Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure-Activity Relationship and Target Identification Studies

Article abstract of DOI:10.1021/acs.jmedchem.7b01347

A BioFocus DPI SoftFocus library of ～35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc₁ complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG-3193 and BCG-3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.

Full text of DOI:10.1021/acs.jmedchem.7b01347

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Article
Novel Anti-tubercular 6-Dialkylaminopyrimidine Carboxamides from
Phenotypic Whole-Cell High Throughput Screening of a SoftFocus
Library: Structure Activity Relationship and Target Identification Studies
Colin R. Wilson, Richard K. Gessner, Atica Moosa, Ronnett Seldon, Digby F. Warner, Valerie
Mizrahi, Candice Soares de Melo, Sandile B. Simelane, Aloysius T Nchinda, Efrem Abay, Dale
Taylor, Mathew Njoroge, Christel Brunschwig, Nina Lawrence, Helena I. M. Boshoff, Clifton E. Barry,
Frederick A. Sirgel, Paul van Helden, C. John Harris, Richard Gordon, Sonja Ghidelli-Disse, Hannah
Pflaumer, Markus Boesche, Gerard Drewes, Olalla Sanz, Gracia Santos, Maria José Rebollo-
Lopez, Beatriz Urones, Carolyn Selenski, Maria Jose Lafuente-Monasterio, Matthew Axtman, Joël
Lelièvre, Lluis Ballell, Rudolf Mueller, Leslie J. Street, Sandeep R. Ghorpade, and Kelly Chibale
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01347 • Publication Date (Web): 17 Nov 2017
Downloaded from http://pubs.acs.org on November 18, 2017
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Santos, Gracia; Cellzome GmbH
Rebollo-Lopez, Maria José; GlaxoSmithKline, Diseases of the Developing
World
Urones, Beatriz; GlaxoSmithKline, Diseases of the Developing World
Selenski, Carolyn; GLAXOSMITHKLINE MEDICINES RESEARCH CENTRE
Lafuente-Monasterio, Maria; GlaxoSmithKline, Diseases of the Developing
World
Axtman, Matthew; GlaxoSmithKline, Chemical Biology
Lelièvre, Joël; GlaxoSmithKline, Diseases of the Developing World
Ballell, Lluis; GlaxoSmithKline - Director of Diseases of the Developing
World (DDW), - Tres Cantos (Madrid), Spain, Diseases of the Developing
World
Mueller, Rudolf ; University of Cape Town, Institute of Infectious Disease
and Molecular Medicine
Street, Leslie; University of Cape Town, H3-D centre, Department of
hemistry
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Ghorpade, Sandeep; University of Cape Town, H3-D centre, Department of
hemistry
Chibale, Kelly; University of Cape Town, Department of Chemistry
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Novel Antiꢀtubercular 6ꢀDialkylaminopyrimidine
Carboxamides from Phenotypic WholeꢀCell High
Throughput Screening of a SoftFocus Library:
Structure Activity Relationship and Target
Identification Studies
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a,
a‡
c
a,d
c,d
Colin R. Wilson , Richard K. Gessner , Atica Moosa , Ronnett Seldon , Digby F. Warner ,
c,d
a
a
a
Valerie Mizrahi , Candice Soares de Melo , Sandile B. Simelane , Aloysius Nchinda , Efrem
b
b
b
b
b
Abay , Dale Taylor , Mathew Njoroge , Christel Brunschwig , Nina Lawrence , Helena I. M.
e
e
f
f
g
Boshoff , Clifton E. Barry III , Frederick A. Sirgel , Paul van Helden , C John Harris , Richard
h
i
i
i
i
Gordon , Sonja Ghidelli-Disse , Hannah Pflaumer , Markus Boesche , Gerard Drewes , Olalla
j
j
j
j
j
Sanz , Gracia Santos , Maria José Rebollo-Lopez , Beatriz Urones , Carolyn Selenski , Maria
j
j
j
j
a
Jose Lafuente-Monasterio , Matthew Axtman , Joël Lelièvre , Lluis Ballell , Rudolf Mueller ,
a
a
a,d,k
Leslie J. Street , Sandeep R. Ghorpade ,* and Kelly Chibale
*
a
Drug Discovery and Development Centre (H3D), Department of Chemistry, University of Cape
b
Town, Rondebosch 7701, South Africa; Drug Discovery and Development Centre (H3D),
Division of Clinical Pharmacology, Department of Medicine, University of Cape Town,
c
Observatory, 7925, South Africa; SAMRC/NHLS/UCT Molecular Mycobacteriology Research
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Unit, Department of Pathology, University of Cape Town, Rondebosch 7701, South Africa;
d
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch
e
7701, South Africa; Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases;
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National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
f
Maryland 20892, United States; DST/NRF Centre of Excellence for Biomedical TB Research,
SA MRC Centre for TB Research, Division of Molecular Biology and Human Genetics, Faculty
g
of Health Sciences, Stellenbosch University, Tygerberg, South Africa; CJH Consultants, Ford
h
Cottage, South Weirs, Brockenhurst, Hampshire SO42 7UQ, UK, United Kingdom; Strategic
Health Innovation Partnerships (SHIP), South African Medical Research Council, Parow Valley,
i
South Africa; Cellzome GmbH, Molecular Discovery Research, GlaxoSmithKline,
j
Meyerhofstrasse 1, Heidelberg, Germany; Diseases of the Developing World, GlaxoSmithKline,
k
Calle Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain; South African Medical Research
Council Drug Discovery and Development Research Unit, Department of Chemistry, University
of Cape Town, Rondebosch 7701, South Africa
ABSTRACT: A BioFocus DPI SoftFocus library of ~35,000 compounds was screened against
Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antiꢀtubercular activity.
