Synthetic studies to help elucidate the metabolism of the preclinical candidate TBAJ-876—a less toxic and more potent analogue of bedaquiline

Article abstract of DOI:10.3390/molecules25061423

Bedaquiline is a novel drug approved in 2012 by the FDA for treatment of drug-resistant tuberculosis (TB). Although it shows high efficacy towards drug-resistant forms of TB, its use has been limited by the potential for significant side effects. In particular, bedaquiline is a very lipophilic compound with an associated long terminal half-life and shows potent inhibition of the cardiac potassium hERG channel, resulting in QTc interval prolongation in humans that may result in cardiac arrhythmia. To address these issues, we carried out a drug discovery programme to develop an improved second generation analogue of bedaquiline. From this medicinal chemistry program, a candidate (TBAJ-876) has been selected to undergo further preclinical evaluation. During this evaluation, three major metabolites arising from TBAJ-876 were observed in several preclinical animal models. We report here our synthetic efforts to unequivocally structurally characterize these three metabolites through their independent directed synthesis.

Full text of DOI:10.3390/molecules25061423

molecules
Article
Synthetic Studies to Help Elucidate the Metabolism
of the Preclinical Candidate TBAJ-876—A Less Toxic
and More Potent Analogue of Bedaquiline
Peter J. Choi 1,2,
*
, Hamish S. Sutherland 1, Adrian Blaser 1 , Amy S.T. Tong 1,
Christopher B. Cooper 3 , Anna M. Upton 3, Brian D. Palmer 1,2 and William A. Denny 1,2
1
Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand; h.sutherland@auckland.ac.nz (H.S.S.);
a.blaser@auckland.ac.nz (A.B.); amytong0606@gmail.com (A.S.T.T.); b.palmer@auckland.ac.nz (B.D.P.);
b.denny@auckland.ac.nz (W.A.D.)
Maurice Wilkins Centre, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
Global Alliance for TB Drug Development, 40 Wall St, New York, NY 10005, USA;
2
3
christopher.cooper@tballiance.org (C.B.C.); anna.upton@tballiance.org (A.M.U.)
*
Correspondence: p.choi@auckland.ac.nz
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 February 2020; Accepted: 19 March 2020; Published: 20 March 2020
Abstract: Bedaquiline is a novel drug approved in 2012 by the FDA for treatment of drug-resistant
tuberculosis (TB). Although it shows high efficacy towards drug-resistant forms of TB, its use has
been limited by the potential for significant side effects. In particular, bedaquiline is a very lipophilic
compound with an associated long terminal half-life and shows potent inhibition of the cardiac
potassium hERG channel, resulting in QTc interval prolongation in humans that may result in cardiac
arrhythmia. To address these issues, we carried out a drug discovery programme to develop an
improved second generation analogue of bedaquiline. From this medicinal chemistry program,
a candidate (TBAJ-876) has been selected to undergo further preclinical evaluation. During this
evaluation, three major metabolites arising from TBAJ-876 were observed in several preclinical animal
models. We report here our synthetic efforts to unequivocally structurally characterize these three
metabolites through their independent directed synthesis.
Keywords: bedaquiline; TMC207; Sirturo; bedaquiline analogues; TBAJ-876; mycobacterium
tuberculosis; tuberculosis; drug development
1. Introduction
Tuberculosis (TB) is one of the top ten leading causes of death in the world and is the leading cause
of death from a single infectious agent. In fact, TB is more lethal than HIV/AIDS globally [
estimated TB caused 1.5 million deaths in 2018, with 10 million new cases of TB estimated for that year
alone. The often late detection of TB [ ], coupled with the need to use long and complex multi-drug
1]. It was
2
treatment regimens to treat it, has led to an alarming increase in cases which are resistant to the
standard front-line drugs for drug sensitive TB (i.e., rifamicin, isoniazid, pyrazinamide, and ethambutol).
In 2017, about 558,000 people developed TB that was resistant to rifampicin, and of these, 82% had
multidrug-resistant TB (MDR TB), and this proportion has been rising rapidly [1].
The discovery and regulatory approval of the novel antitubercular agent bedaquiline
Sirturo; Figure 1) has been of great significance within the TB community. It has a unique diarylquinoline
(DARQ) structure and a novel mechanism of action (inhibition of the mycobacterial ATP synthase [ ]),
1 (TMC207,
3
and has shown significant activity against drug-resistant tuberculosis strains (MDR TB, and XDR TB).
When added to standard background therapy used for MDR TB, it demonstrated more rapid bactericidal
Molecules 2020, 25, 1423; doi:10.3390/molecules25061423
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1423
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activity than standard therapy alone [
4
] and was approved by the FDA for treatment of MDR TB in
2012, making it the first TB drug with a novel mode of action in 40 years. However, bedaquiline does
have some significant draw-backs. In a registration trial, bedaquiline showed an increased incidence
of death compared to the placebo treatment group, possibly due to several pharmacological factors [
It is very lipophilic (measured log P of 7.25), and binds to fatty tissues in the body and has been
associated with an increased incidence of phospholipidosis [ ]. This lipophilicity also likely contributes
to its long terminal half-life of 5–6 months [ ]. It inhibits the cardiac potassium hERG channels to
5].
6
7
cause QTc interval prolongation (resulting in delayed ventricular repolarization). This is a serious
consideration for combination regimens with other anti-TB drugs such as the fluoroquinolones or
clofazimine, which also exhibit such cardiovascular side effects [8]. Due to such potential toxicities, the
WHO advises caution when using bedaquiline, and recommends that strict monitoring procedures be
put in place for patients taking bedaquiline [9].
With such limitations present with bedaquiline, the identification of new analogues with similar
or better anti-bacterial potency but lower clogP and diminished inhibition of hERG channel activity
would be of tremendous interest. In light of this, an exploration of the diarylquinolines to identify
improved second generation analogues of bedaquiline was initiated [10
–15]. After extensive drug
discovery efforts, TBAJ-876 was selected for preclinical evaluation as a more potent, less lipophilic
2
analogue with lower cardiotoxic potential than bedaqiline 1 (Table 1) [16].
Figure 1. Structure of bedaquiline 1 and preclinical candidate TBAJ-876 2.
Table 1. Physical and biological properties of bedaquiline 1 and TBAJ-876 2.
Bedaquiline
Property
TBAJ-876 (2)
(1)
Lipophilicity (cLogP) a
Solubility pH 7.4 (µM)
7.25
<0.06
44
0.04
0.08
1.6
5.15
<0.02
26
0.004
0.006
>30
Mouse PO t 1 (hr)
/2
MIC (MABA) (µg/mL) b
MIC (LORA) (µg/mL) b
hERG IC50 (µM) c
a
clogP calculated by ChemDraw v19.0.0.22. (CambridgeSoft); b MIC90
(
µg/mL); minimum inhibitory concentration
for 90% inhibition of growth of M.tb strain H37Rv, determined under aerobic (replicating; MABA) or non-replicating
(LORA) conditions; c Inhibition of hERG (IC50 in µM).
The metabolic fate of TBAJ-876
2 was evaluated over the course of several preclinical in vivo
studies. In particular, TBAJ-876 was administered orally at 10.0 mg/kg QD to Sprague Dawley rats
2
for 5 days, and plasma samples were obtained at the conclusion of the final dosing period. While the
majority of the test article remained intact, several circulating metabolites were identified in pooled rat
plasma, and their structures were tentatively assigned by mass spectrometry fragmentation. From this
in vivo experiment, three major metabolites were identified and assigned structures 3–5 as shown in
Scheme 1. There were no species difference between rat and mouse metabolism of TBAJ-876 and rats
were selected as they were well represented across various preclinical studies.

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Scheme 1. Proposed metabolic pathways of TBAJ-876 2 in rat plasma.
Metabolite
(D-chain) of TBAJ-876
had two sites of demethylation, mono-demethylated in the D-chain, together with a second
3
is conjectured to arise from mono-demethylation in the dimethylamino sidechain
2
while metabolite represents the bis-demethylated analogue. Metabolite
4
5
mono-demethylation of one of the other methoxy groups in the molecule. Such metabolites are
important factors to consider when conducting drug safety studies for new pharmaceutical drugs.
The identification and characterization of major metabolites of TBAJ-876 would help establish their
impacts on safety and efficacy of TBAJ-876 for further preclinical studies. Synthetic studies towards
these possible metabolites to help elucidate the metabolism of TBAJ-876, and to provide authentic
standards for mass spectrometry and for metabolite profiling studies, are outlined below.
2. Results and Discussion
2.1. Synthesis of Metabolite 3
The chemistry to access metabolite 3 is shown in Scheme 2.

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O
N
O
O
O
O
N
O
O
H
N
Pg
N
Deprotection
OH
OH
Br
Br
H
O
N
OMe
N
N
O
N
O
Metabolite 3
6
O
Coupling
O
N
N
O
O
Pg
O
N
H
Br
+
8
7
O
O
N
O
Scheme 2. Proposed synthesis of Metabolite 3.
The A/B unit
which, following protecting group removal would yield the desired metabolite
the protecting group for Mannich base be acid labile, as base labile protecting groups would be difficult
7
[14] would be coupled with an appropriately protected Mannich base
8 to give 6,
3. It was imperative that
8
to remove cleanly. Diarylquinoline analogues of bedaquiline are unstable under basic conditions
leading to retro-aldol reactions and reversion to their respective starting units [17]. The protecting
group would also need to be stable enough to survive the LDA used in the standard DARQ coupling
conditions. After several trials, dimethoxybenzyl (DMB) was chosen as the most appropriate protecting
group (other acid labile protecting groups such as BOC and silyl protecting groups had failed to yield
Mannich base 8). Commercially available acid 9 was converted to 10 using sodium methoxide at
130 ◦C for 18 h (Scheme 3). It was important to form the sodium methoxide in situ using methanol
and sodium metal as premade commercial grade reagents gave varying results including incomplete
reaction and mono displacement of methoxide. 10 was converted to the Weinreb amide 11 using
oxalyl chloride, followed by addition of hydroxylamine hydrochloride salt and pyridine. With 11
in hand, it was added to vinyl magnesium bromide to provide vinyl ketone intermediate 12 in situ.
The addition of amine 14 and water gave a 68% yield of the desired protected Mannich base 15.
Gratifyingly, Mannich base 15 underwent coupling with A/B unit
7 under standard DARQ
coupling conditions [10 12] to give coupled product DARQ 16 in 69% yield as a 1:1 mixture of
–
diastereomers (Scheme 4). The direct removal of the DMB protecting group using various acidic
conditions (TFA, 2M HCl) was unsuccessful. Instead, we resorted to converting DMB group to
trifluoroacetate 17 by reacting 16 with TFAA in DCM and then removing the trifluoroacetate group
using a mild base such as cesium carbonate [18] despite our previous experience that this could lead to
retro-coupling. Treatment of 17 in MeOH with cesium carbonate at r.t. did indeed cause retro-coupling
to yield A/B unit
of the reaction conditions by carrying out the reaction at
minimal retro-coupling.
7
with no signs of desired metabolite
3
−
. However, after the careful optimisation
20 ◦C gave the desired metabolite
with
3

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O
N
O
COOH
COOH
O
O
i
ii
iii
O
N
O
Cl
N
Cl
O
N
11
O
N
O
9
10
12
CHO
HN
O
iv
O
O
14
13
O
O
O
N
O
15
O
N
O
Scheme 3. Synthesis of Mannich base 15. Reagents and conditions: (i) NaOMe, MeOH, 130 ◦C, 18 h,
87%; (ii) (COCl)2, cat. DMF, DCM, then MeNH(OMe).HCl, pyridine, 20 ◦C, 18 h, 83%; (iii) vinylMgBr,
THF, 0 ◦C then 14, H2O, 68%; (iv) MeNH2, NaBH4, MeOH, 2 h, 100%.
Scheme 4. Synthesis of metabolite
3
. Reagents and conditions: (i) LDA, THF, ,
75 ◦C, 1.5 h then 15
−
−75 ◦C, 5 h, 69%; (ii) TFAA, DCM, 1 h; (iii) Cs2CO3, MeOH, −20 ◦C, 12 h, (49%).
2.2. Synthesis of Metabolite 4
The chemistry to access metabolite 4 is outlined in Scheme 5 below.

