Development of inhibitors against mycobacterium abscessus tRNA (m1G37) Methyltransferase (TrmD) Using Fragment-Based Approaches

Article abstract of DOI:10.1021/acs.jmedchem.9b00809

Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m¹G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.

Full text of DOI:10.1021/acs.jmedchem.9b00809

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Article
Development of inhibitors against Mycobacterium abscessus tRNA
m1G37) methyltransferase (TrmD) using fragment-based approaches
(
Andrew Whitehouse, Sherine E Thomas, Karen P Brown, Alexander
Fanourakis, Daniel Chan, M. Daben J. Libardo, Vitor Mendes, Helena I. M.
Boshoff, R Andres Floto, Chris Abell, Tom L. Blundell, and Anthony G Coyne
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00809 • Publication Date (Web): 08 Jul 2019
Downloaded from pubs.acs.org on July 10, 2019
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Development of inhibitors against Mycobacterium
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abscessus tRNA (m G37) methyltransferase (TrmD)
using fragment-based approaches
Andrew J. Whitehouse,†,⊥ Sherine E. Thomas,‡,⊥ Karen Brown, Alexander Fanourakis, Daniel
§
†
†
‖
‡
‖
§
S. H. Chan, M. Daben J. Libardo, Vitor Mendes, Helena I. M. Boshoff, R. Andres Floto,
,†
,‡
,†
Chris Abell,* Tom L. Blundell,* and Anthony G. Coyne*
†Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW,
UK.
‡Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2
1
GA, UK.
§Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory
of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2
0
QH, UK; Cambridge Centre for Lung Infection, Royal Papworth Hospital, Cambridge, CB23
RE, UK.
3
‖Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National
Institute of Allergy and Infectious Disease, National Institutes of Health, 9000 Rockville Pike,
Bethesda, Maryland 20892, USA.
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KEYWORDS
Cystic fibrosis; nontuberculous mycobacteria; NTM; Mycobacterium abscessus; Mycobacterium
1
tuberculosis; Mtb; Mycobacterium leprae; tRNA (m G37) methyltransferase; TrmD; SpoU-
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TrmD; SPOUT; reading frame; fragment-based; fragment-merging; differential scanning
fluorimetry; DSF; isothermal titration calorimetry; ITC; native mass spectrometry; native MS.
ABSTRACT
Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant
nontuberculous mycobacteria (NTM) that has emerged as a growing threat to individuals with
cystic fibrosis, and other pre-existing chronic lung diseases. Mab pulmonary infections are
difficult, or sometimes impossible, to treat and result in accelerated lung function decline and
premature death. There is therefore an urgent need to develop novel antibiotics with improved
1
efficacy. tRNA (m G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It
is essential in Mab and other mycobacteria, improving reading frame maintenance on the
ribosome to prevent frameshift errors. In this work a fragment-based approach was employed
with the merging of two fragments bound to the active site, followed by structure-guided
elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these
compounds exhibit promising activity against mycobacterial species, including Mycobacterium
tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a
target for the development of antimycobacterial compounds.
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INTRODUCTION
Mycobacterium abscessus (Mab) is an opportunistic human pathogen responsible for a wide
range of lung, skin and soft tissue infections. Although acquisition was thought to occur through
environmental exposure (from soil and/or water), it has become apparent that indirect person-to-
person transmission may be an important route of infection in cystic fibrosis (CF) patients (via
fomite or long-lived infectious aerosol spread).1,2 In CF patients, Mab infection results in
accelerated inflammatory lung damage, and impaired quality and quantity of life.3–5 Mab is
challenging to treat due to its unique combination of drug modifying enzymes, high number of
efflux pumps and genetic polymorphism of target genes, in addition to its complex and
impermeable multilayered cell envelope. As a result, Mab possesses both intrinsic and acquired
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resistance to currently available antibiotics, and is therefore currently difficult or sometimes
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impossible to treat. Consequently, there is an acute need for novel antibiotics with improved
efficacy against Mab infection.
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tRNA (m G37) methyltransferase (TrmD), a member of the SpoU-TrmD (SPOUT) RNA
methyltransferase family, catalyzes the transfer of a methyl group from S-adenosyl methionine
1
(
SAM) to the N position of guanosine 37 in bacterial tRNA when preceded by another
7
guanosine in the sequence. The addition of this marker immediately adjacent to the anticodon
acts to improve reading frame maintenance on the ribosome, preventing frameshift errors that
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would result in truncated and inactive peptides. TrmD has been shown to be essential for growth
in a range of bacterial species from Staphylococcus aureus and Pseudomonas aeruginosa to
mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mab.9–12
The X-ray crystal structure of Mab TrmD shows a fold consistent with that of previously
described TrmD enzymes,13–15 with a homodimeric structure exhibiting deep trefoil knots for
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SAM binding in two symmetry-related sites. These sites are formed by contributions from the N-
and C-terminal domains of alternate subunits, which are separated by catalytically-relevant
interdomain linkers that show organization upon tRNA binding.16 The analogue of TrmD in
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archaea and eukaryotes, Trm5, is structurally distinct with a differing SAM binding mode. This
reduces the chance of an inhibitor of Mab TrmD binding off-site in the human host, making this
essential enzyme an attractive target for drug development.