The hits were evaluated in biology triage assays to exclude compounds suggested to function via
frequently encountered promiscuous mechanisms of action including inhibition of the QcrB
subunit of the cytochrome bc complex, disruption of cellꢀwall homeostasis, and DNA damage.
1
Among the hits which passed this screening cascade, a 6ꢀdialkylaminopyrimidine carboxamide
series was prioritized for hit to lead optimization. Compounds from this series were active
against clinical Mtb strains while no crossꢀresistance to conventional antiꢀtuberculosis drugs was
observed. This suggested a novel mechanism of action, which was confirmed by
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chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative
targets of the series with unknown function. Initial structureꢀactivity relationship studies have
resulted in compounds with moderate to potent antiꢀtubercular activity and improved
physicochemical properties.
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INTRODUCTION
Tuberculosis (TB) is one of the world’s most deadly infectious diseases, leading to 1.4 million
1
deaths in 2015. The disease is caused by Mycobacterium tuberculosis (Mtb) and primarily
affects lower to middle income countries such as those in subꢀSaharan Africa. A standard firstꢀ
line 4ꢀdrug regimen of rifampin, isoniazid, pyrazinamide, and ethambutol is used to treat drugꢀ
2
susceptible TB. Although these drugs form the core of TB treatment regimens, they have
become ineffective against resistant Mtb strains. TB that does not respond to at least isoniazid
and rifampin, the two frontline drugs, is defined as multiꢀdrug resistant (MDR) TB. MDR TB
that develops additional resistance to any fluoroquinolone and at least one of the three injectable
secondꢀline drugs (amikacin, kanamycin, or capreomycin) is referred to as extensively drug
3
resistant (XDR) TB and is virtually untreatable. One of the main drivers behind the rise of
resistance is the 6ꢀmonth long treatment required for drugꢀsensitive disease, resulting in poor
patient adherence. Thus, the main goal of many TB drug discovery programs is the identification
of compounds that will potentially contribute to treatment shortening; albeit there is no clear
4
evidence as to how this can be accomplished. One possible approach is to identify compounds
that inhibit novel targets, and do not show crossꢀresistance to currently used antiꢀTB drugs.
Various novel drug combinations can then be evaluated for their potential towards treatment
shortening while establishing clinical effectiveness. Identifying compounds which exert their
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antiꢀTB effect through novel targets is not a trivial process. This is exemplified by the approval
in 2012 of bedaquiline, the first new drug approved for the treatment of TB in more than 40
years with a novel mechanism of action (MOA) that involves inhibition of the mycobacterial
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ATP synthase.
Two hitꢀgeneration approaches that have been traditionally used are targetꢀbased and wholeꢀ
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cell screening. Historically, there has been extremely limited success with the targetꢀbased
approach owing to the lack of translation from target activity to wholeꢀcell activity.
Nevertheless, efforts to discover and validate novel drug targets using various approaches must
continue. This is exemplified by the recently reported validation of CoaBC as a bactericidal
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target using a chemical biology approach. On the other hand, a more successful approach has
been phenotypic wholeꢀcell highꢀthroughput screening (HTS) of chemical libraries to identify
novel chemotypes whose target is then identified retrospectively. There are several compounds
in the TB drug pipeline that have been identified through wholeꢀcell HTS. Examples include
Q203 (Figure 1), an inhibitor of the QcrB subunit of the cytochrome bc complex which is
1
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involved in electron transport. Another example is SQ109 (Figure 1) whoseprecise MOA
remains to be elucidated but is believed to involve the Mycobacterial Membrane Protein Large 3,
1
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MmpL3, a mycolic acid transporter involved in cell wall synthesis,.
Similarly, some
14
inhibitors of decaprenylphosphorylꢀβꢀDꢀribose 2′ꢀepimerase (DprE1) e.g. pBTZ, and azaindoles
1
5,16
TBA7371
are poised to enter clinical trials (Figure 1). While these compounds show promise
as new antiꢀTB agents, their targets are yet to be clinically validated. This necessitates the need
to continuously identify new chemical matter with alternate novel MOAs towards populating the
TB drug pipeline.
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Figure 1. New TB drug Development candidates identified from whole cell screening
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In line with this, we initiated a HTS campaign using a diverse SoftFocus™ compound library
acquired from BioFocus and complemented this with a biology triage process that aimed
specifically to exclude compounds acting on targets such as QcrB and the cell wall (e.g. MmpL3
and DprE1), as well as DNA damaging agents. The rationale was to exclude targets known for
18,19
their promiscuity, with a large proportion of HTS hits having these as part of their MOA.
