Bisubstrate inhibitors of nicotinamide N-methyltransferase (NNMT) with enhanced activity

Article abstract of DOI:10.1021/acs.jmedchem.9b00413

Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor previously developed in our group, we prepared a diverse library of inhibitors to probe the different regions of the enzyme's active site. This investigation revealed that incorporation of a naphthalene moiety, intended to bind the hydrophobic nicotinamide binding pocket via π-πstacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors (half-maximal inhibitory concentration 1.41 μM). These findings are further supported by isothermal titration calorimetry binding assays as well as modeling studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-dependent inhibitory effect on the cell proliferation of the HSC-2 human oral cancer cell line.

Full text of DOI:10.1021/acs.jmedchem.9b00413

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Article
Bisubstrate Inhibitors of Nicotinamide N-
Methyltransferase (NNMT) with Enhanced Activity
Yongzhi Gao, Matthijs van Haren, Ed E. Moret, Johannes J.M. Rood, Davide Sartini, Alessia
Salvucci, Monica Emanuelli, Pierrick Craveur, Nicolas Babault, Jian Jin, and Nathaniel I. Martin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00413 • Publication Date (Web): 02 Jul 2019
Downloaded from http://pubs.acs.org on July 3, 2019
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Bisubstrate Inhibitors of Nicotinamide
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N-Methyltransferase (NNMT) with Enhanced Activity
a, ‡
a, ‡
b
c
d
Yongzhi Gao, Matthijs J. van Haren, Ed E. Moret, Johannes J.M. Rood, Davide Sartini,
d
d
e
e,f
f
Alessia Salvucci, Monica Emanuelli, Pierrick Craveur, Nicolas Babault, Jian Jin,
a,*
Nathaniel I. Martin
‡
These authors contributed equally
a
Biological Chemistry Group, Institute of Biology Leiden, Leiden University, Sylviusweg 72,
b
2333 BE Leiden, The Netherlands; Chemical Biology & Drug Discovery Group, Utrecht
Institute for Pharmaceutical Sciences, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands;
c
Pharmacoepidemiology & Clinical Pharmacology Group, Utrecht Institute for Pharmaceutical
d
Sciences, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands; Department of Clinical
e
Sciences, Universitá Politecnica delle Marche, Via Ranieri 65, 60131, Ancona, Italy; Synsight,
f
Genopole Entreprises, 4 Rue Pierre Fontaine, 91000, Évry, France; Center for Chemical Biology
and Drug Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch
Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY10029, United States;
E-mail: n.i.martin@biology.leidenuniv.nl
ABSTRACT: Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of
nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a
variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for
NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor
previously developed in our group, we prepared a diverse library of inhibitors to probe the
different regions of the enzyme’s active site. This investigation revealed that incorporation of a
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naphthalene moiety intended to bind the hydrophobic nicotinamide binding pocket via π-π
stacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors
(IC50 1.41 µM). These findings are further supported by ITC binding assays as well as modeling
studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-
dependent inhibitory effect on the cell proliferation of an HSC-2 oral cancer cell line.
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INTRODUCTION
Nicotinamide N-methyltransferase (NNMT) is an important metabolic enzyme that catalyzes
the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto its
various substrates, most notably nicotinamide (NA) and other pyridines, to form 1-methyl-
1
–3
nicotinamide (MNA) or the corresponding pyridinium ions.
The past decade has seen a
renewed interest in the biological function of NNMT in a range of human diseases. While it was
previously assumed that NNMT’s primary roles were limited to nicotinamide metabolism and
xenobiotic detoxification of endogenous metabolites, broader roles for NNMT in human health
4
and disease are becoming clearer. NNMT has been found to be overexpressed in a variety of
5
–7
8,9
10–14
diseases, including metabolic disorders , cardiovascular disease , cancer
and Parkinson’s
15,16
disease . In general, overexpression of NNMT has been linked to disease progression in the
aforementioned afflictions, with the exception of its role in Parkinson’s disease where NNMT
1
7,18
seems to be neuroprotective.
Collectively, NNMT appears to play a unique role in the
regulation of post-translational modifications and signal transduction, making it an attractive and
viable therapeutic target.
Despite growing interest, few small molecule NNMT inhibitors have been described to date.
Among these structures, the product of the enzymatic reaction, MNA, is a known inhibitor of
19
NNMT and has generally been used in biochemical activity assays. Recently, Cravatt and
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coworkers reported chloroacetamide-based covalent NNMT inhibitors that react with cysteine
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C165 in the SAM-binding pocket of the enzyme. Notably, Sanofi researchers also recently
reported a series of nicotinamide analogues that inhibit NNMT activity, leading to decreased
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MNA production, stabilization of insulin levels, glucose regulation, and weight loss in mouse
21,22
models of metabolic disorders.