The attractiveness of TrmD as a target is reflected in the recent application of a high-
throughput screen against TrmD from Pseudomonas aeruginosa to identify low-micromolar
18
inhibitors, However, there are currently on-going efforts to validate the in-vivo mechanism of
these compounds before they are further developed as antibiotics. Structure-driven fragment-
based methods provide an alternative methodology for the efficient design of potent inhibitors
from low-molecular weight starting points and are now firmly established in both academia and
1
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industry. A fragment-based approach was used in a prior study against Haemophilus influenzae
TrmD to develop selective inhibitors that ordered the interdomain linker in a similar manner to
tRNA.20 Disappointingly, in general these compounds only displayed weak activity when
screened against a range of Gram-positive and Gram-negative pathogens, including efflux
mutant strains of Escherichia coli and Haemophilus influenzae. In this work a fragment-based
approach was employed to design potent inhibitors against Mab TrmD that show growth
inhibition across a range of pathogenic mycobacteria.
RESULTS AND DISCUSSION
A fragment library of 960 fragments was screened against TrmD using differential scanning
fluorimetry (DSF) as a primary screen. This resulted in fifty-three hits with a thermal shift cut-
off value of 3 standard deviations from the negative control. These hits were then carried forward
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for soaking experiments using X-ray crystallography. Of these hits, density was observed for
twenty-seven fragments, all of which were shown to bind at the SAM binding pocket of Mab
TrmD. The remaining twenty-six fragments did not show any electron density.12 This work
herein describes the use of fragment-growing and merging strategies on fragment hits to develop
novel compounds to inhibit Mab TrmD. Using the fragment-merging strategy, compounds have
been developed that afford up to a 4-order of magnitude improvement in affinity against Mab
TrmD, combined with inhibition of Mab growth in vitro and in a human macrophage infection
model. A number of these key compounds display potent inhibition of Mtb growth in vitro, while
one of the lead molecules also exhibits growth inhibition of intracellular Mycobacterium
leprae.12
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Fragment-Growing Strategy. The SAM co-factor has been shown by X-ray crystallography
to bind to Mab TrmD simultaneously in the two symmetry-related active sites, with the adenine
ring ‘anchoring’ the molecule in place through hydrogen bonds to the backbone amides of
12
residues Ile133, Gly134, Tyr136 and Leu138 (Figure 1a). This is formed from the loop of
Val131 to Leu138, bordered behind and above by Pro83 and Pro85, and below by Ile133,
Tyr136, Leu138 and Ala144 encompasses the ‘adenine binding pocket’. Fragment hit 1 (K 89
d
µM, LE 0.55, Table 1) was observed by X-ray crystallography to occupy the adenine binding
pocket, with the pyrazole ring hydrogen bonding to the backbone amides of Gly134 and Ile133,
and the carbonyl oxygen engaging the backbone amide of Leu138 (Figure 1b). This highly
ligand-efficient fragment was chosen as a starting point for a fragment-growing strategy (Table
1
). Differential scanning fluorimetry (DSF) screening was used as a first line screen with the
elaborated compounds as a guide to prioritize compounds for isothermal titration calorimetry
(ITC). The ester group of 1 was shown to be resistant to replacement, with the amide analogue 2
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giving a thermal shift below +0.5 °C at 5 mM (+4.0 °C for 1) by DSF. From the X-ray crystal
structure, the ethyl chain of 1 extends into the region occupied by the ribose portion of SAM, the
‘ribose binding pocket’ (Figures 1a and 1b). This was used as a vector for a fragment-growing
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strategy. The phenyl ring introduced in 3 (K 33 µM, LE 0.41) was shown by X-ray
d
crystallography to occupy the ribose-binding pocket (Figure S1a). This induced a movement in
Tyr111 (2.5 - 5.1 Å at C-α depending on the active site), similar to that shown in the SAM-bound
Mab TrmD X-ray crystal structure. From the synthesized analogues of 3 with varying
substituents, 7 (K 14 µM, LE 0.39) demonstrated a ligand-efficient improvement in binding
d
affinity. The X-ray crystal structures of 7- and 3-bound Mab TrmD both showed evidence of a
second ligand molecule in the active site bound to the backbone amide nitrogen of Glu180
through its carbonyl oxygen (Figures 1c and S1a).
Figure 1. a) X-ray crystal structures of Mab TrmD in complex with SAM (PDB code 6NW6),12
illustrating both the whole dimer (individual protomers in blue/ gray) with the positions of the
active sites highlighted and one of the active sites in detail, and Mab TrmD in complex with b) 1
12
(
PDB code 6QOS) and c) 7 (PDB code 6QRE), illustrating one of the active sites.
a)
b)
c)
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Table 1. The change in the melting temperatures (T
(LE) of fragment hit 1 and compounds 2-7.
m
), affinities (K ) and ligand efficiencies
d
a
K
(µM) LEb
Compound T (°C)
R
d
m
1
2
3
4
5
6
7
+0.4c
NDd
+3.1
+0.4
+0.9
+2.8
+3.5
-
89 ± 4
ND
0.55
-
-
H
33 ± 2
ND
0.41
-
4-NO
2
4-CO
2
Me ND
-
3-CN
30 ± 1
14 ± 1
0.36
0.39
3-OMe
a
100 μM ligand and 10 μM Mab TrmD.
b
-1
kcal mol per heavy atom.
