Design and Synthesis of Brain Penetrant Trypanocidal N-Myristoyltransferase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01255

N-Myristoyltransferase (NMT) represents a promising drug target within the parasitic protozoa Trypanosoma brucei (T. brucei), the causative agent for human African trypanosomiasis (HAT) or sleeping sickness. We have previously validated T. brucei NMT as a promising druggable target for the treatment of HAT in both stages 1 and 2 of the disease. We report on the use of the previously reported DDD85646 (1) as a starting point for the design of a class of potent, brain penetrant inhibitors of T. brucei NMT.

Full text of DOI:10.1021/acs.jmedchem.7b01255

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Article
Design and Synthesis of Brain Penetrant
Trypanocidal N-Myristoyltransferase Inhibitors
Tracy Bayliss, David A. Robinson, Victoria C. Smith, Stephen Brand, Stuart P
McElroy, Leah S. Torrie, Chido Mpamhanga, Suzanne Norval, Laste Stojanovski,
Ruth Brenk, Julie A Frearson, Kevin D Read, Ian H. Gilbert, and Paul G. Wyatt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01255 • Publication Date (Web): 10 Nov 2017
Downloaded from http://pubs.acs.org on November 11, 2017
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Design and Synthesis of Brain Penetrant Trypanocidal N-
Myristoyltransferase Inhibitors
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Tracy Bayliss, David A. Robinson, Victoria C. Smith, Stephen Brand, Stuart P. McElroy,
Leah S. Torrie, Chido Mpamhanga, Suzanne Norval, Laste Stojanovski, Ruth Brenk, Julie A.
Frearson, Kevin D. Read, Ian H. Gilbert, Paul G. Wyatt*.
Drug Discovery Unit, College of Life Sciences, University of Dundee, Sir James Black
Centre, Dundee, DD1 5EH, UK.
ABSTRACT: NꢀMyristoyltransferase (NMT) represents a promising drug target within the
parasitic protozoa Trpanosoma brucei (T. brucei), the causative agent for human African
trypanosomiasis (HAT) or sleeping sickness. We have previously validated T. brucei NMT as
a promising druggable target for the treatment of HAT in both stage 1 and 2 of the disease.
We report on the use of the previously reported DDD85646 (1) as a start point for the design
of a class of potent, brain penetrant inhibitors of T. brucei NMT.
INTRODUCTION
Human African trypanosomiasis (HAT) or sleeping sickness is prevalent in subꢀSaharan
1
2
Africa with an estimated “at risk” population of 65 million . The causative agents of HAT
are the protozoan parasites Trypanosoma brucei gambiense and Trypanosoma brucei
3ꢀ4
rhodesiense transmitted through the bite of an infected tsetse fly. HAT progresses through
two stages. In the first stage (stage 1), the parasites proliferate solely within the bloodstream.
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In the second, late stage (stage 2), the parasite infects the central nervous system (CNS)
causing the symptoms characteristic of the disease, such as disturbed sleep patterns and often
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death . Currently there are a number of treatments available for HAT, though none are
without issues, including toxicity and inappropriate routes of administration for a disease of
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rural Africa .
Research has revealed enzymes and pathways that are crucial for the survival of T. brucei,
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ꢀ10
and based on these studies, a number of antiꢀparasitic drug targets have been proposed . T.
brucei Nꢀmyristoyltransferase (TbNMT) is one of the few T. brucei druggable targets to be
7, 11ꢀ12
genetically and chemically validated in both in vitro and in rodent models of HAT
.
NMT is a ubiquitous essential enzyme in all eukaryotic cells. It catalyses the coꢀ and postꢀ
translational transfer of myristic acid from myristoylꢀCoA to the Nꢀterminal glycine of a
variety of peptides. Protein Nꢀmyristoylation facilitates membrane localization and biological
1
1, 13
activity of many important proteins
.
NMT has been extensively investigated as a potential target for the treatment of other
1
4
15
16ꢀ17
parasitic diseases including malaria , leishmaniasis and Chagas’ disease
resulting in the
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identification of multiple chemically distinct small molecule inhibitors . NMT has also been
shown to be a potential therapeutic target for human diseases such as autoimmune disorders
1
9
20ꢀ21
and cancer
.
7, 22ꢀ23
Previously we have reported the discovery of compound 1 (Figure 1)
which showed
excellent levels of inhibitory potency for TbNMT and T. brucei brucei (T. br. brucei)
proliferation in vitro and was used as a model compound to validate TbNMT as a druggable
7
, 22
target for stage 1 HAT . However, 1 is not bloodꢀbrain barrier penetrant, a requirement for
stage 2 activity. Two approaches were taken to increase the brain penetration of 1. A classical
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lead optimisation approach is described elsewhere . This paper describes a second approach
that used a minimum pharmacophore of 1 aiming to derive a structurally distinct series of
potent TbNMT inhibitors with brain penetration, as leads for the identification of suitable
candidates for the treatment of stage 2 HAT.
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Figure 1. Compound 1 *Potencies were determined against recombinant TbNMT and HsNMT1, and against
bloodstream form T. brucei brucei (T. br. brucei) and MRCꢀ5 proliferation studies in vitro using 10 point curves
a
TM
b
replicated ≥ 2. Calculated using Optibrium STARDROP software. Ligand efficiency (LE), calculated as
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0
.6*ln(IC50)/(heavy atom count) using T. brucei NMT IC50 potency . IC50 values are shown as mean values of
c
two or more determinations. Standard deviation was typically within 2ꢀfold from the IC50. Enzyme selectivity
calculated as HsNMT1 IC50 (ꢀM)/ TbNMT IC50 (ꢀM).