There is also the question around whether or not compounds targeting cell wall metabolism can
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contribute to the much sought after treatment shortening. Compounds that induce DNA damage
were also excluded in order to avoid general cytotoxicity, as well as targets such as DNA gyrase
whose clinically used inhibitors are exemplified by the fluoroquinolones. Along with an
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aminopyrazolo[1,5ꢀa]pyrimidine chemical series , a 6ꢀdialkylaminopyrimidine carboxamide
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scaffold was identified as a hit series with a potentially novel MOA, based on the initial biology
triage process (Figure 2). Herein, we describe the synthesis, structure activity relationship (SAR),
in vitro absorption, distribution, metabolism and excretion (ADME), in vivo pharmacokinetic
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(PK) and biological profiles of this chemical series.
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H9/ADC MIC99
20 µM
10 µM
20 µM
>20 µM
20 µM
287 µM
<5 µM
4.07
GAST/Fe MIC99
∆
∆
cyd GAST/Fe MIC99
cyd 7H9 GLU ADC MIC99
qcrBA317T GAST/ Fe MIC99
Vero IC50
Kinetic Solubility (PBS pH 7.4)
cLogP
Figure 2. Compound 1 and biology triage data.
RESULTS AND DISCUSSION.
Phenotypic whole-cell HTS and biology triage.
A screen of a BioFocus DPI SoftFocus library of ~35,000 compounds against virulent Mtb
H37Rv conducted at the National Institute of Allergy and Infectious Diseases of the National
Institutes of Health (NIAID/NIH, USA) led to a number of confirmed hits, which included
compound 1 with a moderate Minimum Inhibitory Concentration (MIC) value of 20 ꢀM in
albuminꢀdextrose complex (ADC) medium containing ~0.4% Bovine serum albumin (BSA). The
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compound was slightly more potent in GASTꢀFe (Glycerolꢀalanineꢀsalts) minimal medium,
likely driven by the absence of BSA which allowed for higher free concentration of the
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compound under the assay conditions. Hence, GASTꢀFe was used alongside the 7H9/ADC
medium during further SAR exploration of the series. Compound 1 was tested in the three
biology triage assays in order to assess activity against promiscuous targets. The compound did
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not show hypersensitivity against a cytochrome bd oxidase knockout mutant strain (cydKO)
A317T
and also retained activity against a QcrB mutant (qcrB
), thereby eliminating this as potential
target. Compound 1 also did not elicit a positive response in two standard bioluminescence
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reporter assays: PiniBꢀLUX , which is designed to detect compounds targeting Mtb cell wall
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biosynthesis, and PrecAꢀLUX which allows detection of genotoxic compounds (data in
supporting information). Similar results were obtained with other analogues in the series
obtained during SAR studies (data in supporting information). These results, along with
chemoproteomics and crossꢀresistance studies (see biology results below), indicate that the 6ꢀ
dialkylaminopyrimidine carboxamide series of compounds exemplified by compound 1
possesses a novel MOA that is not shared with known TB drugs.
SAR exploration strategy. Compound 1 was identified as a suitable hit with a moderate
minimum inhibitory concentration (MIC ) of 20 µM in 7H9/ADC medium, low toxicity against
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the mammalian Vero cell line (IC of 287 µM) yet poor kinetic solubility (KS, < 5 µM). The
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poor solubility was assumed to be related to the high lipophilicity and flat aromatic character of
the molecule. Since there were very few active near neighbors identified from the screen, very
limited information was available on further optimization scope with the scaffold to improve
antiꢀTB activity while addressing the poor physicochemical properties and, potential safety
issues, which may be associated with the scaffold. A detailed SAR plan as summarized in figure
3
was embarked upon. The molecule was divided into three parts for convenience, the central
pyrimidine core, rightꢀhand side (RHS) amide and leftꢀhand side (LHS) hydrophobic Nꢀbenzyl
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moiety (Figure 3). The major SAR exploration focus was on understanding key hydrogen bond
(HB) donorꢀacceptor interactions critical for activity, shape and size of the central core, and
scope for substitution/modification on either the RHS or LHS parts of the molecule to improve
potency. The scope for addition of heteroatoms or polar groups into the molecule was also
explored with the aim of reducing lipophilicity along with addressing related issues such as poor
physicochemical properties and structure alerts such as the presence of potentially AMES
positive anilines upon cleavage of the amide group.
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Figure 3: SAR exploration strategy
Synthesis. Compounds 1ꢀ20 were synthesized to explore the SAR around the Nꢀmethylꢀ1ꢀ
phenylmethanamine motif (SAR on LHS, Table 1), compounds 21ꢀ30 were synthesized to
explore the central pyrimidine core of the scaffold (SAR on central core, Table 2), and
compounds 31ꢀ54 were synthesized to explore the SAR around the amide portion of the scaffold
(SAR on RHS, Table 3). In general, target compounds were synthesized using either one of two
routes. Scheme 1 was used to synthesize an advanced intermediate with a specific amide in place
allowing for the exploration of the LHS of the molecule. The same route was also used to
synthesize several of the core modifications. The route involved a straightforward 2ꢀstep
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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
procedure starting from a chloroꢀheterocyclicꢀcarboxylic acid, where coupling with
corresponding
amine
using
1ꢀ[Bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀ
b]pyridinium 3ꢀoxide hexafluoroꢀphosphate (HATU) was first performed, followed by
nucleophilic displacement of the relevant chloro group by an appropriate amine. Thus, synthesis
of compounds 1ꢀ20 and 39 (Table 1) was achieved by coupling of 6ꢀchloropyrimidineꢀ4ꢀ
carboxylic acid with aniline, 4ꢀmethoxyaniline or 4ꢀaminopyridine to give intermediates 55, 56
and 57 respectively, followed by displacement of 6ꢀchloro group with various amines.