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c
Determined to be +4.0 °C with 5 mM ligand and 10 μM Mab TrmD.12
d
Determined to be +0.4 °C with 5 mM ligand and 10 μM Mab TrmD.
Compound 7 was used as the basis for further derivatives (Table 2), initially from the use of
the 3-methoxy group as a vector for growth (see Figure S1). The use of aromatic rings in 9 (T
2.5 °C) and 11 (T +3.0 °C), an ester group in 8 (K 10 µM, LE 0.32) or chain extension in 10
11 µM, LE 0.38) did not result in either a significant improvement in binding affinity or,
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m
+
m
d
(
K
d
when this was not determinable by ITC, an increase in thermal shift. The observation of a second
ligand molecule for 7 in the active site was exploited for a fragment linking approach, with the
attachment of a molecule of 1 to the scaffold of 7 through a propynyl linker in 12 (K 4.6 µM,
d
LE 0.24), however whilst an improved binding affinity was achieved the ligand efficiency was
adversely affected. The 5-position of the phenyl ring of 7 was also utilized as a vector for growth
for the insertion of ring systems into the volume bordered by residues Glu112, Val137, Arg154
and Glu180. However, whilst the added pyrrolidinyl and pyridyl groups were tolerated in 13 (K
0 µM, LE 0.31) and 14 (K 6.7 µM, LE 0.31), a significant improvement in affinity was again
not achieved, including with the addition of a methylene linker in 15 (K 12 µM, LE 0.28).
d
1
d
d
Protein X-ray crystallography showed that the added groups in 8 and 14 did reach the desired
regions of the active site in Mab TrmD (Figures S1b and S1c), however attempts to improve the
affinity using the stated strategies proved challenging. The lack of increase in affinity and the
liability of the ester functional group in these compounds pointed to the need to develop an
alternative strategy to target Mab TrmD.
Table 2. The change in the melting temperatures (T
(LE) of compounds 8-15.
m
), affinities (K ) and ligand efficiencies
d
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a
R1
R2
H
H
H
H
LEb
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-
Compound T (°C)
K (µM)
d
m
8
9
1
1
+2.6
+2.5
+3.1
+3.0
CO
2
Me
10 ± 1
ND
0
1
Me
H
11 ± 1
NDc
0.38
-
12
+9.6
4.6 ± 0.7 0.24
13
+3.0
+3.8
H
H
10 ± 2
0.31
1
1
4
5
6.7 ± 0.6 0.31
+3.0
H
12 ± 1
0.28
a
b
c
1
00 μM ligand and 10 μM Mab TrmD.
-1
kcal mol per heavy atom.
Not determined due to solubility.
Fragment-Merging Strategy. In a similar way to compound 1 (K
d
89 µM, LE 0.55, Table 2),
the aminopyrazole ring system of fragment hit 16 (K
d
0.17 mM, LE 0.37) occupied the adenine
binding pocket, with the pyrazole ring hydrogen bonding to the backbone amides of Tyr136 and
Leu138 and the amino group engaging Gly134 and the alcohol side chain of Ser132 (Figure 2a).
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In contrast, the 4-methoxyphenyl ring of 16 extended into the ribose binding pocket. The
methoxy group showed no direct interactions with the surrounding residues, however the
corresponding phenyl (17) and 4-tolyl (18) analogues lacking the methoxy group afforded
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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
relatively low T values (1.4 – 1.5 °C). The methoxy group of 16 was also shown to possess a
m
preference for the 4-position of the phenyl ring, with the 3-methoxyphenyl analogue 19
providing a negative thermal shift (Table 3). The ribose binding pocket was shown to be
occupied by the indole ring of fragment hit 20 (K 0.26 mM, LE 0.41), where the indole nitrogen
d
indirectly interacts with the backbone carbonyl of Leu138 through a water molecule. The 6-
boronic acid group of 20 partially extends into the adenine binding pocket, engaging the
backbone amides of residues Tyr136 and Leu138 through hydrogen bonds, as well as two water
molecules that occupy the remaining space in the pocket, interacting with the backbone amides
of Val131, Ile133 and Gly134 in addition to the side chain of Ser132 (Figure 2b). The isosteric
replacement of the boronic acid group with a carboxylic acid, as shown in fragment 21, was
shown to not be tolerated, with no T observed. Further, examination of the structure with 5-
m
boronic acid isomer 22 bound using X-ray crystallography, showed the indole ring ‘flipping’ to
maintain the hydrogen bonding interactions of the boronic acid, with the indole nitrogen
interacting with an extensive hydrogen-bonded water network in the rear of the active site
(
Figure S2a). Even though the boronic acid of 22 was shown to form the same hydrogen-bonding
water network in the adenine binding pocket as 20, it gave a lower T (Table 3). The overlap of
m
the respective 4-methoxyphenyl and indole ring systems of 16 and 20 (Figure 2c), and their
spanning of the adenine and ribose binding pockets, offers the possibility of a fragment-merging
strategy. This was explored successfully with 23 (K 0.11 mM, LE 0.36), providing a new
d
aminopyrazole-indole scaffold with both improved affinity and prospects for further elaboration
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relative to the parent fragments (Figure 2d). The compound 23 was shown by X-ray
crystallography to maintain the hydrogen-bonding interactions of the original fragment 16, which
remained unaffected throughout all subsequent additions to the scaffold, with the indole nitrogen
providing a vector for elaboration that the fragment hit 16 lacked.
