Variations in the C-unit of bedaquiline provides analogues with improved biology and pharmacology

Article abstract of DOI:10.1016/j.bmc.2019.115213

Analogues of the anti-tuberculosis drug bedaquiline, bearing a 3,5-dimethoxy-4-pyridyl C-unit, retain high anti-bacterial potency yet exert less inhibition of the hERG potassium channel, in vitro, than the parent compound. Two of these analogues (TBAJ-587 and TBAJ-876) are now in preclinical development. The present study further explores structure-activity relationships across a range of related 3,5-disubstituted-4-pyridyl C-unit bedaquiline analogues of greatly varying lipophilicity (clogP from 8.16 to 1.89). This broader class shows similar properties to the 3,5-dimethoxy-4-pyridyl series, being substantially more potent in vitro and equally active in an in vivo (mouse) model than bedaquiline, while retaining a lower cardiovascular risk profile through greatly attenuated hERG inhibition.

Full text of DOI:10.1016/j.bmc.2019.115213

Journal Pre-proofs
Variations in the C-unit of bedaquiline provides analogues with improved bi-
ology and pharmacology
Hamish S. Sutherland, Amy S.T. Tong, Peter J. Choi, Adrian Blaser, Scott G.
Franzblau, Christopher B. Cooper, Anna M. Upton, Manisha Lotlikar,
William A. Denny, Brian D. Palmer
PII:
S0968-0896(19)31505-6
https://doi.org/10.1016/j.bmc.2019.115213
BMC 115213
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 September 2019
6 November 2019
10 November 2019
Please cite this article as: H.S. Sutherland, A.S.T. Tong, P.J. Choi, A. Blaser, S.G. Franzblau, C.B. Cooper, A.M.
Upton, M. Lotlikar, W.A. Denny, B.D. Palmer, Variations in the C-unit of bedaquiline provides analogues with
improved biology and pharmacology, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.bmc.2019.115213
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© 2019 Published by Elsevier Ltd.

Variations in the C-unit of bedaquiline provides analogues with improved biology and
pharmacology
Hamish S. Sutherlanda, Amy S.T. Tonga, Peter J. Choia, Adrian Blasera, Scott G. Franzblauc,
Christopher B. Cooperd, Anna M. Uptond, Manisha Lotlikard, William A. Dennya,b,*, Brian
D. Palmera,b,
Affiliations aAuckland Cancer Society Research Centre, School of Medical Sciences, and
bMaurice Wilkins Centre, University of Auckland, Private Bag 92019, Auckland 1142, New
Zealand; cInstitute for Tuberculosis Research, College of Pharmacy, University of Illinois at
Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA; dGlobal Alliance for TB
Drug Development, 40 Wall St, New York, New York 10005, USA
Keywords
CFU; colony-forming units; hERG, the alpha subunit of a K+ channel that contributes to the
electrical activity of the heart; HPLC, high-peformance liquid chromatography; LDA, lithium
diisopropylamide; LiTMP, lithium tetramethylpiperidide; LORA, low oxygen recovery assay;
MABA, microplate alamar blue assay; MIC90, minimum concentration for 90% inhibition;
M.tb, Mycobacterium tuberculosis; TB, tuberculosis
Abstract
Analogues of the anti-tuberculosis drug bedaquiline, bearing a 3,5-dimethoxy-4-pyridyl C-
unit, retain high anti-bacterial potency yet exert less inhibition of the hERG potassium
channel, in vitro, than the parent compound. Two of these analogues (TBAJ-587 and TBAJ-
876) are now in preclinical development. The present study further explores structure-activity
relationships across a range of related 3,5-disubstituted-4-pyridyl C-unit bedaquiline
analogues of greatly varying lipophilicity (clogP from 8.16 to 1.89). This broader class shows
similar properties to the 3,5-dimethoxy-4-pyridyl series, being substantially more potent in
vitro and equally active in an in vivo (mouse) model than bedaquiline, while retaining a lower
cardiovascular risk profile through greatly attenuated hERG inhibition.
1. Introduction
The introduction of the mycobacterial ATP synthase inhibitor bedaquiline (1) for the
treatment of multidrug-resistant tuberculosis (MDR-TB) is seen as a considerable step
forward, although it has been noted1 that the promising results to date have been based
largely on culture conversion2 rather than durable therapeutic response. One issue with
bedaquiline is its long in vivo half-life (due significantly to its very high lipophilicity; clogP
7.25), which may increase the risk of acquired resistance.3 In addition, it shows potent (IC50
1.6 µM) inhibition of the hERG potassium channel, which can contribute to the risk of
cardiac QTc prolongation.4 Although this can be ameliorated by the co-administration of
verapamil,5 it is still a risk factor that must be considered in the formulation of multi-drug
treatment regimens that include bedaquiline.6

With this in mind, we have recently reported7,8 systematic studies towards the development of
less lipophilic and less (potentially) cardiotoxic second-generation analogues of 1. These
studies have shown that, while there is a broad positive correlation between clogP and anti-
mycobacterial potency, compounds such as 2 and 3, with calculated clogP values two logs
lower than 1, can have equivalent or even superior biological activity. This is true both in
vitro (MIC90 value against replicating M.tb strain H37Rv) and in vivo (reduction in colony-
forming units in mice after daily treatment with 20 mg/kg for 12 days) (Figure 1).7,8 We have
also shown that C-unit pyridyl analogues6 (and particularly C-unit 3,5-dialkoxy-4-pyridyl
analogues7) have a greatly-reduced inhibitory activity towards the hERG cardiac potassium
channel protein. Compounds 2 (TBAJ-587)8 and 3 (TBAJ-876)8 are now in preclinical
development.9-11
Figure 1. Properties of bedaquiline (BDQ) and 2 advanced analogues
F
MeO
MeO
N
OMe
B
D
MeO
OH
OH
OH
A
Br
NMe2
Br
NMe2 Br
OMe
NMe2
OMe
N
O
N
O
N
O
C
N
N
MeO
MeO
2
3
1
bedaquiline ( )
clogP
7.25
1.6
0.03
4.5–6.1
5.80
13
0.006
4.8
5.15
>30
0.004
>5.5
hERG IC50 (µM)
M.tb MIC90 (µM)
log10CFUreduction
In the present paper we follow up this work by exploring bedaquiline analogues with a wider
range of 3,5-disubstituted 4-pyridyl C-units, further extending the structure-activity
relationships for these variations for their effects on drug lipophilicity, anti-mycobacterial
potency and, particularly, hERG channel inhibition.
2. Results and Discussion
2.1 Chemistry
The new 3,5-disubstituted 4-pyridyl analogues were prepared by the general route outlined in
Scheme 1. The A/B units were assembled as described previously,7,8,12 from 6-bromo-2-
methoxyquinoline (A) and the required aldehydes (B), or by palladium mediated coupling of
a quinolone boronic acid (A1) and a (halomethyl)arene (B1). The C/D-units were prepared in
high yields via the Weinreb amides, as shown in Scheme 2 and Table 1. Details of the
preparation of the required acids (I) in Scheme 2 are given in Supplementary Information.
The LDA mediated coupling of the benzylquinoline A/B-units and 1-(2,6-disubstituted
pyridin-4-yl)-3-(dimethylamino)propan-1-one (Mannich base) C/D-units (Scheme 1) gave the
compounds of Table 2, as a racemic mixture of four diastereomers. The desired RS,SR
diastereomer (depicted) was then isolated by super-critical fluid HPLC at BioDuro LLC
(Beijing). The 6-cyano compounds were prepared from the corresponding 6-bromo analogues
by direct cyanation under previously reported conditions.7,8 The C-unit SO2Me and SO2Et

analogues 23 and 43 were prepared from the corresponding SMe and SEt compounds 21 and
42 respectively by oxidation with N-methylmorpholine N-oxide/OsO4.
Scheme 1: Synthesis of 3,5-disubstituted 4-pyridyl analogues of 1
Y
Y
N
D
Br
O
NMe2
OH
R1
X
NMe2
R2
Br
N
+
O
R
i
A
v
+
Y
O
N
O
R1
N
C
R2
B
A/B
N
X=Br
X=CN
OHC
R=OH
R=H
vi
ii or iii
Y
Br
B(OH)2
O
Br
N
+
iv
A1
B1
Y
N
O
A/B
Hal
(Hal = Cl, Br)
Reagents and conditions: (i) LiTMP, THF, -75 oC, 1.5 h then the appropriate aldehyde B, -75 oC, 4 h;
(ii) Et3SiH, TFA, DCM; (iii) MsCl, Et3N, DMF, then NaBH4; (iv) Cs2CO3, Pd(PPh3)4, PhMe/DMF,
110 oC (sealed tube), 5 h; (v) LDA, THF, -75 oC, 1.5 h then the appropriate ketone C/D, then HOAc;
(vi) Zn/Zn(CN)2, Pd2(dba)3/P(o-tol)3, DMF, 50 oC, then separation of the diastereomers by SFC
HPLC.
Scheme 2: Synthesis of pyridyl 3-(dimethylamino)propan-1-one Mannich bases
Me
O
N
O
OH
O
NMe2
OMe
i
ii
R1
N
R2
R1
N
R2
R1
N
R2
III
II
I
Reagents and conditions: (i) (COCl)2, cat. DMF, DCM, then MeNH(OMe).HCl, pyridine; (ii)
vinylMgBr, THF; then Me2NH, water.
Table 1. New 3,5-disubstituted Mannich bases
R1
R2
OEt
Yields I-II /
II-III
OMe
97 / 67

OMe
OMe
OMe
OMe
OMe
OMe
OEt
OEt
SMe
SEt
OiPr
97 / 99
91 / 99
85 / 99
81 / 99
98 / 99
86 / 72
97 / 99
96 / 98
82 / 81
96 / 66
86 / 81
OnPr
OcBu
OCF2H
SMe
NMe2
OiPr
OnPr
SMe
SEt
SEt
NMe2
Li-mediated coupling of the benzylquinoline A/B-units and 1-(2,6-disubstituted pyridin-4-
yl)-3-(dimethylamino)propan-1-one (Mannich base) C/D-units (Scheme 1) gave the
compounds of Table 2, as a racemic mixture of four diastereomers. The desired RS,SR
diastereomer (depicted) was then isolated by super-critical fluid HPLC at BioDuro LLC
(Beijing).
2.2 Structure-activity relationships
In earlier work7, we showed that while analogues of bedaquiline bearing a wide variety of
methyl- and methoxy-substituted pyridyls as the C-unit were less lipophilic but equally potent
M.tb inhibitors, there was little improvement in their hERG liability. In contrast, as noted
above, analogues bearing symmetrical 3,5-dimethoxy- or 3,5-diethoxy-4-pyridyl C-units
retained high potency against both replicating13 and non-replicating13,14 cultures of M.tb,
together with marked improvements in hERG liability8 (Fig. 1). In the present work, we
explore this particular substitution pattern in more detail, with a wider variety of both
symmetric and asymmetric 3,5-diethoxy-4-pyridyl C-units, to extend our understanding of
how these changes affect both anti-tubercular activity and hERG liability (Table 2).
Table 2. Structural and in vitro data on 3,5-disubstituted pyridyl analogues of
bedaquiline
Y
OH
X
NMe2
R2
N
O
N
R1