Compound 21 was synthesized by coupling of 4ꢀchloropicolinic acid with aniline to form
intermediate 58 followed reaction with 1ꢀ(4ꢀfluorophenyl)ꢀNꢀmethylmethanamine.
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
Scheme 1. General route for the synthesis of 6-Amino-pyrimidine-4-carboxamides as well
as alternative cores.
Reagents and conditions: (i) Amine, DIPEA, HATU, DMF, 25°C (ii) Amine, iPrOH, DIPEA,
1
00 °C
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Scheme 2. Synthetic routes used to access core modifications
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
Reagents and conditions: (i) 1ꢀ(4ꢀfluorophenyl)ꢀNꢀmethylmethanamine, Et N, Dioxane, 60 °C
3
(
ii) Aniline, Mo(CO) , Et N, TBAC, Toluene, Tetraglyme, 150 °C (iii) a) POCl , 110 °C b)
6
3
3
Aniline, Et N (iv) 4ꢀFluorobenzylamine, Et N, Dioxane, microwave, 125 °C (v) 4ꢀ
3
3
Fluorobenzylamine, H O:EtOH (1.5:1), 25°C (vi) LiOH, MeOH (vii) Aniline, HATU, DMF,
2
Et N (viii) 4ꢀFluorobenzaldehyde, NaBH , EtOH (ix) MeI, K CO , DMF.
3
4
2
3
Synthesis of compounds 22ꢀ26 (Table 2) with changes in the central core ring are summarized in
scheme 2. Compound 22 was synthesized by nucleophilic displacement of 59 with 1ꢀ(4ꢀ
fluorophenyl)ꢀNꢀmethylmethanamine followed by carbamoylation according to the procedure
2
4
described by Ren et al. Compound 23 was synthesized in a similar manner except the initial
nucleophilic displacement step of 60 resulted in a mixture of the intended 4ꢀchloroꢀNꢀ(4ꢀ
fluorobenzyl)ꢀNꢀmethylpyrimidinꢀ2ꢀamine (61) as well as the 2ꢀchloro regioisomer in a 1:2 ratio.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The pyridazine core replacement analogue was synthesized by heating 6ꢀoxoꢀ1,6ꢀ
dihydropyridazineꢀ4ꢀcarboxylic acid with POCl3 followed by quenching of the 6ꢀ
chloropyridazineꢀ4ꢀcarbonyl chloride intermediate with aniline to afford 63. Intermediate 63 was
subsequently subjected to nucleophilic displacement with 1ꢀ(4ꢀfluorophenyl)ꢀNꢀ
methylmethanamine under microwave heating to deliver the final compound 24. The pyridone
analogue (25) was synthesized from the commercially available precursor, methyl coumalate
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
(64), which was reacted with 4ꢀfluorobenzylamine at room temperature (25 °C ) to give the Nꢀ
substituted pyridinoneꢀester (65). The ester was then hydrolyzed using LiOH followed by an
amide coupling to afford 25. The thiazole analogue 26 was synthesized by reductive amination
of methyl 2ꢀaminothiazoleꢀ4ꢀcarboxylate 66 with 4ꢀfluorobenzaldehyde and NaBH , followed by
4
methylation under basic conditions to afford 67. Intermediate 67 was then subjected to the same
hydrolysis and amide coupling conditions as 65, to afford 26.
(x) a) KOH, EtOH, b) HCl, (ii) SOCl , DMF (iii) 1ꢀ(4ꢀfluorophenyl)ꢀNꢀmethylmethanamine,
2
K CO , DMF (iv) LiOH, H O:THF (2:1), 25 °C (v) HATU, DIPEA, DMF, 25 °C (vi) (COCl) ,
2
3
2
2
EtOAc, Aniline (vii) 1ꢀ(4ꢀChlorophenyl)ꢀNꢀmethylmethanamine, iPrOH, DIPEA, 100 °C.
The 2ꢀ and 5ꢀmethylpyrimidine analogues 27 and 28 (Table 2) were synthesized in a similar
fashion by condensation of the appropriate 1,4ꢀdiethyl ester and imidamide 68 and 69 to form the
corresponding dihydropyrimidine carboxylic ester and acid intermediates, 70 and 71,
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respectively. Intermediate 70 was chlorinated using SOCl2 followed by nucleophilic
displacement of the 6ꢀchloro with 1ꢀ(4ꢀfluorophenyl)ꢀNꢀmethylmethanamine delivered 72, which
was then hydrolyzed under standard basic conditions followed by an amide coupling with HATU
to produce 27. Intermediate 71 was subjected to chlorination of both the hydroxyl and carboxylic
acid groups using oxalyl chloride. The reaction was then quenched with aniline to afford 73
which was subsequently aminated with 1ꢀ(4ꢀchlorophenyl)ꢀNꢀmethylmethanamine to afford 28.