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Scheme 5. Proposed synthesis of metabolite 4.
The synthetic plan to access metabolite was similar to that for metabolite
4
3, but instead of using
mono-protected Mannich base 15, a suitably bis-protected Mannich base 19 was needed (Scheme 5).
The benzophenone imine protected Mannich base 21 was considered to be a suitable protecting group
as it could be readily cleaved in acidic conditions and yet also be stable during the coupling reaction
conditions. Synthesis began with Weinreb amide 11, to which was added vinyl magnesium bromide
to form vinyl ketone intermediate 12, which was then added to benzophenone imine 20 followed by
addition of water (Scheme 6). However, instead of generating desired ketone 21, it formed two major
by-products 22 and 23. This could be explained by the cleaved N,O-dimethylhydroxylamine in the
reaction mixture from 11 reacting with vinyl ketone intermediate 12 to yield by-product 22. 23 is a
quaternary salt formed from another equivalent of vinyl ketone 12 being added to by-product 22.
Scheme 6. Attempted synthesis of intermediate 21. Reagents and conditions: (i) vinylMgBr, THF, 0 ◦C
then 20, H2O.

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This problem was overcome by isolating the vinyl ketone intermediate 12 from the reaction
mixture instead of using it in situ, hence avoiding the presence of competing nucleophiles, such as
cleaved N,O-dimethylhydroxylamine. By using a modified reported procedure [19], Mannich base
24 [14] was treated with methyl iodide for 72 h to form quaternary ammonium iodide salt which
was washed with water and subsequent elimination liberated pure vinyl compound 12 (Scheme 7).
With 12 isolated, it was dissolved in THF, added benzophenone imine 20 and stirred at r.t. for 30 min.
After work up and silica chromatography, desired Mannich base 21 was isolated along with ~10%
of benzophenone 25 which could be removed during purification on the next step. Several other
protecting groups were also investigated, however were unsuccessful in forming bis-BOC protected or
bis-DMB protected Mannich base 19.
Compound 21 was then coupled successfully with A/B unit
7 to yield DARQ 26. Removal of
benzophenone imine protecting group was achieved using hydroxylamine and sodium carbonate in
DCM to give metabolite 4 in 53% yield.
Scheme 7. Synthesis of intermediate 21 and metabolite 4. Reagents and conditions: (i) MeI, DCM, 14 h,
41%; (ii) benzophenone imine 20, THF, 0.5 h, 68%; (iii) LDA, THF,
35%; (iv) NH2OH-HCl, Na2CO3, DCM, 48 h, 53%.
−
75 ◦C, 1.5 h then 21
,
75 ◦C, 5 h,
−

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2.3. Synthesis of Metabolite 5
The exact structure of metabolite
5
was unknown. From mass spectral evidence, it had two sites
of demethylation, being mono-demethylated in the D-chain and mono-demethylated somewhere in
one of the methoxy groups in the molecule. This leaves five possible sites of demethylation and the
possible structures for metabolite 5 are outlined in Figure 2.
Figure 2. Five possible structures of metabolite 5.
2.3.1. Metabolite 5A
Metabolite 5A has a demethylated D-chain with a demethylation at the methoxy group on the
3-position of the pyridine B-ring (Scheme 8). Metabolite 5A was synthesized from suitably protected
A/B unit 27, which was coupled with the previously synthesized Mannich base 15.

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Scheme 8. Proposed synthesis of metabolite 5A.
Synthesis of A/B unit 27 began with boronic acid 30 which was converted to alcohol 31 in 90% yield
(Scheme 9). Alcohol 31 was protected with an ethoxymethyl ether (EOM) protecting group to yield
32. Formylation of 32 gave aldehyde 33 which was reduced to alcohol 34 using sodium borohydride
in good yields. The conversion of alcohol 34 to a mesylate group which was displaced by lithium
bromide furnished bromide 35. Suzuki reaction between bromide 35 and boronic acid 28 produced
desired A/B unit 36. It was important to carry out the Suzuki reaction at 35 ◦C as higher temperatures
(80 ◦C) led to degradation of products. With 36 in hand, it was coupled with Mannich base 15 to give
37 as 1:1 mixture of diastereomers in 69% yield. The subsequent removal of the DMB-protecting group
using previously established chemistry followed by the removal of the EOM-protecting group using
1M HCl in dioxane yielded desired metabolite 5A.

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Scheme 9. Synthesis of key intermediate 36 and metabolite 5A. Reagents and conditions: (i) 32%
peracetic acid in acetic acid, THF, 2 h, 90%; (ii) NaH (60% in mineral oil), chloromethyl ethyl ether, DMF,
2 h, 93%; (iii) LDA, THF, DMF, 3 h 51%; (iv) NaBH4, MeOH, 2 h, 93%; (v) MsCl, DCM, 0.5 h then LiBr,
acetone, 100 ◦C, 99%; (vi) Cs2CO3, Pd(PPh3)4, PhMe/DMF, 35 ◦C (sealed tube), 1 h, 76%; (vii) LDA,
THF,
−
75 ◦C, 1.5 h then 15
,
−
75 ◦C, 5 h, 69%; (viii) TFAA, DCM, 1 h; (ix) Cs2CO3, MeOH, 20 ◦C, 72 h,
−
49%; (x) 1M HCl, dioxane, 24 h, Isomer A: 20%, Isomer B: 27%.
2.3.2. Metabolite 5B
Metabolite 5B has a demethylated D-chain with a demethylation at the methoxy group on
the 6-position of the pyridine B-ring (Scheme 10). Metabolite 5B was synthesized from suitably
protected A/B unit 40 which was coupled with previously synthesized Mannich base 15. A selective
mono-demethylation of A/B unit
of the B-ring in would be activated. Although methoxy group at the 2-position is also activated,
steric hindrance from methoxy group at the 3-position would hinder demethylation from occurring.
7 was proposed to yield 41 as 6-methoxy group adjacent to the nitrogen
7

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H
O
N
O
O
PgO
N
O
O
O
O
Coupling
O
N
OH
NH
Br
Br
O
+
N
O
N
40
O
O
N
O
15
N
Metabolite 5B
O
Protection
H
O
N
N
O
O
O
7
O
N
N
O
O
Selective
demethylation
Br
Br
41
O
Scheme 10. Proposed synthesis of metabolite 5B.
The selective demethylation of
7 was achieved using lithium chloride and pTSA in DMF [20]
(Scheme 11). This reaction condition was a mild alternative which gave a far superior yield (90%)
compared to BBr3 (15%), a common demethylation reagent. The structure of 41 was confirmed by 1H
NMR and 2D NOESY experiments. A/B unit
7 shows a clear NOE signal between the 5-H and the
adjacent CH3 on the 6-methoxy group in the pyridine B-ring while for product 41, NOE signal clearly
disappears, confirming it is structure 41 as this signal would still be present if it was structure 42
It was pleasing demethylation had occurred selectively at the desired 6-position of the pyridine B-ring
with excellent yields. With structure of 41 confirmed, it was dissolved in DCM, added DIPEA and
chloromethyl ethyl ether and stirred at r.t. for 18 h to give EOM protected A/B unit 43 in adequate yields.
43 was submitted to our standard coupling reaction conditions with DMB protected Mannich base 15
to yield DARQ 44. Using similar chemistry as the previous metabolites, treatment of 44 with TFAA led
to deprotection of both the EOM and the DMB group to furnish trifluoroacetamide 45. Subsequent
mild basic hydrolysis conditions yielded metabolite 5B as pure high and low Rf diastereomers.
.

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Scheme 11. Synthesis of key intermediate 43 and metabolite 5B. Reagents and conditions: (i) LiCl,
pTSA, DMF, 120 ◦C, 1 h, 90%; (ii) DIPEA, chloromethyl ethyl ether, DCM, 18 h, 49%; (iii) LDA, THF,
75 ◦C, 1.5 h then 15 75 ◦C, 5 h, 83%; (iv) TFAA, DCM, 1 h; (v) Cs2CO3, MeOH, 20 ◦C, 48 h, Isomer
−
,
−
−
A: 19%, Isomer B: 21%.
2.3.3. Metabolite 5C
Metabolite 5C has a demethylated D side chain with mono demethylation on the methoxy group
at the 2 position in the B-ring (Scheme 12). Metabolite 5C was accessed from a coupling reaction
between suitably protected A/B unit 46 with Mannich base 15. A/B unit 46 could be synthesized from
bromide 47 which would undergo a Suzuki reaction with boronic acid 28.

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Scheme 12. Proposed synthesis of metabolite 5C.
The synthesis of suitably protected bromide 47 began with commercially available pyridine 48
,
which underwent protection with chloromethyl ethyl ether to yield 49 (Scheme 13). Formylation of 49
with N-formylpiperidine gave 50 followed by removal of EOM protecting group and methylation using
iodomethane gave aldehyde 52. Reactive aldehyde functionality needed to be protected before N-oxide
chemistry could be attempted. Aldehyde 52 was protected with 1,2-ethanediol and pyridinium tosylate
to give dioxalane 53 in good yields. With aldehyde protected, oxidation to N-oxide using m-CPBA gave
54 in 70% yield. Upon refluxing 54 in acetic anhydride, it gave 55 in 71% yield. Dioxalane protecting
group and acetate group was removed using trifluoroacetic acid to yield desired aldehyde 56.
Scheme 13. Synthesis of intermediate 56. Reagents and conditions: (i) NaH, chloromethyl ethyl ether,
DMF, 2 h, 87%; (ii) LDA, THF, 0 ◦C, 3 h then n-formylpiperidine, 56%; (iii) 3M HCl, THF, 0 ◦C, 3 h, 64%;
(iv) K2CO3, MeI, DMF, 0 ◦C, 3 h, 99%; (v) 1,2-ethanediol, PPTS, PhMe, 0 ◦C, 4 h, 88%; (vi) m-CPBA,
DCM, 18 h, 70%; (vii) Ac2O, 120 ◦C, 4 h, 71%; (viii) TFA, CHCl3, 18 h, 85%.
The next step was EOM protection of 56 (Scheme 14). Using our standard EOM protection
procedure, 56 was dissolved in DCM and added DIPEA and chloromethyl ethyl ether. This reaction
gave a mixture of EOM-protected products 57 and 58 where EOM alkylation had occurred on both
the nitrogen and the oxygen of the pyridone 56. N-alkylation was favoured yielding 4:1 ratio of 58
:57.
(Table 2, condition 1). Although it did not matter which position the protecting group went as it would