X
Y
R1
R2
Ylda
MIC90
(MABA) b (LORA)b
hERGc
clogPd
1
bedaquiline
0.03
0.006
0.004
0.06
0.01
0.006
1.6
13
>30
7.25
5.80
5.15
2e
3e
Br 2-F, 3-OMe
Br 4-aza, 2,3,5-
triOMe
OMe OMe
OMe OMe
4
5
6
7
8
9
Br 2-F, 3-OMe
Br 3-Me
Br 4-aza, 2,3-diOMe OMe OEt
OMe OEt
OMe OEt
51
<0.01
<0.02
<0.02
<0.02
<0.01
0.01
0.01
0.01
<0.004
<0.01
0.02
0.01
0.01
0.04
<0.02
<0.004
<0.02
<0.02
0.02
<0.01
<0.02
<0.02
0.03
<0.01
0.01
0.02
0.06
0.01
<0.01
0.03
0.03
0.03
<0.02
<0.02
<0.004 >10
0.04
<0.02
0.04
0.73
0.02
0.06
<0.02
<0.02
<0.02
0.05
<0.02
<0.02
0.03
0.06
0.17
0.05
0.21
<0.02
0.20
0.06
0.06
0.05
0.03
5.0
>30
>10
3.3
>30
17
>10
>10
7.8
>10
>10
4.8
6.33
6.83
5.30
6.64
5.61
7.13
7.35
5.83
5.68
6.17
4.81
80
43
70
18
22
45
24
Br 2-F, 3-OMe
OMe OiPr
Br 4-aza, 2,3-diOMe OMe OiPr
Br 3-Me
OMe OiPr
OMe OnPr
10 Br 3-Me
11 Br 4-aza, 2,3-diOMe OMe OnPr
12 Br 4-aza, 2,3-diOMe OMe OcBu
13 Br 2,3-O(CH2)2O-
14 CN 2,3-O(CH2)2O-
15 Br 2-F, 3-Me
16 CN 2-F, 3-Me
17 Br 2-F, 3-OMe
18 CN 2-F, 3-OMe
OMe OCF2H 34
OMe OCF2H 73f
OMe OCF2H 56
OMe OCF2H 58f
OMe OCF2H 49
OMe OCF2H 49f
3% / 10g 6.88
>30
>30
5.3
5.53
6.25
4.89
5.21
3.86
6.14
4.79
4.73
5.08
3.73
6.14
6.89
6.46
5.10
5.43
5.69
5.36
19 Br 4-aza, 2,3-diOMe OMe OCF2H 60
20 CN 4-aza, 2,3-diOMe OMe OCF2H 68f
7.0
21 Br 2-F, 3-OMe
22 CN 2-F, 3-OMe
23 Br 2-F, 3-OMe
24 Br 4-aza, 2,3-diOMe OMe NMe2
25 CN 4-aza, 2,3-diOMe OMe NMe2
26 Br 4-aza, 2,3-diOMe OEt
27 Br 4-aza, 3,5-diOMe OEt
28 Br 2-F, 3-OMe
29 CN 2-F, 3-OMe
OMe SMe
OMe SMe
46
>30
>10
1.8
ND
3.4
>10
>10
>10
5.1
51f
OMe SO2Me 87h
0.46
0.45
0.06
68
84f
56
59
61
88f
67
38
67f
63
88f
55
53
70
82f
87
62
58f
79f
OiPr
OiPr
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SEt
<0.02
<0.02
<0.02
0.03
<0.02
<0.02
0.01
<0.004
0.02
0.02
0.04
<0.02
0.10
<0.02
0.01
<0.02
0.06
SMe
SMe
30 Br 4-aza, 2,3-diOMe SMe
2.8
>10
5.9
31 Br 2,3-O(CH2)2O-
32 CN 2,3-O(CH2)2O-
33 Br 2-F, 3-Me
34 CN 2-F, 3-Me
35 Br
SMe
SMe
SMe
SMe
SEt
13% / 3g 7.10
>10
>30
9.2
5.74
8.18
7.30
7.52
6.16
8.16
6.39
5.36
5.13
1.89
H
36 Br 4-aza, 2, 3-diOMe SEt
SEt
37 Br 2-F, 3-OMe
38 CN 2-F, 3-OMe
39 CN 2-F, 3-Me
40 Br 2,3-O(CH2)2O-
41 CN 2,3-O(CH2)2O-
42 CN 4-aza, 2,3-diOMe SEt
43 CN 4-aza, 2,3-diOMe SO2Et SO2Et
44 Br 3-Me
45 CN 3-Me
46 Br 4-aza, 2,3-diOMe SEt
SEt
SEt
SEt
SEt
SEt
SEt
SEt
SEt
SEt
>10
>10
>10
>10
>10
1.5
SEt
SEt
76f,h 4.4
iND
SEt
SEt
NMe2
NMe2
NMe2
43
0.14
0.12
0.17
0.57
23% / 3g 7.46
81f,h 0.12
51% / 3g 6.10
37
0.04
>10
5.93

Footnotes for Table 2
aYields in the AB/CD coupling step to give bedaquiline analogues (as racemic mixtures). bMIC90
(µg/mL); minimum inhibitory concentration for 90% inhibition of growth of M.tb strain H37Rv,
determined under aerobic, replicating (MABA) (ref. 13) or non-replicating (LORA) (ref. 14)
conditions, determined at the Institute for Tuberculosis Research, University of Illinois at Chicago.
cInhibition of hERG (IC50 in µM); dclogP calculated by ChemDraw Ultra v12.0.2. (CambridgeSoft);
eData from ref. 7; fYields for the Br/CN conversion; ghERG single concentration determination; %
inhibition at the stated dose (µM); hYield for oxidation of the corresponding SMe or SEt analogue
with N-methylmorpholine N-oxide/OsO4; iND; not done due to poor MIC value.
Overall, while the analogues covered a wide range of lipophilicity, from 8.16 to 1.89
(calculated logP (clogP) values), there was relatively little variation in MIC with changing
lipophilicity. All compounds except the very hydrophilic 43 retained high potency against M.
tb in vitro (in many cases beyond the range of the assay) and mostly lower potency against
hERG blockade compared to bedaquiline (Table 2).
Compounds 4-12 explored asymmetric 3,5-dialkoxy-4-pyridyl units. Replacing one methoxy
substituent in 2 (TBAJ-587) with an ethoxy (4) or isopropoxy (7) had minimal effect on MIC
values but showed an improvement in hERG IC50, albeit with increased lipophilicity.
Increasing the steric bulk around one alkoxy group while retaining the remainder of the
molecular structure (compounds 6, 8, 11 and 12) yielded little change in either MIC or hERG
values were observed, suggesting some bulk tolerance in this position for both activities. The
retention of MIC activity in the 3-ethoxy-5-isopropoxy-4-pyridyl derivative (27) (<0.02
µg/mL) provides further evidence for steric tolerance around the pyridyl group.
The mildly electron-withdrawing -OCF2H group (compounds 13-20) had no effect on MIC,
and most of these compounds also showed low hERG inhibition. In contrast, the introduction
of a strongly electron-withdrawing SO2Me substituent in compound 23 resulted in significant
loss of MIC potency and a hERG IC50 of 1.8 µM. Since compounds of similarly overall low
lipophilicity (e.g., 14, 18, 22) retained good anti-tubercular potency, the electronic effects of
substituents appear important.
Compounds 21-23 looked at mixed OMe/SMe pyridyl units. The SMe group is moderately
electron donating, but has the potential of metabolic oxidation to likely less effective -SO2Me
analogues. However, 21 and 22 retained good MIC and hERG values and in vivo activity
(Table 3). Retention of potency was also observed when an OMe group was replaced with the
strongly electron donating NMe2 group in compounds 24 and 25. However, 25 does not
present an improvement over bedaquiline in hERG potency (IC50 3.4 µM) compared to many
of the other compounds.
Compounds 28-34 bear 3,5-diSMe-4-pyridyl C-units, while compounds 44-46 bear 2-SEt, 5-
NMe2-4-pyridyl C-units. Unsurprisingly, in light of the results for 21 and 22 (OMe/SMe

substituents), these compounds also retain good MIC potency (~0.02 µg/mL) and most have
similar hERG properties (IC50s 5 to >10 µM). Compounds 35-42 have analogous 3,5-diSEt-
4-pyridyl C-units, and their results are broadly similar in terms of MIC (~0.02 µg/mL) and
(except 42) hERG (>10 µM) properties, although these compounds are significantly more
lipophilic (clogP from 6-8). Chemical oxidation of 42 gave the extremely polar 43 (clogP
1.89), with very electron-withdrawing diSO2Et substitution, which not surprisingly had
significantly attenuated in vitro MIC values (5 µg/mL).
A substantial number of the compounds, representative of the sub-classes studied, were
evaluated for a series of relevant pharmacological and in vivo antibacterial properties, in an
endeavour to discern structure-activity relationships for these analogues. The data are shown
in Table 3. The compounds were additionally tested for mammalian cell toxicity (with Vero
green monkey-derived epithelial kidney cells),14,15 where all compounds had IC50s >10
µg/mL, except for compounds 28 (IC50 of 9.6 µg/mL), 37 (IC50 of 6.5 µg/mL), and 43 (not
tested) (data not shown). In comparison, the value for 1 in repeat assays was between 4-16
µg/mL
Table 3. Additional biological data on selected analogues of Table 1
Mouse Acute TB
efficacy
Microsomal
clearance
Mouse PK parameters
No
Log reduction in
mouse lung CFU
H Clint M Clint
IV Cl
Vz
AUCinf
F
µg*h/
mLd
17.4
1.7
5.6
4.0
9.4
12.7
9.0
5.6
10.9
8.4
3.2
8.6
14.4
9.8
3.0
4.6
1.1
3.3
32.0
Test
Beda-
quilineg
4.5-6.1
6.1
>5.5
4.6
5.5
5.6
4.6
4.6
μL/min/mg
ml/min
/kgb
L/kgc
(%)e
compd f
proteina
1
3.0
2.2
2.1
4.1
4.4
3.8
3.4
6.4
8.2
2.5
1.6
0.6
0.1
2.1
2.5
4.8
16
7.3
18
22
7.0
48
13
19
12
9.5
8.9
6.8
6.9
11
27
13
9.7
8.7
24
13
42
26
3.1
22
95
31
51
45
28
34
19
44
33
41
38
18
27
40
32
54
14
9.1
56
48
44
46
65
70
43
22
50
56
51
66
83
50
43
31
28
50
58
--
4.8
>5.5
4.1
0.7
5.4
4.1
2.6
5.0
5.2
5.1
>5.2
4.4
4.3
5.2
4.7
>4.3
5.2
4.7
2g
3g
4
14
5
6
7
8
7.8
7.6
6.0
6.7
6.5
4.6
15
5.9
6.4
4.9
11
11
12
14
16
17
19
20
21
22
25
26
5.5
4.9
4.6
4.9
6.1
>5.5
>5.5
4.5h
4.9
4.9
4.5h
20
36
8.4
1.8
16
1.6