COMPOUND RATIONALE AND DESIGN
To aid compound design, and to significantly lower molecular weight and polar
surface area (PSA), the chlorines and the sulfonamide moieties of 1 were removed to define a
minimum pharmacophoric scaffold (Figure 2 panel A). This scaffold was chosen because the
piperidine makes a key interaction through the formation of a salt bridge with NMT’s
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terminal carboxylate. This interaction is highly conserved across the binding modes of
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NMT inhibitors covering multiple chemotypes including 1 (Figure 2 panel B) ; known antiꢀ
fungal NMT inhibitors such as Roche’s (2ꢀbenzofurancarboxylic acid, 3ꢀmethylꢀ4ꢀ[3ꢀ[(3ꢀ
26ꢀ27
pyridinylmethyl)amino]propoxy]ꢀethyl ester (ROꢀ09ꢀ4609)
, Searle’s Nꢀ[2ꢀ[4ꢀ[4ꢀ(2ꢀ
methylꢀ1Hꢀimidazolꢀ1ꢀyl)butyl]phenyl]acetyl]ꢀLꢀserylꢀNꢀ(2ꢀcyclohexylethyl)ꢀ Lꢀlysinamide,
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(SCꢀ58272)
(Figure
2
panel
C)
and
Pfizer’s
2ꢀ((1r,4r)ꢀ4ꢀ
(aminomethyl)cyclohexanecarboxamido)ꢀN,Nꢀdimethylbenzo[d]thiazoleꢀ6ꢀcarboxamide
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(
UKꢀ370,485) . Attempts to crystallize TbNMT had proved to be unsuccessful therefore, the
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4, 30
fungal Aspergillus fumigatus NMT (AfNMT)
were used as a surrogate model for TbNMT
in this study. AfNMT is 42% identical to TbNMT, however within the peptide binding groove
the level of identity is 92%. Previously, a selection of molecules from series 1 were assayed
against AfNMT and TbNMT using the SPA biochemical assay and pIC50 values compared
using linear regression analysis. The pIC50 values were shown to be correlated with an Rꢀ
squared value of 0.73 suggesting that AfNMT is a suitable surrogate system for study within
this chemical series (see Supporting Information).
This minimum pharmacophoric scaffold had low molecular weight (237) and low
2
PSA (12 Å to maximise the potential for CNS penetration) from which we could design
varied chemistry (Figure 2 panel A) to either access the serine pocket (occupied by the
pyrazole moiety in 1) or the peptide recognition region, as seen in the peptomimetic
compound highlighted in red (Figure 2 panel C).
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Figure 2. Development of the chemistry scaffold. A) 2D interaction map of 1 bound to AfNMT leading to the
design of the minimal scaffold. B) Crystal structure of 1 bound; key recognition residues are highlighted and
labelled. C) Proposed minimal scaffold (C atoms gold) docked into the crystal structure of AfNMT overlaid with
peptomimetic compound PDB 2NMT (C atoms cyan); the key S/T K peptide recognition region is highlighted
red.
Compound design. The adopted compound design strategy covered both compounds
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based on 1 (where common sulfonamide bioisosteres and pyrazole mimics were included)
and compounds based on the binding pocket structural features, probing these with diverse
hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) groups. We employed high
throughput chemistry, using technologies and techniques such as scavengers and solid
supported reagents enabling arrays to be made in parallel. Three different but complementary
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chemistries of Suzuki couplings, amidations and Mitsunobu reactions were chosen to explore
all positions around ring A (Figure 3).
Crossing the bloodꢀbrain barrier (BBB) was an essential part our chemistry design and
presented its own challenges. Improving the BBB permeation of molecules have been widely
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studied and in silico prediction methods developed based on known CNS penetrant and nonꢀ
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penetrant compounds
. Examination of the physicochemical properties of molecules and
their influence on affecting BBB permeability has suggested some guiding principles and a
physicochemical property range to increase the probability of improving the BBB
3
3
permeability . The top 25% CNS penetrant drugs sold in 2004 were found to have mean
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values of PSA (Å ) 47, HBD 0.8, cLogP 2.8, cLogD (pH 7.4) 2.1 and MW 293. They
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suggested the following maximum limits when designing compounds as PSA <90 Å , HBD <
3, cLogP 2ꢀ5, cLogD (pH 7.4) 2ꢀ5, MW <500. As this was the first round of compound
2
design we restricted the compounds to the following parameters: PSA 40ꢀ70 Å , HBD < 3,
cLogP 2ꢀ4.5, MW 250ꢀ400.
Virtual libraries of all possible compounds that could be constructed from our inꢀhouse
chemical inventory were constructed, and minimised to ensure that a wide region of chemical
space was explored and structures were not biased to one region. Reaction schemes,
intermediates and examples of compounds made are described in the supporting information.
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a
Figure 3. Scaffold Array Chemistry and Design. Filter parameters calculated using the Optibrium
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STARDROP software.
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RESULTS AND DISCUSSION
Table 1. Array Chemistry Selected Results for the Amide, Homologated Amides and Ether
Series
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TbNMT
TbNMT
TbNMT
IC50
ꢀM)
IC50
(ꢀM)
IC50
(ꢀM)
b
b
b
(
Homologated
Amides
Amides
Ether
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3
1.7
8*
0.4
17
>100
a
a
4*
>100
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9*
0.60
0.07
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9.5
1.6
a
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10*
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a
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>100
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11*
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a
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0.60
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1.4
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1.2
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0.002
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0.5
a
*
Compounds greater than 90% pure, compounds >95% pure
b
IC50 values are shown as mean values of two or more determinations. Standard deviation was typically within 2ꢀfold from the IC50. nd =
not determined.