In another approach, the group of Watowich focused on the
development of inhibitors based on NNMT’s alternative substrate quinoline. Their compounds
2
3
showed improvement of symptoms in diet-induced obese mice. Previous work in our group has
focused on bisubstrate inhibitors designed to mimic the transition state of the methylation
reaction catalyzed by NNMT with compound 1 (Fig. 1) showing activity on par with the known
2
4
general methyltransferase inhibitor sinefungin.
Designing bisubstrate analogues as inhibitors is an established and effective strategy that has
been applied to a range of methyltransferase enzymes including catechol O-methyltransferase
2
5-26
27
28–30
(COMT),
histone lysine methyltransferases, arginine methyltransferases,
and more
2
4,31
recently nicotinamide N-methyltransferase.
A recently published co-crystal structure of a
bisubstrate inhibitor bound to NNMT (PDB ID: 6CHH) clearly delineates key interactions with
residues in the enzyme active site, providing valuable information for further optimization of
3
1
improved bisubstrate-like inhibitors. The work here described builds on our previous findings
for “trivalent” inhibitor 1, which is assumed to simultaneously bind in the adenosine, amino acid,
and nicotinamide binding pockets of the NNMT active site. Based upon insights provided by
recent NNMT crystal structures, we have designed new inhibitors wherein the nicotinamide
moiety is replaced by other aromatic substituents accompanied by variation in the length of the
linker connecting the amino acid moiety. Based on the high conservation of the residues in the
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adenosine binding pocket, no changes were made to the adenosine group. A schematic overview
of the design strategy is been presented in Figure 1.
NH2
N
N
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N
O
N
HO
HO
NH2
O
n
n
n=1,2
O
N
n
HO
O
H N
O
2
n
OH
O
H N
OH
2
n=1,2
O
compound 1 (n=1)
compound 2 (n=2)
O
Figure 1. Schematic overview of the design strategy of the second generation of inhibitors based
2
4
31
on trivalent bisubstrate compounds 1 and 2
RESULTS AND DISCUSSION
Design: The ternary crystal structure of NNMT (PDB ID: 3ROD) reveals the interactions of
3
2
nicotinamide and S-Adenosyl-L-homocysteine (SAH) with the active site residues. The active
site can be roughly divided into three binding regions for the adenosine group, the amino acid
moiety, and the nicotinamide unit. The starting point was trivalent bisubstrate compound 1,
which was designed to bind all three binding regions. In order to find the optimal substitutions, a
systematic approach was applied, where variations were made to the nicotinamide mimic on the
one hand and the amino acid moiety on the other. The benzamide group, representing
nicotinamide, was also replaced by methyl benzoate or benzoic acid moieties. Notably, the
crystal structure of the NNMT−nicotinamide−SAH ternary complex reveals π-π stacking
32
between tyrosine residue Y204 and the nicotinamide substrate. We therefore also prepared an
analogue bearing a naphthalene unit in the presumed nicotinamide position with the aim of
introducing stronger π-π stacking with the tyrosine residues of the NNMT active site. We also
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explored variation of the amino acid moiety as part of our design strategy: in some analogues the
amine of the amino acid unit was omitted to reduce charge and in others the carboxylic acid
replaced by the corresponding primary amide. In addition, variation in the length of the carbon
1
1
1
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chain linking the amino acid moiety was examined. Furthermore, inspired by the structure of
33
histone methyltransferase DOTL1 inhibitor pinometostat,
we also investigated the
incorporation of an isopropyl group to replace the amino acid moiety entirely.
Synthesis: Key aldehyde intermediates (compounds 6, 8, 9, 16, 17, 22, 23, 27, 28) required for
the synthesis of the various bisubstrate analogues pursued were prepared from commercially
available materials, in good overall yields, as summarized in Schemes 1-3. The trivalent
inhibitors were then prepared via a convenient double reductive amination strategy starting from
the commercially available 2’-3’-O-isopropylidene-6-aminomethyl-adenosine starting material
and the corresponding aldehydes (Schemes 4 and 5).
The preparation of aromatic aldehydes 6, 8, and 9 began with the selective mono-deprotection
34
of dimethyl isophthalate using sodium hydroxide (Scheme 1). Monomethyl isophthalate (3)
was subsequently transformed into trityl-protected amide 4 using tritylamine via its acid chloride
intermediate and reduced by diisobutylaluminum hydride (DIBAL-H) to give alcohol 5. The
alcohol was oxidized to aldehyde 6 using pyridinium dichromate (PDC). For aldehydes 8 and 9,
the carboxylic acid of 3 was selectively reduced using a mixture of sodium borohydride and
3
5
boron trifluoride diethyl etherate. The resulting alcohol (7) was oxidized using PDC to yield
the corresponding aldehyde (8). Following hydrolysis of the methyl ester in 8 and subsequent
3
6
conversion to the tert-butyl ester, aldehyde 9 was obtained.