27
28
29
30
34
35
37
38
39
40
1.1
3.4
15
8.8
9.2
2.3
4.9
17
2.3
17
20
10
13
17
12
27
33
12
2.7
13
44
18
22
11
3.6
29
9.4
3.4
20
15
53
64
79
30
14
93
41
9.4
33.0
8.4
1.1
3.9
2.4
4.0
11.9
1.4
4.0
5.5
58
66
28
41
31
24
25
24
22
11
4.5
5.0
2.0
4.1
3.9
1.8
4.2
1.7
2.9
2.9
>5.5
5.6
4.5h
5.6
4.9
4.6
5.6
5.0
5.6
5.6
9.1
9.5
Footnotes for Table 3
aClearance of compound by human or mouse liver microsomes (μL/min/mg protein); bIV clearance in
mice (µg*h/mL); cIV apparent volume of distribution during terminal phase, mouse (L/kg); dOral
exposure, area under the curve 0 to infinity, in mice (µg*h/mL); eOral bioavailability in mice; fLog
reduction in colony-forming units (CFU) compared to vehicle when dosed at 20 mg/kg daily in mice
for 12 days, beginning 10 days after M.tb inoculation; gResults for bedaquiline (positive control)
under the same conditions in the same assay. hBedaquiline test concentration was 16.7 mg/kg
The data in Table 2 show that the two “lead” unit C 3,5-dimethoxy compounds 2 and 39-11 do
not show much improvement in the very slow human clearance of bedaquiline (1); However,
compounds with pyridyl sulfur substituents do achieve this; e.g., 21 and 22 (SMe/OMe), 28-
34 (SMe/SMe) and 35-40 (SEt/SEt). The last group have an average 3-fold higher Hclint value
than 1. In contrast, the mixed dialkoxy compounds 4-12 were no better than 1, and the
OCF2H analogues 14-20 had even slower clearances than 1.
Pharmacokinetic studies in male CD-1 mice used single oral gavage dosing at 10 mg/kg or
single intravenous bolus injection at 2–3 mg/kg. Oral bioavailability was generally good;
mostly in the 40-60% range typical of 1 and with little apparent correlation with lipophilicity.
Again, compounds with sulfur-bearing pyridyl subunits seemed on average to have the lowest
F values, maybe reflecting sulfur oxidative metabolism. Volumes of distribution varied
widely, with no clear correlation to specific structural features. As demonstrated previously
for 116 and its analogues8, human plasma protein binding was always >99.5%.
The compounds were evaluated for in vivo efficacy in female BALB/c mice (n = 7 per
group), dosed by oral gavage at 20 mg/kg daily for 12 days, beginning 10 days after the
aerosol inoculation of M.tb H37Rv, as previously described.8 Compounds were formulated in
20% hydroxy-beta-cyclodextrin. Efficacy was determined by the reduction in colony-forming
units (CFU) of M. tb bacteria recovered from lung homogenates from compound-treated
mice, relative to the bacterial burden in mice administered with vehicle only, compared to the
bacterial reduction shown by treatment with 1 as a positive control.
The average CFU for vehicle-only treated control groups was approximately 5.5-8 logs; thus
a CFU reduction in the treated groups of >4.5 log units shows clearance of bacteria in the
lungs of mice within the limit of detection (30 CFU/mouse) after 12 days of treatment. Ten of

the new compounds in Table 3 (6, 11, 12, 14, 16, 20, 26, 28) achieved this high level of
efficacy, with log CFU reduced to ~1.5 CFU, which reflecting essentially similar efficacy to
1, given the assay-to-assay variation of these experiments.
3. Conclusions
Previous work8 showed that analogues of 1 bearing 3,5-dimethoxy-4-pyridyl C-units in place
of the naphthalene possessed good in vitro and in vivo anti-mycobacterial activity and
greatly-reduced hERG potassium channel blockade (a potential liability of 1) and identified
two preclinical candidates (2 and 3). The present study broadens the scope of this SAR,
showing that analogues with a wide variety of other 3,5-disubstituted-4-pyridyl units retain
similarly-high antibacterial efficacy and similar or better in vitro hERG safety. Of the eight
“best” new in vivo compounds (Table 3), five had a unit B 3,5-dialkoxy substitution pattern.
The “best” unit C substitutions for in vivo activity were 3,5-diOalkyl (four), 3-OMe, 5-CF2H
(three) and 3,5-siSMwe (one). Effective suppression of hERG channel stimulation (IC50 >10
µM) was much less dependent on structure, with the great majority of the Table 3
compounds, bearing a cross-section of the unit C substitutions, meeting this criterion. As
noted above, many of these compounds (e.g., 12, 14, 16, 20) also have much faster clearance
rates than 1, making them further potential backup candidates.
4. Experimental
4.1. Chemistry
Compounds were analysed for purity by reverse-phase HPLC (Alltima C18 5 µm column,
150 × 3.2 mm; Alltech Associated, Inc., Deerfield, IL) using an Agilent HP1100 equipped
with a diode-array detector. Mobile phases were gradients of 80% CH3CN/20% H2O (v/v) in
45 mM NH4HCO2 at pH 3.5 and 0.5 mL/min. Purity was determined by monitoring at 330 ±
50 nm and was ≥95% for all final products. NMR spectra were obtained on a Bruker Avance
400 spectrometer at 400 MHz for 1H. Low-resolution atmospheric pressure chemical
ionization (APCI) mass spectra were measured for organic solutions on a ThermoFinnigan
Surveyor MSQ mass spectrometer, connected to a Gilson autosampler. Melting points were
determined on an Electrothermal 9100 melting point apparatus.
4.1.1. Scheme 1. New A/B units
4.1.1.1. Scheme 1: 6-bromo-3-((2,6-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (AB-
7) for compound 27 of Table 1

OMe
MeO
N
MeO
N
OMe
MeO
N
OMe
MeO
N
OMe
Br
CO2H
N
OMe
OH
Br
S3
AB-7
S1
S2
Borane-dimethylsulfide (4.60 mL, 48.5 mmol) and trimethylborate (5.58 mL, 49.1 mmol)
were added to a solution of 2,6-dimethoxyisonicotinic acid (S1) (3.00 g, 16.4 mmol) in THF
(100 mL, dist. Na) at 0 °C, the solution was then stirred at r.t. for 18 h, cooled to 0 °C,
quenched with MeOH and evaporated. The residue was partitioned between EtOAc and water
and the organic fraction was dried and evaporated. Column chromatography (2:1
hexanes:EtOAc) gave (2,6-dimethoxypyridin-4-yl)methanol (S2) (2.72 g, 98%). 1H NMR
(CDCl3) δ 6.30 (s, 2H), 4.64 (d, J = 5.8 Hz, 2H), 3.91 (s, 6H), 1.75 (t, J = 6.1 Hz, 1H). Found:
[M+H]=170.1.
A solution of S2 (1.57 g, 9.30 mmol) in DCM (40 mL, anhydrous) at 0 °C was treated with
triethylamine (2.60 mL, 18.6 mmol) then mesyl chloride (1.08 mL, 13.9 mmol), the mixture
was stirred at 0 °C for 1 h then partitioned between DCM and water. The organic fraction was
dried and evaporated and the residue was dissolved in acetone (50 mL). LiBr (4.04 g, 46.5
mmol) was added and the mixture was refluxed for 1 h then evaporated. The residue was
partitioned between DCM and water and the organic fraction was dried and evaporated.
Column chromatography (DCM) gave 4-(bromomethyl)-2,6-dimethoxypyridine (S3) (1.96 g,
91%). 1H NMR (CDCl3) δ 6.32 (s, 2H), 4.29 (s, 2H), 3.91 (s, 6H). Found: [M+H] = 232.0
A mixture of (6-bromo-2-methoxyquinolin-3-yl)boronic acid (2.20 g, 7.80 mmol), S3 (1.93 g,
8.30 mmol), Cs2CO3 (5.10 g, 15.7 mmol) and Pd(PPh3)4 (0.45 g, 0.39 mmol) in DMF (10 mL)
and toluene (20 mL) was purged with nitrogen, then heated to 80 °C for 4 h under nitrogen.
The mixture was partitioned between EtOAc and water and the organic fraction was dried and
evaporated. Column chromatography (3:1 hexanes:DCM) eluted non polar impurities, elution
with DCM gave 6-bromo-3-((2,6-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (AB-7)
(1.95 g, 64%). 1H NMR (CDCl3) δ 7.78 (d, J = 2.1 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.63 (dd,
J = 8.9, 2.2 Hz, 1H), 7.57 (s, 1H), 6.17 (s, 2H), 4.06 (s, 3H), 3.92 (s, 2H), 3.89 (s, 6H). Found:
[M+H] = 389.1
4.1.2. New C/D units of Table 1
4.1.2.1. 3-(Dimethylamino)-1-(2-ethoxy-6-methoxypyridin-4-yl)propan-1-one (CD-1)
OMe
O
OMe
O
N
O
OMe
O
OH
O
N
EtO
N
OMe
EtO
N
OMe
HO
N
OMe
EtO
N
OMe
EtO
N
OMe
S4
S6
CD-1
S7
S5
A suspension of 2,6-dihydroxyisonicotinic acid (10.00 g, 64.5 mmol) in MeOH (60 mL) was
treated dropwise with H2SO4 (10 mL, 18.4 M, 184 mmol). The solution was refluxed for 72 h
and then evaporated. The residue was treated with sat. aq. NaHCO3 to pH 8 and extracted with

EtOAc (3 x 200 mL). The organic extracts were washed with sat. aq. NaHCO3 and brine, then
dried and evaporated to give methyl 2-hydroxy-6-methoxyisonicotinate (S4) (3.55 g, 30%). 1H
NMR (DMSO-d6) δ 11.2 (bs, 1H), 6.61 (bs, 2H), 3.84 (s, 3H), 3.83 (s, 3H). Found:
[M+H]=184.2.
A solution of S4 (6.96 g, 38.0 mmol) in DMF (100 mL, anhydrous) was treated with K2CO3
(6.57 g, 47.6 mmol) and then iodoethane (3.85 mL, 47.6 mmol). The mixture was stirred at r.t.
for 24 h, partitioned between EtOAc and water and the aqueous layer was extracted with
EtOAc. The organic fractions were washed with water, dried and evaporated, chromatography
(DCM) gave methyl 2-ethoxy-6-methoxyisonicotinate (S5) (6.20 g, 77%). 1H NMR (CDCl3) δ
6.84 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 3.91 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). Found:
[M+H]=212.1.
A solution of LiOH (2.10 g, 87.7 mmol) in water (60 mL) was added to a solution of S5 (6.20
g, 29.4 mmol) in MeOH (60 mL) and THF (60 mL), the solution was stirred at r.t. for 18 h and
then evaporated. The residue was dissolved in water (150 mL) and acidified to pH 3 with 2 M
HCl. The precipitate was filtered and dried to give 2-ethoxy-6-methoxyisonicotinic acid (S6)
(5.61 g, 97%) as a white solid. 1H NMR (DMSO-d6) δ 13.54 (bs, 1H), 6.73 (d, J = 1.0 Hz, 1H),
6.71 (d, J = 1.0 Hz, 1H), 4.32 (q, J = 7.0 Hz, 2H), 3.87 (s, 3H), 1.33 (t, J = 7.0 Hz, 3H). Found:
[M+H]=198.2.
Oxalyl chloride (2.18 mL, 25.8 mmol) was added to a suspension of S6 (4.23 g, 21.5 mmol) in
DCM (100 mL, anhydrous) and DMF (0.3 mL) at r.t.. The mixture was stirred at r.t. for 1 h to
give a colourless solution which was cooled to 0 °C. N,O-Dimethylhydroxylamine
hydrochloride (2.51 g, 25.8 mmol) and pyridine (5.2 mL, 64.3 mmol) were added sequentially
and the mixture was stirred at r.t. for 18 h, then partitioned between DCM and sat. aq. NaHCO3.
Column chromatography with 3:1 hexanes:EtOAc gave 2-ethoxy-N,6-dimethoxy-N-
methylisonicotinamide (S7) (5.02 g, 97%). 1H NMR (CDCl3) δ 6.46 (s, 1H), 6.45 (s, 1H), 4.35
(q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 3.59 (bs, 3H), 3.32 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). Found:
[M+H]=241.1.
Vinylmagnesium bromide (16.6 mL, 1 M, 16.6 mmol) was added to a solution of S7 (2.00 g,
8.30 mmol) in THF (100 mL, dist. Na) at 0 °C, the brown solution was warmed to r.t. for 1 h
then dimethylamine in THF (2M, 16.6 mL, 33.2 mmol) and water (25 mL) were added. The
solution was stirred at r.t. for 1 h, then partitioned between EtOAc and water. The solution was
dried and evaporated, column chromatography of the residue (95:5 DCM:MeOH) gave 3-
(dimethylamino)-1-(2-ethoxy-6-methoxypyridin-4-yl)propan-1-one (CD-1) (1.40 g, 67%). 1H
NMR (CDCl3) δ 6.73 (s, 1H), 6.72 (s, 1H), 4.37 (q, J = 7.0 Hz, 2H), 3.93 (s, 3H), 3.06 (t, J =
7.4 Hz, 2H), 2.72 (t, J = 7.4 Hz, 2H), 2.27 (s, 6H), 1.41 (t, J = 7.0 Hz, 3H). Found:
[M+H]=253.2.
4.1.2.2. 3-(Dimethylamino)-1-(2-isopropoxy-6-methoxypyridin-4-yl)propan-1-one (CD-2)
OMe
O
OMe
O
OMe
O
N
O
N
O
OH
iPrO
N
OMe
iPrO
N
OMe iPrO
N
OMe
iPrO
N
OMe
HO
N
OMe
S10
CD-2
S4
S9
S8