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be removed in the final step to give the same product, we ideally wanted O-protection (57) as similar
O-protected A/B units (46, Scheme 12) were known to undergo successful DARQ coupling reactions.
Interestingly, solvent polarity had a huge influence on the ratio of O and N-alkylated products
being formed. The product formation of O-alkylation and N-alkylation could be reversed by using a
range of solvents with different polarities. Using a non-polar solvent such as benzene (condition 2)
promoted more N-alkylation 58 over O-alkylation 57 (9.5:5) while carrying out the reaction with a
polar solvent like DMF promoted more O-alkylation 57 over N-alkylation 58 (4:1). The addition of
silver carbonate to form silver salt of 2-pyridones, which is reported to promote O-alkylation under
non-polar solvents [21] did not yield any O-alkylated product, instead only yielding N-alkylated
product 58 (conditions 4 and 5).
Scheme 14. Synthesis of intermediates 57 and 58.
Table 2. Summary of conditions explored for protection of 56 with chloromethyl ethyl ether.
Solvent
DCM
Base, Additives
DIPEA
Time
24 h
24 h
24 h
24 h
24 h
Product Ratio (57:58)
1
2
3
4
5
1:4
Benzene
DMF
DIPEA
0.5:9.5
4:1
DIPEA
Benzene
Benzene
Ag2CO3
58 only
58 only
Ag2CO3, DIPEA
With both 57 and 58 in hand, using similar chemistry as before, they were successfully modified to
A/B units 65 and 61 respectively (Schemes 15 and 16). However only O-alkylated A/B unit 65 underwent
a successful coupling reaction with Mannich base 15 to yield 66 (Scheme 16). A/B unit 61 did form the
characteristic dark-purple/wine coloured reaction mixture once LDA was added, which suggested
the successful formation of anion at the benzylic position of 61 (a common phenomenon in most
DARQ coupling reactions). However, it is presumed the benzylic anion formed is stabilized by the
changed electronics in the B-ring to prevent reaction with Mannich base 15. Failed coupling reactions
were commonly observed with A/B units with B-rings which stabilized the anion formed at the
benzylic position.

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Scheme 15. Synthesis of intermediate 61 and attempted coupling reaction with 15. Reagents and
conditions: (i) NaBH4, MeOH, 2 h, 98%; (ii) MsCl, DCM, 0.5 h then LiBr, acetone, 100 ◦C, 93%;
(iii) Cs2CO3, Pd(PPh3)4, PhMe/DMF, 35 ◦C (sealed tube), 1 h, 61%; (iv) LDA, THF,
−75 ◦C, 5 h.
−
75 ◦C, 1.5 h then 15
,
Gratifyingly, A/B unit 65 was successfully coupled with 15 to yield DARQ 66. Following established
chemistry as for the previous metabolites, the DMB group deprotection was carried out to give
trifluoroacetamide 67 followed by DARQ 68. Final removal of EOM group was achieved using
anhydrous 2M HCl in diethyl ether to give metabolite 5C as 1:1 mixture of diastereomers (isomer A)
and some as pure low Rf diastereomers (isomer B).

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Scheme 16. Synthesis of key intermediate 65 and metabolite 5C. Reagents and conditions: (i) NaBH4,
MeOH, 2 h, 92%; (ii) MsCl, DCM, 0.25 h then LiBr, acetone, 0 ◦C, 98%; (iii) Cs2CO3, Pd(PPh3)4,
◦
◦
◦
PhMe/DMF, 50 C (sealed tube), 1 h, 81%; (iv) LDA, THF,
−
75 C, 1.5 h then 15
,
−
75 C, 5 h, 59%;
(v) TFAA, DCM, 1 h; (vi) Cs2CO3, MeOH,
54%, Isomer B: 23%.
−
20 ◦C, 48 h, 67%; (vii) 2M HCl in Et2O, 0 ◦C, 0.5 h, Isomer A:
Comparison of 1H NMR run in deuterated chloroform of A/B units 36
,
43 and 65 which were used
to synthesize its corresponding metabolites 5A
, 5B and 5C, respectively, showed a clear difference
1
between the H NMR spectrums (Figure 3). A close up of the 3.7 to 5 ppm region showed a clear
distinction in the positions of the three methoxy peaks and the position of the benzylic CH2 group.
It was comforting to confirm that the three A/B units were indeed distinguishable with each isomer
synthesized from its corresponding starting material.

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1H NMR of AB unit 36 which led to Metabolite
OCH3
OCH3
OCH3
CH2
CH3CH2
1H NMR of AB unit 43 which led to Metabolite
OCH3
OCH3
OC
CH2
CH3CH2
1H NMR of AB unit 65 which led to Metabolite
OCH3
OCH3
OCH3
CH3CH2
CH2
Figure 3. 1H NMR spectrums of A/B units 36
5B and 5C respectively.
, 43 and 65 which were used to synthesize metabolites 5A,
2.3.4. Metabolite 5D
Metabolite 5D was synthesized from coupling reaction between previously synthesized A/B unit
7 and a suitably bis-protected Mannich base 69 (Scheme 17).
O
O
N
O
O
O
O
O
N
O
N
Coupling
Pg1
H
OH
Br
N
+
Br
N
O
O
N
69
OPg2
N
7
O
NH
Metabolite 5D
O
Scheme 17. Proposed synthesis of metabolite 5D.
Synthesis of suitably protected Mannich base 69 began with pyridine 70, which was converted to
Weinreb amide 71 (Scheme 18). After experimenting with various protecting groups, p-methoxybenzyl
(PMB) protecting group was used to protect 71 to yield 72. Addition of vinyl magnesium bromide to
72 followed by addition of dimethoxy benzyl amine 14 and water gave desired Mannich base 74.
A/B unit 7 and Mannich base 74 were coupled using standard coupling conditions (Scheme 19).
It gave desired DARQ 75 in 67% yield. Removal of DMB group using established conditions gave
77 in 53% over 2 steps. The final deprotection of the PMB group using a TFA:DCM (1:4) mixture gave
desired metabolite 5D in 95% yield.

Molecules 2020, 25, 1423
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Scheme 18. Synthesis of intermediate 74. Reagents and conditions: (i) EDCI, HOBT, HNMeOMe.HCl.
TEA, 24 h, 70%; (ii) NaH, DMF, PMB-Cl, 18 h, 32%; (iii) vinylMgBr, THF, 0 ◦C then 14, H2O, 64%.
Scheme 19. Synthesis of metabolite 5D. Reagents and conditions: (i) LDA, THF,
75 ◦C, 5 h, 67%; (ii) TFAA, DCM, 1 h; (iii) Cs2CO3, MeOH, 20 ◦C, 48 h, 53%; (iv) TFA:DCM (1:4),
15 min, 95%.
−
75 ◦C, 1.5 h then 55
,
−
−

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2.3.5. Metabolite 5E
Metabolite 5E was demethylated on the methoxy group on the quinoline A-ring and
mono-demethylated in the dimethylamine D-chain (Scheme 20). Metabolite 5E was synthesized
from coupling reaction between A/B unit 78 and Mannich base 15. A/B unit 78 was accessed via Suzuki
reaction between boronic acid 79 and bromide 80.
Scheme 20. Proposed synthesis of metabolite 5E.
The synthesis of A/B unit 78 began with boronic acid 31 which was demethylated using 1M
BBr3 solution in DCM (Scheme 21). The reaction was carried out in DMF to give 91% yield of 81
DCM and acetonitrile were also screened but only provided low to modest yields of 81 most likely due
to solubility issues. 81 was protected with EOM group to form 82 in 69% yield. The Suzuki reaction
.
between 82 and bromide 80 [14] proceeded well to yield A/B unit 83 in 71% yield. The coupling reaction
between 83 and Mannich base 15 was successful which gave DARQ 84. Deprotection of the DMB
group was achieved using standard conditions to yield 86 in 63% over 2 steps.

Molecules 2020, 25, 1423
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Scheme 21. Synthesis of key intermediates 83 and 86. Reagents and conditions: (i) 1M BBr3 in DCM,
DMF, 24 h, 91%; (ii) DIPEA, chloromethyl ethyl ether, DMF, 66 h, 69%; (iii) Cs2CO3, Pd(PPh3)4,
PhMe/DMF, 50 ◦C (sealed tube), 2.5 h, 71%; (iv) LDA, THF,
(v) TFAA, DCM, 1 h; (vi) Cs2CO3, MeOH, −20 ◦C, 96 h, 63%.
−
75 ◦C, 1.5 h then 15
,
75 ◦C, 5 h, 53%;
−
The removal of the EOM protecting group of 86 was problematic (Scheme 22). EOM group
deprotection is normally carried out in acidic conditions (e.g., using 2M HCl in diethyl ether at room
temperatures). However, the EOM group off substrate 86, was not cleaved even in strong acidic
conditions at refluxing temperatures (Table 3, conditions 1, 2). Various other conditions were screened
(Table 3). Conditions 3–6 led either to decomposition or gave back unreacted starting materials. When a
1M BBr3 solution in DCM was used (conditions 7, 8), it was possible to remove the EOM protecting
group to yield metabolite 5E. However, it also generated byproducts 87 and 88 in which a methoxy
group was also removed. After the careful optimisation of the reaction temperature and the equivalence
of BBr3, reaction mixture containing higher ratio of the desired product 5E along with demethylated
byproducts 87 and 88 could be obtained (condition 8). The mixture products 5E, 87 and 88 were
separated into pure fractions using preparative supercritical fluid chromatography to give a pure batch
of metabolite 5E.