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O
O
O
O
HO
O
H
O
O
a
c
b
d
H
N
H
H
N
N
O
OH
Trt
Trt
Trt
O
3
O
4
O
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e
O
O
O
O
O
O
d
f, g
OH
H
H
7
8
O
9
O
Scheme 1. Synthetic route for aldehydes 6, 8 and 9. Reagents and conditions: (a) NaOH, MeOH,
rt, 16 h (95%); (b) i) SOCl , reflux, 2h, ii) tritylamine, CH Cl , 0°C-rt, 2 h (72%); (c) DIBAL-H,
-78°C-rt, 2 h (85%); (d) PDC, CH Cl , rt, 2 h (53-64%); (e) NaBH
(89%); (f) LiOH, THF/H O (2:1); (g) 2-tert-butyl-1,3-diisopropylisourea, CH
39% over 2 steps).
2
2
2
.
2
2
4
, BF
3
Et
2
O, THF, 0°C-rt, 2 h
Cl , tert-butanol
2
2
2
(
Aliphatic aldehydes 16 and 17 containing trityl-protected amide functionalities were prepared
from succinimide and glutarimide respectively (Scheme 2). The cyclic amides were first trityl-
protected and subsequently ring-opened using potassium hydroxide. Reduction to the
37,38
corresponding alcohols and oxidization using PDC gave aldehydes 16 and 17.
In analogous
fashion, aldehydes 22 and 23, both containing tert-buytl ester moieties, were prepared by ring
opening of succinic or glutaric anhydride with tert-butyl alcohol to obtain mono-esters 18 and
3
9,40
1
9.
The carboxylic acid functionalities were reduced to alcohols 20 and 21 and then oxidized
using PDC to yield aldehydes 22 and 23.
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R
N
O
O
O
O
O
b
Trt
OH
c
d
Trt
H
Trt
OH
N
H
N
H
N
n
n
n
H
n
O
O
R=H, n=1,2
n=1, 12
n=2, 13
n=1, 14
n=2, 15
n=1, 16
n=2, 17
a
R=Trt, n=1, 10
R=Trt, n=2, 11
1
1
1
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O
O
O
O
O
O
b
OH
c
OH
d
H
O
O
O
n
n
n
n
O
O
n=1,2
n=1, 18
n=2, 19
n=1, 20
n=2, 21
n=1, 22
n=2, 23
Scheme 2. Synthetic route for aldehydes 16, 17, 22 and 23. Reagents and conditions: (a) TrtCl,
CH
BF
3
.
CN, K
Et O, THF, 0°C-rt, 2 h (64-81%); (d) PDC, CH
N, toluene, overnight (25-93%).
2
CO
3
, rt, 48 h (20-28%); (b) KOH, EtOH, reflux, overnight (37-93%) (c) NaBH
4
,
3
2
2
Cl , rt, 2 h (65-78%); (e) tert-butanol,
2
DMAP, N-Hydroxysuccinimide, Et
3
Aldehydes 27 and 28, both containing protected amino acid functionalities, were prepared
starting from the appropriately protected aspartic acid and glutamic acid building blocks
(
Scheme 3). Conversion of the side chain carboxylates to their corresponding Weinreb amides
yielded intermediates 24 and 25. Reduction of aspartate-derived 24 with DIBAL-H gave amino
acid aldehyde 27 in high yield. For the preparation of aldehyde 28, a similar route was followed
with the addition of a second Boc-protection of intermediate 25 to avoid an intramolecular
2
4,41
cyclization side reaction.
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O
O
O
n
a
n
c
n
HO
N
H
OtBu
NHBoc
O
OtBu
NRBoc
OtBu
NRBoc
O
O
O
n=1,2
n=1, R=H 24
n=2, R=H 25
n=1, R=H 27
n=2, R=Boc 28
b
n=2, R=Boc, 26
0
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Scheme 3. Synthetic route for aldehydes 27 and 28. Reagents and conditions: (a)
CH NHOCH ·HCl, BOP, Et N, CH Cl , rt, 2 h (85-88%); (b) (Boc) O, Et N, DMAP, CH Cl
(94%); (c) DIBAL-H in hexanes (1 M), THF, -78°C, assumed quant.