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Scaffold array results. No compounds made in the Suzuki chemistry (1, Figure 3) derived
series had a potency <10
made). Table 1 shows the potency against TbNMT for selected examples from the amide (3-
), homologated amide (8-12) and ether series (17-24). The most potent compound in the
amide series was 3 (TbNMT IC50 1.7 M). Amides directly linked to the phenyl ring in the 3ꢀ
ꢀM against TbNMT, (see supplementary info for compounds
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ꢀ
position were found to be more potent than the corresponding 4ꢀsubstituted analogues (5 vs. 4
and 7 vs. 6). The homologated amide series in comparison to the amide series were on the
whole >3ꢀfold more potent (6 vs. 12) with the most potent compound achieving a TbNMT
IC50 value of 0.07 ꢀM (10). In the homologated amides series the 4ꢀposition amides showed
greater potency than the corresponding 3ꢀposition analogues (the opposite trend to the amide
series). Further optimisation of both the directly linked and homologated amide series failed
to improve the potency or the pharmacokinetic properties.
The ether array produced compounds with good levels of activity against TbNMT, the most
active of these achieved an IC50 value 0.5
substituted in the 4ꢀposition, on average showing around 10ꢀfold greater potency over their 3ꢀ
position analogues e.g. 3ꢀ compound 22 (35 M) vs. 4ꢀ compound 23 (1.2 M) or 3ꢀ
compound 20 (13 M) vs. 4ꢀ compound 21 (1.4 M). Interestingly, the replacement of the
sulfonamide in structure 1 with an ether linkage (17) was completely inactive against TbNMT
IC50 > 100 M). This was surprising, as methyl ethers are considered possible sulfonamide
ꢀM (24). The more potent compounds were
ꢀ
ꢀ
ꢀ
ꢀ
(
ꢀ
31
bioisosteres . Compound 24 was not selective over human NMT (HsNMT1) but exhibited an
M in the T. br. brucei proliferation assay, with good microsomal stability and
EC50 of 2
ꢀ
moderate levels of selectivity against proliferating human MRCꢀ5 cells (Figure 4).
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The crystal structure of 24 bound to AfNMT (Figure 4 panel A), shows the ligand binds in
the peptide binding groove in an overall U–shaped conformation, with the ligand wrapping
round the side chain of Phe157. The central aryl rings of 24 lie perpendicular to each other
allowing the ligand to sit in the cleft formed by the side chain of Tyr263, Tyr393 and Leu436.
The cleft is formed by the movement of the side chain of Tyr273; a feature observed in
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binding mode of benzofuran ligands
and subsequent derivatives .
The pyridyl nitrogen of 24 forms an interaction with Ser378 in a similar orientation as the
trimethylꢀpyrazole group of 1 and the piperidine moiety interacts directly with the Cꢀterminal
carboxyl group of Leu492.
Compound 24 does not interact with His265, an interaction formed by the sulfonamide in 1
(overlaid with 24, Figure 4 panel B), which potentially explained the drop off in potency
between 1 (TbNMT 0.002
ꢀM) and (24, 0.5ꢀM). Despite this loss of activity, 24 had
3
5
comparable ligand efficiency (LE) 0.33 to 1 LE 0.36, and in combination with the observed
binding mode gave us confidence that the design strategy was valid.
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N
O
Compound 24
TbNMT, IC50 0.5 ꢀM
HsNMT1, IC50 0.3 ꢀM
T. br. brucei, EC50 2 ꢀM
MRC-5, EC50 6.7 ꢀM
Cint (mouse) 2.3 ml/min/g
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PSA 34 Å2 a
Molecular weight 358a
logP 4.25a
LE 0.33b
NH
Enzyme Selectivity = 0.6c
Figure 4. Binding mode of 24. A) Compound 24 (C atoms gold) bound to AfNMT (C atoms
grey) PDB 5T5U. Hꢀbonds are shown as dashes lines and key residues labelled. B) A
comparison of the binding mode of 24 with 1 bound to AfNMT (PDB 4CAX) highlighting the
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movement of Tyr273. Compound 1 and the side chain of Tyr273 (4CAX) are shown with
cyan C atoms.
Optimization of compound 24. With the aim of increasing potency against TbNMT the
diphenyl piperidine ring was replaced with the dichlorophenylꢀpyridylꢀpiperidine moiety of
1. This change reduced the logP by ~ 1 log unit from 4.3 for 24, with an increase in PSA
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
from 34 Å (19) to 50 Å which was within the acceptable guidance limits for BBB
32ꢀ33
permeability
to give 29 (synthesis shown in Scheme 1).
Compound 29 (Figure 5) exhibited a 4ꢀfold improvement in potency against TbNMT (IC50
.1 M), and improved efficacy in the T. br. brucei proliferation assay (EC50 0.7 M), whilst
0
ꢀ
ꢀ
retaining good microsomal stability (1.4 ml/min/g) and LE 0.33. Encouragingly 29 showed
good levels of brain penetration (brain:blood, 0.4), a significant improvement over 1
2
2
(brain:blood <0.1) indicating that the strategy of reducing MW and PSA was a valid
approach (1, PSA 101 Å, MW 495). The crystal structure of 29 bound to AfNMT (Figure 6)
was determined showing the ligand adopted a conformation similar to 1 with the biaryl
system sitting in plane with the 2,6ꢀdichlorophenyl ring stacking in plane with the side chain
of Tyr273. Key interactions between the piperidine N to Ser378 and the piperazine to the Cꢀ
terminal carboxyl group are retained from 24.
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a
Scheme 1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (a) Polymer supportedꢀPPh
3
3 4
, DIAD, alcohol, THF; (b) dioxane/1 M aq K PO ,
Pd(PPh ) ; (c) TFA, DCM; (d) 2 M HCl in diethyl ether.
3
4
a
TM
b
Figure 5. Compound 29 Profile. Values calculated using the Optibrium STARDROP software. Ligand
2
5
efficiency (LE), calculated as 0.6*ln(IC50)/(heavy atom count) using T. brucei NMT IC50 potency . IC50 values
are shown as mean values of two or more determinations. Standard deviation was typically within 2ꢀfold from
c
the IC50. nd = not determined. Enzyme selectivity calculated as HsNMT1 IC50 (µM)/ TbNMT IC50 (µM).