A solution of S4 (5.04 g, 27.5 mmol) in DMF (100 mL, anhydrous) was treated with K2CO3
(4.75 g, 34.4 mmol) and then 2-iodopropane (3.43 mL, 34.4 mmol). The mixture was stirred at
r.t. for 24 h, more 2-iodopropane (3.43 mL, 34.3 mmol) was added and the mixture was stirred
for a further 72 h, then partitioned between EtOAc and water and the aqueous layer was
extracted further with EtOAc. The organic fractions were washed with water, dried and
evaporated. Chromatography (DCM) gave methyl 2-isopropoxy-6-methoxyisonicotinate (S8)
(6.21 g, 100%). 1H NMR (CDCl3) δ 6.81 (s, 2H), 5.24 (sp, J = 6.2 Hz, 1H), 3.92 (s, 3H), 3.90
(s, 3H), 1.36 (d, J = 6.2 Hz, 6H). Found: [M+H]=226.2.
A solution of LiOH (1.98 g, 82.7 mmol) in water (60 mL) was added to a solution of S8 (6.20
g, 27.5 mmol) in MeOH (60mL) and THF (60 mL) and the solution was stirred at r.t. for 18 h
and then evaporated. The residue was dissolved in water (150 mL) and acidified to pH 3 with
2 M HCl. The precipitate was filtered and dried to give 2-isopropoxy-6-methoxyisonicotinic
acid (S9) (5.33 g, 92%) as a white solid. 1H NMR (DMSO-d6) δ 13.50 (bs, 1H), 6.70 (d, J =
1.0 Hz, 1H), 6.66 (d, J = 1.0 Hz, 1H), 5.20 (sp, J = 6.2 Hz, 1H), 3.86 (s, 3H), 1.31 (d, J = 6.2
Hz, 6H). Found: [M+H]=212.1.
Oxalyl chloride (1.75 mL, 20.7 mmol) was added to S9 (3.65 g, 17.3 mmol) in DCM (100 mL,
anhydrous) and DMF (0.3 mL) at r.t.. The mixture was stirred at r.t. for 1 h to give a colourless
solution which was cooled to 0 °C. N,O-Dimethylhydroxylamine hydrochloride (2.03 g, 20.8
mmol) and pyridine (4.2 mL, 51.9 mmol) were added sequentially and the mixture was stirred
at r.t. for 18 h, then partitioned between EtOAc and water. Column chromatography with 3:1
hexanes:EtOAc gave 2-isopropoxy-N,6-dimethoxy-N-methylisonicotinamide (S10) (4.58 g,
1
100%) which was used directly. H NMR (CDCl3) δ 6.42 (s, 1H), 6.41 (s, 1H), 5.24 (sp, J =
6.2 Hz, 1H), 3.90 (s, 3H), 3.60 (bs, 3H), 3.32 (bs, 3H), 1.35 (d, J = 6.2 Hz, 6H). Found:
[M+H]=255.2.
Vinylmagnesium bromide (36 mL, 1 M, 36 mmol) was added to a solution of S10 (4.54 g, 17.9
mmol) in THF (200 mL, dist. Na) at 0 °C, the brown solution was warmed to r.t. for 1 h, then
dimethylamine in THF (36 mL, 2 M, 72 mmol) and water (30 mL) were added. The solution
was stirred at r.t. for 1 h and then partitioned between EtOAc and water. Column
chromatography using a gradient of 97.5:2.5 DCM:MeOH to 95:5 DCM:MeOH gave 3-
(dimethylamino)-1-(2-isopropoxy-6-methoxypyridin-4-yl)propan-1-one (CD-2) (3.58 g,
75%). 1H NMR (CDCl3) δ 6.70 (d, J = 1.0 Hz, 1H), 6.69 (d, J = 1.0 Hz, 1H), 5.25 (sp, J = 6.2
Hz, 1H), 3.92 (s, 3H), 3.05 (t, J = 7.4 Hz, 2H), 2.72 (t, J = 7.4 Hz, 2H), 2.26 (s, 6H), 1.36 (d, J
= 6.2 Hz, 6H). Found: [M+H]=267.2.
4.1.2.2. 3-(Dimethylamino)-1-(2-methoxy-6-propoxypyridin-4-yl)propan-1-one (CD-3)
OMe
MeO
O
O
N
O
N
MeO
O
HO
O
HO
N
OMe
PrO
N
OMe
PrO
N
OMe
PrO
N
OMe
PrO
N
OMe
S4
S11
S12
CD-3
S13
A solution of S4 (6.00 g, 32.8 mmol) in DMF (100 mL, anhydrous) was treated with K2CO3
(6.80 g, 49.2 mmol) and then 1-iodopropane (4.8 mL, 49.2 mmol). The mixture was stirred at
r.t. for 48 h, partitioned between EtOAc and water and the aqueous layer was extracted with

EtOAc. The organic fractions were washed with water, dried and evaporated. Column
chromatography (DCM) gave methyl 2-methoxy-6-propoxyisonicotinate (S11) (6.59 g, 89%).
1H NMR (CDCl3) δ 6.85 (d, J = 1.0 Hz, 1H), 6.83 (d, J = 1.0 Hz, 1H), 4.24 (t, J = 6.7 Hz,
2H), 3.93 (s, 3H) , 3.91 (s, 3H), 1.80 (qt, J = 7.4, 6.7 Hz, 2H), 1.02 (t, J = 7.4 Hz, 3H). Found:
[M+H]=226.1.
A solution of LiOH (2.04 g, 85.2 mmol) in water (60 mL) was added to a solution of S11 (6.40
g, 28.4 mmol) in THF (60 mL) and MeOH (60 mL), the solution was stirred at r.t. for 18 h and
then evaporated. The residue was dissolved in water (200 mL) and acidified to pH 3 with 2 M
HCl. The precipitate was filtered and dried to give 2-methoxy-6-propoxyisonicotinic acid (S12)
(5.39 g, 90%). 1H NMR (DMSO-d6) δ 13.53 (bs, 1H), 6.73 (d, J = 1.0 Hz, 1H), 6.72 (d, J = 1.0
Hz, 1H), 4.23 (t, J = 6.6 Hz, 2H), 3.87 (s, 3H), 1.73 (qt, J = 7.4, 6.6 Hz, 2H), 0.96 (t, J = 7.4
Hz, 3H). Found: [M+H]=212.1.
Oxalyl chloride (2.76 mL, 32.6 mmol) was added to S12 (5.74 g, 27.2 mmol) in DCM (100
mL, anhydrous) and DMF (0.4 mL, 5.2 mmol) at r.t.. The mixture was stirred at r.t. for 1 h to
give a colourless solution which was cooled to 0 °C. N,O-Dimethylhydroxylamine
hydrochloride (3.18 g, 32.6 mmol) and pyridine (6.6 mL, 81.6 mmol) were added sequentially
and the mixture was stirred at r.t. for 18 h, then partitioned between DCM and water. Column
chromatography
on
alumina
with
DCM
gave
N,2-dimethoxy-N-methyl-6-
1
propoxyisonicotinamide (S13) (6.29 g, 91%). H NMR (CDCl3) δ 6.42 (s, 1H), 6.41 (s, 1H),
5.24 (sp, J = 6.2 Hz, 1H), 3.90 (s, 3H), 3.59 (bs, 3H), 3.32 (s, 3H), 1.35 (d, J = 6.2 Hz, 6H).
Found: [M+H]=255.1.
Vinylmagnesium bromide (43 mL, 1 M in THF, 43 mmol) was added to a solution of S13 (5.78
g, 21.7 mmol) in THF (100 mL, dist. Na) at 0 °C, the brown solution was stirred at 0 °C for 1
h and then dimethylamine (43 mL, 2 M in THF, 86 mmol) and water (40 mL) were added. The
solution was stirred at r.t. for 1 h then partitioned between EtOAc and water. The solution was
dried and evaporated to give 3-(dimethylamino)-1-(2-methoxy-6-propoxypyridin-4-yl)propan-
1-one (CD-3) (5.75 g, 100%). 1H NMR (CDCl3) δ 6.73 (d, J = 1.1 Hz, 1H), 6.72 (d, J = 1.1 Hz,
1H), 4.26 (t, J = 6.7 Hz, 2H), 3.93 (s, 3H), 3.06 (t, J = 7.4 Hz, 2H), 2.72 (J = 7.4 Hz, 2H), 2.27
(s, 6H), 1.80 (qt, J = 7.4, 6.7 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H). Found: [M+H]=267.2.
4.1.2.3. 1-(2-Cyclobutoxy-6-methoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (CD-4)
OMe
O
OH
O
N
O
OMe
O
N
MeO
O
O
N
OMe
O
N
OMe
O
N
OMe
O
N
OMe
HO
N
OMe
CD-4
S14
S15
S16
S4
A solution of S4 (3.00 g, 16.4 mmol) in DMF (50 mL, anhydrous) was treated with K2CO3
(4.52 g, 32.7 mmol) and then bromocyclobutane (2.00 mL, 25.0 mmol). The mixture was
stirred at r.t. for 48 h, partitioned between EtOAc and water and the aqueous layer was
extracted with EtOAc. The organic fractions were washed with water, dried and evaporated.
Column chromatography (DCM) gave methyl 2-cyclobutoxy-6-methoxyisonicotinate (S14)
(2.21 g, 57%). 1H NMR (CDCl3) δ 6.84 (d, J = 1.0 Hz, 1H), 6.79 (d, J = 1.0 Hz, 1H), 5.08