3
3
3
2
2
2
3
2
2
With the necessary aldehyde building blocks in hand, assembly of the bisubstrate inhibitors
was performed in each case starting from commercially available 2’-3’-O-isopropylidene-6-
aminomethyl-adenosine (Scheme 4). Using a reliable reductive amination approach, aromatic
aldehydes 6, 8, 9, and commercially available 2-naphthaldehyde were each coupled to the
protected adenosine species to yield intermediates 29-32. These intermediates were next
connected with aliphatic aldehydes 16, 17, 22, 23, 27, 28 or acetone via a second reductive
amination step to give the corresponding protected tertiary amine intermediates 33-56 (Scheme
2 2
5). Global deprotection of the acid-labile protecting groups was carried out in CH Cl /TFA (1:1)
with isopropylidene group cleavage facilitated by subsequent addition of water. The crude
products were purified by preparative HPLC to yield bisubstrate analogues 1, 57-60.
NH2
NH2
O
O
R = NHTrt, 6
R = OMe, 8
R = OtBu, 9
1
N
N
N
N
N
N
H
R
1
1
N
O
N
O
R = ArCONHTrt, 29
R = ArCOOMe, 30
R = ArCOOtBu, 31
R = naphthalene, 32
1
a
+
O
or
O
O
1
1
H
1
O
O
NH2
NH
R1
Scheme 4. Synthesis of intermediate compounds 29-32. Reagents and conditions: (a)
NaBH(OAc) , AcOH, DCE, rt, overnight (50-74%).
3
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NH2
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
O
N
O
O
O
HO
O
a
b
O
O
HO
NH
N
N
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
R2
R2
H
N
H
N
H N
2
Trt
Trt
O
O
O
R = butanamide, 57
R = pentanamide, 58
R = butanoic acid, 59
2
R = pentanoic acid, 60
R = 2-amino-butanoic acid, 1
R2= isopropyl, 61
2
9
R = N-(trityl)butanamide, 33
2
R = N-(trityl)pentanamide, 34
R = tert-butyl butanoate, 35
R = tert-butyl pentanoate, 36
R = tert-butyl-2-(Boc-amino)butanoate, 37
R = isopropyl, 38
2
2
2
2
2
2
2
2
2
Scheme 5. Representative scheme for the synthesis of the final compounds, shown for
compounds 1 and 57-61. The same procedure was used starting from aldehydes 30-32 to form
intermediate compounds 39-56 and 80 and final compounds 62-79 and 81 as detailed in the
experimental section. Reagents and conditions: (a) aldehyde, NaBH(OAc)
overnight (49-77%); (b) i) TFA, CH Cl , rt, 2h, ii) H O, rt, 30 min (47-73%).
3
, AcOH, DCE, rt,
2
2
2
Inhibition Studies: The bisubstrate analogues were next tested for their NNMT inhibitory
2
activity using a method recently developed in our group. This assay employs Ultra High
Performance (UHP) Hydrophilic Liquid Interaction Chromatography (HILIC) coupled to
Quadrupole Time-Of-Flight Mass Spectrometry (QTOF-MS) to rapidly and efficiently assess
NNMT inhibition by analysis of the formation of MNA. The NNMT inhibition of all compounds
was initially screened at a fixed concentration of 250 µM for all of the compounds. In cases
where at least 50% inhibition was detected at this concentration full inhibition curves were
measured in triplicate to determine the corresponding IC50 values. As reference compounds, we
included the well-established and general methyltransferase inhibitors sinefungin and SAH. In
addition, we also synthesized two recently described NNMT inhibitors, compound 2 and 6-
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7
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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
(
methylamino)-nicotinamide, following the procedures described in the corresponding
2
1,31
publications.
The structures of these reference compounds are provided in Figure 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
NH2
N
N
O
N
N
O
NH2
O
NH2
HO
OH
N
H
N
HO
NH2
sinefungin
6-methylamino-NA
O
NH2
NH2
NH2
N
N
N
N
N
N
N
N
O
O
O
NH2
O
HO
HO
S
N
OH
OH
HO
HO
NH2
SAH
2
Figure 2. Chemical structures of the reference compounds used in NNMT inhibition studies
The results of the NNMT inhibition studies are summarized in Table 1 and clearly show that
only minor adjustments to the functional groups found in the enzyme’s natural substrates are
tolerated. Among the compounds studied, the most potent inhibition was observed when the
aliphatic moiety corresponded to the same length in the amino acid side chain as present in the
methyl donor SAM. Notably, the preferred aromatic moiety was found to be the naphthalene
group, an apparent confirmation of our hypothesis that increased π-π stacking can lead to
enhanced binding in the nicotinamide pocket. The bisubstrate analogue containing both of these
elements (compound 78), displayed the highest inhibitory activity against NNMT with an IC50 of
1
.41 µM. Interestingly, the amino acid and naphthyl moieties were also found to independently
enhance the activity of the other inhibitors prepared. In this way a suboptimal moiety at one
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position can be compensated – to an extent – by including either the SAM amino acid motif or
the naphthalene unit at the other position. For example, bisubstrate analogues containing the
benzamide, benzoic acid, or methyl benzoate groups only show inhibitory activity if they also
contain the amino acid motif (compounds 1, 2, 66, 72) with IC50 values of 4.36-23.4 µM
respectively. On the other hand, among the bisubstrate analogues lacking the amino acid motif,
inclusion of the naphthalene moiety (compounds 74-79) enhances NNMT inhibition albeit with
moderate IC50 values in the range of 52.6-129.9 µM.