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 2. X-ray crystal structures of Mab TrmD in complex with a) 16 (PDB code 6QOT), b) 20
(
(
PDB code 6QOU), c) 16 and 20 (overlay of PDB code 6QOT and PDB code 6QOU) and d) 23
PDB code 6QQS), illustrating one of the active sites.12
a)
b)
c)
d)
Table 3. The change in the melting temperatures (T
m
), affinities (K ) and ligand efficiencies
d
(
LE) of fragment hits 16 and 20, their structural analogues 17-19 and 21-22, and compound 23.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
Compound R1
R2
a
LEb
T (°C) K (M)
m
+3.0
+1.5
+1.4
-0.5
d
16c
17
4-OMe
-
170 ± 10
ND
0.37
H
-
-
18
4-Me
-
ND
-
19
3-OMe
-
ND
-
20c
21
-
-
-
-
6-B(OH)
+4.0
-0.6
260 ± 20
ND
0.41
2
2
6-CO
2
H
-
22
5-B(OH)
-
+1.3
+4.8e
ND
-
23d
110 ± 10
0.36
a
b
c
d
e
5 mM ligand and 10 μM Mab TrmD.
-1
kcal mol per heavy atom.
T
m
, K
and LE previously reported.12
Determined to be +1.1 °C with 100 µM ligand and 10 μM Mab TrmD.
d
and LE previously reported.12
K
d
Elaboration of Compound 23. Elaboration of 23 was initially carried out from the indole
nitrogen with the introduction of a 4-methoxybenzyl group in 24a (K 59 µM, LE 0.24) (Table
d
4). Whilst the gain in affinity was less than desired, the added group reached the portion of the
active site occupied by the methionine moiety of SAM defined by Pro85, Glu112, Val137,
Arg154 and Glu180 (Figure 3a). The synthesis of the 3-methoxybenzyl 24b (K
and benzyl 24c (K 19 µM, LE 0.29) derivatives demonstrated improved affinities (GE 0.14 and
.15 respectively). Whilst the 2-benzamide 24e gave a lower ΔT of +1.4 °C, further
d
13 µM, LE 0.28)
d
0
m
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improvements in affinity were realized relative to 24c with the synthesis of the 2-cyanobenzyl
2
4d (K
d
12 µM, LE 0.28) and 2-picolyl 24f (K 12 µM, LE 0.30) analogues. Attention was also
d
focused on elaboration from the 4-position of the pyrazole ring of 23. This vector was shown by
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
X-ray crystallography to face a narrow and elongated pocket formed by Pro83, Thr84, Val131,
Ser132, Ile133 and Ala144 (Figure 2d). The addition of a nitrile group in 25 (K
d
5.0 µM, LE
0
.43) afforded a jump in affinity from 23 (K 0.11 mM, LE 0.36), with the added functionality
d
highly group efficient (GE 0.91). An X-ray crystal structure of 25 bound to TrmD showed that
the nitrile extended into the pocket, engaging the backbone amides of Thr84 and Ile133, with no
major changes in the ligand binding orientation or conformation of neighboring residues (Figure
3e). The nitrile was subsequently incorporated into 24f to afford 26f (K 0.50 µM, LE 0.36), with
d
the added nitrile functional group affording a similar jump in affinity as in 25 (GE 0.95 and 0.91
respectively).
Figure 3. X-ray crystal structures of Mab TrmD in complex with a) 24a (PDB code 6QQT), b)
1
2
2
4b (PDB code 6QRC), c) 24c (PDB code 6QQW), d) 24f (PDB code 6QQX), e) 25 (PDB
1
2
code 6QQU) and f) 26f (PDB code 6QQY), illustrating one of the active sites.
a)
b)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c)
d)
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
e)
f)
Table 4. The change in the melting temperatures (T
compounds 24a-f, 25, 26c and 26f.
m
), affinities (K ) and efficiency metrics of
d
Compound R1
R2
H
a
K
d
(µM)
LEb
GE (R ) GE (R )
b
1
b
2
T (°C)
m
2
2
4a
4b
+3.4
59 ± 17
13 ± 2
0.24 0.04
0.28 0.14
-
-
H
+4.6
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2
2
4c
4d
H
H
+3.4
+4.4
19 ± 1
12 ± 2
0.29 0.15
0.28 0.15
-
-
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
2
2
4e
H
H
+1.4
+3.1
ND
-
-
-
-
4fc
12 ± 1
0.30 0.18
2
2
5
H
CN +4.4
5.0 ± 2.1
NDd
0.43
-
-
-
0.91
-
6c
CN +10.1
2
6fc
CN +8.3
0.50 ± 0.14 0.36 0.20
0.95
a
100 μM ligand and 10 μM Mab TrmD.
b
c
d
-1
kcal mol per heavy atom.
and LE previously reported.12
K
d
Not determined due to solubility.