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
Scheme 3. General route for the synthesis of 6-Amino-pyrimidine-4-carboxamides as well
as selected core modifications.
o
Reagents and conditions: (i) RꢀCH NHCH , THF, 0 C (ii) 10% Pd/C, H , MeOH (iii) LiOH,
2
3
2
MeOH
(iv)
Amine,
DIPEA,
HATU,
DMF
(v)
LiOH,
THF:H
O (1:1) (vi) NH or cyclopropylamine, iꢀPrOH, Et N, 100 °C
3
3
In order to access an advanced intermediate that would allow for exploration of the RHS of the
molecule (31, 33ꢀ38, 40-52), another relatively straightforward approach (Scheme 3), involving
4
steps from the commercially available methyl 2,4ꢀdichloropyrimidineꢀ6ꢀcarboxylate was used.
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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
Briefly, quantitative amination by selective displacement of the 6ꢀchloro group using an
o
appropriate Nꢀmethylꢀ1ꢀphenylmethanamine at 0 C in THF, followed by hydrogenation with
10% Pd/C and subsequent hydrolysis using LiOH in methanol afforded the carboxylic acid (78ꢀ
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
8
0). Lastly, amide coupling with appropriate amines using HATU gave the desired carboxamide
final compound. The intermediate as represented by 74 could be utilized to access 2ꢀamino
substiuted analogues such as 29 and 30. Intermediate 74 was hydrolyzed using LiOH in
THF/MeOH (1:1) followed by amide coupling with HATU, and substitution of the 2ꢀchloro
group with an appropriate amine through heating in 2ꢀpropanol in the presence of triethylamine
in a sealed tube, affording compounds 29 and 30.
Scheme 4. Synthetic schemes used to access reverse amide and amide isosteres.
o
Reagents and conditions: (i) BzCl, DIPEA, THF, RTꢀ50 C (ii) 1ꢀ(4ꢀFluorophenyl)ꢀNꢀ
methylmethanamine, iꢀPrOH, Et N, 100 °C (iii) HATU, Et N, N,Oꢀdimethylhydroxylamine,
3
3
DMF, RT (iv) a) TMSꢀCF , Toluene, 20% CsF b) TBAF, H O, 50 °C (v) 4ꢀFluroaniline,
3
2
NaCNBH , Et N, TiCl , MeOH, DCM (50:50), N (vi) Benzonitrile, K CO , BuOH, 120 °C.
3
3
4
2
2
3
The reverse amide 32 was synthesized by reacting 6ꢀchloropyrimidinꢀ4ꢀamine, 82 with benzoyl
chloride in the presence of a base followed by nucleophilic substitution of the 6ꢀchloro with 1ꢀ(4ꢀ
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fluorophenyl)ꢀNꢀmethylmethanamine (Scheme 4). The trifluoromethylketone, 83, was
synthesized from the free acid, 78, via formation of a Weinreb amide followed by
2
5
trifluoromethylation using TMSꢀCF . Reductive amination using NaCNBH in the presence of
3
3
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
TiCl then afforded compound 53. Finally, triazolyl compound 54 was synthesized by heating
4
the hydrazide 84 with benzonitrile in refluxing butanol in the presence of potassium carbonate.
SAR at LHS. After ascertaining that the 4ꢀmethoxy substituent of the phenyl carboxamide was
not crucial for activity (Table 1; compound 2; R1 = H) the LHS (Table 1) of the scaffold was
varied to identify the key pharmacophore needed for activity whilst keeping the central
pyrimidine core unchanged and the RHS constant as 4ꢀmethoxyphenyl or phenyl.
Table 1: SAR on the LHS
Compou
nd
R
R1
MIC99
7H9/A GAST/Fe
MIC99
Solubility Cytotoxicity
a
b
(µM)
(µM)
DC
(µM)
(µM)
1
OMe 20
10
10
40
<5
287
2
3
4
H
H
H
20
<5
314
178
ND
>50
>160
ND
ND
ND
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
6
H
H
>160
80
ND
ND
<5
<5
ND
ND
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
7
H
>160
ND
<5
ND
8
9
1
1
1
1
H
H
H
H
H
>160
>50
6.25
5
>160
40
<5
5
ND
272
297
207
258
272
0
1
2
3
1.56
2.3
5
<5
<5
<5
5
5
OMe 37
5
1
4
H
H
20
10
<5
<5
ND
ND
15
>160
ND
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1
1
6
7
H
H
>50
20
<5
10
ND
ND
c
c
>125
>125
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
8
H
H
H
>160
ND
ND
150
<5
ND
ND
258
c
c
19
48
37
2
0
37
10
a
b
c
Kinetic solubility at pH 7.4; IC on Vero cell lines; MIC from GFP strain based assay; ND –
50
99
Not determined
Replacement of the aryl ring with a saturated cyclohexyl ring (compound 3) led to considerable
loss of activity, suggesting essentiality of an aromatic ring on the LHS. Similarly, replacement of
the NꢀCH linker with “O” or “NH” led to inactive compounds 4 and 5 respectively, suggesting
3
the critical importance of a tertiary nitrogen at the linker position or intolerance toward more
polar “O” or “NH” groups. Furthermore, linker modifications such as addition of a methylene
group (Nꢀmethylphenethyl, 6) or removal of CH (Nꢀmethylaniline, 7), cyclization of the N onto
2
the phenyl ring as tetrahydroisoquinoline moiety (8) or extension of Nꢀmethyl to Nꢀethyl (9), all
led to loss or deterioration of MICs. This all indicated stringent shape requirements at this
presumably hydrophobic part of the molecule.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Realizing the essential nature of the aryl ring and the linker, we focused efforts on exploring
substitution on the phenyl ring to improve potency and introduce polarity. Paraꢀhalogenated
compounds, 10 (4ꢀF), 11 (4ꢀCl) and 12 (4ꢀCF ) were at least ≥ 4ꢀfold and ≥ 2ꢀfold more potent in
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
7
H9/ADC and GAST/Fe, respectively, compared to 1. This was encouraging and the first sign of
a SAR trend emerging, and leading to the conclusion that a paraꢀhalogenated phenyl ring gave
the best activity with 4ꢀF phenyl compound 10 showing the most promise. Compounds with 3ꢀF
(13) and 3ꢀCl (14) were less potent compared to corresponding 4ꢀhalogenated analogues whereas
the 2ꢀCl analogue (15) was inactive up to the highest concentration tested, once again suggesting
stringent shape requirements on the LHS. Addition of more polar groups such as 4ꢀCN (16) and
4
ꢀSO Me (17) led to deterioration or loss of MICs.