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
Other notable findings were the results obtained with the reference compounds. The general
methyltransferase inhibitors sinefungin and SAH showed inhibitory activities in line with those
2
4
previously reported. Interestingly, the 6-methylamino-NA compound recently described by
21
Sanofi to be a submicromolar inhibitor, gave an IC50 of 19.8 µM in our assay. The recently
published bisubstrate analogue 2 exhibited good activity (IC50 4.4 µM) on par with published
3
1
values. Given the potent inhibition measured for both compound 2 and 78, we also prepared
and tested compound 81, an analogue of 78 bearing the same naphthyl moiety but with the amino
acid motif containing an additional methylene unit as in 2. Somewhat surprisingly, this linker
elongation resulted in a complete loss of inhibitory activity (IC50>250 µM).
To gain insight into the selectivity of compound 78, we also tested its activity against
representative members of both the arginine and lysine families of methyltransferases, PRMT1
and NSD2 respectively. In both cases compound 78 was tested at a concentration of 50 µM and
showed no significant inhibition (>50% of the enzyme’s activity remained), see Table S1.
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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
Table 1. Tabulated overview of the chemical structures and inhibition results of the final
compounds and reference compounds
a
IC50 values (µM)
6-methylamino-
nicotinamide
Reference compounds
Sinefungin
SAH
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
IC50 (µM)
12.51 ± 2.11
35.30 ± 5.48
19.81 ± 2.50
NH2
R
1
N
N
O
O
O
N
N
H N
MeO
HO
2
O
R1
N
O
R2
O
n
O
7
4:
5
5
5
6
7 (n=1): >250
8 (n=1): >250
9 (n=1): >250
0 (n=1): >250
62: >250
63: >250
64: >250
65: >250
68: >250
69: >250
70: >250
71: >250
NH2
111.50 ± 28.79
O
75: 52.62 ± 9.08
NH2
O
76: >250
77: >250
OH
O
R
2
OH
1
1
(m=1, n=1):
4.90 ± 2.07
78 (m=1, n=1):
1.41 ± 0.16
81 (m=2, n=2):
>250
O
66 (m=1, n=1):
17.45 ± 2.65
72 (m=1, n=1):
23.41 ± 4.86
OH
m
2 (m=2, n=2):
4
NH2
.36 ± 0.27
6
>
1 (n=1):
250
79:
6
7: >250
73: >250
129.90 ± 14.80
a
Assays performed in triplicate on at least six different inhibitor concentrations. Standard errors of the mean reported.
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ITC binding studies: To further evaluate the binding interactions of the most active bisubstrate
analogues with NNMT, isothermal titration calorimetry (ITC) studies were performed,
Compounds, 1, 66, 72 and 78, all containing the amino acid moiety but with varying aromatic
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
substituents, were investigated. As illustrated in Figure 3, the dissociation constants (K
measured for these compounds track very well with the IC50 values measured in the in vitro
assay. Compounds 1 and 66 display similar binding to NNMT with K values of 36 µM and 25
µM respectively while compound 72 binds less tightly with a K of 124 µM. In good agreement
with the results of the inhibition assay, the most active inhibitor, compound 78, also displayed
the highest binding affinity for NNMT with a K of 5.6 µM. As expected, the inhibitors were
d
)
d
d
d
each found to bind the enzyme with a 1:1 stoichiometry.
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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
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 3. ITC isotherms and thermograms including thermodynamic binding parameters
measured for compounds 1, 66, 72, and 78 with hNNMT.