Following the development of 26f, the corresponding 3-pyridyl isomer 26g (K
d
0.18 µM, LE
0
.38, Table 5) afforded a greater than 2-fold improvement in binding affinity without additional
interactions being observed in the X-ray crystal structure of ligand-bound Mab TrmD (Figures 3f
and 4a). The late stage functionalization of 26g through addition of a chlorine atom on the 3-
position of its indole ring in 27g (K 8.5 µM, LE 0.28) was not tolerated (Table 5). Whilst this
d
modification of 26g did not result in a significant shift in the indole ring, one of the ordered
water molecules in the rear of the active site bound to Pro83 was displaced (Figure 4b). 2-
Hydroxypyridyl/pyridone derivatives of 26g, 26j (K
d
3.2 µM, LE 0.30) and 26k (K
d
1.3 µM, LE
0
.32), were synthesized to engage neighboring residues Arg154 and Glu180 respectively through
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1
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the added oxygen atoms. Whilst the X-ray crystal structure of Mab TrmD with 26j bound did
show evidence of a hydrogen bond between the added oxygen atom of the ligand and Arg154
(Figure 4c), neither derivative possessed an improved binding affinity. In contrast, the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
exploration of the alternative 4-pyridyl isomer 26l (K
d
0.12 µM, LE 0.39) resulted in a 4-fold
decrease of K value relative to 26f, with the nitrogen atom of its pyridyl ring lying 2.9 Å from
d
the carboxylate group of Glu180 (Figure 4d).
Modification of the pyridyl ring of 26l to the corresponding 4-quinolyl ring system in 26m
afforded a significant thermal shift (ΔT +10.3 °C at 100 µM), suggesting that the bicyclic ring
m
was tolerated (Table 5). However, this modification adversely affected solubility, precluding the
determination of binding affinity by ITC at the desired concentrations. The replacement of the
pyridyl ring with more soluble saturated alternatives resulted in the development of racemic
compounds N-methyl piperidin-2-yl 26n (K
(K 0.36 µM, LE 0.35), whose ITC isotherms could be fitted by a one-site binding model
Figures S7d and S8a). However, racemic mixtures can afford deceptively simple isotherms
under certain circumstances, such as when the enthalpies of binding of the individual
d
0.59 µM, LE 0.34) and N-methyl piperidin-3-yl 26o
d
(
21
enantiomers are equivalent, with the curve mimicking the K of the weaker component. Hence,
d
a reverse ITC titration was also performed for 26o. This afforded a simple curve that could be
fitted by similar parameters to the forward titration (Figure S8b), implying that the enantiomers
of 26o are equipotent. The X-ray crystal structure of 26n showed that the nitrogen atom of its
piperidinyl ring interacts with the backbone carbonyl of Tyr111 (Figure S2c), whilst that of 26o
formed electrostatic interactions with either of Glu112 or Glu180 depending on the active site
(
Figures 4e and 4f).
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Figure 4. X-ray crystal structures of Mab TrmD in complex with a) 26g (PDB code 6QQZ), b)
2
7g (PDB code 6QR1), c) 26j (PDB code 6QR2) and d) 26l (PDB code 6QR0), illustrating one
of the active sites, and Mab TrmD in complex with 26o (PDB code 6QR3), illustrating active
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
sites 1 (e) and 2 (f).
a)
b)
c)
d)
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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
e)
f)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. The change in the melting temperatures (T
compounds 26g, 26j-o and 27g.
m
), affinities (K ) and efficiency metrics of
d
Compound R1
26g
R2 T (°C)
a
K
d
(µM)
LEb
GE (R )
b
1
m
H
+10.5
+5.5
0.18 ± 0.06 0.38 0.28
2
6j
H
3.2 ± 0.3
1.3 ± 0.1
0.30 0.03
0.32 0.10
26k
26l
H
H
H
+8.3
+11.5
+10.3
0.12 ± 0.02 0.39 0.31
NDc
26m
-
-
2
6n
H
+8.8
0.59 ± 0.23 0.34 0.16
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2
2
6o
7g
H
+10.9
0.36 ± 0.05 0.35 0.20
Cl +5.8
8.5 ± 1.0
0.28
-
a
b
c
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
100 μM ligand and 10 μM Mab TrmD.
-1
kcal mol per heavy atom.
Not determined due to solubility.