2
Up to this point, all compounds tested had poor aqueous solubility of ≤ 5 µM. In an attempt to
improve solubility by introducing polarity, the phenyl ring was replaced with a pyridyl ring.
Compound 18 with a 2ꢀpyridyl ring was found to be inactive whereas compound 19 with a 3ꢀ
pyridyl ring was equipotent to compound 1 albeit with a much improved aqueous solubility of
1
4
50 µM at pH 7.4. Further substitution of the 3ꢀpyridyl ring as in compound 20 with a 3ꢀpyridylꢀ
ꢀtrifluoromethyl group retained similar activity, but now with loss of solubility albeit this
substitution improved the in vivo pharmacokinetic properties as will be discussed in the DMPK
section.
SAR at the central core: Exploration of the SAR around the central core of the scaffold was
aimed at understanding whether or not the 4,6–disubstituted pyrimidine was essential for activity
whilst retaining a 4ꢀhalo group on the LHS phenyl ring and a 4ꢀmethoxy, halogen or hydrogen
on the RHS phenyl ring. Initial efforts looked at shuffling the nitrogen atoms around the core
ring, including the pyridine (21), pyrazine (22), and pyrimidine (23), with one of the nitrogen
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atoms shifted between the aminobenzyl and carboxamide groups, and the pyridazine (24),
analogues. Only the pyridine retained partial activity at 10 µM in the GAST/Fe medium, which
was not sufficient to warrant a scaffold hop. Compounds based on the pyrimidinone isostere
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
(25), and thiazole (26) all lost activity. Lastly 5ꢀ, and 2ꢀsubstitutions (27, 28, 29 and 30) on the
pyrimidine core were evaluated and found to be similarly inactive. It was thus concluded that the
original pyrimidine core was optimal for activity and could not be replaced with other diazine
and heteroaromatic rings.
Table 2: SAR on the central core
Com R
poun
d
R1
Core
change
MIC99 MIC99 Solubility Cytotoxicity
a
b
7H9/
ADC /Fe
µM) (µM)
GAST (µM)
(µM)
(
2
1
H
ND
>50
>50
>50
10
<5
<5
<5
40
298
22
F
>50
>50
>50
282
273
279
2
3
OMe
H
24
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
5
6
H
H
>50
>50
>50
>50
170
<5
310
279
F
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
7
F
>50
37
<5
271
ND
ND
ND
28
H
>160 >160 ND
2
3
9
0
OMe
OMe
>50
>50
>160 <5
>160
5
a
b
Kinetic solubility at pH 7.4; IC on Vero cell lines; ND – Not determined
5
0
SAR on the RHS: An extensive SAR exploration on the RHS was undertaken in order to
evaluate the scope for optimization of potency and physicochemical properties (Table 3). The
free amide NH was deemed critical for potency as Nꢀmethylation (31) led to a complete loss in
activity. Similarly, reversal of the amide functionality led to inactive compound 32. For further
exploration of substitution on the aryl ring of the amide, a 4ꢀhalophenyl or 4ꢀCF ꢀ3ꢀpyridyl was
3
fixed on the LHS of the scaffold. Replacement of the phenyl ring with an aliphatic chain such as
isopropyl (33) was detrimental to activity, while replacement with a saturated cyclohexyl ring
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(
34) resulted in retention of activity in the GAST/Fe medium albeit it was completely inactive in
7
H9/ADC. The overall trend suggested a preference for an aryl ring on the RHS. Since the
original hit contained a methoxy group, additional ethers were explored in the para position,
including isopropoxy (36), 2ꢀmethoxyethoxy (37), and a N,Nꢀdimethylꢀ2ꢀethoxy ether (38).