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Modeling studies:
To further investigate the way in which the inhibitors bind within the NNMT active site
modeling studies were performed. Working from the available crystal structure of NNMT
3
2
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
protein bounded to nicotinamide and SAH (PDB ID: 3ROD) , compounds 1, 2, 78, and 81 were
modeled in the binding pocket. In an attempt to explain the significant difference in activity of
7
8 and 81 additional molecular dynamic simulations were also performed for compounds 1, 2, 78
and 81. While these simulations suggest differences in the binding interaction of the compounds
(supplemental figure S1), the calculated binding energies for each are all very similar
(supplemental table S2). In terms of their active site orientations, compounds 1, 2, 78 and 81 are
all predicted to position their three branches roughly in the same regions of the active site,
however their orientations and interactions are quite different.
From the modeling data, two distinct features are apparent. First, when the chain linking the
amino acid moiety is shorter (as in compounds 1 and 78), the formation of an intramolecular
hydrogen bond interaction was observed between the carboxylate of the amino acid moiety and
the protonated tertiary amine (see Figure 4). This intramolecular interaction is highly stable for
compound 78 and less stable for compound 1. This additional interaction reduces the entropic
energy of the ligand, thereby potentially stabilizing its binding, and re-orients the amino acid part
in the pocket, preventing the polar interactions with neighboring residues (e.g. Y25, D61, Y69,
and T163) observed when the chain is longer (as present in compounds 2 and 81). This
intramolecular hydrogen bond may explain the difference in activity observed between
compounds 78 and 81. The second distinct feature is the tyrosine rich environment around the
naphthalene moiety of 78 compared to the nicotinamide unit of 1. The orientation of the tyrosine
residues surrounding this part of the molecule leads to π-π stacking interactions with the
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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
naphthalene and hint at an explanation for the strong inhibition and high affinity of compound 78
with NNMT protein (Fig. 4).
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 4. Modeling results for compound 78 in the NNMT active site (PDB ID: 3ROD).
Molecular dynamics simulation indicates the presence of an intramolecular hydrogen bond
(2.7Å, shown in cyan) specific to compound 78 (in green) that would be expected to reduce the
entropic energy of the ligand and potentially stabilize binding to NNMT (in white). Proposed
intermolecular hydrogen bond network (in yellow) and π-π stacking interactions with Tyr
residues (in purple) stabilize compound 78 in the NNMT active site (hydrogens omitted for
clarity).
Cell based assays
To evaluate the cellular activity of the bisubstrate inhibitors, the compounds were tested for
their effect on cell proliferation in the human oral cancer cell line HSC-2. We have shown
recently that knockdown of NNMT in this particular cell line has a marked effect on cell
42
proliferation and apoptosis. As shown in Figure 5 there were no significant differences in cell
proliferation rate between HSC-2 cells treated with DMSO at 0.1% concentration and cells
grown with culture medium only, at any time of each performed assay. Upon treatment with the
NNMT inhibitors, cell proliferation was not significantly inhibited by compounds 1, 2 and 81
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(
Figure 5). On the contrary, relative to the DMSO control, treatment with compound 78 led to a
notable decrease in cell proliferation. In particular, cell proliferation was significantly (p < 0.05)
inhibited by compound 78 at 10µM (20% reduction), 50 µM (21% reduction) and 100 µM (27%
reduction) concentrations, 48 hours after treatment. Interestingly, at the longest 72 hours time
point taken, treatment with compound 78 lead to an even greater and significant (p < 0.01)
decrease in cell proliferation (44% reduction), at the highest concentration (100 µM) (Figure 5).
We next investigated the effect of compound 78 on cellular NNMT activity by assessing its
impact on MNA production in the same HSC-2 cell line. Cells were treated with 100 µM of 78
and MNA levels determined after 0, 1, 2, and 3 days. Cells treated with compound 78 show a
significant (p < 0.01) decrease in the levels of MNA (50% reduction) compared to controls after
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
4
8 hours. Interestingly, at 72 hours an increase in cellular MNA production was detected,
however, the same effect was also observed in the DMSO control (but not in the untreated
control) suggesting an effect attributable to longer-term DMSO exposure. The results of the
cellular MNA analysis are presented in supplemental Figure S2.