The targeting of Glu112 and Glu180 through electrostatic interactions with saturated
heterocyclic ring systems was continued, with the pyridyl ring of 26f providing a good starting
point for the presentation of amines at the top of the active site (Table 6). The implementation of
a similar strategy against Haemophilus influenzae TrmD with engagement of the catalytic
2
0
residue Asp169 resulted in improved inhibition and the ordering of the interdomain linker. The
addition of a pyrrolidinyl ring through a methylene linker to 26f in 28 (K 92 nM, LE 0.32)
showed that the strategy was applicable to this lead series. Modification of the scaffold through
replacement of the pyridyl ring of 28 with a phenyl ring in 29a (K 27 nM, LE 0.34) was
beneficial to the binding affinity, in contrast to the difference in affinities between the phenyl and
pyridyl derivatives 24c (K 19 µM, LE 0.29) and 24f (K 12 µM, LE 0.30). The X-ray crystal
d
d
d
d
structure of Mab TrmD in complex with 29a showed the pyrrolidinyl ring occupying the binding
sites in two conformations, thereby interacting with the carboxylates of either Glu112 or Glu180,
whilst Asp169 and the rest of the interdomain linker remained unstructured (Figures 5a and 5b).
Replacement of the nitrile group on the 4-position of the pyrazole ring of 29a was also explored
through the methyl-substituted alternative 30a (K
binding affinity than the unsubstituted derivative 31a (K
with alternative substituents in place of its pyrrolidinyl ring were trialed, including 29b (K
d
2.0 µM, LE 0.27), which showed a higher
0.49 µM, LE 0.31). Derivatives of 29a
70
d
d
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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
nM, LE 0.31) whose piperidinyl ring was shown to interact with Glu112 in preference to Glu180
(
Figure 5c). The use of a morpholinyl ring resulted in a significant attenuation of binding affinity
in 29c (K 0.19 µM, LE 0.30), possibly due to the impact of reduced basicity of the morpholinyl
d
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
nitrogen on its interactions with Glu112 and Glu180. Protein X-ray crystallography showed that
compound 29c adopted a similar binding pose as 29b, but with the morpholinyl ring rotated and
orienting its oxygen atom towards Pro57 (Figure 5d). In contrast to 29c, the N-methyl
piperazinyl ring of 29d (K 73 nM, LE 0.30) was oriented away from Glu112 with its methyl
d
group extending further into the pocket defined by the loop from Ala176 to Glu180 (Figure 5e).
The methyl substituent on the piperazinyl ring of 29d was expanded to an isopropyl group in 29e
(K 0.10 µM, LE 0.28), whose electron density maps suggest two alternative binding poses with
d
the isopropyl group oriented towards either Glu112 or the loop from Ala176 to Glu180 at the top
of the active site. (Figure 5f).
Figure 5. X-ray crystal structures of Mab TrmD in complex with 29a (PDB code 6QR6),12
illustrating active sites 1 (a) and 2 (b), and Mab TrmD in complex with c) 29b (PDB code
1
2
6
QR7), d) 29c (PDB code 6QR9), e) 29d (PDB code 6QR8) and f) 29e (PDB code 6QRD),
illustrating one of the active sites.
a)
b)
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c)
d)
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
e)
f)
Table 6. The change in the melting temperatures (T
compounds 28, 29a-e, 30a and 31a-b.
m
), affinities (K
d
) and efficiency metrics of
Compound
28c
X
N
R1
R2
T (°C)
a
K
d
(nM)
LEb
GE (CH
b
2
R1)
m
CN +12.0
CN +12.9
92 ± 18
27 ± 4
0.32 0.17
0.34
2
9ac
CH
-
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
2
9b
CH
CH
CH
CH
CN +12.6
CN +12.6
CN +12.0
CN +11.9
70 ± 29
0.31
0.30
0.30
0.28
-
-
-
-
29c
190 ± 23
73 ± 30
2
2
9dc
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
9e
0a
100 ± 24
3
CH
CH
CH
Me +6.1
2000 ± 310 0.27
-
31ac
1b
H
H
+7.0
+7.2
490 ± 210
710 ± 90
0.31 0.36
0.29 0.28
3
a
b
c
1
00 μM ligand and 10 μM Mab TrmD.
-1
kcal mol per heavy atom.
and LE previously reported.12
K
d
Native Mass Spectrometry. Native mass spectrometry (native MS) was used to investigate
the structure and interactions of Mab TrmD. The protein alone presented as the dimer with an
observed mass of 53,187 Da, centered around the 15+ charge state (Figure 6). The observed mass
is slightly higher than the theoretical mass of 52.52 kDa due to the presence of solvent and buffer
molecules that remain weakly bound to the protein under the soft ionization conditions
2
2
employed. In the presence of 5% DMSO, the native mass spectrum of Mab TrmD shifted to a
lower charge state distribution (Figure 6). This is consistent with previous studies on the effect of
DMSO on protein charging in electrospray ionization (ESI).23,24
Figure 6. Native mass spectrometry of Mab TrmD with compounds 24c, 26c and 29a.