Encouragingly, compounds 36 and 37, were some of the most active compounds synthesized up
to this point, but were poorly soluble (solubility pH 7.4 ≤5 ꢀM). Compound 38 with the basic
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
dimethylaminoethoxy sideꢀchain (predicted pKa 8.77) was less active (7H9/ADC MIC 25 ꢀM)
even though it showed considerably improved aqueous solubility of 155 µM. Nonetheless, these
results suggested scope for substitutions on the aryl ring to modulate physicochemical properties
whilst retaining potency. Replacement of the phenyl ring with a 4ꢀpyridyl ring, as in compound
3
9, was tolerated indicating a wider scope for SAR exploration at this position with heterocycles
and weakly basic substituents. Addition of a less basic Nꢀmethylpiperazine ring at the 4ꢀposition
of the phenyl ring (predicted pKa 7.85) led to potent compound 40 which was poorly soluble at
pH 7.4 but sufficiently soluble at a slightly acidic pH of 6.5 (refer DMPK section). Solubility
could be improved further by combining Nꢀmethylpiperazinylphenylamide on the RHS with a 4ꢀ
CF ꢀ3ꢀpyridyl group on the LHS as in compound 41 with some loss in potency (MIC 12.5 ꢀM,
3
solubility pH 7.4 165 ꢀM). Moving the Nꢀmethylpiperazine ring to the 3ꢀposition of the phenyl
ring (42) was detrimental to activity with MIC values dropping by 10ꢀ30 fold. Similarly, 3ꢀ
pyridylꢀ4ꢀ(Nꢀmethylpiperazinyl) analogue 43 was found to be much less potent (7H9/ADC MIC
>
50 ꢀM; GAST/Fe MIC 19 ꢀM). Compounds with neutral substituents at the 4ꢀposition of the
phenyl ring like Nꢀacetylpiperazinyl (44) or, morpholino (compound 45), retained good MICs
<5 ꢀM) but were much less soluble. Interestingly, the 4ꢀmorpholinoꢀ2ꢀpyridyl analogue (46)
(
retained good MIC activity (2ꢀ3 ꢀM) indicating scope for the introduction of a nitrogen into the
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5
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phenyl ring to address potential reactivity issues of electronꢀrich anilines during further
optimization.
A small set of amides with 5ꢀmembered heterocycles was synthesized to evaluate the scope for
further SAR exploration at this position. Compounds with Nꢀmethylpyrazole (47), Nꢀ
methyltriazolyl (48) and isoxazolyl (49) substituents were either inactive or weakly active.
Compound 50 containing a 4ꢀmethylthiazolyl group showed a GAST MIC equivalent to its
phenyl counterpart (5 ꢀM). However, its MIC in 7H9/ADC medium (37 ꢀM) was weaker and
0
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9
0
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9
0
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9
0
had a poor solubility (<5 ꢀM). Similarly, compounds containing 1,3,4ꢀthiadiazole moieties (51
and 52) showed reasonable MIC in GAST/Fe medium (~5 ꢀM), but had weaker potency in
7
H9/ADC medium (12.5 and 50 ꢀM respectively). Unfortunately compounds with these moieties
were cytotoxic in vitro and were also found to be toxic at higher doses in animal studies. Hence,
these modifications were abandoned even though more leadꢀlike DMPK properties were
achieved with these compounds.
2
6
27
Lastly, two amide isosteres CF ꢀamine (53) and triazole (54) were synthesized in an attempt
3
to explore the scope for variation and in line with metabolite identification studies
(supplementary information Table S1). Metabolite identification, performed on 12 and 51,
indicated cleavage of the amide bound on the RHS as a primary route of metabolism compared
to Nꢀdemethylation on the LHS. Even though both these modifications resulted in compounds
showing some activity, there was no improvement in other properties like solubility and hence,
were not explored further. Overall, as summarized in Figure 4, the SAR around the LHS and the
central core was narrow with fewer modifications tolerated; SAR around the RHS was more
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dynamic allowing scope for further optimization. From this, several compounds were identified
for further profiling both in vitro and in vivo.
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: SAR on the RHS (R)
Compound Y
X R
MIC99 MIC99
Solubility Cytotoxicity
a
b
7
H9/ GAST/Fe (µM)
ADC (µM)
µM)
(µM)
(
3
1
H
F
C
C
>160 ND
ND
ND
32
N/A
>50
>160
5
ND
3
3
3
3
4
5
CF
CF
F
3
C
C
C
>160
13
5
217
ND
c
c
>125 12
3
5
5
2.5
1.2
62
60.48
254*
3
6
F
C
5
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4
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5
5
5
5
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3
3
7
8
F
F
C
C
2.3
1.56
12.5
<5
244
ND
25
155
0
1
2
3
4
5
6
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8
9
0
1
2
3
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5
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9
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8
9
0
1
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8
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0
1
2
3
4
5
6
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8
9
0
3
4
9
0
Cl
C
C
ND
10
<5
<5
282
161
CF
3.13
1.25
3
41
CF
F
3
N
C
12.5
37
12.5
37
165
74
45.2
118
4
4
2
3
F
C
N
>50
4.7
19
150
<5
126
>50
44
CF
3
2.3
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F
C
N
0.78
0.60
2.3
<5
<5
237
211
CF
3
3.13
1
1
1
1
1
1
1
1
1
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2
2
2
2
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2
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4
4
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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
4
4
7
8
F
C
C
C
C
>50
50
40
10
160
5
90
ND
28
CF
F
173
ND
<5
3
49
>50
37
ND
ND
5
5
0
1
F
F
C
N
C
12.5
50
4.7
5
128
191
<5
21.9
28
52
CF
3
5
3
4
F
37
19
41.7
5
F
C
>50
25
<5
277
a
b
c
Kinetic solubility at pH 7.4; IC on Vero cell lines; MIC from MtbꢀGFP strain based assay
ND – Not determined
5
0
99
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2
2
2
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2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4: Summary of SAR
Biology profiling. A spontaneous resistant mutant of Mtb H37Rv was successfully raised against
compound 13. Most of the analogues from the series were crossꢀresistant against this mutant
except the thiadiazole analogues 50 and 51, indicating additional targets and/or a different MOA
(Table 4). Additionally, these mutants were not crossꢀresistant to any known TB drugs
suggesting a potential novel MOA for the series. Unfortunately, whole genome sequencing of the
mutant did not reveal any genetic polymorphism(s) suggestive of the target or MOA. Hence,
further studies using different target identification approaches were explored in order to identify
the molecular target of these compounds. Representative compounds were screened against drugꢀ
sensitive clinical isolates and were found to retain MICs similar to those in H37Rv (Table 5).