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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
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 5. Results of the MTT cell viability assay on HSC-2 human oral cancer cells. Only
compound 78 showed a significant effect on cell proliferation after 48 and 72 hours
CONCLUSION
24
Building from our earlier findings with first reported ternary bisubstrate NNMT inhibitor 1, we
designed and prepared a focused library of novel inhibitors to provide new structure-activity
insights. In doing so, various structural motifs were investigated for the ability to enhance
inhibitor activity and binding within the NNMT active site. By probing the SAM and NA
binding pockets with different spacers and functional groups, we found that the optimal ligands
are the endogenous amino acid side-chain and the naphthalene moiety. Among the naphthalene-
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containing bisubstrate analogues prepared, compound 78 showed the most potent NNMT
inhibition. In this way the activity of our initial NNMT inhibitor 1 (IC50 14.9 µM) was improved
10-fold with compound 78 displaying an IC50 value of 1.41 µM. Notably, using an assay
designed to directly measure NNMT product formation, compound 78 was shown to be more
potent than most other NNMT inhibitor reported to date. ITC-based binding studies provided
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
d
additional insights in the affinity of the inhibitors for the enzyme with measured K value
following a trend similar to that observed for the IC50 data obtained in the in vitro inhibition
assays. From modeling studies, the improved activity of compound 78 can be rationalized by the
apparent presence of an intramolecular hydrogen bonding interaction predisposing the compound
to an active conformation with lower entropic cost. In addition, the modeling indicates that the
naphthalene group in 78 is properly oriented so as to benefit from additional π-π stacking
interactions with several tyrosine residues in the nicotinamide binding pocket of the enzyme. The
cellular data obtained for compound 78 shows a significant inhibitory effect on cell proliferation
in HSC-2 oral cancer cells. These promising results provide important new insights for the
design and further optimization of potent NNMT inhibitors.
EXPERIMENTAL PROCEDURES
General Procedures: All reagents employed were of American Chemical Society (ACS) grade
or finer and were used without further purification unless otherwise stated. For compound
1
characterization, H NMR spectra were recorded at 400 MHz with chemical shifts reported in
2 3
parts per million (ppm) downfield relative to tetramethylsilane (TMS), H O (δ 4.79), CHCl
1
(
7.26) or DMSO (δ 2.50). H NMR data are reported in the following order: multiplicity (s,
singlet; d, doublet; t, triplet; q, quartet and m, multiplet), coupling constant (J) in hertz (Hz) and
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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
the number of protons. Where appropriate, the multiplicity is preceded by br, indicating that the
1
3
signal was broad. C NMR spectra were recorded at 101 MHz with chemical shifts reported
1
3
relative to CDCl
3
(δ 77.16), methanol (δ 49.00) or DMSO (δ 39.52). The C NMR spectra of 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
compounds recorded in D
2
O could not be referenced. High-resolution mass spectrometry
24
31
34
34
36
(
HRMS) analysis was performed using a Q-TOF instrument. Compounds 1, 2, 3, 7, 8,
3
7
43
38
38
44
40
40
45
46
47
40
41
41
48
41
48
9
, 10, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 were
prepared as previously described and had NMR spectra and mass spectra consistent with the
assigned structures. Purity was confirmed to be ≥ 95% by analytical RP-HPLC using a
Phenomenex Kinetex C18 column (5 µm, 250 × 4.6 mm) eluted with a water–acetonitrile
gradient moving from 0% to 100% CH
purified via preparative HPLC using a Reprosil-Pur C18-AQ column (10 µm, 250 × 22 mm)
eluted with a water–acetonitrile gradient moving from 0% to 50% CH CN (0.1% TFA) over 60
3
CN (0.1% TFA) in 30 minutes. The compounds were
3
−
1
minutes at a flow rate of 12.0 ml min with UV detection at 214 nm and 254 nm.
Methyl 3-(tritylcarbamoyl)benzoate (4). Mono-methylisophthalate 3 (0.98 g, 5.4 mmol) was
refluxed in 10 mL SOCl
solution). The SOCl was removed under reduced pressure and the acid chloride intermediate
was redissolved in 15 mL dry CH Cl and transferred to a cooled (ice-bath) solution of
tritylamine (1.41 g, 5.4 mmol) and 2 mL triethylamine in 30 mL CH Cl . The reaction was
stirred overnight under N atmosphere, allowing the mixture to warm to room temperature. After
the reaction was completed (monitored by TLC (petroleum ether / CH Cl = 1:1)), the reaction
mixture was washed with water and brine and the organic phase dried over Na SO and
2
at 90 °C for about one hour (until the reaction mixture is a clear
2
2
2
2
2
2
2
2
2
4
concentrated. The crude product was purified by column chromatography (petroleum ether /
1
CH
2
Cl
2
= 2:1) to give compound 4 as a white powder (1.64 g, 72% yield). H NMR (400 MHz,
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Page 21 of 65
Journal of Medicinal Chemistry
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4
5
6
7
8
9
CDCl
3
) δ 8.45 (t, J = 1.6 Hz, 1H), 8.18 (m, 1H), 8.03 (m, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.41 –
1
3
7
3
.26 (m, 15H), 3.94 (s, 3H). C NMR (101 MHz, CDCl ) δ 166.3, 165.4, 144.5, 135.6, 132.5,
131.7, 130.6, 128.9, 128.7, 128.1, 128.1, 127.6, 127.2, 71.0, 52.4. HRMS (ESI): calculated for
+
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
C
28
H
23NO
3
[M+Na] 444.1576, found 444.1581.