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3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Binding mode of 29 (C atoms gold) bound to AfNMT (PDB 5T6C). Binding mode of 1 (C atoms
cyan) is shown for comparison.
Replacement of the 2,6-dichlorophenyl ring. Optimisation of 29 focused on
modifications to the central 2,6ꢀdichlorophenyl ring to increase enzymatic selectivity relative
to HsNMT1 (0.3 ꢀM, 3ꢀfold compared to TbNMT IC50). These modifications were made
employing the same chemistry as outlined in Scheme 1, by varying the starting substituted
bromophenol used in the Mitsunobu step. These 2,6ꢀdichlorophenyl modifications are
detailed in Table 2.
None of the core modifications improved potency against TbNMT when compared to 29
(
Table 2) nor LE and enzyme selectivity, although some demonstrated increased levels of
potency against HsNMT1 (37, HsNMT1, IC50 0.01 ꢀM). The reason for this increase in
HsNMT1 activity was not explained using the available crystal structure data. Certainly
20
inhibitors of human NMT such as 37 are of potential interest in the treatment of cancer and
further elaboration of the core could be further explored.
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Table 2. Modifications to the 2,6ꢀdichorophenyl central ring of compound 29.
T.br.
brucei
Tryps
MRC-
5
EC50
TbNMT HsNMT1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R
IC50
IC50
b
Enzyme
c
LE
a
a
(ꢀM)
(ꢀM)
Selectivity
EC50 (ꢀM) (ꢀM)
2
9
0.1
0.3
0.7
3.7
0.33
3
36
0.6
0.09
2.8
5.0
0.31
0.15
3
3
3
4
4
7
8
9
0
1
0.8
1.2
2.0
2.0
16
0.01
0.06
0.03
0.08
1.8
2.2
2.4
2.8
2.2
2.7
1.5
3.3
2.5
8.7
8.9
0.29
0.29
0.27
0.28
0.23
0.01
0.05
0.02
0.04
0.11
a
IC50 values are shown as mean values of two or more determinations. Standard deviation was typically within 2ꢀfold from the IC50. nd = not
b
25 c
determined. Ligand efficiency (LE), calculated as 0.6*ln(IC50)/(heavy atom count) using T. brucei NMT IC50 potency . Enzyme selectivity
calculated as HsNMT1 IC50 (ꢀM)/ TbNMT IC50 (ꢀM).
Pyridyl head group SAR. The next phase of optimisation focused on modifications to the
ether pyridyl ring of 29 shown in Table 3. These compounds were made using the same
common phenol intermediate (Scheme 2), applying solid phase reagents such as polystyrene
bound triphenylphosphine, and running reactions and purifications in parallel using
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
commercially available alcohols, or alcohols derived from commercially available carboxylic
acids or esters after reduction with borane or lithium aluminium hydride (see supporting
information).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Pyridyl Head Group SAR of Compound 29
T. br.
MRC-
TbNMT HsNMT1
brucei
Tryps
EC50
5
R
IC50
(ꢀM)
IC50
(ꢀM)
b
Enzyme
c
EC50
LE
a
a
Selectivity
(
ꢀM)
(ꢀM)
29
0.1
0.3
0.7
3.7
0.33
3
45
5.4
1.6
>1
>25
>1
>1
>1
0.26
0.28
nd
46
0.1
0.67
47
0.1
0.8
0.3
0.8
1.3
4.6
6.5
1.3
0.2
>1
0.4
0.6
0.5
>1
4.6
2.8
2.3
>1
>1
>1
0.34
0.28
0.31
0.29
0.28
0.25
65
1.6
0.7
nd
4
8
9
4
50
5
1
2
0.5
0.7
>10
>10
0.4
0.15
5
a
IC50 values are shown as mean values of two or more determinations. Standard deviation was typically within 2ꢀfold from the IC50. Nd =
b
25 c
not determined. Ligand efficiency (LE), calculated as 0.6*ln(IC50)/(heavy atom count) using T. brucei NMT IC50 potency . Enzyme
selectivity calculated as HsNMT1 IC50 (ꢀM)/ TbNMT IC50 (ꢀM).
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9
Modifications to the pyridyl head group showed encouraging results with 47 equipotent to
9 (IC50 ~ 0.1 µM) but with ~ 65ꢀfold selectivity over HsNMT1, equivalent activity in the T.
2
br. brucei proliferation assay, and promising microsomal stability (Cint 4.2 ml/min/g).
Compound 30 though had equivalent activity to 29 in the MRCꢀ5 counter screen, indicating
that HsNMT1 activity may not have been driving the MRCꢀ5 toxicity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Homologation of the linker to the pyridyl group did not improve potency, as did groups on
the pyridyl ring at the 6ꢀ (48) or 4ꢀ (49) position though both 48 and 49 showed equivalent
activity in the T. br. brucei proliferation assay to 29. The crystal structure of 29 overlaid with
the trimethylpyrazole of 1 suggested that additions of methyl substitution may have been
beneficial to potency (Figure 7 panel A), because the trimethyl substitution of pyrrole in 1
was essential for activity. Subsequent crystal structures of 48 showed the binding pocket the
pyridyl head group accesses is small and that these substituents in the case of 49 forced the
ether pyridyl ring to twist in the pocket to avoid steric clashes with its dichlorophenyl ring
and for 48 the 4ꢀmethyl forces the pyridyl ring out of the pocket. In both cases the direct
hydrogen bond from the pyridyl nitrogen to the serine was broken, but 48 still formed an
interaction, though this was now water mediated (Figure 7 panel B).
