6)
>40
>40
>40
>40
>40
3.2
3.8
3.0
4.8
4.4
51
51
51
51
41
1.4
-
O
6
7d
O
6
8
>40
-
O
6
9d
>40
-
70
13 (0.8) 34 (1.0)
22 (5.1) 27 (9.5)
6.3 (0.6) 14 (4.0)
0.5
H
N
7
7
1
2
2.9
3.5
54
45
1.8
1.7
N
a
EC data represents means and SDs for three or more independent experiments measuring
50
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
b
23
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
c
d
Calculated using the LTR EC50 value.
Analogues both have 4-chloro-3-methyl
substitution on the phenoxy ring. 1 is included for comparison.
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We further explored the three-carbon chain, between the phenoxy and the
carboxamide, concentrating on modification of the carbonyl alpha-carbon (Table 7). The
addition of a methyl substitution (73) displayed a 2-fold loss in LTR reporter potency (EC50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3 µM) and maintained CMV selectivity (EC >40 µM), compared to the comparator 28
5
0
(EC 38 µM). Gem dimethyl substitution of the carbonyl alpha-carbon (74) resulted in a
5
0
complete loss of LTR activity. Replacing the carbonyl alpha-carbon with a nitrogen atom
to form a urea in (75) improved LTR activity 8-fold (EC 2.8 µM) but resulted in an increase
5
0
in CMV reporter activity (EC 5.0 µM). Noting the effect of the urea moiety on LTR
5
0
activity, we next explored the configuration of the amide (Table 7). Methylation of the
amide NH (76) and removal of the amide carbonyl (77) resulted in loss of LTR reporter
activity. In addition, reversing the configuration of the amide bond (78) also eliminated
LTR activity, highlighting the importance of both the presence and configuration of the
amide moiety.
Table 7. The activity of acyl variations in the FlpIn.FM dual reporter cell assay.
LTR
EC50
CMV
EC50
(SD)
N
S
cLogP
PSA
(Å )
O
Cl
LipE c
Cmpd
28
b
2
b
(SD)
a
a
M
M
O
O
1
3
38
4.4
4.9
5.5
51
51
51
0.5
-0.3
-
N
H
(2.9)
23
(8.0)
7
3
>40
>40
N
H
O
74
>40
N
H
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Page 25 of 122
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
O
2.8
(1.1)
5.0
(1.4)
7
7
7
7
5
6
7
8
3.9
4.2
4.8
3.6
63
42
34
51
1.7
N
H
N
H
> 40
> 40
> 40
> 40
> 40
> 40
-
-
-
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H
H
N
O
a
EC data represents means and SDs for three or more independent experiments measuring
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
Calculated using the LTR EC value. 28 is included for comparison.
5
0
b
23
c
5
0
To improve the aqueous solubility and reduce overall cLogP of the 2-acylaminothiazole
series we next replaced the hydrophobic phenoxy aryl ring with differentially substituted
pyridines, concentrating on substitution in the meta position (Table 8) to mimic 3-chloro,
3
-trifluoromethyl or 3-nitrile phenoxy systems (6, 8, 10) that conferred to greatest LTR
activity (Table 1). Analogues 79-88 were generated with the endocyclic nitrogen in 2-, 4-,
- and 6- positions with either chloro, trifluoromethyl or nitrile in the 3-position. The 3-
5
chloro substituted analogues with the heterocyclic nitrogen in the 6-, 2- and 5- positions
(79, 80 and 82 respectively) displayed comparable LTR activity (EC 2.7, 4.7 and 2.9 µM)
5
0
to the non-endocyclic nitrogen ortholog 6 (EC 1.3 µM) while maintaining 10-fold greater
5
0
activity window over the CMV reporter (EC 34, 39 and >40 µM). The 3-chloro substituted
5
0
4
-endocyclic nitrogen derivative 81 exhibited no LTR reporter activity. The 3-
trifluoromethyl and 3-nitrile pyridinyl analogues (83-85 and 87-88) all displayed no LTR
activity, except for the 3-trifluoromethyl 5-endocyclic nitrogen derivative (86, LTR EC50
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Journal of Medicinal Chemistry
Page 26 of 122
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1 µM) with no observable CMV activity (EC >40 µM). This data demonstrated the SAR
5
0
between the 3-substituted phenoxy and the pyridinyl analogues was not entirely
conserved. Although cLogP of the pyridinyl analogue (79, 80 and 82) was improved
compared to phenoxy compound 6, the LipE was only notably enhanced in the case of 82.
Furthermore, aqueous solubility of the pyridinyl analogues 80 and 82 at both pH 2 and 6.5
was not significantly improved compared to the phenoxy comparator 6 (<1 µM) (Table 10).
Metabolism was also not improved with 80, but an improvement in intrinsic clearance was
observed with the pyridinyl analogue 82 (CLint human 86; mouse 340 μL/min/mg protein)
compared to 6 (CLint human 340; mouse >866 μL/min/mg protein) (Table 10). Overall,
incorporating a heterocyclic nitrogen in the phenoxy ring was not beneficial for LTR
activity and did not dramatically improve physicochemical properties, and therefore
integration of other pyridinyl or heterocyclic systems were not further considered.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 8. Activity of heterocyclic aryl analogues in the Flp.FM dual reporter cell assay.