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Scheme 22. Synthesis of metabolite 5E. Reagents and conditions: (i) 1M BBr3 in DCM, DCM,
75 ◦C,
−
3 h then −20 ◦C, 72 h, 14%.
Table 3. Summary of conditions explored for removal of EM protecting group from 86.
Reagents
2M HCl diethyl ether
4M HCl diethyl ether
TFA
Solvent
DCM
Temp
150 ◦C
150 ◦C
110 ◦C
Time
24 h
24 h
24 h
Products (% Yield)
1
2
3
-
-
-
benzene
DCM
Pyridinium
p-toluenesulfonate
4
butanol
110 ◦C
24 h
Trace of 5E
Tetra-n-butylammonium
5
6
7
DCM
i-PrOH
DCM
110 ◦C
110 ◦C
0 ◦C
24 h
24 h
24 h
-
-
bromide, TMS-Cl
CBr4
5E:87:88
(~10%:8%:9%)
1M BBr3 (5 eq.)
−
78 ◦C then
5E:87:88
(~14%:5%:10%)
8
1M BBr3 (3 eq.)
DCM
3 h then 72 h
−20 ◦C
3. Materials and Methods
General
Melting points were determined on an Electrothermal 2300 melting point apparatus. NMR spectra
were obtained on a Bruker Avance 400 spectrometer at 400 MHz for 1H and 100 MHz for 13C spectra
and are referenced to Me4Si. Chemical shifts and coupling constants are recorded in units of ppm and
Hz, respectively. Low resolution atmospheric pressure chemical ionization mass spectra ([M + H])
of intermediates were measured for methanol solutions on a ThermoFinnigan Surveyor MSQ mass
spectrometer. Thin-layer chromatography was carried out on aluminium-backed silica gel plates
(Merck 60 F254) with visualization of components by UV light (254 nm) and/or exposure to I2. Column
chromatography was carried out on silica gel (Merck 230–400 mesh) unless stated otherwise. Alumina
for column chromatography was Merck aluminium oxide 90 (standardised). Analysis of the final test
compounds was carried out on an Agilent 1200-6110 LCMS system, using the following conditions;

Molecules 2020, 25, 1423
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Column: Sunfrie C-18, 4.6
×
50 mm; Mobile phase: ACN (0.05%TFA)-water (0.05%TFA); Gradient:
5% ACN to 95% ACN in 1.0 min, hold 1.0 min, total 2.5 min; flow rate: 1.8 mL/min; LC detector:
UV 214 nm, 254 nm; MS ([M + H]): atmospheric pressure electrospray ionisation; MS cone voltage:
(V) Positive 4000, Negative 3000. All test compounds were determined to have >95% purity.
(6-Bromo-2-methoxyquinolin-3-yl)boronic acid (28). A solution of 2,2,6,6-tetramethylpiperidine (14.4 mL,
84.8 mmol) in THF (100 mL, dist. Na) at
82.5 mmol), the solution was then warmed to
of 6-bromo-2-methoxyquinoline (10.0 g, 42.0 mmol) and triisopropylborate (20.0 mL, 87.2 mmol) in
THF (100 mL, dist. Na) was added dropwise and the orange solution was stirred for 3 h at
78 ◦C,
warmed to
40 ◦C and then quenched with sat. aq. NH4Cl (500 mL). The mixture was diluted with
water (1 L) and the white precipitate was filtered, triturated with hexanes and dried to give 28 (11.17 g,
94%) as a white solid [22]. 1H NMR (DMSO-d6)
8.44 (s, 1H), 8.15–8.18 (m, 3H), 7.76 (dd, J = 8.8,
−
78 ◦C was treated with n-BuLi (33 mL, 2.5 M in hexanes,
−
20 ◦C for 20 min and then cooled to 78 ◦C. A solution
−
−
−
δ
2.3 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 3.99 (s, 3H). LRMS: calculated for C10H9BBrNO3: 281.0; found:
[M + H − OH + OCH3] = 296.2.
N,2,6-Trimethoxy-N-methylisonicotinamide (11). Oxalyl chloride (1.34 mL, 15.8 mmol) was added to a
suspension of 2,6-dimethoxyisonicotinic acid (2.41 g, 13.2 mmol) in DCM (70 mL) and DMF (0.20 mL,
2.6 mmol) at r.t.. The mixture was stirred for 1 h to give a colourless solution which was cooled to 0 ◦C.
N,O-dimethylhydroxylamine hydrochloride (1.42 g, 14.6 mmol) and pyridine (3.51 mL, 28.9 mmol)
were added sequentially and the mixture was stirred at r.t. for 18 h, then partitioned between EtOAc
and sat. aq. NaHCO3. Column chromatography with hexanes:EtOAc (2:1) gave 11 as a light yellow
1
oil (2.49 g, 83%) [22]. H NMR (CDCl3)
δ
6.47 (s, 2H), 3.93 (s, 6H), 3.58 (br s, 3H), 3.32 (s, 3H). LRMS:
calculated for C10H14N2O4: 226.1; found: [M + H] = 227.2.
3-((2,4-Dimethoxybenzyl)(methyl)amino)-1-(2,6-dimethoxypyridin-4-yl)propan-1-one (15). Vinylmagnesium
bromide (17.7 mL of a 1N solution in THF, 17.7 mmol) was added to a solution of 11 (2.00 g,
◦
8.84 mmol) in dry THF (30 mL) at 0 C. The brown solution was warmed to r.t. for 1 h then a
solution of N-methyl-2,4-dimethoxybenzylamine (4.00 g, 22.0 mmol) in THF (10 mL), and water (10
mL) was 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 a brown oil, which was chromatographed. Elution with
EtOAc/hexanes gave fore fractions, then elution with EtOAc gave 15 (2.27 g, 68%) as a light yellow
1
oil [22]. H NMR (CDCl3)
δ
7.13 (d, J = 8.9 Hz, 1H), 6.73 (s, 2H), 6.44–6.41 (m, 2H), 3.59 (s, 6H), 3.88
(s, 3H), 3.53 (s, 3H), 3.50 (s, 2H), 3.12 (t, J = 7.0 Hz, 1H), 2.84 (t, J = 7.0 Hz, 2H), 2.26 (s, 3H). LRMS:
calculated for C20H26N2O5: 374.2; found: [M + H] = 375.3.
General Coupling Procedure. n-BuLi (0.91 mL of a 2N solution in cyclohexane, 1.81 mmol) was
added at
dry THF (6 mL) and the solution was stirred at this temperature for 10 min, then cooled to
A solution of (0.63 g, 1.51 mmol) in dry THF (6 mL) was added dropwise and the mixture was stirred
at
78 ◦C for 90 min, to give a dark, wine-red colored solution. A solution of 15 (0.56 g, 1.51 mmol) in
−
30 ◦C under dry nitrogen to a solution of dry diisopropylamine (0.25 mL, 1.81 mmol) in
−
78 ◦C.
7
−
dry THF (7 mL) was added and the reaction mixture was stirred at this temperature for 5 h. Water
(100 mL) was added and the mixture was extracted with EtOAc (2x). The combined organic extract
was washed with sat. aq. NaHCO3 solution, and brine, then dried (Na2SO4) and the solvent removed
under reduced pressure. The residue was purified by flash column chromatography.
1-(6-Bromo-2-methoxyquinolin-3-yl)-4-((2,4-dimethoxybenzyl)(methyl)amino)-2-(2,6-dimethoxypyridin-4-yl)-1-
(2,3,6-trimethoxypyridin-4-yl)butan-2-ol (16). The product was prepared from
7 and 15 using the General
Coupling Procedure above. Column chromatography with EtOAc:hexanes (1:1) gave fore fractions,
then 15 as a mixture of isomers (75%), as a yellow foam which were used crude for the next step.
LRMS: calculated for C39H45BrN4O9: 792.2; found: [M + H] = 793.2.
1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-(methylamino)-1-(2,3,6-trimethoxypyridin-
4-yl)butan-2-ol (3
). To a solution of 16 (1.38 g, 1.87 mmol) in DCM (10 mL) cooled to 0 ◦C, was added

Molecules 2020, 25, 1423
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triethylamine (0.57 mL, 4.11 mmol) and trifluoroacetic anhydride (0.52 mL, 3.73 mmol). The reaction
mixture was stirred for 1 h, poured onto sat. aq. NaHCO3 (50 mL), extracted with DCM (3 × 10 mL).
The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced
pressure to obtain a yellowish residue. The crude material was dissolved in MeOH (100 mL) and
◦
cooled to
stirred at
−
78 C. Cesium carbonate (1.82 g, 5.59 mmol) was added and the reaction mixture was
−
20 ◦C for 45 h. Reaction mixture was added water (100 mL) and extracted with EtOAc
(3
×
50 mL). The combined organic layers washed with brine (100 mL), dried over Na2SO4, filtered
and concentrated under reduced pressure to obtain a yellow residue. Column chromatography with
DCM/MeOH (9:1) gave 3* as a foamy solid (0.74 g, 63%). H NMR (CDCl3, 400 MHz)
1
δ
8.73 (s, 1H),
8.12 (s, 1H), 7.83 (dd, J = 4.7, 2.2 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.60 (dd, J = 8.9, 2.2 Hz, 1H), 7.54–7.51
(m, 2H), 7.22 (s, 1H), 6.55 (br s, 3H), 6.49 (s, 2H), 5.49 (s, 1H), 5.38 (s, 1H), 4.21 (s, 3H), 4.01 (s, 3H), 3.93
(s, 3H), 3.87 (s, 9H), 3.82 (s, 9H), 3.81 (s, 3H), 3.80 (s, 3H), 2.63–2.51 (m, 2H), 2.32–2.23 (m, 2H), 2.21
(s, 3H), 2.13 (s, 3H), 1.88–1.75 (m, 2H), 1.64–1.53 (m, 2H). (no OH, NH observed). Found: [M + H]+
= 643.2. HRMS: calculated for C30H35BrN4O7: 642.1689; found: 642.1701.
1-(2,6-Dimethoxypyridin-4-yl)-3-(dimethylamino)propan-1-one (24). Vinylmagnesium bromide (32 mL of a
1N solution in THF, 32 mmol) was added to a solution of 11 (2.45 g, 10.8 mmol) in dry THF (100 mL) at
0 ◦C. The brown solution was warmed to r.t. for 1 h then dimethylamine (32 mL of a 2N solution in
THF, 64 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 and column chromatography
with DCM:MeOH (95:5) eluted impurities while DCM:MeOH (9:1) gave 24 as an oil (0.81 g, 31%) [22].
1H NMR (CDCl3)
δ 6.74 (s, 2H), 3.95 (s, 6H), 3.06 (t, J = 7.0 Hz, 2H), 2.72 (t, J = 7.0 Hz, 2H), 2.27 (s, 6H).
LRMS: calculated for C12H18N2O3: 238.1; found: [M + H] = 239.1.
1-(2,6-Dimethoxypyridin-4-yl)prop-2-en-1-one (12). To a solution of 24 (3.00 g, 11.0 mmol) in DCM (50 mL)
was added iodomethane (15.5 g, 109.0 mmol). The reaction mixture was stirred at r.t. for 14 h. Reaction
mixture was washed with water (3 × 50 mL). The organic layer was collected and washed with brine
(100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow
1
residue. Column chromatography with hexanes:EtOAc (2:1) gave 12 as an oil (0.87 g, 41%). H NMR
(CDCl3)
δ 7.26 (s, 1H), 6.96 (dd, J = 17.2, 10.6 Hz, 1H), 6.43 (dd, J = 13.3, 1.4 Hz, 1H), 6.00 (dd, J = 10.6,
1.4 Hz, 1H), 3.96 (s, 6H). Found: [M + H] = 194.1. HRMS: calculated for C10H11NO3: 193.0739; found:
193.0730.
1-(2,6-Dimethoxypyridin-4-yl)-3-((diphenylmethylene)amino)propan-1-one (21). To a solution of 12 (0.87 g,
4.45 mmol) in THF (10 mL), benzophenone imine (2.42 g, 13.4 mmol) was added. The reaction mixture
was stirred at r.t. for 0.5 h. Water (50 mL) was then added to the reaction mixture and extracted with
EtOAc (3
×
50 mL). The combined organic fractions were dried over Na2SO4, filtered and concentrated
under reduced pressure to obtain a yellow residue. Column chromatography with hexanes:EtOAc (9:1)
gave 21 as an oil (1.13 g, 68%). H NMR (CDCl3)
1
δ
7.60–7.15 (m, 10H), 6.75 (s, 2H), 3.94 (s, 6H), 3.76
(t, J = 6.8 Hz, 2H), 3.29 (t, J = 6.8 Hz, 2H). Found: [M + H] = 375.2. HRMS: calculated for C23H22N2O3:
374.1630; found: 374.1638.
1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-4-((diphenylmethylene)amino)-1-(2,3,6-
trimethoxypyridin-4-yl)butan-2-ol (26). The product was prepared from
7 and 21 using the General
Coupling Procedure above. Column chromatography with EtOAc:hexanes (9:1) gave fore fractions,
1
then 26 as a mixture of isomers (35%), as a yellow foam. H NMR (CDCl3, 400 MHz)
δ
8.85 (s, 1H), 8.22
(s, 1H), 7.92–7.87 (m, 2H), 7.69–7.30 (m, 21H), 7.18–7.06 (m, 2H), 6.96–6.87 (m, 4H), 6.65–6.42 (m, 3H),
5.56 (s, 1H), 5.44 (s, 1H), 4.18 (s, 3H), 4.00 (s, 3H), 3.92 (s, 3H), 3.88 (s, 3H), 3.86 (s, 6H), 3.84 (s, 3H), 3.82
(s, 3H), 3.82 (s, 3H), 3.80 (s, 6H), 3.40 (s, 3H), 3.22–3.12 (m, 2H), 3.01–2.40 (m, 2H), 2.11–1.85 (m, 2H),
1.70–1.40 (m, 2H). Found: [M + H] = 793.2. HRMS: calculated for C42H41BrN4O7: 792.2159; found:
792.2160.