Figure 7. Binding mode of pyridyl head group modifications. Panel A shows binding mode
of 49 (C atoms aquamarine) PDB 5T6H compared with 29 (C atoms gold). The side chain of
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Phe278 rotates to accommodate the 4ꢀmethyl group. Panel B shows the binding mode of 48
(C atoms aquamarine) PDB 5T6E; the interaction with Ser378 is now a water bridged
interaction. Panel C shows 48 compared with 29.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 2
a
Reagents and conditions: a) Boc O, NEt , THF; (b) 4ꢀbromoꢀ2,6ꢀdichlorophenol, MeCN/1M aq K PO ,
2
3
3
4
2 3
Pd(dppf) Cl2; (c) PSꢀPPh , DIAD, alcohol, THF; (d) TFA, DCM.
Alternative non-pyridyl head groups SAR. To advance the series, two regions within the
structure were modified with the aim to improve potency, firstly examining pyridyl
replacements and modifications to the pyridyl ring and replacement of the piperazinoꢀ
pyridine moiety. Firstly, the pyridyl ring was replaced with a range of 5ꢀmembered
heterocycles, mainly thiazoles, with various substitutions, see Table 4. The most potent of
these showed levels of promising activity against TbNMT IC ~ 0.05ꢀ0.06 ꢀM (58 and 57).
5
0
The SAR around 57 was tight. The removal of either methyl groups (60 and 65) lost activity
against TbNMT; in addition substitution of the 2ꢀmethyl group with either ethyl (64) or
isopropyl (66) lost all activity in the T. br. brucei proliferation assay. Compound 57 showed
good stability to microsomal turnover (Cint 2.4 ml/min/g) but also improved selectivity over
MRCꢀ5 cytotoxicity. Both 58 and 57 showed equivalent levels of potency against HsNMT1
(IC50 ~0.03ꢀ0.08 ꢀM) and again showed very different MRCꢀ5 activities again indicating that
MRCꢀ5 toxicity may not be entirely driven by HsNMT1 activity.
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Table 4. Pyridyl Head Group Replacements.
T. br.
TbNMT HsNMT1 brucei MRC-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R
IC50
ꢀM)
IC50
(ꢀM)
Tryps 5 EC50
b
Enzyme
Selectivity
LE
c
a
a
(
EC50
(ꢀM)
(
ꢀM)
2
9
0.1
0.3
0.7
3.7
0.33
3
5
5
6
7
0.3
4.4
1.1
3.5
2.2
0.32
0.34
15
2
0.05
0.08
0.03
0.33
5
5
6
6
8
9
0
1
0.06
2.9
1.3
0.3
0.5
0.2
0.8
>100
2.8
>10
>100
3.1
0.34
0.34
0.26
0.29
0.33
0.33
0.5
>34
11
46
20
5
>100
15
14
>10
>100
>1
>10
>1
62
9.9
0.9
6
6
3
4
>100
0.3
2.0
3.5
0.3
0.9
3.8
>100
>100
>15
>1
>1
2.0
0.30
0.29
0.24
1
0.5
1
65
6
6
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
7
4.2
12
>10
>10
0.24
3
a
IC50 values are shown as mean values of two or more determinations. Standard deviation was typically within 2ꢀfold from the IC50. nd = not
b
25 c
determined. Ligand efficiency (LE), calculated as 0.6*ln(IC50)/(heavy atom count) using T. brucei NMT IC50 potency . Enzyme
selectivity calculated as HsNMT1 IC50 (ꢀM)/ TbNMT IC50 (ꢀM).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Replacement of the piperazino-pyridine moiety. We had previously validated TbNMT
as a druggable target in the stage 2 model for HAT in mice using 68 as a model compound
24
(
Figure 8 panel A) . Compound 68 showed good potency in the T. br. brucei proliferation
assay at EC50 0.001µM and improved levels of selectivity over MRCꢀ5 cells when compared
to 1. We examined hybridising the 4ꢀC chain derivative of 68, 69, which showed equally
good efficacy and potency and 29 to increase efficacy in the T. br. brucei proliferation assay.
Compound 71 (synthesis Scheme 3) showed increased selectivity over MRCꢀ5 cells and
HsNMT1, but showed a significant drop off in efficacy in the T. br. brucei proliferation
assay. This was potentially caused by the significant increase in lipophilicity of 71 (logP 5.5)
compared to 29 (logP 3.4) resulting in an increase level of nonꢀspecific protein/membrane
binding. Given the more favourable logP of 29, further optimization focused on derivatives of
2
9 rather than 71. Compound 70 (Scheme 3), the NH piperidine of 71 showed no in vitro
activity against TbNMT.
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8
9
10
11
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13
14
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18
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21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. Hybridization Approach
a
Scheme 3
a
Reagents and conditions: a) 9ꢀBBN, THF; b) Pd(PPh
BH, CHCl
3 3 4 2 2
),K PO , H O, DMF; c) TFA, DCM; d) CH O,
Na(OAc
3
)
3
3
.
Pyridyl head group optimisation. The crystal structure of 29 (Figure 6) indicated that the
methyl substituent on the pyridyl ring was pointing into a small pocket. Chemistry was
developed to explore this pocket with various hydrophobic and polar groups as detailed in
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 4. Using a common intermediate (ethyl 2ꢀchloronicotinate, 72), Suzuki and Negishi
reactions were used to install aromatics rings (73) and alkyl groups (74a-c) respectively.
Amines were installed through displacement of the chlorine of 72. After reduction of the
ethyl esters (73-75) to the corresponding alcohols (76-78), they were reacted using standard
Mitsunobu conditions (Scheme 2) to give final products detailed in Table 5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 4
a
Reagents and conditions: (a) H
2
4 3 4 3 4 4
SO , EtOH; (b) Phenylboronic acid, 1 M K PO /dioxane, Pd(PPh ) ; (c) LiAlH
o
t
2
M in THF, 0 C; (d) Pd( BuP) , 0.5 M isobutylzinc bromide, anhydrous THF.