(pd, J = 7.4, 0.8 Hz, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 2.42-2.52 (m, 2H), 2.12-2.24 (m, 2H),
1.80-1.90 (m, 1H), 1.62-1.75 (m, 1H). Found: [M+H]=238.2.
A solution of LiOH (0.71 g, 29.6 mmol) in water (20 mL) was added to a solution of S14
(2.20 g, 9.29 mmol) in MeOH (20mL) and THF (20 mL); the solution was stirred at r.t. for 18
h and then evaporated. The residue was dissolved in water (80 mL) and acidified to pH 3 with
2 M HCl. The precipitate was filtered and dried to give 2-cyclobutoxy-6-methoxyisonicotinic
acid (S15) (2.02 g, 97%). 1H NMR (DMSO-d6) δ 13.56 (bs, 1H), 6.74 (d, J = 1.0 Hz, 1H),
6.67 (d, J = 1.0 Hz, 1H), 5.07 (pd, J = 7.1, 0.7 Hz, 1H), 3.85 (s, 3H), 2.37-2.46 (m, 2H), 2.14-
2.22 (m, 2H), 1.74-1.83 (m, 1H), 1.59-1.72 (m, 1H). Found: [M+H]=224.2.
Oxalyl chloride (0.45 mL, 5.32 mmol) was added to a suspension of 173*S15 (1.00 g, 4.48
mmol) in DCM (50 mL, anhydrous) and DMF (0.2 mL) at r.t.. The mixture was stirred at r.t.
for 1 h to give a colourless solution which was cooled to 0 °C. N,O-Dimethylhydroxylamine
hydrochloride (0.52 g, 5.33 mmol) and pyridine (1.09 mL, 13.5 mmol) were added
sequentially and the mixture was stirred at r.t. for 18 h, then partitioned between DCM and
sat. aq. NaHCO3. Column chromatography on alumina with DCM gave 2-cyclobutoxy-N,6-
dimethoxy-N-methylisonicotinamide (S16) (1.01 g, 85%). 1H NMR (CDCl3) δ 6.46 (s, 1H),
6.40 (d, J = 0.6 Hz, 1H), 5.08 (pd, J = 7.4, 0.9 Hz, 1H), 3.90 (s, 3H), 3.58 (bs, 3H), 3.32 (s,
3H), 2.41-2.51 (m, 2H), 2.12-2.23 (m, 2H), 1.80-1.90 (m, 1H), 1.62-1.74 (m, 1H). Found:
[M+H]=267.2.
Vinylmagnesium bromide (8.0 mL, 1 M, 8.0 mmol) was added to a solution of S16 (1.02 g,
3.84 mmol) in THF (50 mL, dist. Na) at 0 °C, the brown solution was warmed to r.t. for 1 h
then dimethylamine in THF (8.0 mL, 2M, 16.0 mmol) and water (10 mL) were added. The
solution was stirred at r.t. for 1 h, and then partitioned between EtOAc and water. The
solution was dried and evaporated, column chromatography (95:5 DCM:MeOH) gave 1-(2-
cyclobutoxy-6-methoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (CD-4) (1.05 g, 97%).
1H NMR (CDCl3) δ 6.73 (d, J = 1.1 Hz, 1H), 6.68 (d, J = 1.2 Hz, 1H), 5.10 (pd, J = 7.4, 1.1
Hz, 1H), 3.91 (s, 3H), 3.05 (t, J = 7.0 Hz, 2H), 2.72 (t, J =7.0 Hz, 2H), 2.42-2.50 (m, 2H),
2.27 (s, 6H), 2.13-2.23 (m, 2H), 1.80-1.90 (m, 1H), 1.62-1.74 (m, 1H). Found: [M+H]=279.2.
4.1.2.4.
1-(2-(Difluoromethoxy)-6-methoxypyridin-4-yl)-3-(dimethylamino)propan-1-one
(CD-5)
O
OH
O
N
O
N
O
OMe
O
O
HF2C
HF2C
HF2C
HF2C
O
N
O
O
N
O
N
O
O
N
O
S17
S19
CD-5
S18
To a solution of S4 (3.00 g, 16.4 mmol) in DMF (40 mL) was added sodium
chlorodifluoroacetate (7.50 g, 49.2 mmol) and K2CO3 (2.73 g, 21.3 mmol). The reaction
mixture was stirred at 80 °C for 72 h, then washed with water (50 mL) and extracted with
EtOAc (3 x 30 mL). The combined organic layers were washed with brine (50 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue.
Purification by flash column chromatography using hexanes:EtOAc (1:1) gave ethyl 2-
1
(difluoromethoxy)-6-methoxyisonicotinate (S17) as a white solid (2.02 g, 52%). H NMR

(CDCl3) δ 7.40 (t, J = 73.0, 1H), 7.10 (d, J = 1.0 Hz, 1H), 7.00 (d, J = 1.0 Hz, 1H), 3.94 (s, 3H),
3.93 (s, 3H). Found: [M+H]=234.5.
To a solution of S17 (2.02 g, 8.66 mmol) in MeOH:THF:H2O (60 mL, 1:1:1) was added lithium
hydroxide (0.62 g, 26.0 mmol) and the reaction mixture was stirred at r.t. for 72 h. The solvent
was removed under reduced pressure, water (50 mL) was added and the mixture was washed
with EtOAc (50 mL) which was discarded. 2M HCl (50 mL) was added to the aqueous layer,
which was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with
brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain
2-(difluoromethoxy)-6-methoxyisonicotinic acid (S18) as a white solid (1.90 g, 99%). 1H NMR
(DMSO-d6) δ 13.85 (bs, 1H), 7.76 (t, J = 72.5 Hz, 1H), 7.03 (d, J = 0.96 Hz, 1H), 6.95 (d, J =
0.92 Hz, 1H), 3.91 (s, 3H). Found: [M+H]=220.6.
To a solution of S18 (1.70 g, 8.53 mmol), hydroxybenzotriazole (1.29 g, 9.54 mmol), N,O-
dimethylhydroxylamine hydrochloride (1.27 g, 13.0 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (1.48 g, 9.54 mmol) in DCM (50 mL) was added DMF
(4.83 mL, 34.7 mmol). The reaction mixture was stirred at r.t for 24 h. The reaction mixture
was washed with water (100 mL) and extracted with DCM (3 x 50 mL). The organic phase was
dried with Na2SO4 and concentrated to give a yellow residue. Purification by flash column
chromatography using hexanes:EtOAc (1:1) gave 2-(difluoromethoxy)-N,6-dimethoxy-N-
1
methylisonicotinamide (S19) as a yellow oil (1.85 g, 81%). H NMR (CDCl3) δ 7.41 (t, J =
73.1 Hz, 1H), 6.73 (d, J = 0.68 Hz, 1H), 6.65 (d, J = 0.64 Hz, 1H), 3.92 (s, 3H), 3.58 (s, 3H),
3.34 (s, 3H). Found: [M+H]=263.5.
To a solution of S19 (1.85 g, 7.06 mmol) in THF (100 mL) at 0 °C was added vinylmagnesium
bromide (1M solution in THF, 14.8 mL, 14.8 mmol) and the solution stirred at 0 °C for 3 h.
Dimethylamine (2M solution in THF, 14.8 mL, 29.7 mmol) was added followed by water (40
mL). After 30 minutes stirring at r.t., the reaction mixture was concentrated under reduced
pressure to obtain a brownish residue. This was extracted with EtOAc (3 x 200 mL). The
combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure to give 1-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)-
3-(dimethylamino)propan-1-one (CD-5) as a brown oil (1.92 g, 99%). 1H NMR (CDCl3) δ 7.40
(t, J = 73.0 Hz, 1H), 6.97 (d, J = 1.1 Hz, 1H), 6.89 (d, J = 1.1 Hz, 1H), 3.95 (s, 3H), 3.07 (t, J
= 7.0 Hz, 2H), 2.73 (t, J = 7.3 Hz, 2H), 2.27 (s, 6H). Found: [M+H]= 275.6.
4.1.2.5. 3-(Dimethylamino)-1-(2-(dimethylamino)-6-methoxypyridin-4-yl)propan-1-one (CD-
6)
O
N
O
OMe
O
OH
O
N
O
O
S
N
S
N
O
S
N
O
S
N
O
S21
S22
CD-6
S20
To a mixture of methyl 2-methoxy-6-(methylthio)isonicotinate (S20) (WO 2010/036632) (2.07
g, 9.71 mmol) in MeOH:THF:H2O (60 mL, 1:1:1) was added lithium hydroxide (0.697 g, 29.1
mmol). The reaction mixture was stirred at r.t. for 24 h. The solvent was removed under
reduced pressure, water (50 mL) was added and the mixture was washed with EtOAc (50 mL)

which was discarded. 2M HCl (50 mL) was added to the aqueous layer, which was extracted
with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (100 mL), dried
over Na2SO4, filtered and concentrated under reduced pressure to obtain 2-methoxy-6-
1
(methylthio)isonicotinic acid (S21) as a white solid (1.70 g, 88%). H NMR (DMSO-d6) δ
13.67 (s, 1H), 7.22 (d, J = 0.92 Hz, 1H), 6.86 (d, J = 0.92 Hz, 1H), 3.90 (s, 3H), 2.55 (s, 3H).
Found: [M+H]=200.5.
To a solution of S21 (1.70 g, 8.53 mmol) in DCM (200 mL) was added DMF (0.13 mL),
followed by dropwise addition of oxalyl chloride (0.88 mL, 10.2 mmol). The mixture was
stirred at r.t. for 2 h. The reaction mixture was cooled to 0 °C and N,O-dimethylhydroxylamine
hydrochloride (0.915, 9.38 mmol) followed by pyridine (2.27 mL, 28.2 mmol) were added and
resulting mixture was stirred at r.t. for 18 h. The mixture was poured onto sat. NaHCO3 (150
mL) and extracted with DCM (150 mL) and EtOAc (100 mL). The combined organic phase
was dried with Na2SO4 and concentrated to give a yellow residue. Purification by flash column
chromatography using hexanes:EtOAc (4:1) gave N,2-dimethoxy-N-methyl-6-
(methylthio)isonicotinamide (S22) as yellow oil (2.07 g, 99%). 1H NMR (CDCl3) δ 6.93 (d, J
= 0.72 Hz, 1H), 6.59 (s, 1H), 3.97 (s, 3H), 3.57 (s, 3H), 3.32 (s, 3H), 2.57 (s, 3H). Found:
[M+H]=243.5.
To a solution of S22 (2.07 g, 8.54 mmol) in THF (100 mL) at 0 °C was added vinylmagnesium
bromide (1M solution in THF, 17.9 mL, 17.9 mmol) and the solution was stirred at 0 °C for 3
h. Dimethylamine (2M solution in THF, 17.9 mL, 35.9 mmol) was added followed by water
(40 mL). After 30 minutes stirring at r.t. the reaction mixture was concentrated under reduced
pressure to obtain a brownish residue. This was extracted with EtOAc (3 x 200 mL). The
combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure to give 3-(dimethylamino)-1-(2-methoxy-6-
(methylthio)pyridin-4-yl)propan-1-one (CD-6) as a brown oil (2.17 g, 99%). 1H NMR (CDCl3)
δ 7.19 (d, J = 1.2 Hz, 1H), 6.82 (d, J = 1.1 Hz, 1H), 3.98 (s, 3H), 3.04 (t, J = 7.0 Hz, 2H), 2.72
(t, J = 7.3 Hz, 2H), 2.58 (s, 3H), 2.27 (s, 6H). Found: [M+H]=255.6.
4.1.2.6. 3-(Dimethylamino)-1-(2-(dimethylamino)-6-methoxypyridin-4-yl)propan-1-one (CD-
7)
O
N
O
O
O
OH
O
O
O
N
O
N
O
N
N
O
N
N
O
N
N
O
N
Cl
O
N
S24
S25
S26
S23
CD-7
To a glass tube was charged ethyl 2-chloro-6-methoxyisonicotinate (S23) (WO 2009/024905)
(4.01 g, 18.60 mmol), diphenylphosphino-1,1'-binaphthol (1.85 g, 2.976 mmol) and cesium
carbonate (8.49 g, 26.10 mmol) under continuous nitrogen flow. Anhydrous toluene (72 mL)
was added. The mixture was purged with nitrogen for 5 min. Palladium acetate (0.33 g, 1.488
mmol) was added, the mixture was purged again with nitrogen. Dimethylamine in
tetrahydrofuran (2M, 11.2 mL, 22.3 mmol) was added and the mixture was sealed in the tube
and heated at 80 °C for 19.5 hours. The mixture was filtered through Celite, washing with
EtOAc. The filtrate was concentrated in vacuo to yield the crude product as a dark red liquid.