LTR
EC50
CMV
EC50
2
cLog PSA (Å ) LipE
P
Cmpd
b
b
c
(
SD) M (SD) M
a
a
Cl
Cl
7
9
2.7 (0.9) 34 (6.8)
4.7 (0.3) 39 (2.0)
3.2
3.4
2.8
2.6
64
64
64
64
2.4
1.9
-
N
N
80
Cl
Cl
8
1
>40
>40
>40
N
82
2.9 (0.6)
2.9
N
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Page 27 of 122
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CF3
CF3
8
3
>40
>40
>40
3.5
3.9
3.3
2.9
2.5
1.9
64
64
64
64
88
88
-
-
N
N
84
>40
>40
>40
>40
>40
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CF3
CF3
8
5
>40
-
N
86
11 (2.4)
>40
2.1
-
N
CN
CN
8
7
N
88
>40
-
N
a
EC data represents means and SDs for three or more independent experiments measuring
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
compounds in a 10-point dilution series for 48 h. Calculated using ChemAxon software.
5
0
b
23
c
Calculated using the LTR EC value.
5
0
Earlier SAR established 3-chloro, 3-nitrile or 3-trifluoromethyl substitution on the
phenoxy aryl group was important for enhancing LTR activity (Tables 1 and 2). In addition,
chloro, nitrile or trifluoromethyl substitutions on the 5-position of the aminothiazole ring
also increased LTR activity (Table 3). To improve LTR activity we next examined
combining substituents that conferred the greatest LTR activity in the 5-position of the
thiazole and the 3-position of the phenoxy group (Table 9). The analogues generated (89-
9
7) all possessed EC values between approximately 1 and 4 µM for the LTR reporter except
5
0
for 92 and 95 which exhibited 30 and 10-fold selectivity over the CMV reporter. This data
indicates there were no significant improvements in LTR activity from combining the
substituents from the 3-phenoxy or the 5-thiazole positions compared to orthologs in Table
1
and 3. The majority of analogues (89-97) displayed CMV reporter activity comparable to
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Journal of Medicinal Chemistry
Page 28 of 122
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the LTR reporter. To improve LTR activity and selectivity, 3,4-disubstitution on the
phenoxy aryl group (from Table 2) was combined with both the 5-chloro and the 5-
trifluromethyl thiazole to give analogues 98-102 (Table 9). These analogues also displayed
EC values between approximately 1 and 4 µM for the LTR reporter. Analogues 98 and 101
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
displayed comparable LTR activity to the CMV reporter, but analogues 99, 100 and 102
exhibited a 20- to 10-fold increase in LTR activity compared to the CMV reporter. Overall, the
combination of substitution on the 5-position of the thiazole and the 3-substituted phenoxy aryl
moiety was not beneficial for LTR activity nor LTR selectivity compared to analogues
incorporating substitution only on the 3-position of the phenoxy group, such as 6 or 8 (Table
1
) or analogues incorporating substitution on the 5-position of the thiazole, such as 36 and 37
(Table 3). Furthermore, the physicochemical properties (cLogP, PSA), LipE and aqueous
solubility of the analogues with a combination of substituents were not improved compared
to their comparators (Table 10). However, metabolic stability in the presence of human and
mouse liver microsomes was significantly improved with the analogues 96 (CLint human
2
2; mouse 56 μL/min/mg protein) and 97 (CL human 30; mouse 78 μL/min/mg protein)
int
compared to the hit compound 1 and the other analogues in Table 10.
Table 9. Activity of combinations of substitution in the Flp.FM dual reporter cell assay.
Cmpd
LTR
EC50
CMV
EC50 cLogP
PSA
(Å )
LipE
R1
R3
R4
b
2
b
c
(
SD)
(SD)
a
a
M
M
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Page 29 of 122
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
CH3
Cl
H
H
H
24 (3.6) >40
3.2
4.0
51
51
1.4
1.9
1
(0.1)
0.9
.3
2.9
(0.1)
2.0
8
9
0
Cl
9
CN
CF3
Cl
Cl
Cl
H
H
3.2
4.2
3.2
2.4
3.5
4.2
3.5
4.5
4.6
4.5
4.5
4.7
75
51
75
99
75
51
75
51
51
51
51
51
2.8
1.3
2.7
3.0
2.6
1.0
2.0
1.2
1.0
1.2
1.1
0.7
(
0.3)
.0
1.1)
.4
(0.03)
.7
1.3)
.8
0.2)
.7
(0.3)
.1
0.6)
.2
0.6)
.6
(0.5)
.9
0.3)
.7
0.8)
.3
(0.7)
.6
0.9)
(1.0)
3.9
(2.1)
3
(
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
91
9
9
2
3
CN
CN
CN
CF3
CF3
CF3
Cl
H
>40
3
(
4.0
(2.8)
2.7
CN
CF3
Cl
H
0
(
4
94
H
(0.8)
9
9
5
6
H
>40
3
(
5.7
(3.2)
15
(6.0)
4.1
CN
CF3
Cl
H
2
(
2
97
H
9
9
8
9
Cl
Cl
CH3
Cl
(0.6)
1
(
Cl
CH3
Cl
>40
2
(
4
31.0
(6.6)
11.1
(4.9)
26
100
01
102
Cl
1
CF3
CH3
3
Br
CH3
Cl
4.7
51
0.8
(
(3.5)
a
EC data represents means and SDs for three or more independent experiments measuring
bioluminescence of in the HEK293 FlpIn.FM dual reporter cell line following exposure to
compounds in a 10-point dilution series for 72 h. Calculated using ChemAxon software.
5
0
b
23
c
Calculated using the LTR EC value. 1 is included for comparison.
5
0
Table 10. Physicochemical and metabolism properties of selected compounds.
Solubility a
Human liver microsomes
Mouse liver microsomes
Cmpd
pH 6.5
(µM)
pH 2.0 Half- in vitro Predicte Half-
in vitro
CLint
Predicte PSA cLog
d EH
b
b
2
c
c
(µM)
life
CLint
min (μL/min
d EH
life
(Å )
P
(
(min (μL/min/
)
)
/mg
protein)
mg
protein)
ACS Paragon Plus Environment