2
Table 5. Pyridyl Substitutions
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9
T. br. MRC-
TbNMT HsNMT1
brucei
EC50
5
Enzyme
c
R
IC50
ꢀM)
IC50
(ꢀM)
b
EC50
LE
Selectivity
a
a
(
(ꢀM)
(ꢀM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
7
9
9
0.1
0.3
0.7
3.7
0.33
3
>50
>50
>50
>50
8
8
0
1
0.1
0.04
0.04
0.2
>10
0.1
0.1
14
1.5
2.1
0.28
0.33
0.28
0.4
2
0.02
0.08
82
3
8
8
3
4
0.02
0.06
0.04
0.18
0.3
0.5
>1
0.34
0.29
2
3
1.4
a
IC50 values are shown as mean values of two or more determinations. Standard deviation was typically within 2ꢀfold from the IC50. nd = not
b
25 c
determined. Ligand efficiency (LE), calculated as 0.6*ln(IC50)/(heavy atom count) using T. brucei NMT IC50 potency . Enzyme selectivity
calculated as HsNMT1 IC50 (ꢀM)/ TbNMT IC50 (ꢀM).
Good levels of inhibition of TbNMT were observed for all compounds prepared (except
79), some with improved potency over 29. The loss of activity of 79 was most probably
caused by the alkoxyꢀgroup reducing the basicity of the pyridine ring, making the ring
nitrogen a poorer HBA. Compounds 81 and 82 showed promising potency against the
parasite (EC = 0.1 ꢀM) with good selectivity compared to MRCꢀ5 cells (81) and had good
50
microsomal stability (81 1.2 ml/min/mg, 82 1.6 ml/min/mg). Compound 81 (Figure 9)
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
showed significant levels of brain penetration (brain:blood ratio 1.9), a significant
improvement on 29 (brain:blood ratio 0.4), and 1 (brain:blood ratio <0.1). Compound 81
represents a good lead for further optimization to identify candidates for development for
stage 2 HAT.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
NH
O S O
O
Cl
Cl
N
Cl
Cl
N
N
N
NH
NH
1: DDD85646
81
TbNMT, IC50 0.002 M
HsNMT1, IC50 0.004 M
T. br. brucei, EC50 0.002 M
MRC-5, EC50 0.4 M
TbNMT, IC50 0.02 M
HsNMT1, IC50 0.04 M
T. br brucei, EC50 0.12 M
MRC-5, EC50 1.5 M
Cint (mouse) 0.6 ml/min/g
Cint (mouse) 1.2 ml/min/g
Brain : Blood < 0.1
PSA 101 Å2
Brain : Blood 1.9
PSA 50 Å2
Molecular weight 495
logP 3.1
Molecular weight 471
logP 4.2
LE 0.36
LE 0.33
Enzyme Selectivity = 2
Enzyme Selectivity = 2
Figure 9. Comparison of 1 and 81
CONCLUSIONS
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Using 1 as a start point to identify alternative TbNMT inhibitor scaffolds with
physicochemical properties suitable for penetration into the brain to treat stage 2 HAT, we
identified an ether linker as a replacement of the sulfonamide of 1. This modification reduced
molecular weight and polar surface area, producing a viable alternative series with excellent
levels of brain penetration. This work highlights the importance of decreasing the PSA as a
way of increasing the probability of brain penetration. Further optimization identified
compounds with good levels of TbNMT and T. br. brucei antiꢀproliferative activity, and
microsomal stability. Though in comparison with the original structure 1, further potency
gains against the enzyme and in the parasite proliferation assay are required. This series
presents good leads to identify potential development candidates for stage 2 HAT.
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11
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Experimental Section
Synthetic Materials and Methods. Chemicals and solvents were purchased from the
Aldrich Chemical Company, Fluka, ABCR, VWR, Acros, Fisher Chemicals and Alfa Aesar
and were used as received unless otherwise stated. Airꢀ and moistureꢀsensitive reactions were
carried out under an inert atmosphere of argon in ovenꢀdried glassware. Analytical thinꢀlayer
chromatography (TLC) was performed on preꢀcoated TLC plates (layer 0.20 mm silica gel 60
with fluorescent indicator UV254, from Merck). Developed plates were airꢀdried and
analyzed under a UV lamp (UV254/365 nm). Flash column chromatography was performed
using preꢀpacked silica gel cartridges (230ꢀ400 mesh, 40–63 ꢀm, from SiliCycle) using a
1
Teledyne Presearch ISCO Combiflash Companion 4X, or Combiflash Retrieve. H NMR and
1
3
1
C NMR spectra were recorded on a Bruker Avance II 500 spectrometer ( H at 500.1 MHz,
13
1
C at 125.8 MHz) or a Bruker DPX300 spectrometer ( H at 300.1 MHz). Chemical shifts (δ)
are expressed in ppm recorded using the residual solvent as the internal reference in all cases.