Flash column chromatography of the crude product using 3-10% Et2O in hexanes provided
ethyl 2-(dimethylamino)-6-methoxyisonicotinate (S24) as a yellow crystalline solid. Yield =
3.36 g, 80%. 1H NMR (CDCl3)  6.61 (1H, d, J = 0.96 Hz), 6.52 (1H, d, J = 0.88 Hz), 4.35
(2H, q, J = 7.1 Hz), 3.90 (3H, s), 3.1 (6H, s), 1.38 (3H, t, J = 7.2 Hz). Found: [M+H]=225.5.
To a solution of S24 (6.77 g, 30.2 mmol) in EtOH (150 mL) was added at r.t. NaOH (30.2
mL, 60.4 mmol). The mixture was stirred at r.t. for 2 hours and then concentrated in vacuo to
a light yellow solution, which was further diluted in water. The aqueous mixture was
acidified to pH ~2 with 2M HCl, when bright yellow solids precipitated out. These were
collected by filtration, washed with water and dried under ambient conditions to yield 2-
(dimethylamino)-6-methoxyisonicotinic acid (S25) as a bright yellow powder. Yield = 5.60
g, 95%. 1H NMR (DMSO-d6)  13.25 (1H, br s), 6.54 (1H, d, J = 0.9 Hz), 6.33 (1H, d, J = 0.8
Hz), 3.82 (3H, s), 3.04 (6H, s).
To a suspension of S25 (0.75 g, 3.81 mmol) in anhydrous DMF (22 mL) was added
triethylamine (1.6 mL, 11.4 mmol), the mixture was cooled to 2 °C. Ethyl chloroformate
(0.62 mL, 4.19 mmol) was added dropwise and fumes were evolved. The mixture was stirred
from 2 °C to r.t. for 2.5 hours and then treated with N,O-dimethylhydroxylamine
hydrochloride (0.557 g, 5.71 mmol) at 2 °C under nitrogen. The mixture was stirred to r.t.
overnight. The mixture was diluted in water, and the aqueous mixture was extracted with
EtOAc (4x) and the organic extract was washed with water, brine, dried (MgSO4) and
concentrated to afford the crude product. This was purified by repeated flash chromatography
eluting with mixtures of 4:1 then 2:1 hexanes/EtOAc as eluent, yielding 2-(dimethylamino)-
N,6-dimethoxy-N-methylisonicotinamide (S26) as a yellow oil. Yield = 0.70 g, 77%. 1H
NMR (CDCl3)  6.17 (1H, s), 6.11 (1H, s), 3.89 (3H, s), 3.62 (3H, br s), 3.31 (3H, s), 3.08
(6H, s).
To a solution of S26 (2.53 g, 10.60 mmol) in freshly distilled THF (96 mL) was added at 2
°C under nitrogen vinylmagnesium bromide in THF (1N, 21.1 mL, 21.10 mmol) dropwise.
The mixture was stirred at 2 °C for 20 min, then at r.t. for 75 min. Dimethylamine in THF
(2N, 21.1 mL, 42.20 mmol) was added, followed by water (40 mL). The mixture was stirred
at r.t. for 1 hour. The aqueous mixture was partitioned between water and EtOAc and the
organic phase was separated and the aqueous phase was extracted with EtOAc (2x). The
organic extract was washed with brine, dried (Na2SO4) and concentrated in vacuo to give the
crude product as a yellow-brown oil. Flash chromatography of the product using 2-8%
MeOH in DCM as eluent gave 3-(dimethylamino)-1-(2-(dimethylamino)-6-methoxypyridin-
4-yl)propan-1-one (CD-7) as a yellow oil which crystallised at -20 °C. Yield = 1.91 g, 72%.
1H NMR (CDCl3)  6.47 (1H, d, J = 1.0 Hz), 6.39 (1H, d, J = 1.0 Hz), 3.91 (3H, s), 3.10 (6H,
s), 3.06 (2H, t, J = 7.0 Hz), 2.72 (2H, t, J = 7.1 Hz), 2.27 (6H, s).
4.1.2.7. 3-(Dimethylamino)-1-(2-ethoxy-6-isopropoxypyridin-4-yl)propan-1-one (CD-8)
OMe
O
OEt
O
OEt
O
N
O
OH
O
N
OiPr
EtO
N
OiPr
EtO
N
OiPr
EtO
N
OH
EtO
N
OiPr
EtO
N
S27
S28
CD-8
S29
S30

A suspension of 2,6-dihydroxyisonicotinic acid (40.00 g, 258 mmol) in EtOH (300 mL) was
treated dropwise with H2SO4 (40 mL, 18.4 M, 752 mmol). The solution was refluxed for 72 h
then evaporated; the residue was treated with sat. aq. NaHCO3 to pH 8 and then extracted with
EtOAc (3 x 500 mL). The organic extracts were washed with sat. aq. NaHCO3, brine, then
dried and evaporated to give ethyl 2-ethoxy-6-hydroxyisonicotinate (S27) (10.86 g, 20%). 1H
NMR (DMSO-d6) δ 11.15 (bs, 1H), 6.59 (d, J = 1.0 Hz, 1H), 6.57 (bs, 1H), 4.29 (q, J = 7.1 Hz,
2H), 4.25 (q, J = 7.1 Hz, 2H), 1.298 (t, J = 7.1 Hz, 3H), 1.296 (t, J = 7.1 Hz, 3H). Found:
[M+H]=212.2.
A solution of S27 (10.82 g, 51.2 mmol) in DMF (125 mL, anhydrous) was treated with K2CO3
(8.65 g, 62.5 mmol) and then 2-iodopropane (6.4 mL, 64 mmol). The mixture was stirred at r.t.
for 48 h, K2CO3 (8.65 g, 62.5 mmol) and 2-iodopropane (6.4 mL, 64 mmol) were added and
the mixture was stirred for a further 24 h, partitioned between EtOAc and water and the aqueous
layer was extracted with EtOAc. The organic fractions were washed with water, dried and
evaporated. Chromatography (DCM) gave ethyl 2-ethoxy-6-isopropoxyisonicotinate (S28)
(11.56 g, 89%). 1H NMR (DMSO-d6) δ 6.81 (s, 2H), 5.23 (sp, J = 6.2 Hz, 1H), 4.20-4.38 (m,
4H), 1.35-1.42 (m, 12H). Found: [M+H]=254.1.
A solution of LiOH (3.25 g, 136 mmol) in water (60 mL) was added to a solution of S28 (11.42
g, 45.1 mmol) in THF (60 mL) and MeOH (60 mL), the solution was stirred at r.t. for 18 h and
then evaporated. The residue was dissolved in water (150 mL) and acidified to pH 3 with 2 M
HCl. The precipitate was filtered and dried to give 2-ethoxy-6-isopropoxyisonicotinic acid
(S29) (10.03 g, 99%). 1H NMR (DMSO-d6) δ 13.48 (bs, 1H), 6.66 (d, J = 0.9 Hz, 1H), 6.64 (d,
J = 0.9 Hz, 1H), 5.17 (sp, J = 6.2 Hz, 1H), 4.29 (q, J = 7.0 Hz, 2H), 1.32 (t, J = 7.0 Hz, 3H),
1.30 (d, J = 6.2 Hz, 6H). Found: [M+H]=226.1.
Oxalyl chloride (3.13 mL, 37 mmol) was added to S29 (6.95 g, 30.8 mmol) in DCM (100 mL,
anhydrous) and DMF (5.2 mmol) at r.t.. The mixture was stirred at r.t. for 1 h to give a
colourless solution which was cooled to 0 °C. N,O-Dimethylhydroxylamine hydrochloride
(3.61 g, 37.0 mmol) and pyridine (7.5 mL, 92.7 mmol) were added sequentially and the mixture
was stirred at r.t. for 18 h, then partitioned between EtOAc and water. The organic fractions
were washed with water, dried and evaporated. Column chromatography with 95:5
DCM:EtOAc gave 2-ethoxy-6-isopropoxy-N-methoxy-N-methylisonicotinamide (S30) (7.98
g, 97%). 1H NMR (CDCl3) δ 6.40 (s, 1H), 6.39 (s, 1H), 5.22 (sp, J = 6.2 Hz, 1H), 4.33 (q, J =
7.1 Hz, 2H), 3.60 (bs, 3H), 3.31 (s, 3H), 1.39 (t, J = 6.2 Hz, 3H), 1.34 (d, J = 7.1 Hz, 6H).
Found: [M+H]=269.2.
Vinylmagnesium bromide (40 mL, 1 M in THF, 40 mmol) was added to a solution of S30 (5.33
g, 19.9 mmol) in THF (100 mL, dist. Na) at 0 °C, the brown solution was stirred at 0 °C for 1
h and then dimethylamine (40 mL, 2 M in THF, 80 mmol) and water (40 mL) were added. The
solution was stirred at r.t. for 1 h then partitioned between EtOAc and water. The solution was
dried and evaporated, to give 3-(dimethylamino)-1-(2-ethoxy-6-isopropoxypyridin-4-
yl)propan-1-one (CD-8) (5.57 g, 100%) as a brown oil. 1H NMR (CDCl3) δ 6.68 (d, J = 1.0 Hz,
1H), 6.67 (d, J = 1.0 Hz, 1H), 5.22 (sp, J = 6.2 Hz, 1H), 4.33 (q, J = 7.0 Hz, 2H), 3.05 (t, J =
7.5 Hz, 2H), 2.72 (t, J = 7.5 Hz, 2H), 2.26 (s, 6H), 1.40 (t, J = 6.2 Hz, 3H), 1.36 (d, J = 7.0 Hz,
6H). Found: [M+H]=281.7.
4.1.2.9. 3-(Dimethylamino)-1-(2-ethoxy-6-propoxypyridin-4-yl)propan-1-one (CD-9)

O
N
O
N
CO2Et
CO2Et
CO2H
O
ii
I
iv
iii
OnPr
100%
75%
EtO
N
OnPr
OnPr
EtO
N
OH
EtO
N
OnPr
EtO
N
EtO
N
S31
S32
S33
CD-9
S34
A mixture of ethyl 2-ethoxy-6-hydroxyisonicotinate (S31) (10.0 g, 47.3 mmol) and potassium
carbonate (5.80 g, 59.2 mmol) in DMF (100 mL) was treated with 1-iodopropane (8.17 mL,
59.4 mmol) and then stirred at room temperature for 72 h. The mixture was partitioned with
EtOAc and water. The organic fractions were washed with water, dried and evaporated.
Column chromatography on silica with DCM gave ethyl 2-ethoxy-6-propoxyisonicotinate
1
(S32) (8.98 g, 75%). H NMR (CDCl3) δ 6.84 (d, J = 1.1 Hz, 1H), 6.83 (d, J = 1.1 Hz, 1H),
4.36 (q, J = 7.2 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.23 ( t, J = 6.7 Hz, 2H), 1.80 (qt, J = 7.4, 6.8
Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H). Found:
[M+H]=269.2.
A solution of lithium hydroxide (2.53, 106.0 mmol) in water (60 mL) was added to a solution
of S32 (35.4 mmol) in methanol (60mL) and THF (60 mL), the solution was stirred at room
temperature for 18 h and then evaporated. The residue was dissolved in water (150 mL) and
acidified with 2 M HCl to pH 3. The precipitate was filtered, dissolved in EtOAc, dried and
evaporated to give 2-ethoxy-6-propoxyisonicotinic acid (S33) as a white solid (7.96 g, 100%).
1H NMR (DMSO-d6) δ 13.52 (bs, 1H), 6.70 (d, J = 1.0 Hz, 1H), 6.69 (d, J = 1.0 Hz, 1H), 4.30
(q, J = 7.0 Hz, 2H), 4.20 (t, J = 6.6 Hz, 2H), 1.78 (qt, J = 7.4, 6.8 Hz, 2H), 1.32 (t, J = 7.0 Hz,
3H), 0.95 (t, J = 7.4 Hz, 3H). Found: [M+H]= 226.1.
Oxalyl chloride (1.51 mL, 17.8 mmol) was added to S33 (3.35 g, 14.9 mmol) in DCM (100
mL, anhydrous) and DMF (0.4 mL, 5.2 mmol) at r.t.. The mixture was stirred at r.t. for 1 h to
give a colourless solution which was cooled to 0 °C. N,O-Dimethylhydroxylamine
hydrochloride (1.74 g, 17.8 mmol) and pyridine (3.61 mL, 44.6 mmol) were added sequentially
and the mixture was stirred at r.t. for 18 h, then partitioned between EtOAc and water. The
organic fractions were washed with water, dried and evaporated. Column chromatography with
DCM on alumina gave 2-ethoxy-N-methoxy-N-methyl-6-propoxyisonicotinamide (S34) (3.82
g, 96%). 1H NMR (CDCl3) δ 6.44 (s, 1H), 6.42 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.22 (t, J =
6.7 Hz, 2H), 3.59 (bs, 3H), 3.32 (s, 3H), 1.79 (qt, J = 7.4, 6.8 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H),
1.01 (t, J = 7.4 Hz, 3H). Found: [M+H]=254.2.
Vinylmagnesium bromide (28.0 mL, 1 M in THF, 28.0 mmol) was added to a solution of S34
(3.78 g, 14.0 mmol) in THF (150 mL, dist. Na) at 0 °C, the brown solution was stirred at 0 °C
for 1 h and then dimethylamine (28.0 mL, 2 M in THF, 56.0 mmol) and water (30 mL) were
added. The solution was stirred at r.t. for 1 h then partitioned between EtOAc and water. The
solution was dried and evaporated, to give 3-(dimethylamino)-1-(2-ethoxy-6-propoxypyridin-
4-yl)propan-1-one (CD-9) (3.86 g, 98%) as a brown oil. 1H NMR (CDCl3) δ 6.72 (d, J = 1.1
Hz, 1H), 6.70 (d, J = 1.1 Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 4.24 (t, J = 6.7 Hz, 2H), 3.05 (q, J
= 7.0 Hz, 2H), 2.71 (q, J = 7.0 Hz, 2H), 2.27 (s, 6H), 1.80 (qt, J = 7.4, 6.8 Hz, 2H), 1.40 (t, J =
7.1 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H). Found: [M+H]=281.7.
4.1.2.10. 1-(2,6-Bis(methylthio)pyridin-4-yl)-3-(dimethylamino)propan-1-one (CD-10)