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4-Amino-1-(6-bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-1-(2,3,6-trimethoxypyridin-4-
yl)butan-2-ol (
4
). To a solution of 29 (0.42 g, 0.528 mmol) in DCM (10 mL) was added s◦odium carbonate
(0.40 g, 3.75 mmol) and hydroxylamine hydrochloride (0.279 g, 4.01 mmol) at 0 C. The reaction
mixture was stirred at r.t. for 48 h. The reaction mixture was added water (50 mL) and extracted with
EtOAc (3
under reduced pressure to obtain a yellow residue. Column chromatography with DCM:MeOH (9:1)
gave 4* as foamy solid (0.175 g, 53%) as a mixture of isomers. 1H NMR (DMSO, 400 MHz)
8.81
×
50 mL). The combined organic fractions were dried over Na2SO4, filtered and concentrated
δ
(s, 1H), 8.22–8.16 (m, 3H), 7.72–7.51 (m, 4H), 7.19 (s, 1H), 6.52 (s, 1H), 6.46–6.41 (m, 4H), 5.42 (s, 1H),
5.31 (s, 1H), 4.17 (s, 3H), 3.93 (s, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.73
(s, 3H), 3.73 (s, 9H), 3.31 (s, 3H), 2.57–2.45 (m, 2H), 2.26–2.13 (m, 2H), 1.85–1.76 (m, 2H), 1.68–1.40
(m, 2H) (no OH, NH observed). Found: [M + H] = 629.1. HRMS: calculated for C29H33BrN4O7:
628.1533; found: 628.1542.
(2,6-Dimethoxypyridin-3-yl)boronic acid (30). To a solution of 2,6-dimethoxypyridine (10 g, 71.84 mmol)
and N,N-diisopropylamine (0.50 mL, 3.59 mmol) in THF (200 mL, dist. Na) at
was added n-BuLi (43.10 mL, 86.21 mmol) dropwise. The resultant solution was stirred at
5 min, and then warmed to 0 ◦C and stirred at this temperature for a further 3 h. The solution was then
again cooled to
40 ◦C, and triisopropylborate (24.87 mL, 107.76 mmol) was added dropwise, and the
−
40 ◦C under nitrogen
40 ◦C for
−
−
mixture stirred at r.t. for another 1 h. Water (50 mL) was added and the solvent was removed in
vacuo. To the residue, 1M NaOH (100 mL) was added and the aqueous layer was washed with EtOAc
(
2 × 100 mL). The aqueous layer was then acidified to pH 3 and a solid precipitated. This solid was
1
filtered and dried to afford the product 30 (8.10 g, 61%) [22]. H NMR (DMSO-d6)
δ 7.87 (d, J = 7.9 Hz,
1H), 6.36 (d, J = 7.9 Hz, 1H), 3.90 (s, 3H), 3.87 (s, 3H). LRMS: calculated for C7H10BNO4: 183.1; found:
Found: [M + H] = 184.2.
2,6-Dimethoxypyridin-3-ol (31). To a solution of 30 (8.00 g, 43.49 mmol) in THF (150 mL, dist. Na)
at 0 ◦C was added dropwise 32% peracetic acid in acetic acid (21.53 mL, 86.98 mmol) over 10 min.
The resultant solution was stirred at r.t. for 2 h. A 10% solution of sodium sulfite (75 mL) was then
added and the mixture stirred at r.t. for 0.5 h. The solvent was evaporated and the residue partitioned
between EtOAc and water. The aqueous layer was extracted twice and the organic layer dried and
evaporated. Column chromatography with 9:1 hexanes/EtOAc afforded the product 31 (6.05 g, 90%) as
1
white solid [22]. H NMR (CDCl3)
δ
7.12 (d, J = 8.3 Hz, 1H), 6.21 (d, J = 8.2 Hz, 1H), 4.90 (s, 1H), 7.00
(s, 3H), 3.86 (s, 3H). LRMS: calculated for C7H9NO3: 155.1; found: [M + H] = 156.7.
3-(Ethoxymethoxy)-2,6-dimethoxypyridine (32). To a solution of 31 (6.45 g, 40.97 mmol) in DMF (70 mL,
anhydrous) at 0 ◦C was added 60% sodium hydride in mineral oil (41.97 g, 9.16 mmol) in portions.
The mixture was warmed to r.t. and stirred for 1 h. 1-Chloro-2-methoxyethane (4.37 mL, 47.11 mmol)
was then added, and the resultant mixture stirred at r.t. for a further 2 h. The reaction was diluted
with brine (100 mL) and extracted with EtOAc three times. The organic layer was washed with brine
three times, dried and evaporated. Column chromatography with 19:1 hexanes/EtOAc afforded the
1
product 32 (8.14 g, 93%) as an oil [22]. H NMR (CDCl3)
δ
7.41–7.33 (m, 1H), 6.26–6.17 (m, 1H), 5.15
(d, J = 1.9 Hz, 2H), 3.98 (d, J = 1.8 Hz, 3H), 3.87 (d, J = 2.0 Hz, 3H), 3.77 (dq, J = 1.8, 7.1 Hz, 2H), 1.22
(dt, J = 2.9, 7.0 Hz, 3H). LRMS: calculated for C10H15NO4: 213.1; found: [M + H] = 214.1.
3-(Ethoxymethoxy)-2,6-dimethoxyisonicotinaldehyde (33). To a solution of 32 (4.00 g, 18.78 mmol) and
N,N-diisopropylamine (0.13 mL, 0.94 mmol) in THF (60 mL, dist. Na) at
40 ◦C under nitrogen was
−
added n-BuLi (14.09 mL, 28.17 mmol) dropwise. The resultant solution was stirred at
−
40 ◦C for 5 min,
◦
and then warmed to 0 C and stirred at this temperature for a further 3 h. The solution was then
again cooled to
−
40 ◦C, and 1-formylpiperidine (3.75 mL, 33.80 mmol) was added dropwise, and the
mixture stirred at r.t. for another 1 h. Acetic acid (7.5 mL) was added and the solvent was removed in
vacuo. The resultant mixture was partitioned between EtOAc and water, and the organic fraction dried
and evaporated. Column chromatography with 19:1 hexanes/EtOAc afforded the product 33 (2.30 g,

Molecules 2020, 25, 1423
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1
51%) [22]. H NMR (CDCl3)
δ
10.39 (s, 1H), 6.61 (s, 1H), 6.19 (s, 2H), 4.02 (s, 3H), 3.88 (s, 3H), 3.78
(q, J = 10.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). LRMS: calculated for C11H15NO5: 241.1; found: [M + H]
= 242.2.
(3-(Ethoxymethoxy)-2,6-dimethoxypyridin-4-yl)methanol (34). A mixture of 33 (1.20 g, 4.98 mmol) in MeOH
(30 mL, anhydrous) at 0 ◦C was added sodium borohydride (0.38 g, 9.96 mmol). The reaction mixture
was stirred at r.t. for 2 h. The solvent was then removed and the residue partitioned between EtOAc
1
and water. The organic layer was dried and evaporated to afford the product 34 (1.12 g, 93%). H NMR
(CDCl3)
δ 6.31 (s, 1H), 5.08 (s, 2H), 4.59 (d, J = 6.5 Hz, 2H), 3.96 (s, 3H), 3.88 (s, 3H), 3.81–3.75 (m, 2H),
3.03 (t, J = 6.6 Hz, 1H), 1.26–1.22 (m, 3H). Found: [M + H] = 244.2. HRMS: calculated for C11H17NO5:
243.1107; found: 243.1110.
4-(Bromomethyl)-3-(ethoxymethoxy)-2,6-dimethoxypyridine (35). To a solution of 34 (1.12 g, 4.61 mmol)
and triethylamine (1.03 mL, 7.37 mmol) in DCM (20 mL, anhydrous) at r.t., mesyl chloride (0.43 mL,
5.53 mmol) was added dropwise. After 30 min, the reaction was diluted with DCM (20 mL) and the
organic layer washed with sat. aq. NaHCO3, dried and evaporated. The residue was dissolved in
acetone (40 mL, anhydrous), lithium bromide (1.20 g, 14.0 mmol) added, and the mixture heated at
reflux for 30 min. The solution was then cooled and the solvent evaporated, and the residue partitioned
between EtOAc and water. The aqueous layer was extracted twice with EtOAc and the organic layer
1
was dried and evaporated to give the product 35 (1.40 g, 99%). H NMR (CDCl3)
δ
6.32 (s, 1H), 5.14
(s, 2H), 4.45 (s, 2H), 3.95 (s, 3H), 3.87 (s, 3H), 3.83 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). Found:
[M + H] = 306.1. HRMS: calculated for C11H16BrNO4: 305.0263; found: 305.0265.
6-Bromo-3-((3-(ethoxymethoxy)-2,6-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (36). A mixture of
28 (1.84 g, 6.56 mmol), 35 (2.00 g, 6.56 mmol) and cesium carbonate (4.49 g, 14.0 mmol) in toluene
(40 mL, anhydrous) and DMF (20 mL, anhydrous) was purged with nitrogen. Pd(PPh3)4 (0.38 g,
0.33 mmol) was then added, the mixture purged with nitrogen then heated to 35 ◦C under nitrogen
for 1 h. The reaction was partitioned between EtOAc and water and the organic fraction was dried
1
and evaporated. Column chromatography (9:1 hexanes/EtOAc) gave the product 36 (3.01 g, 76%). H
NMR (CDCl3)
(s, 1H), 6.02 (s, 1H), 5.07 (s, 2H), 4.07 (s, 3H), 4.05 (s, 2H), 3.97 (s, 3H), 3.83 (s, 3H), 3.75 (q, J = 7.1 Hz,
2H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3)
161.1, 158.2, 155.4, 145.4, 144.4, 136.3, 132.8, 132.2,
δ 7.76 (d, J = 2.2 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.61 (dd, J = 8.9, 2.2 Hz, 1H), 7.53
δ
129.4, 128.7, 126.9, 125.2, 117.3, 101.5, 97.4, 65.7, 53.9, 53.8, 53.7, 30.1, 15.3. Found: [M + H] = 463.1.
HRMS: calculated for C21H23BrN2O5: 462.0790; found: 462.0794.
1-(6-Bromo-2-methoxyquinolin-3-yl)-4-((2,4-dimethoxybenzyl)(methyl)amino)-2-(2,6-dimethoxypyridin-4-yl)-
1-(3-(ethoxymethoxy)-2,6-dimethoxypyridin-4-yl)butan-2-ol (37). The product was prepared from 36 and
15 using the General Coupling Procedure above. Column chromatography with EtOAc:hexanes (1:1)
gave fore fractions, then 37 as a mixture of isomers (69%), as a yellow foam which were used crude for
the next step. LRMS: calculated for C41H49BrN4O10: 836.3; found: [M + H] = 837.2.
1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-1-(3-(ethoxymethoxy)-2,6-dimethoxypyridin-
4-yl)-4-(methylamino)butan-2-ol (39). To a solution of 37 (1.87 g, 2.24 mmol) in DCM (100 mL) cooled to
0 ◦C, was added triethylamine (0.69 mL, 4.93 mmol) and trifluoroacetic anhydride (0.62 mL, 4.47 mmol).
The reaction mixture was stirred for 1 h, poured onto sat. aq. NaHCO3 (150 mL), extracted with DCM
(3
×
100 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under
reduced pressure to◦obtain a yellowish residue. The crude material was dissolved in MeOH (100 mL)
and cooled to
−
78 C. Cesium carbonate (1.82 g, 5.60 mmol) in water (3 mL) was added and the
◦
reaction mixture was stirred at
extracted with EtOAc (3
over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue. Column
chromatography with DCM:MeOH (9:1) gave 39 (0.75 g, 49%) as a mixture of isomers. 1H NMR
−
20 C for 72 h. Reaction mixture was added water (150 mL) and
×
100 mL). The combined organic layers washed with brine (100 mL), dried
(CDCl3, 400 MHz) δ 8.69 (s, 1H), 8.04 (s, 1H), 7.83–7.80 (m, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.60 (dd, J = 8.9,