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1
1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
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Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), pentet
(p), multiplet (m), broad (br), or a combination thereof. Coupling constants (J) are quoted to
the nearest 0.1 Hz. LCꢀMS analyses were performed with either an Agilent HPLC 1100
series connected to a Bruker Daltonics micrOTOF, or an Agilent Technologies 1200 series
HPLC connected to an Agilent Technologies 6130 quadrupole LCꢀMS, where both
instruments were connected to an Agilent diode array detector. LCꢀMS chromatographic
separations were conducted with a Waters Xbridge C18 column, 50 mm x 2.1 mm, 3.5 ꢁm
particle size; mobile phase, water/acetonitrile +0.1% HCOOH, or water/acetonitrile + 0.1%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
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7
8
9
0
NH ; linear gradient 80:20 to 5:95 over 3.5 min, and then held for 1.5 min; flow rate 0.5 mL
3
ꢀ1
min . All assay compounds had a measured purity of ≥95% (by TIC and UV) as determined
using this analytical LCꢀMS system, a lower purity level is indicated. High resolution
electrospray measurements were performed on a Bruker Daltonics MicrOTOF mass
spectrometer. Microwaveꢀassisted chemistry was performed using a Biotage Initiator
Microwave Synthesizer.
tert-Butyl-4-(3-bromophenyl)piperidine-1-carboxylate (14). A solution of 4ꢀ(3ꢀ
2
bromophenyl)piperidine.hydrochloride (13) (5.1 g, 18.4 mmol, 1 eq), Boc O (4.4 g, 20.2
mmol, 1.1eq), and triethylamine (3.87 mL, 27.8 mmol, 1.5 eq) in THF (50 mL) was stirred at
room temperature for 16 h. The reaction was filtered, the filtrate washed with dilute 10%
citric acid and extracted into ethyl acetate. The ethyl acetate layer was washed with water,
then dried over MgSO
4
, filtered and evaporated to give an offꢀwhite solid (14) (6.13 g, 98%
δ1.51 (s, 9H), 1.57ꢀ1.66 (m, 2H), 1.81ꢀ1.86 (m, 2H), 2.64
1
yield). H NMR, 500MHz, CDCl
3
(tt, J = 3.70, 12.21, 1H), 2.77ꢀ2.85 (m, 2H), 4.22ꢀ4.32 (m, 2H), 7.14ꢀ7.22 (m, 2H), 7.35ꢀ7.39
+
(
m, 2H). [M+H] = 388.4
tert-Butyl 4-(3'-hydroxy-[1,1'-biphenyl]-3-yl)piperidine-1-carboxylate (15). tertꢀButyl 4ꢀ(3ꢀ
bromophenyl)piperidineꢀ1ꢀcarboxylate (14) (2 g, 5.88 mmol, 1 eq), 3ꢀhydroxyphenyl boronic
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3 4
acid (974 mg 7.06 mmol, 1.2 eq), anhydrous dioxane (10 mL) and 1 M aq K PO (6 mL)
were combined in a microwave vessel and argon bubbled through the mixture for 5 min.
Pd(PPh
3
)
4
(136 mg, 0.118 mmol, 2%), was added and the reaction degassed again for a
o
further 5 min before microwaving at 140 C for 15 min. The resulting solution was extracted
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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into dichloromethane, washed with sat. aq. NaHCO , and passed through a phase separation
3
cartridge, the organic layer was then absorbed onto silica and purified by flash column
chromatography running a gradient from 0% ethyl acetate/hexane to 50% ethyl
1
acetate/hexane, to give 15 as a colourless oil (1.76 g, 85% yield). H NMR 500MHz, CDCl
3
δ
1
.53 (s, 9H), 1.60ꢀ1.76 (m, 2H), 1.83ꢀ1.91 (m, 2H), 2.71 (tt, J = 3.68, 12.33, 1H), 2.70ꢀ2.91
(m, 2H), 4.24ꢀ4.35 (m, 2H), 6.36 (s, 1H), 6.88 (dd, J = 2.50, 8.11, 1H), 7.10ꢀ7.20 (m, 3H),
.28ꢀ7.46 (m, 4H).
7
tert-Butyl 4-(4'-hydroxy-[1,1'-biphenyl]-3-yl)piperidine-1-carboxylate (16). Made using 4ꢀ
hydroxyphenylboronic acid (183 mg, 1.32 mmol, 1 eq), tertꢀbutyl 4ꢀ(3ꢀ
bromophenyl)piperidineꢀ1ꢀcarboxylate (14) (450 mg, 1.32 mmol, 1 eq), Pd(PPh ) (30 mg)
3
4
1
and K PO (1 eq) in DMF:H O (3:1, 4 ml) to afford 16 (326 mg, 70% yield). H NMR
3
4
2
5
2
7
00MHz, DMSO, δ 1.56 (s, 9H), 1.68ꢀ1.74 (m, 2H), 1.86ꢀ1.96 (m, 2H), 2.72ꢀ2.80 (m, 1H),
.80ꢀ3.00 (m, 2H), 4.29ꢀ4.32 (br. s, 2H), 5.02 (s, 1H), 6.96ꢀ6.99 (m, 2H), 7.18ꢀ7.21 (m, 1H),
+
t
.39ꢀ7.48 (m, 3H), 7.52ꢀ7.56 (m, 2H). [M+H] = 354.2331, 298.1651 (product – Bu)
4-(3'-((1,3,5-Trimethyl-1H-pyrazol-4-yl)methoxy)-[1,1'-biphenyl]-3-yl)piperidine (17). tertꢀ
Butyl 4ꢀ(3'ꢀhydroxyꢀ[1,1'ꢀbiphenyl]ꢀ3ꢀyl)piperidineꢀ1ꢀcarboxylate (15) (100 mg, 0.28 mmol,
1
eq), (1,3,5ꢀtrimethylpyrazole)methanol (44 mg, 0.31 mmol, 1.1 eq), polystyrene boundꢀ
PPh (PPh = triphenylphosphine, 1.84 mmol/g loading, 228 mg, 0.42 mmol, 1.5 eq),
3
3
diisopropyl azodicarboxylate (DIAD, 66 uL, 0.34 mmol, 1.2 eq) in anhydrous dioxane (5
o
mL) in a capped test tube was heated at 60 C for 16 h. The reaction was absorbed onto silica
and purified by flash column chromatography running a gradient from 0% ethyl
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acetate/hexane to 100% ethyl acetate. The resulting product was evaporated in vacuo before
dissolving in dichloromethane (10 mL), addition of trifluoroacetic acid (10 eq) and stirring at
RT for 3 h. The reaction was then evaporated in vacuo before dissolving in dichloromethane
and loading onto a preꢀwashed SCX cartridge. The SCX cartridge was washed with
dichloromethane (3 x 10 mL) and MeOH (3 x 10 mL) before eluting the product with 7 N
0
1
2
3
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9
0
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0
1
ammonia in methanol. This was evaporated to give 17 (64 mg, 61% yield). H NMR
500MHz, CDCl
3
δ 1.66 (s, 3H), 1.67ꢀ1.75 (m, 2H), 1.87ꢀ1.92 (m, 2H), 2.70ꢀ2.77 (m, 1H),
2.80ꢀ2.89 (m, 2H), 3.76 (s, 3H), 4.22ꢀ4.35 (m, 2H), 4.90 (s, 2H), 6.39 (br. s, 1H), 6.98ꢀ7.01
(m, 1H), 7.20ꢀ7.23 (m, 3H), 7.37ꢀ7.47 (m, 4H). [M+H] = 427.2.