Experiments with ligands contain 5% DMSO.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
Native MS was used to study the interaction between the small molecules and Mab TrmD. A
titration experiment performed with 24c revealed increasing occupancy of the protein as the
concentration of the ligand was increased (Figure 6). At 20 µM of Mab TrmD and 12.5 µM of
2
4c (with 5% DMSO), a single molecule of the ligand could be observed to bind to the 13+
charge state of the protein, although the majority of that charge state remained unbound. At 25
µM of 24c, both singly and doubly-bound Mab TrmD could be detected at the 13+ charge state.
The proportion of doubly-bound Mab TrmD increased at 50 µM of 24c, while at 100 µM the 13+
charge state of the protein was entirely doubly-bound. A similar trend could be observed for the
14+ charge state, except that the degree of occupancy of the protein was lower at each
concentration of 24c due to the greater Coulombic repulsion experienced by the protein-ligand
complexes at 14+ compared to at 13+. The lack of triply-bound Mab TrmD observed even at
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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
excess concentrations of 24c suggests that little, if any, of the small molecule bound non-
specifically to the protein dimer.
Compounds 26c and 29a were also screened against Mab TrmD using native MS at a ligand
concentration of 100 µM (Figure 6). Interestingly, despite its tight binding affinity for Mab
TrmD as measured by ITC, complexes formed with 29a showed low gas-phase stabilities in
native MS. Dissociation of 29a from Mab TrmD led to a shift in the charge state distribution of
the protein to lower charge states, which was confirmed for individual precursor charge states by
MS/MS (data not shown). This could be rationalized by the dissociation of positively-charged
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
9a molecules from Mab TrmD, leading to a reduction of the overall positive charge of the
protein. In contrast 24c and 26c, being potentially less cationic than 29a, did not exhibit this
charge reduction behavior. Overall, the native MS data confirms the direct binding of the ligands
to Mab TrmD, and suggests that the interaction is specific with each Mab TrmD dimer binding to
two molecules of the ligand.
Synthetic Chemistry. Synthesis of compound 2 (Scheme 1) began with addition of a benzyl
protecting group to fragment hit 1 through heating at reflux with benzyl bromide in acetone
(
93% yield), followed by alkaline hydrolysis of the ester group of 32 to a carboxylic acid (94%
yield). Acid 33 was converted by the action of EDC to the amide 34 (84% yield), then
debenzylated to 2 through transfer hydrogenation with palladium on carbon, formic acid and tert-
butanol (64% yield). The production of esters 3-7 (Scheme 1) started with hydrolysis of 1 to the
corresponding carboxylic acid 35 (96% yield), whose pyrazole was protected with a
tetrahydropyran ring through treatment with 3,4-dihydro-2H-pyran under acidic conditions to
give 36 (43% yield). Carboxylic acid 36 was used to synthesize the final ester compounds 3-7 by
two alternative two step protocols, involving either initial treatment with a benzyl halide and
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9
base in DMF or EDC-mediated ester coupling with a benzyl alcohol. Both protocols ended with
removal of the tetrahydropyran protecting group by TFA (5-57% yield overall).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthesis of Compounds 2-7.
…
……………………………………………………………………………………………………
Reagents and Conditions: (a) BnBr, K
50 min; (c) EtNH .HCl, DIPEA, DMAP, EDC.HCl, DCM, 15 h; (d) Pd/C (10 wt. % loading),
formic acid, BuOH, 50 °C, 2 h; (e) NaOH, H O, reflux, 2 h; (f) 3,4-dihydro-2H-pyran,
TsOH·H O, EtOAc, DMF, overnight; (g) (i) 4-nitrobenzyl alcohol, DIPEA, DMAP, EDC.HCl,
2
CO
3
, acetone, reflux, 7 h; (b) NaOH, THF, H O, 70 °C,
2
1
2
t
2
2
DCM, overnight (ii) TFA, 5 min; (h) (i) RCH
min.
2
Br, DIPEA, DMF, 90 min to 2 h (ii) TFA, 10 to 12
The routes for synthesis of compounds 8-11 (Scheme 2) involved protection of phenol 37 with
a triisopropylsilyl protecting group (81% yield), followed by reduction of the ester group of 38 to
the corresponding alcohol 39 using lithium aluminium hydride (83% yield). Alcohol 39 was put
through an EDC-mediated ester coupling reaction with 36, with the silyl protecting group
removed using TBAF (28% yield overall). Phenol 40 was treated with an alkyl bromide and base
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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
in DMF, then the tetrahydropyran protecting group removed with TFA to afford 8 and 9 (50-53%
yield overall). In contrast, 10 and 11 were synthesized from 40 through initial coupling with an
alcohol under Mitsunobu conditions with triphenylphospine and diisopropyl azodicarboxylate,
followed by treatment with TFA (24-46% yield overall). Compounds 12-15 all utilized
bromoarene 44 in their respective syntheses (Scheme 2), itself synthesized by a sequence
involving methyl iodide alkylation of 41 (97% yield), followed by lithium aluminium hydride
reduction of ester 42 to alcohol 43 (71% yield) that was coupled with 36 using EDC (63% yield).