Molecules 2020, 25, 1423
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2.2 Hz, 1H), 7.56–7.49 (m, 2H), 7.28–7.26 (m, 2H), 6.58 (s, 2H), 6.48 (s, 2H), 5.49 (s, 1H), 5.38 (s, 1H),
5.21 (q, J = 4.6 Hz, 2H), 4.82–4.73 (m, 2H), 4.18 (s, 3H), 4.01–3.98 (m, 2H), 3.98 (s, 6H), 3.87 (s, 3H),
3.85 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.80 (s, 6H), 3.79 (s, 3H), 3.79–3.73 (m, 2H), 2.65–2.53 (m, 2H),
2.32–2.22 (m, 2H), 2.22 (s, 3H), 2.12 (s, 3H), 1.92–1.82 (m, 2H), 1.63–1.55 (m, 2H), 1.39 (t, J = 7.1 Hz,
3H), 1.23 (t, J = 7.1 Hz, 3H) (no OH, NH observed). Found: [M + H] = 687.2. HRMS: calculated for
C32H39BrN4O8: 686.1951; found: 686.1955.
4-(1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-2-hydroxy-4-(methylamino)butyl)-2,6-
dimethoxypyridin-3-ol (5A). To a solution of 39 (0.70 g, 1.02 mmol) in dioxane (5 mL) was added
1 M HCl (5 mL). The reaction mixture was stirred for 24 h, poured onto sat. aq. NaHCO3 (10 mL),
extracted with EtOAc (3
×
10 mL). The combined organic layers were dried over Na2SO4, filtered
and concentrated under reduced pressure to obtain a yellowish residue. Column chromatography
with EtOAc:MeOH (9:1) gave fore fractions, followed by isomer A of 5A* (0.13 g, 20%). Elution with
EtOAc:MeOH (4:1) gave isomer B of 5A* (0.18 g, 27%). Isomer A: 1H NMR (CDCl3, 400 MHz)
δ 8.48
(s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.68–7.60 (m, 2H), 6.51 (s, 2H), 6.08 (s, 1H), 5.00 (s, 1H), 4.15 (s, 3H),
3.88 (s, 9H), 3.61 (s, 3H), 2.65–2.60 (m, 1H), 2.40–2.32 (m, 1H), 2.19 (s, 3H), 1.90–1.85 (m, 1H), 1.77–1.69
(m, 1H), (no OH, NH observed) Found: [M + H] = 629.2. HRMS: calculated for C29H33BrN4O7:
628.1533; found: 628.1543. Isomer B: 1H NMR (CDCl3, 400 MHz)
δ 8.28 (s, 1H), 7.78 (d, J = 1.9 Hz, 1H),
7.58–7.51 (m, 2H), 6.32 (s, 2H), 6.18 (s, 1H), 5.04 (s, 1H), 4.01 (s, 3H), 3.83 (s, 6H), 3.81 (s, 3H), 3.77
(s, 3H), 2.95–2.90 (m, 1H), 2.74–2.68 (m, 1H), 2.51 (s, 3H), 2.20–2.02 (m, 2H) (no OH, NH observed)
Found: [M + H] = 629.2. HRMS: calculated for C29H33BrN4O7: 628.1533; found: 628.1543.
4-((6-Bromo-2-methoxyquinolin-3-yl)methyl)-5,6-dimethoxypyridin-2(1H)-one (41). To a solution of 7 (0.60 g,
1.44 mmol) in DMF (5 mL) was added lithium chloride (0.30 g, 7.18 mmol) and p-toluenesulfonic acid
(1.24 g, 7.18 mmol). The reaction mixture was heated at 120 ◦C for 1 h. Reaction mixture was washed
with water, extracted with EtOAc (3 × 20 mL). The organic fractions were collected and washed with
brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a
1
yellow residue. Column chromatography with EtOAc gave 41 (0.52 g, 90%). H NMR (DMSO)
δ 12.0
(s, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.58 (dd, J = 8.7, 2.2 Hz, 1H), 7.55 (s, 1H), 7.24 (d, J = 8.8 Hz, 1H), 6.16
(s, 1H), 3.91 (s, 3H), 3.78 (s, 3H), 3.76 (s, 2H), 3.64 (s, 3H). Found: [M + H] = 405.1. HRMS: calculated
for C18H17BrN2O4: 404.0372; found: 404.0374.
6-Bromo-3-((6-(ethoxymethoxy)-2,3-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (43). To a solution of
41 (0.52 g, 1.29 mmol) in DCM (25 mL) was added diisopropylethylamine (0.34 mL, 1.94 mmol) followed
by chloromethyl ethyl ether (0.24 mL, 2.58 mmol). The mixture was warmed to 50 ◦C and stirred for
18 h. The reaction was washed with water (50 mL) and extracted with EtOAc (3 × 50 mL). The organic
fractions were collected and washed with brine (100 mL), dried over Na2SO4, filtered and concentrated
under reduced pressure to obtain a yellow residue. Column chromatography with hexanes:EtOAc (9:1)
1
gave 43 (0.30 g, 49%). H NMR (CDCl3)
δ 7.77 (d, J = 2.1 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.63–7.60
(m, 2H), 6.04 (s, 1H), 5.75 (s, 2H), 4.02 (s, 2H), 4.00 (s, 3H), 3.84 (s, 3H), 3.72 (s, 3H), 3.70 (q, J = 7.1 Hz,
2H), 1.22 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3)
δ
159.7, 157.8, 155.7, 144.7, 144.2, 136.9, 135.8, 132.4,
129.3, 129.0, 127.0, 125.2, 117.7, 101.4, 91.2, 65.9, 60.7, 53.8, 53.7, 30.2, 15.4. Found: [M + H] = 463.1.
HRMS: calculated for C21H23BrN2O5: 462.0790; found: 462.0788.
1-(6-Bromo-2-methoxyquinolin-3-yl)-4-((2,4-dimethoxybenzyl)(methyl)amino)-2-(2,6-dimethoxypyridin-4-yl)-1-
(6-(ethoxymethoxy)-2,3-dimethoxypyridin-4-yl)butan-2-ol (44). The product was prepared from 43 and 15
using the General Coupling Procedure above. Column chromatography with EtOAc:hexanes (1:1)
gave fore fractions, then 44 as a mixture of isomers (83%), as a yellow foam which were used crude for
the next step. LRMS: calculated for C41H49BrN4O10: 836.3; found: [M + H] = 837.2.
4-(1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-2-hydroxy-4-(methylamino)butyl)-5,6-
dimethoxypyridin-2(1H)-one (5B). To a solution of 44 (0.44 g, 0.53 mmol) in DCM (50 mL) cooled to 0 ◦C,
was added triethylamine (0.16 mL, 1.16 mmol) and trifluoroacetic anhydride (0.15 mL, 1.06 mmol).