Compounds 14-20 were made in an analogue manner to 17 from 16, see Supporting
Information for analytical data.
Prototypical Mitsunobu reaction of a pyridyl alcohol and a substituted phenol (Scheme 1)
(
See Supporting Information for the synthesis of intermediates 30-35 for compounds 36-41,
Table 2).
-((4-Bromo-2,6-dichlorophenoxy)methyl)-2-methylpyridine (27). DIAD (diisopropyl
3
azodicarboxylate, 5 mL, 24.8 mmol, 1.2 eq) was added to a suspension of 4ꢀbromoꢀ2,6ꢀ
dichlorophenol (25) (5.0 g, 20.7 mmol, 1 eq) and 2ꢀmethylꢀ3ꢀhydroxymethylpyridine (26)
(3.1 g, 24.8 mmol, 1.2 eq), polystyrene boundꢀPPh
3
(1.84 mmol/g loading, 16.2 g, 29.8
o
mmol, 1.2 eq), in anhydrous THF (20 mL) then heated at 70 C for 4 h. After cooling the
reaction mixture was filtered, the beads washed with MeOH and dichloromethane, and the
filtrate concentrated in vacuo. The resulting residue was triturated with MeOH to give 27 as a
1
white solid (5.46 g, 76% yield). H NMR 500MHz, CDCl
3
δ 2.68 (s, 3H), 5.03 (s, 2H), 7.18
(dd, J = 4.90, 7.68Hz, 1H), 7.49 (s, 3H), 7.82 (dd, J = 1.68, 7.68Hz, 1H), 8.50 (dd, J = 1.68,
+
4
.90Hz, 1H). LCMS [M+H] = 347.9.
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tert-Butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperazine-1-
carboxylate (28). A solution of 1ꢀ(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridinꢀ2ꢀ
yl)piperazine (2.97 g, 10.27 mmol, 1 eq), diꢀtertꢀbutylꢀdicarbonate (Boc
.1 eq), in THF (20 mL) and triethylamine (2.1 mL, 15.4 mmol, 1.5 eq) was stirred at RT
overnight. The resulting reaction was extracted into dichloromethane, and then washed with
2
O, 2.5 g, 11.3 mmol,
1
10
11
12
13
14
15
16
17
18
19
20
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27
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4
10% citric acid and then water. The dichloromethane layer was dried over MgSO , filtered
1
and evaporated to give (28) as a white solid (4 g, 100% yield). H NMR 500MHz, CDCl
3
δ
1.28 (s, 12H), 1.42 (s, 9H), 3.45ꢀ3.50 (m, 8H), 6.90 (d, J = 4.91, 1H), 6.97 (s, 1H), 8.14 (dd, J
+
=
1.02, 4.89, 1H). [M+H] = 389.45
Prototypical Suzuki reaction of an aryl bromide and a boronate ester (Compounds 29-35).
-(4-(3,5-Dichloro-4-((2-methylpyridin-3-yl)methoxy)phenyl)pyridin-2-yl)piperazine
1
dihydrochloride salt (29).tertꢀButyl 4ꢀ(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
yl)pyridinꢀ2ꢀyl)piperazineꢀ1ꢀcarboxylate (28) (67 mg, 0.23 mmol, 1eq), 3ꢀ((4ꢀbromoꢀ2,6ꢀ
dichlorophenoxy)methyl)ꢀ2ꢀmethylpyridine (27) (80 mg, 0.23 mmol, 1eq), and potassium
phosphate.trihydrate (49 mg, 0.231 mmol, 1 eq) in DMF:H O (1:1, 4 mL) was combined in a
2
3 4
microwave vessel and degassed with argon for 5 min, before the addition of Pd(PPh ) (14
o
mg, 0.012 mmol, 5%), and reaction degassed again, then microwaved at 100 C for 40 min.
Reaction was concentrated in vacuo, extracted into dichloromethane and then washed with
aq. NaHCO . The two phase system was passed through a phase separation cartridge, the
3
filtrate concentrated in vacuo and the title compound purified by flash column
3
chromatography using 8% MeOH/ethyl acetate + 1% aq NH as the eluent. The residue was
taken up in dichloromethane, ethereal HCl was added (2 M, 2 mL), concentrated and the diꢀ
hydrochloride salt of 29 triturated with ether, filtered and washed with ether. (104 mg, 71%
1
yield). H NMR 500MHz, d6ꢀDMSO δ 2.82 (s, 3H), 3.17ꢀ3.23 (m, 4H), 3.85ꢀ3.90 (m, 4H),
5.29 (s, 2H), 7.16 (d, J = 5.20Hz, 1H), 7.27ꢀ7.30 (m, 1H), 7.78ꢀ7.86 (m, 1H), 8.06 (s, 2H),
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