Table 4: Crossꢀscreening against 13ꢀresistant mutant
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4
day GAST/Fe 14 day GAST/Fe MIC99
MIC99 (µM) against (µM)
WT H37Rv (ꢀM)
Against
13ꢀ
resistant mutant (ꢀM)
1
20
10
>160
>160
>160
>160
>160
10
1
0
3
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
2.5ꢀ5
10
20
36
1.25ꢀ5
5
51
Rif
0.012
0.18
1.62
2.3
0.012
0.36
1.62
2.3
INH
STREP
ETB
LEVO
KAN
0.64
0.32
1.3
0.32
Rif: Rifampicin; INH: Isoniazid; STREP: Streptomycin; ETB: Ethambutol;
LEVO: Levofloxacin; KAN: Kanamycin
Table 5: Activity against clinical isolates
MIC on
Referen
ce strain
a
a
µM
MICs against Clinical Isolates µM
Compou H37Rv SAWC11 SAWC39 SAWC33 SAWC32 SAWC33 SAWC39
nd
25
33
85
0.31
1.25
<0.08
10
00
88
06
36
1.25
1.25-2.5
2.5
2.5
2.5
<0.08
1.25
0.31
0.31
4
5
5
0
1
2
2.5ꢀ5
<0.08
2.5
1.25 ꢀ 2.5
0.625
10ꢀ20
1.25 ꢀ 2.5 0.31ꢀ0.63
<0.08
2.5ꢀ5
0.15
10
<0.08
2.5ꢀ5
50
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a
MIC readout from MABA assay in Middlebrook 7H9 Medium; SAWC 1125 ꢀTypical Beijing
9
0
(
(
lng 2.2); SAWC 3933 –T1 (lng 4.2); SAWC 3385 (Cas 1/Delhi (lng 3); SAWC ꢀ 3200 ꢀ X1
lng 4.9); SAWC 3388 ꢀ T1, Tuscany, T5/Rus (lng4.3); SAWC 3906 ꢀAtypic Beijing (lng 2)
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0
Chemoproteomics. In order to supplement our MOA identification efforts, we used a
chemoproteomics approach to identify the potential target proteins of the 6ꢀ
dialkylaminopyrimidine carboxamides. This affinityꢀbased approach was recently successfully
2
8–30
employed for the direct target identification of antitubercular compounds.
This approach is
based on the modification of the compound 40 with a linker for covalent immobilization on
sepharoseꢀbeads by Nꢀhydroxysuccinamide (NHS) coupling. The modified compound 85 (Figure
5
, synthesis scheme S1) retained antimycobacterial activity (MIC90 18.6 ꢀM as determined
against M. bovis BCG) suggesting that the derivatization with the linker moiety did not interfere
with target binding. The 85ꢀbeads were incubated with Mycobacterium bovis BCG extract and
proteins captured by the beads were digested with trypsin, labeled with isobaric mass tags (TMT
10plex), and quantitatively identified by liquidꢀchromatographyꢀtandem mass spectrometry (LCꢀ
MS/MS). In order to distinguish true targets from nonspecific background binding, the bacterial
extracts were incubated prior to the beadꢀincubation step with either the active test compound 40,
the inactive test compound 25, or with a vehicle (DMSO only) control. Only the active
compound should bind to its target proteins in the lysate and thus reduce the binding of these
proteins to the beads, but not the inactive compound. Indeed, two proteins, BCG_3193 (Rv3169)
and BCG_3827 (Rv3768), showed reduced binding to the compoundꢀbeads in the presence of
competing “free” 40 (Fig. 3A), but not with inactive 25, which suggests that these proteins are
the potential targets of 40. In order to estimate inhibitor potency, we performed the affinityꢀstep
in the presence of different concentrations of active compound 40 in the protein extract, which
app
app
allowed the determination of an apparent dissociation constant Kd (Fig. 3C). The Kd takes
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into consideration the affinity of the target for the beadꢀimmobilized ligand. The latter effect can
3
1
be deduced by measuring the depletion of the target protein by the beads. The apparent
dissociation constant of compound 40 for BCG_3193 was determined in two separate
experiments as 0.61 µM and 0.69 µM and for BCG_3827 as 2.1 µM and 3.8 µM (Fig. 3C). Both
potential novel target proteins, BCG_3193/Rv3169 and BCG_3827/Rv3768 (contains a
Polyketide cyclase SnoaLꢀlike domain, IPR009959), are conserved hypothetical proteins of
unknown function. Both proteins are nonꢀessential but notably found using Triton Xꢀ114
detergentꢀphase separation for extraction of lipophilic proteins, suggesting they are associated
1
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3
2
with the cell envelope despite lacking detectable transꢀmembrane helices.
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