Molecules 2020, 25, 1423
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The reaction mixture was stirred for 1 h, poured onto sat. aq. NaHCO3 (150 mL), extracted with
DCM (3 50 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated
×
under reduced pressure to obtain a yellowish residue. The crude material was dissolved in MeOH
◦
(100 mL) and cooled to
−
78 C. Cesium carbonate (0.52 g, 1.58 mmol) in water (3 mL) was added
and the reaction mixture was stirred at
and extracted with EtOAc (3 50 mL). The combined organic layers washed with brine (100 mL),
dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue.
Column chromatography with EtOAc:MeOH (9:1) gave fore fractions, followed by isomer A of
5B* (0.064 g, 19%). Elution with EtOAc:MeOH (4:1) gave isomer B of 5B* (0.069 g, 21%). Isomer
A: 1H NMR (DMSO, 400 MHz)
−
20 ◦C for 48 h. Reaction mixture was added water (150 mL)
×
δ
8.34 (s, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.12
(d, J = 8.8 Hz, 1H), 6.56 (s, 1H), 6.42 (s, 2H), 5.24 (s, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 3.76 (s, 9H), 2.53–2.48
(m, 1H), 2.36–2.31 (m, 1H), 2.24 (s, 3H), 2.03–1.70 (m, 2H) (no OH, NH observed) Found: [M + H]
= 629.2. HRMS: calculated for C29H33BrN4O7: 628.1533; found: 628.1543. Isomer B: 1H NMR (DMSO,
400 MHz)
δ 8.01 (d, J = 2.2 Hz, 1H), 7.92–7.85 (m, 1H), 7.59 (dd, J = 8.8, 1.9 Hz, 1H), 7.24 (d, J = 8.7 Hz,
1H), 7.10–7.08 (m, 1H), 6.43–6.35 (m, 2H), 5.47 (s, 1H), 3.79 (s, 6H), 3.75 (s, 3H), 3.73 (s, 3H), 3.72 (s, 3H),
2.38–2.22 (m, 1H), 2.14–2.03 (m, 1H), 2.03 (s, 3H), 1.82–1.66 (m, 2H) (no OH, NH observed) Found:
[M + H] = 629.2. HRMS: calculated for C29H33BrN4O7: 628.1533; found: 628.1543.
5-(Ethoxymethoxy)-2-methoxypyridine (49). To a solution of 6-pyridin-3-ol (3.20 g, 25.4 mmol) in DMF
◦
(50 mL, anhydrous) at 0 C was added 60% sodium hydride in mineral oil (1.23 g, 30.5 mmol) in
portions. The mixture was warmed to r.t. and stirred for 1 h. 1-Chloro-2-methoxyethane (2.73 mL,
25.9 mmol) was then added, and the resultant mixture stirred at r.t. for a further 4 h. The reaction was
diluted with brine (100 mL) and extracted with EtOAc three times. The organic layer was washed
with brine three times, dried and evaporated. Column chromatography with 19:1 hexanes/EtOAc
1
afforded the product 49 (4.44 g, 95%). H NMR (CDCl3)
δ
7.95 (d, J = 3.0 Hz, 1H), 7.34–7.31 (m, 1H),
6.66 (d, J = 9.0 Hz, 1H), 5.13 (s, 2H), 3.88 (s, 3H), 3.72 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.0 Hz, 3H). Found:
[M + H] = 184.6. HRMS: calculated for C9H13NO3: 183.0895; found: 183.0899.
5-(Ethoxymethoxy)-2-methoxyisonicotinaldehyde (50). To a solution of 49 (3.64 g, 19.9 mmol) and
◦
N,N-diisopropylamine (0.14 mL, 0.1 mmol) in THF (60 mL, dist. Na) at
−
40 C under nitrogen
was added n-BuLi (15.0 mL, 30.0 mmol) dropwise. The resultant solution was stirred at
5 min, and then warmed to 0 ◦C and stirred at this temperature for a further 3 h. The solution was then
again cooled to
40 ◦C, and 1-formylpiperidine (3.98 mL, 36.0 mmol) was added dropwise, and the
mixture stirred at r.t. for another 1 h. Acetic acid (8 mL) was added and the solvent was removed
in vacuo. The resultant mixture was partitioned between EtOAc and water, and the organic fraction
dried and evaporated. Column chromatography with 9:1 hexanes/EtOAc afforded the product 50
−
40 ◦C for
−
(2.74 g, 65%). 1H NMR (CDCl3)
δ 10.42 (s, 1H), 8.29 (s, 1H), 7.07 (s, 1H), 5.30 (s, 2H), 3.92 (s, 3H),
3.78 (q, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H). Found: [M + MeOH] = 244.2. HRMS: calculated for
C10H13NO4: 211.0845; found: 211.0849.
5-Hydroxy-2-methoxyisonicotinaldehyde (51). A solution of 50 (2.74 g, 13.0 mmol) and 3M hydrochloric
acid (30 mL) in THF (40 mL, dist. Na) was heated at 40 ◦C for 3 h. The solution was then cooled,
diluted with water, and the pH adjusted to 7 using NaHCO3. The aqueous layer was then extracted
with EtOAc three times, and the organic layer dried and evaporated. Column chromatography with
1
4:1 hexanes/EtOAc afforded the product 51 (1.28 g, 64%) [23]. H NMR (CDCl3)
δ
9.97 (s, J = 0.4 Hz,
1H), 9.46 (s, 1H), 8.08 (s, 1H), 6.93 (d, J = 0.5 Hz, 1H), 3.94 (s, 3H). LRMS: calculated for C7H7NO3:
153.0; found: [M + H] = 154.2.
2,5-Dimethoxyisonicotinaldehyde (52). A mixture of 51 (1.28 g, 8.36 mmol) and potassium carbonate
◦
(1.73 g, 12.5 mmol) in DMF (50 mL, anhydrous) was heated at 50 C for 10 min. Methyl iodide
(1.42 g, 10.0 mmol) was then added and the mixture stirred at this temperature for 2 h. The resultant
solution was diluted with EtOAc and washed with brine three times. The organic layer was dried and

Molecules 2020, 25, 1423
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1
evaporated to afford the product 52 (1.39 g, 99%) [23]. H NMR (CDCl3)
δ
10.43 (s, 1H), 8.01 (s, 1H),
7.08 (d, J = 0.6 Hz, 1H), 3.97 (s, 3H), 3.91 (s, 3H). LRMS: calculated for C8H9NO3: 167.1; found: [M + H
= 168.2.
]
4-(1,3-Dioxolan-2-yl)-2,5-dimethoxypyridine (53). A mixture of 52 (0.20 g, 1.20 mmol), pyridinium
p-toluenesulfonate and ethylene glycol (0.37 g, 5.99 mmol) in toluene (6 mL, anhydrous) was heated at
120 ◦C for 6 h. The resultant solution was diluted with EtOAc and washed with sat. NaHCO3 solution.
The organic layer was dried and evaporated. Column chromatography with 9:1 hexanes/EtOAc
1
afforded the product 53 (0.22 g, 88%). H NMR (CDCl3)
δ
7.87 (s, 1H), 6.90 (s, 1H), 6.07 (s, 1H), 4.15–3.89
(m, 4H), 3.89 (s, 3H), 3.88 (s, 3H). Found: [M + H] = 212.2. HRMS: calculated for C10H13NO4: 211.0845;
found: 211.0836.
4-(1,3-Dioxolan-2-yl)-2,5-dimethoxypyridine 1-oxide (54). A mixture of 53 (0.22 g, 1.04 mmol) in DCM
(8 mL, anhydrous) was added 77% m-CPBA (0.47 g, 2.08 mmol) at 0 ◦C. The reaction mixture was
stirred at r.t. for 17 h. The solvent was removed from the reaction mixture and crude product was
purified using silica column chromatography with 9:1 DCM/MeOH afforded the product 54 (0.17 g,
1
70%). H NMR (CDCl3)
δ 8.02 (s, 1H), 7.09 (s, 1H), 6.00 (s, 1H), 4.15–4.02 (m, 4H), 4.05 (s, 3H), 3.85
(s, 3H). Found: [M + H] = 228.2. HRMS: calculated for C10H13NO5: 227.0794; found: 227.0781.
4-(1,3-Dioxolan-2-yl)-3,6-dimethoxypyridin-2-yl acetate (55). A mixture of 54 (1.84 g, 8.10 mmol) in acetic
anhydride (5 mL) was heated at 150 ◦C for 3 h. Acetic anhydride was removed from the reaction
mixture in vacuo and crude product was purified using silica column chromatography with 1:1
1
hexanes/EtOAc afforded the product 55 (1.54 g, 71%). H NMR (CDCl3)
δ 6.81 (s, 1H), 6.05 (s, 1H),
4.12–4.02 (m, 4H), 3.88 (s, 3H), 3.81 (s, 3H), 2.36 (s, 3H). Found: [M + H] = 270.2. HRMS: calculated for
C12H15NO6: 269.0899; found: 269.0887.
3,6-Dimethoxy-2-oxo-1,2-dihydropyridine-4-carbaldehyde (56). A mixture of 55 (1.43 g, 5.32 mmol) in
chloroform (4 mL) was added to TFA (1 mL) and stirred at r.t. for 2 h. Solvent was removed from the
1
reaction mixture in vacuo to give the product 56 (0.83 g, 85%). H NMR (CDCl3)
δ
10.38 (s, 1H), 9.08
(s, 1H), 5.74 (s, 1H), 4.07 (s, 3H), 3.86 (s, 3H). Found: [M + H] = 184.2. HRMS: calculated for C8H9NO4:
183.0532; found: 183.0521.
2-(Ethoxymethoxy)-3,6-dimethoxyisonicotinaldehyde (57). To a solution of 56 (0.59 g, 3.22 mmol) in DMF
(10 mL) was added to diisopropylethylamine (1.12 mL, 6.44 mmol) followed by chloromethyl ethyl
ether (0.914 g, 9.67 mmol). The mixture was stirred at r.t. for 3 h. The reaction was washed with water
(50 mL) and extracted with EtOAc (3
brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow
residue. Column chromatography with hexanes:EtOAc (9:1) gave 57 (0.54 g, 69%). H NMR (CDCl3)
×
50 mL). The organic fractions were collected and washed with
1
δ
10.40 (s, 1H), 6.64 (s, 1H), 5.68 (s, 2H), 3.96 (s, 3H), 3.88 (s, 3H), 3.82 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz
,
3H). Found: [M + H] = 242.2. HRMS: calculated for C11H15NO5: 241.0950; found: 241.0940.
(2-(Ethoxymethoxy)-3,6-dimethoxypyridin-4-yl)methanol (63). To a mixture of 57 (0.54 g, 2.22 mmol) in
MeOH (10 mL, anhydrous) at 0 ◦C, sodium borohydride was added (0.168 g, 4.45 mmol). The reaction
mixture was stirred at r.t. for 0.5 h. The solvent was then removed and the residue partitioned between
EtOAc and water. The organic layer was dried and evaporated to afford the product 63 (0.50 g, 92%).
f1H NMR (CDCl3)
δ 6.36 (s, 1H), 5.65 (s, 2H), 4.68 (s, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 3.80 (q, J = 7.1 Hz,
2H), 1.26 (t, J = 7.1 Hz, 3H). Found: [M + H] = 244.2. HRMS: calculated for C11H17NO5: 243.1107;
found: 243.1110.
4-(Bromomethyl)-2-(ethoxymethoxy)-3,6-dimethoxypyridine (64). To a solution of 63 (0.40 g, 1.65 mmol)
and triethylamine (0.345 mL, 2.47 mmol) in DCM (20 mL, anhydrous) at r.t., mesyl chloride (0.153 mL,
1.98 mmol) was added dropwise. After 30 min, the reaction was diluted with DCM (20 mL) and
the organic layer washed with sat. aq. NaHCO3, dried and evaporated. The residue was dissolved
in acetone (20 mL, anhydrous) and cooled to 0 ◦C and added lithium bromide (0.16 g, 1.82 mmol).