O
OH
O
N
O
N
O
S
S
N
S
S
N
S
S
N
S35
S36
CD-10
To a solution of 2,6-dichloroisonicotinic acid (4.00 g, 20.8 mmol) in DMF (40 mL) at 0 °C was
added sodium thiomethoxide (4.38 g, 65.5 mmol). The reaction mixture was stirred at 150 °C
for 18 h. Water (40 mL) was added to the resultant solution and the pH was adjusted to ~3
using 2M HCl solution. The aqueous solution was extracted with EtOAc (3 x 40 mL), dried
with MgSO4, filtered and concentrated under reduced pressure to give 2,6-
bis(methylthio)isonicotinic acid (S35) as an orange solid, which was recrystallized from
methanol (4.01 g, 90%). 1H NMR (CDCl3) δ 7.44 (s, 2H), 2.61 (s, 6H). Found: [M+H]=216.5.
To a solution of S35 (5.03 g, 23.4 mmol) in DCM (200 mL) was added DMF (0.362 mL),
followed by dropwise addition of oxalyl chloride (2.41 mL, 28.0 mmol). The mixture was
stirred at r.t. for 2 h, cooled to 0 °C and N,O-dimethylhydroxylamine hydrochloride (2.51 g,
25.7 mmol) followed by pyridine (6.22 mL, 77.1 mmol) were added and resulting mixture was
stirred at r.t. for 18 h. The mixture was poured onto sat NaHCO3 (150 mL), extracted with
DCM (150 mL) and EtOAc (100 mL). The organic phase was dried with Na2SO4 and
concentrated to give N-methoxy-N-methyl-2,6-bis(methylthio)isonicotinamide (S36) as yellow
1
oil, which was used without further purification for the next step (4.96 g, 82%). H NMR
(CDCl3) δ 7.04 (s, 2H), 3.56 (s, 3H), 3.33 (s, 3H), 2.60 (s, 6H). Found: [M+H]=259.5.
To a solution of S36 (4.96 g, 19.2 mmol) in THF (90 mL) at 0 °C was added vinylmagnesium
bromide (1M solution in THF, 40.3 mL, 40.3 mmol) and the solution was stirred at 0 °C for 1
h. Dimethylamine (2M solution in THF, 40.3 mL, 80.6 mmol) was added followed by water
(60 mL). After 30 minutes stirring at r.t., the reaction mixture was concentrated under reduced
pressure to obtain a brownish residue. This was extracted with EtOAc (3 x 200 mL). The
combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure to give 1-(2,6-bis(methylthio)pyridin-4-yl)-3-
(dimethylamino)propan-1-one (CD-10) as a brown oil (4.87 g, 94%). 1H NMR (CDCl3) δ 7.26
(s, 2H), 3.04 (t, J = 7.0 Hz, 2H), 2.70 (t, J = 7.2 Hz, 2H), 2.61 (s, 6H), 2.26 (s, 6H). Found:
[M+H]=271.6.
4.1.2.11. 1-(2,6-Bis(ethylthio)pyridin-4-yl)-3-(dimethylamino)propan-1-one (CD-11)
O
OH
O
N
O
N
O
S
S
N
S
S
N
S
S
N
S37
CD-11
S38
To a suspension of sodium hydride (1.82 g, 45.5 mmol) in DMF (20 mL) at 0 °C was added
ethanethiol (3.29 mL, 45.5 mmol) dropwise. The milky foamy solution was stirred at 0 °C for
10 min 2,6-dichloroisonicotinic acid (3.17 g, 13.0 mmol) in DMF (5 mL) was added dropwise
to the solution. The mixture was warmed to 50 °C and stirred for 18 h. Water (40 mL) was

added to the resultant solution and the pH was adjusted to ~3 using 2M HCl solution. The
aqueous solution was extracted with EtOAc (3 x 30 mL), dried with MgSO4, filtered and the
solvent was evaporated to give 2,6-bis(ethylthio)isonicotinic acid (S37) as a yellow solid which
was used for the next step without further purification (2.82 g, 89%). 1H NMR (CDCl3) δ 7.40
(s, 2H), 3.19 (q, J = 7.3 Hz, 4H), 1.39 (t, J = 7.3 Hz, 6H). Found: [M+H]=244.5.
To a solution of S37 (3.24 g, 13.3 mmol) in DCM (150 mL) was added DMF (0.206 mL),
followed by dropwise addition of oxalyl chloride (1.37 mL, 16.0 mmol). The mixture was
stirred at r.t. for 2 h, then cooled to 0 °C and N,O-dimethylhydroxylamine hydrochloride (1.43
g, 14.6 mmol) followed by pyridine (3.55 mL, 43.9 mmol) were added and resulting mixture
was stirred at r.t. for 18 h. The mixture was poured onto sat. NaHCO3 (150 mL), extracted with
DCM (150 mL) and EtOAc (100 mL). The combined organic phase was dried with Na2SO4
and concentrated to give a yellow residue. Purification by flash column chromatography using
hexanes:EtOAc (1:1) gave 2,6-bis(ethylthio)-N-methoxy-N-methylisonicotinamide (S38) as
colourless oil (3.65 g, 96%). 1H NMR (CDCl3) δ 7.02 (s, 2H), 3.56 (s, 3H), 3.32 (s, 3H), 3.19
(q, J = 7.3 Hz, 4H), 1.37 (t, J = 7.3 Hz, 6H). Found: [M+H]=287.5.
To a solution of S38 (3.65 g, 12.7 mmol) in THF (150 mL) at 0 °C was added vinylmagnesium
bromide (1M solution in THF, 31.5 mL, 31.5 mmol) which was then stirred at 0 °C for 4 h.
Dimethylamine (2M solution in THF, 31.5 mL, 63.0 mmol) was added followed by water (60
mL). After 30 minutes stirring at r.t., the reaction mixture was concentrated under reduced
pressure to obtain a brownish residue. This was extracted with EtOAc (3 x 200 mL). The
combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure to give a brown oil. Purification by flash column
chromatography
using EtOAc:MeOH (9:1) gave 1-(2,6-bis(ethylthio)pyridin-4-yl)-3-
(dimethylamino)propan-1-one (CD-11) as a yellow oil (2.51 g, 66%). 1H NMR (CDCl3) δ 7.24
(s, 2H), 3.20 (q, J = 7.4 Hz, 4H), 3.03 (t, J = 7.1 Hz, 2H), 2.71 (t, J = 7.2 Hz, 2H), 2.26 (s, 6H),
1.38 (t, J = 7.4 Hz, 6H). Found: [M+H]=299.6.
4.1.2.12.
3-(Dimethylamino)-1-(2-(dimethylamino)-6-(ethylthio)pyridin-4-yl)propan-1-one
(CD-12)
O
O
O
O
O
N
O
N
O
OH
O
Cl
N
NMe2
EtS
N
NMe2
EtS
N
NMe2
EtS
N
NMe2
EtS
N
NMe2
CD-12
S39
S41
S40
S42
To a solution of methyl 2-chloro-6-(dimethylamino)isonicotinate (S39) (2.44 g, 11.4 mmol),
rac-bis(diphenylphosphino)-1,1'-binaphthal (0.71 g, 1.14 mmol) and cesium carbonate (4.43 g,
13.6 mmol) under continuous nitrogen flow. Anhydrous toluene (30 mL) was added. The
mixture was purged with nitrogen 5 minutes. Palladium acetate (0.26 g, 1.16 mmol) was added,
the mixture was added purged again with nitrogen. Ethanethiol (1.0 mL, 13.6 mmol) was added
and the mixture was sealed in the tube and heated at 150 °C for 22 hours. The mixture was
filtered through celite, washed with ethyl acetate. The filtrate was concentrated in the fume
hood by purging with air. Purification by flash column chromatography using diethyl
ether:hexane (5:95) gave methyl 2-(dimethylamino)-6-(ethylthio)isonicotinate (S40) as a
yellow crystalline solid (2.42 g, 88%). 1H NMR (CDCl3) δ 6.96 (d, J = 1.0 Hz, 1H), 6.73 (d, J

= 1.0 Hz, 1H), 3.89 (s, 3H), 3.16 (q, J = 7.3 Hz, 2H), 3.11 (s, 6H), 1.38 (t, J = 7.3 Hz, 3H).
Found: [M+H]=241.2.
To a solution of S40 (1.50 g, 6.25 mmol) in tetrahydrofuran (30 mL) was added at room
temperature a solution of lithium hydroxide (0.45 g, 18.8) in water (15 mL). The reaction
mixture was stirred at room temperautre overnight. Volatiles were removed in vacuo; the slurry
was treated with 2M hydrochloric acid until pH reached 4. The yellow solids were collected by
filtration, washed with water and dried to give 2-(dimethylamino)-6-(ethylthio)isonicotinic
acid (S41) as a bright yellow solid (1.39 g, 98%). 1H NMR (CDCl3) δ 13.36 (s, 1H), 6.79 (d, J
= 0.8 Hz, 1H), 6.69 (d, J = 0.8 Hz, 1H), 3.11 (q, J = 7.2 Hz, 2H), 3.07 (s, 6H), 1.30 (t, J = 7.2
Hz, 3H). Found: [M+H]=227.2.
To a solution of S41 (2.21 g, 9.77 mmol) in anhydrous DMF (60 mL) was added triethylamine
(4.1 ml, 29.3 mmol). The reaction mixture was cooled to 2 °C, ethyl chloroformate (1.6 ml,
10.7 mmol) was added dropwise. The mixture was stirred at ~4 °C for 1.5 h and was added
N,O-dimethylhydroxylamine hydrochloride (1.43 g, 14.7 mmol). The mixture was stirred at
room temperature for 18 h. The mixture was diluted in water, and the aqueous mixture was
extracted with EtOAc (3 X 200 ml). The organic extract was washed with water, brine, dried
over Na2SO4, filtered and concentrated under reduced pressure to give a crude product.
Purification by flash column chromatography using hexane:EtOAc (2:1) gave 2-
(dimethylamino)-6-(ethylthio)-N-methoxy-N-methylisonicotinamide (S42) as a yellow oil
(4.09 g, 86%). 1H NMR (CDCl3) δ 6.57 (s, 1H), 6.32 (s, 1H), 3.60 (s, 3H), 3.31 (s, 3H), 3.14
(q, J = 7.2 Hz, 2H), 3.09 (s, 6H), 1.38 (t, J = 7.2 Hz, 3H). Found: [M+H]=270.2.
To a solution of S42 (3.95 g, 14.7 mmol) in THF (140 mL) at 0 °C was added vinylmagnesium
bromide (1M solution in THF, 29.3 mL, 29.3 mmol) which was then stirred at 0 °C for 1.5 h.
Dimethylamine (2M solution in THF, 29.3 mL, 29.3 mmol) was added followed by water (50
mL). After 60 minutes stirring at r.t., the reaction mixture was concentrated under reduced
pressure to obtain a brownish residue. This was extracted with EtOAc (3 x 200 mL). The
combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure to give a brown oil. Purification by flash column
chromatography using EtOAc:MeOH (9:1) gave 3-(dimethylamino)-1-(2-(dimethylamino)-6-
(ethylthio)pyridin-4-yl)propan-1-one (CD-12) as a yellow oil (3.35 g, 81%). 1H NMR (CDCl3)
δ 6.83 (d, J = 1.2 Hz, 1H), 6.59 (d, J = 1.2 Hz, 1H), 3.17 (q, J = 7.2 Hz, 2H), 3.12 (s, 6H), 3.06
(t, J = 7.4 Hz, 2H), 2.72 (t, J = 7.6 Hz, 2H), 2.28 (s, 6H), 1.39 (t, J = 7.2 Hz, 6H). Found:
[M+H]=282.2.
O
F
OH
Br
NMe2
S
N
O
N
28
(Table 2)
S
4.1.3. Example of coupling reaction for the bromo compouds of Table 2: 2-(2,6-
bis(methylthio)pyridin-4-yl)-1-(6-bromo-2-methoxyquinolin-3-yl)-4-(dimethylamino)-1-(2-