The Sonogashira cross-coupling of 44 with alkyne 45, itself produced by treatment of fragment
hit 1 with propargyl bromide in a suspension of potassium carbonate and DMF (81% yield),
afforded 46 (43% yield). TFA-mediated deprotection of 46 provided 12 (63% yield). 13 was
synthesized by Buchwald-Hartwig amination of 44 with pyrrolidine, followed by TFA
deprotection (7% yield overall). In contrast, 14 was produced through a Suzuki-Miyaura cross-
coupling reaction of 44 with 3-(bromomethyl)pyridine, followed by TFA deprotection (44%
yield overall). For 15, 44 was converted to the corresponding pinacol boronic ester 47 by a
Miyaura-borylation with bis(pinacolato)diboron (44% yield), which was then cross-coupled with
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
3
-(bromomethyl)pyridine in a Suzuki-Miyaura reaction and deprotected with TFA (18% yield
overall).
Scheme 2. Synthesis of Compounds 8-15.
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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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
…
……………………………………………………………………………………………………
…
……………………………………………………………………………………………………
Reagents and Conditions: (a) TIPSCl, imidazole, DMF, 2 h; (b) LiAlH
°C to rt, 3 h; (c) (i) 36, DIPEA, DMAP, EDC.HCl, DCM, overnight (ii) TBAF (1 M in THF),
0 min; (d) (i) Cs CO , RCH Br (.HBr), DMF, 2 h (ii) TFA, 15 min; (e) (i) PPh , RCH OH,
CO , DMF, 3 d; (g) LiAlH (2.4 M in
O, 0 °C to rt, overnight; (h) 36, DIPEA, DMAP, EDC.HCl, DCM, overnight; (i)
propargyl bromide (80 wt. % solution in toluene), K CO , DMF, 3 d; (j) 44, Pd(dppf)Cl , CuI,
4
(2.4 M in THF), Et O,
2
0
1
2
3
2
3
2
DIAD, THF, 1 h to overnight (ii) TFA, 15 min; (f) MeI, K
THF), Et
2
3
4
2
2
3
2
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1
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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
NEt
3
, DMF, 100 °C, 4 h; (k) TFA, 15 min; (l) (i) pyrrolidine, Cs
2
CO , RuPhos, RuPhos Pd G1
3
t
methyl tert•butyl ether adduct, BuOH, 85 °C, 15 h (ii) TFA, 30 min; (m) (i) 3-pyridinylboronic
acid, Pd(dppf)Cl
Pd(dppf)Cl , KOAc, dioxane, 80 °C, 3 h; (o) (i) 3-(bromomethyl)pyridine HBr, Pd(PPh
CO , DME, H O, 95 °C, 10 h (ii) TFA, 30 min.
2
, K
2
CO
3
, dioxane, H
2
O, 100 °C µW, 30 min (ii) TFA, 15 min; (n) B
2
pin
2
,
,
2
3 4
)
K
2
3
2
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
The synthetic route for the 5-aminopyrazole analogue 19 (Scheme 3) began with nucleophilic
attack of ester 48 by a nitrile anion, generated from n-butyllithium and acetonitrile. This resulted
in the corresponding β-ketonitrile 49 (99% yield), which was heated at reflux with hydrazine in
2
5
ethanol to form 19 (73% yield). The synthesis of 23 (Scheme 3) was similar to 19, using indole
0 as the starting point, which was protected with a tert-butyldimethylsilyl group to form 51
59% yield). After synthesis of the corresponding β-ketonitrile, deprotection was accomplished
5
(
by TBAF without prior purification affording 52 (99% yield overall), which was heated at reflux
with hydrazine in ethanol (59% yield). The deprotected β-ketonitrile 52 was also utilized in the
synthesis of 25 (Scheme 3), with its activated methylene group treated with trichloroacetonitrile
2
6
and sodium acetate. The resultant functionalized compound 53 was purified (87% yield) before
heating at reflux with hydrazine in ethanol to afford 25 (24% yield).
Synthesis of compounds 24a-c and 24f (Scheme 3) utilized formation of β-ketonitriles 55a-c
and 55f from the corresponding methyl esters 54a-c and 54f (84-99% yield) and subsequent
reaction with hydrazine (31-62% yield). Unlike 23, the indole nitrogen was protected by the
benzyl or picolyl groups. In the cases of 54a-d and 54f these were added to indole 50 by heating
at reflux with caesium carbonate and benzyl or picolyl halides in acetonitrile (80-93% yield).
Due to the production of significant amounts of side products in the synthesis of the
corresponding β-ketonitrile from 54d, the resultant product was taken forwards crude in the
synthesis of 24d (18% yield overall) (Scheme 3). The nitrile group of 24d was also hydrolyzed
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to an amide, affording 24e (28% yield) (Scheme 3). Compounds 26c and 26f were synthesized in
a similar manner to 25, using the previously generated β-ketonitriles 55c and 55f from the
production of 24c and 24f (Scheme 3). However, the intermediates from the reaction with
trichloroacetonitrile were taken forwards crude, after aqueous work up, to the subsequent
reaction with hydrazine (45-62% yield overall).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of Compounds 19, 23, 24a-f, 25, 26c and 26f.
…
……………………………………………………………………………………………………
………………………………………………………………………………………………………
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