Molecules 2020, 25, 1423
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The reaction mixture was stirred at 0 ◦C for 3 h. The reaction mixture was partitioned between EtOAc
and water. The aqueous layer was extracted twice with EtOAc and the organic layer was dried and
evaporated to give the product 64 (0.49 g, 98%). 1H NMR (CDCl3)
δ 6.33 (s, 1H), 5.64 (s, 2H), 4.40
(s, 2H), 3.90 (s, 3H), 3.84 (s, 3H), 3.81 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). Found: [M + H] = 306.1
HRMS: calculated for C11H16BrNO4: 305.0263; found: 305.0268.
6-bromo-3-((2-(ethoxymethoxy)-3,6-dimethoxypyridin-4-yl)methyl)-2-methoxyquinoline (65). A mixture of
28 (0.56 g, 1.97 mmol), 64 (0.50 g, 1.65 mmol) and cesium carbonate (1.07 g, 3.30 mmol) in toluene
(20 mL, anhydrous) and DMF (10 mL, anhydrous) was purged with nitrogen. Pd(PPh3)4 (0.095 g, 0.083
mmol) was then added, the mixture purged with nitrogen then heated to 50 ◦C under nitrogen for
0.5 h. The reaction was partitioned between EtOAc and water and the organic fraction was dried and
1
evaporated. Column chromatography (9:1 hexanes/EtOAc) gave the product 65 (0.77 g, 81%). H NMR
(CDCl3)
6.09 (s, 1H), 5.66 (s, 2H), 4.07 (s, 3H), 3.99 (s, 2H), 3.82 (s, 3H), 3.81 (q, J = 7.1 Hz, 2H), 3.75 (s, 3H),
1.25 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3)
161.2, 157.7, 153.9, 145.5, 144.5, 136.4, 135.9, 132.2, 129.3,
δ 7.77 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.61 (dd, J = 8.9, 2.2 Hz, 1H), 7.56 (s, 1H),
δ
128.8, 126.9, 125.2, 117.3, 103.0, 90.8, 65.7, 60.8, 54.0, 53.8, 30.2, 15.4. Found: [M + H] = 463.1. HRMS:
calculated for C21H23BrN2O5: 462.0790; found: 462.0794.
1-(6-Bromo-2-methoxyquinolin-3-yl)-4-((2,4-dimethoxybenzyl)(methyl)amino)-2-(2,6-dimethoxypyridin-4-yl)-1-
(2-(ethoxymethoxy)-3,6-dimethoxypyridin-4-yl)butan-2-ol (66). The product was prepared from 65 and 15
using the General Coupling Procedure above. Column chromatography with EtOAc:hexanes (1:1) gave
fore fractions, then 66 as a mixture of isomers (59%), as a yellow foam which were used crude for the
next step.. Found: [M + H] = 837.2. HRMS: calculated for C41H49BrN4O10: 836.2632; found: 836.2662.
1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-1-(2-(ethoxymethoxy)-3,6-dimethoxypyridin-
4-yl)-◦4-(methylamino)butan-2-ol (68). To a solution of 66 (0.82 g, 0.98 mmol) in DCM (10 mL) cooled
to 0 C, triethylamine (0.30 mL, 2.16 mmol) and trifluoroacetic anhydride (0.25 mL, 1.96 mmol)
were added. The reaction mixture was stirred for 20 min, poured onto sat. aq. NaHCO3 (150 mL),
extracted with DCM (3
concentrated under reduced pressure to obtain a yellowish residue. The crude material was dissolved
in MeOH (30 mL) and cooled to
78 ◦C. Cesium carbonate (0.96 g, 2.94 mmol) was added and the
reaction mixture was stirred at
20 ◦C for 48 h. Water (50 mL) was added to the reaction mixture and
extracted with EtOAc (3 20 mL). The combined organic layers washed with brine (50 mL), dried
×
20 mL). The combined organic layers were dried over Na2SO4, filtered and
−
−
×
over Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue. Column
chromatography with EtOAc:MeOH (9:1) gave 68 (0.45 g, 67%) as a mixture of isomers. 1H NMR
(DMSO, 400 MHz) δ 8.70 (s, 1H), 8.11 (s, 1H), 7.83–7.50 (m, 6H), 7.25–7.22 (m, 1H), 6.64–6.40 (m, 5H),
5.76 (d, J = 6.0 Hz, 1H), 5.58 (d, J = 6.1 Hz, 1H), 5.51–5.43 (m, 3H), 5.37 (s, 1H), 4.20 (s, 3H), 3.97 (s, 3H),
3.88 (s, 6H), 3.82–3.78 (15H), 3.81–3.75 (m, 2H), 3.69–3.62 (m, 2H), 3.42 (s, 3H), 2.62–2.53 (m, 2H),
2.32–2.22 (m, 2H), 2.23 (s, 3H), 2.16 (s, 3H), 1.91–1.77 (m, 2H), 1.74–1.58 (m, 2H), 1.29–1.23 (m, 6H)
(no OH, NH observed). Found: [M + H] = 687.2. HRMS: calculated for C32H39BrN4O8: 686.1951;
found: 686.1960.
4-(1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2,6-dimethoxypyridin-4-yl)-2-hydroxy-4-(methylamino)butyl)-3,6-
dimethoxypyridin-2(1H)-one (5C). Solution of 68 (0.45 g, 0.66 mmol) in THF (10 mL) cooled to 0 ◦C, was
added 2M HCl in diethyl ether (1 mL). The reaction mixture was stirred at 0 ◦C for 30 min. Water
(15 mL) was added to the reaction mixture and extracted with EtOAc (3
organic layers washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced
×
10 mL). The combined
pressure to obtain a yellow residue. Column chromatography with DCM:MeOH (9:1) gave 5C* (0.23 g,
1
54%) as mixture. H NMR (DMSO, 400 MHz)
δ 8.73 (s, 1H), 8.34–8.17 (m, 3H), 7.77–7.63 (m, 3H), 7.53
(d, J = 8.8 Hz, 1H), 6.87 (s, 1H), 6.45–6.32 (m, 4H), 6.26 (s, 1H), 5.43 (s, 1H), 5.34 (s, 1H), 4.18 (s, 3H), 3.82
(s, 12H), 3.73–3.68 (m, 12H), 3.31 (s, 3H), 2.80–2.55 (m, 2H), 2.41–2.05 (m, 9H), 2.00–1.68 (3H) (no OH,

Molecules 2020, 25, 1423
30 of 34
NH observed). Found: [M + H] = 629.2. HRMS: calculated for C29H33BrN4O7: 628.1533; found:
628.1542.
2-Hydroxy-6-methoxyisonicotinic acid (70). Methyl 2-hydroxy-6-methoxyisonicotinate (3.74 g, 18.0 mmol)
in MeOH:THF (1:1, 100 mL) was added LiOH (1.27 g, 53.0 mmol) dissolved in water (50 mL).
The reaction mixture was stirred at r.t. for 3 h. The reaction mixture was concentrated under reduced
pressure and added water (50 mL) and extracted with EtOAc (1
×
20 mL). The aqueous phase was
added 2M HCl until pH was ~1 which led to formation of white precipitates. The solid was filtered
1
and collected to give 70 (3.35 g, 94%). H NMR (DMSO, 400 MHz)
δ
6.59 (s, 2H), 3.82 (s, 3H). Found:
[M + H] = 170.2. HRMS: calculated for C7H7NO4: 169.0375; found: 169.0377.
2-Hydroxy-N,6-dimethoxy-N-methylisonicotinamide (71). 70 (3.35 g, 19.8 mmol) and triethylamine
(9.66 mL, 69.3 mmol) in DMF (30 mL) was stirred at room temperature until homogeneous.
N,O-dimethylhydroxylamine hydrochloride (4.18 g, 22.0 mmol) and hydroxybenzotriazole (2.01 g,
14.9 mmol) were added and the solution was stirred for 2 min. EDCI.HCl (4.18 g, 22.0 mmol) was
added and the mixture was stirred at room temperature for 18 h. The solvent was completely removed
under reduced pressure and the residue was triturated 3 times with acetone. The combined acetone
triturates were concentrated to dryness and the residue was used crude for the next step (2.94 g, 70%).
N,2-Dimethoxy-6-((4-methoxybenzyl)oxy)-N-methylisonicotinamide (72). A solution of 71 (2.94 g, 14.0 mmol)
in DMF (20 mL) was cooled in an ice bath. Sodium hydride 60% w/w in oil (0.83 g, 21.0 mmol) was
added in portions with stirring over 5 min. 1-(Chloromethyl)-4-methoxybenzene (2.28 mL, 17.0 mmol)
was then added and the solution was stirred at room temperature for 18 h. The product was partitioned
between EtOAc and brine, and the combined organic layers washed with brine (50 mL), dried over
Na2SO4, filtered and concentrated under reduced pressure to obtain a yellow residue. Column
chromatography with hexanes:EtOAc (4:1) gave 72 (1.49 g, 32%). 1H NMR (CDCl3, 400 MHz)
δ
7.40–7.36 (m, 2H), 6.92–6.88 (m, 2H), 6.51 (s, 1H), 6.47 (s, 1H), 5.31 (s, 2H), 3.93 (s, 3H), 3.81 (s, 3H), 3.81
(s, 3H), 3.57 (s, 3H), 3.31 (s, 3H). Found: [M + H] = 333.2. HRMS: calculated for C17H20N2O5: 332.1372;
found: 332.1380.
3-((2,4-Dimethoxybenzyl)(methyl)amino)-1-(2-methoxy-6-((4-methoxybenzyl)oxy)pyridin-4-yl)propan-1-one
(
74). Vinylmagnesium bromide (8.97 mL of a 1N solution in THF, 8.75 mmol) was added to a solution
◦
of 72 (1.49 g, 4.48 mmol) in dry THF (30 mL) at 0 C. The brown solution was warmed to r.t. for
1 h then a solution of N-methyl-2,4-dimethoxybenzylamine 14 (2.03 g, 11.2 mmol) in THF (10 mL),
and water (20 mL) were added. The solution was stirred at r.t. for 20 min, then partitioned between
EtOAc and water. The mixture was extracted with EtOAc (4
evaporated to give a brown oil, which was chromatographed. Elution with EtOAc/hexanes gave fore
fractions, then elution with EtOAc gave 74 (1.38 g, 64%) as a light yellow oil. 1H NMR (CDCl3)
×
20 mL). The solution was dried and
δ
7.39–7.37 (m, 2H), 7.13–7.10 (m, 1H), 6.92–6.88 (m, 2H), 6.77 (d, J = 1.1 Hz, 1H), 6.72 (d, J = 1.1 Hz, 1H),
6.42–6.40 (m, 2H), 5.32 (s, 2H), 5.29 (s, 2H), 3.95 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.76 (s, 3H), 3.10
(t, J = 7.0 Hz, 2H), 2.83 (t, J = 7.0 Hz, 2H), 2.24 (s, 3H). Found: [M + H] = 481.3. HRMS: calculated for
C27H32N2O6: 480.2260; found: 480.2272.
1-(6-Bromo-2-methoxyquinolin-3-yl)-4-((2,4-dimethoxybenzyl)(methyl)amino)-2-(2-methoxy-6-((4-methoxybenzyl)
oxy)pyridin-4-yl)-1-(2,3,6-trimethoxypyridin-4-yl)butan-2-ol (75). The product was prepared from 7 and
74 using the General Coupling Procedure above. Column chromatography with EtOAc:hexanes (1:1)
gave fore fractions, then 75 as a mixture of isomers (67%), as a yellow foam which were used crude for
the next step. LRMS: calculated for C46H51BrN4O10: 898.3; found: [M + H] = 899.2.
1-(6-Bromo-2-methoxyquinolin-3-yl)-2-(2-methoxy-6-((4-methoxybenzyl)oxy)pyridin-4-yl)-4-(methylamino)-1-
(2,3,6-trimethoxypyridin-4-yl)butan-2-ol (77). To a solution of 75 (1.43 g, 1.59 mmol) in DCM (30 mL)
cooled to 0 ◦C, triethylamine (0.49 mL, 3.50 mmol) and trifluoroacetic anhydride (0.44 mL, 3.18 mmol)
were added. The reaction mixture was stirred for 30 min, poured onto sat. aq. NaHCO3 (150 mL),
extracted with DCM (3
×
20 mL). The combined organic layers were dried over Na2SO4, filtered