fluoro-3-methoxyphenyl)butan-2-ol (compound 28 of Table 2). n-BuLi (4.79 mL of a 2N
solution in cyclohexane, 9.57 mmol) was added at -40 oC under dry nitrogen to a solution of
dry diisopropylamine (1.46 mL, 9.57 mmol) in dry THF (6 mL) and the solution was stirred at
o
this temperature for 10 min, then cooled to -78 C. A solution of 6-bromo-3-(2-fluoro-3-
methoxybenzyl)-2-methoxyquinoline (AB-10) (3.00 g, 7.97 mmol) in dry THF (10 mL) was
added dropwise and the mixture was stirred at -78 C for 90 min, to give a dark, wine-red
coloured
solution.
A
solution
of
1-(2,6-bis(methylthio)pyridin-4-yl)-3-
(dimethylamino)propan-1-one (CD-10) (2.16 g, 7.97 mmol) in dry THF (7 mL) was added and
the reaction mixture was stirred at this temperature for 5 h. Water (40 mL) was added and the
mixture was extracted with EtOAc (2 X 50 mL). The combined organic extract was washed
with sat. aq. NaHCO3 solution, and brine, then dried with Na2SO4 and the solvent removed
under reduced pressure. The residue was purified by flash column chromatography. Elution
with hexanes:EtOAc (1:1) afforded isomer A of 28(1.77 g, 34%) followed by isomer B of 28
(1.38 g, 27%) as pale yellow foamy solids.
Isomer A. 1H NMR (CDCl3, 400 MHz) δ 8.45 (s, 1H), 8.34 (br s, 1H), 7.83 (d, J = 2.2 Hz, 1H),
7.68 (d, J = 8.9 Hz, 1H), 7.62 (dd, J = 8.9, 2.2 Hz, 1H), 7.58-7.53 (m, 1H), 7.05 (br s, 2H), 6.85
(dt, J = 8.1, 1.5 Hz, 1H), 6.63 (dt, J = 8.1, 1.5 Hz, 1H), 5.35 (s, 1H), 4.15 (s, 3H), 3.71 (s, 3H),
2.55 (s, 6H), 2.28-2.20 (m, 1H), 2.04-1.99 (m, 1H), 2.01 (s, 6H), 1.98-1.91 (m, 1H), 1.71-1.66
(m, 1H). Found: [M+H]= 646.2.
Isomer B. 1H NMR (CDCl3, 400 MHz) δ 8.58 (s, 1H), 8.31 (br s, 1H), 7.79 (d, J = 1.6 Hz, 1H),
7.56-7.50 (m, 2H), 7.28 (dd, J = 7.9, 1.5 Hz, 1H), 7.09 (br s, 2H), 6.95 (dt, J = 8.1, 1.4 Hz, 1H),
6.82 (dt, J = 8.1, 1.4 Hz, 1H), 5.30 (s, 1H), 3.91 (s, 3H) 3.91 (s, 3H), 2.49 (s, 6H), 2.29-2.20
(m, 1H), 2.06-2.01 (m, 1H), 2.04 (s, 6H), 1.99-1.93 (m, 1H), 1.65-1.60 (m, 1H). Found:
[M+H]= 646.2.
The mixture was resolved into its four optical isomers using preparative supercritical fluid
HPLC at BioDuro LLC (Beijing). The data in Table 1 are for the most active R,S-diastereomers.
The other 6-bromo compounds in Table I were prepared and purified similarly.
O
F
OH
NC
NMe2
S
N
O
N
29
(Table 2)
S
4.1.4. Example of cyanation reaction: 3-(2-(2,6-bis(methylthio)pyridin-4-yl)-4-
(dimethylamino)-1-(2-fluoro-3-methoxyphenyl)-2-hydroxybutyl)-2-methoxyquinoline-6-
carbonitrile (compound 29 of Table 2). A solution of Isomer A of 28 (Table 2) (1.06 g, 1.64
mmol) in DMF (10 mL, anhydrous) was purged with nitrogen and heated to 55 °C for 10 min
Tri(o-tolyl)phosphine (0.10 g, 0.328 mmol), zinc dust (0.011 g, 0.164 mmol) and
tris(dibenzylideneacetone)dipalladium(0) (0.150 g, 0.164 mmol) were then added, and the

reaction was again purged with nitrogen and heated for another 10 min at 55 °C. Zinc cyanide
(0.135 g, 1.15 mmol) was then added and the reaction mixture was heated to 65 °C for 5 hours.
The reaction was diluted with water and extracted with EtOAc (3 X 50 mL). The organic layer
was washed with brine, dried with Na2SO4 and evaporated. Column chromatography with 1:1
hexane/EtOAc afforded Isomer A of 29 (0.48 g, 50%) as pale yellow solid. 1H NMR (CDCl3,
400 MHz) δ 8.57 (s, 1H), 8.40 (br s, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H),
7.71 (dd, J = 8.6, 1.9 Hz, 1H), 7.58-7.52 (m, 1H), 7.05 (br s, 2H), 6.87 (dt, J = 8.1, 1.5 Hz, 1H),
6.65 (dt, J = 8.1, 1.5 Hz, 1H), 5.35 (s, 1H), 4.19 (s, 3H), 3.71 (s, 3H), 2.55 (s, 6H), 2.28-2.21
(m, 1H), 2.06-2.01 (m, 1H), 2.01 (s, 6H), 1.95-1.87 (m, 1H), 1.72-1.66 (m, 1H). Found:
[M+H]= 593.2.
A solution of Isomer B of 28 (Table 2) (1.06 g, 1.64 mmol) in DMF (10 mL, anhydrous) was
purged with nitrogen and heated to 55 °C for 10 min Tri(o-tolyl)phosphine (0.10 g, 0.328
mmol), zinc dust (0.011 g, 0.164 mmol) and tris(dibenzylideneacetone)dipalladium(0) (0.150
g, 0.164 mmol) were then added, and the reaction was again purged with nitrogen and heated
for another 10 min at 55 °C. Zinc cyanide (0.135 g, 1.15 mmol) was then added and the reaction
mixture was heated to 65 °C for 5 hours. The reaction was diluted with water and extracted
with EtOAc (3 X 50 mL). The organic layer was washed with brine, dried with Na2SO4 and
evaporated. Column chromatography with 1:1 hexane/EtOAc afforded Isomer B of 29 (0.59 g,
77%) as white solid. 1H NMR (CDCl3, 400 MHz) δ 8.72 (s, 1H), 8.40 (br s, 1H), 8.02 (d, J =
1.7 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.63 (dd, J = 8.6, 1.8 Hz, 1H), 7.31-7.28 (m, 1H), 7.09
(br s, 2H), 6.98 (dt, J = 8.2, 1.4 Hz, 1H), 6.84 (dt, J = 8.1, 1.4 Hz, 1H), 5.28 (s, 1H), 3.94 (s,
3H) 3.91 (s, 3H), 2.49 (s, 6H), 2.31-2.21 (m, 1H), 2.07-2.04 (m, 1H), 2.03 (s, 6H), 2.02-1.93
(m, 1H), 1.65-1.60 (m, 1H). Found: [M+H]= 593.2.
The mixture was resolved into its four optical isomers using preparative supercritical fluid
HPLC at BioDuro LLC (Beijing). The data in Table 1 are for the most active R,S-diastereomers.
The other 6-cyano compounds in Table I were prepared and purified similarly.
4.1.5. 1-(6-Bromo-2-methoxyquinolin-3-yl)-4-(dimethylamino)-1-(2-fluoro-3-methoxyphenyl)-
2-(2-methoxy-6-(methylsulfonyl)pyridin-4-yl)butan-2-ol (compound 23 of Table 2)
O
F
O
F
OH
OH
N
Br
N
Br
O
O
N
O
N
O
N
N
21
23
S O
S
O
A solution of 21 (1.20 g, 1.90 mmol) in THF:water:acetone (2:1:1, 100 ml) was treated with
N-methylmorpholine N-oxide (0.669 g, 5.71 mmol) followed by osmium tetroxide (1.81 g,
0.285 mmol) dropwise. The reaction was stirred for 27 h at room temperature. Reaction was
washed with Na2SO3 (sat) solution, extracted with EtOAc (x 3), dried with Na2SO4, filtered
and the solvent was evaporated to give a yellow oil. Purification by flash column
chromatography with silica using hexanes:EtOAc (1:4) gave 23 as a white solid (1.10 g, 87%).
1H NMR (CDCl3) δ 8.63-8.56 (m, 3H), 7.88 (d, J = 2.0 Hz, 1H), 7.87-7.81 (m, 3H), 7.68 (d, J

= 8.9 Hz, 1H), 7.62 (dd, J = 8.8, 2.2 Hz, 1H), 7.56-7.48 (m, 3H), 7.40-7.33 (m, 1H), 7.16 (s,
1H), 7.02-6.96 (m, 1H), 6.88-6.82 (m, 2H), 6.66-6.60 (m, 1H), 5.39 (s, 1H), 5.35 (s, 1H), 4.13
(s, 3H), 3.95 (s, 3H), 3.91 (s, 6H), 3.90 (s, 3H), 3.68 (s, 3H), 3.13 (s, 3H), 3.07 (s, 3H), 2.22-
2.13 (m, 2H), 2.13-2.05 (m, 4H), 2.04 (s, 6H), 2.02 (s, 6H), 1.82-1.69 (m, 2H). Found:
[M+H]=662.2.

Acknowledgments
This work was supported by the Bill & Melinda Gates Foundation, the U.S. Agency for
International Development (USAID), the U.K. Department for International Development
(DFID), the Directorate General for International Cooperation of the Netherlands (DGIS),
and Irish Aid.
Appendix A. Supplementary data
Supplementary date to this article can be found at *****. These data include MOL files and
1H NMR spectra of the new molecules of Table 1.
References
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Acknowledgements
This work was supported by the Bill & Melinda Gates Foundation (#OPP1017459), the U.S.
Agency for International Development (GHS-A-00-08-00012-00), the U.K. Department for
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Supplementary data
Supplementary data (MOL files for the compound of Tables 2 and 5) associated with this
article can be found in the online version

Graphical abstract
Y
N
OH
OH
Br
NMe2
X
NMe2
R2
N
O
N
O
N
R1
Property
clogP
MIC
hERG
HClint
AUC
Bedaquiline
7.25
0.08
1.6
Table 1 compounds
3.73 – 8.18
<0.004 – 0.46
1.5 - >30
3.0
17.4
56
0.1 – 17
1.1 – 33
11 - 83
F (%)

DARQ 6
Highlights




bedaquiline analogues with 3,5-disubstituted-4-pyridyl C-units
high potency and attenuated hERG inhibition cf bedaquiline
4-6.5 log units of bacterial clearance from lung in mouse model
oral bioavailability generally 40-60%