3
-(Hydroxymethyl)-N-tritylbenzamide (5). Methyl 3-(tritylcarbamoyl)benzoate 4 (0.56 g,
Cl (20 mL) under a N atmosphere, the reaction solution
1
.33 mmol) was dissolved in dry CH
2
2
2
was cooled down to -78 °C, and then diisobutylaluminum hydride (DIBAL-H) (5.5 mL, 1.0 M
hexane solution) was added slowly. The reaction mixture was stirred at -78°C for 2 hours.
Saturated aq. NH
the addition of a saturated Rochelle salt solution (100 mL). The mixture was stirred at room
temperature overnight, extracted with CH Cl and the organic layers dried over Na SO and
4
Cl (50 mL) was added slowly to quench the reaction under -78°C, followed by
2
2
2
4
concentrated under reduced pressure. The crude product was purified by column chromatography
1
(CH
2
Cl
2
/EtOAc = 9:1) to obtain 5 as a white powder (0.44 g, 85% yield). H NMR (400 MHz,
DMSO-d
6
) δ 8.92 (s, 1H), 7.78 (s, 1H), 7.75 – 7.71 (m, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.40 (t, J =
13
7
1
6
.6 Hz, 1H), 7.36 – 7.18 (m, 15H), 5.26 (br, 1H), 4.54 (s, 2H). C NMR (101 MHz, DMSO-d ) δ
67.0, 145.3, 143.0, 135.5, 129.6, 128.9, 128.3, 127.9, 126.7, 126.5, 126.2, 79.6, 69.9, 69.9, 63.0.
+
HRMS (ESI): calculated for C27
H23NO
2
[2M+Na] 809.3355, found 809.3359.
3
-Formyl-N-tritylbenzamide (6). 3-(hydroxymethyl)-N-tritylbenzamide 5 (0.20 g, 0.51
mmol) and pyridinium dichromate (PDC) 0.23 g, 0.61 mmol) were placed in a 50 mL round
bottom flask and 10 mL of dry CH Cl was added under N atmosphere at room temperature. The
reaction was stirred till completion, as monitored by TLC (petroleum ether / CH Cl = 5:1). The
mixture was filtered and the organic layer was washed with brine, dried over anhydrous
Na SO and concentrated under reduced pressure. The resulting crude product was purified by
2
2
2
2
2
2
4
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Journal of Medicinal Chemistry
Page 22 of 65
1
2
3
4
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
2 2
column chromatography (petroleum ether / CH Cl = 9:1) to obtain 6 as a white powder (0.13 g,
1
yield 64%). H NMR (400 MHz, DMSO-d
6
) δ 10.09 (s, 1H), 9.31 (s, 1H), 8.39 (s, 1H), 8.17 (d, J
1
3
= 7.7 Hz, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.41 – 7.17 (m, 15H). C NMR
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
(101 MHz, CDCl
3
) δ 191.5, 165.1, 144.4, 136.5, 136.2, 133.0, 132.5, 129.5, 128.6, 128.5, 128.1,
+
1
27.7, 127.3, 77.2, 71.1. HRMS (ESI): calculated for C27
05.3047.
N-(triphenylmethyl)glutarimide (11). Glutarimide (2.8 g, 25 mmol), triphenylchloromethane
7.4 g, 25 mmol), and potassium carbonate (3.7 g, 25 mmol) were added to 100 mL acetonitrile
and the mixture was stirred at room temperature overnight. Saturated aqueous NaHCO (50 mL)
was added and the mixture was extracted with EtOAc. The combined organic layers were dried
with anhydrous Na SO and the solvent was removed under reduced pressure. The crude product
H
21NO
2
[2M+Na] 805.3042, found
8
(
3
2
4
was purified by column chromatography (petroleum ether / EtOAc = 4:1) to obtain 11 as a white
1
powder (1.8 g, yield 20%). H NMR (400 MHz, DMSO-d
6
) δ 7.45 – 7.35 (m, 6H), 7.20 (t, J =
1
3
7
.8 Hz, 6H), 7.08 (t, J = 7.3 Hz, 3H), 2.66 (t, J = 6.4 Hz, 4H), 2.01 (p, J = 6.5 Hz, 2H). C NMR
(101 MHz, CDCl
3
) δ 172.4, 143.4, 128.5, 127.3, 125.9, 35.5, 16.7. HRMS (ESI): calculated for
+
C
24
H
21NO
2
[M+Na] 378.1470, found 378.1493.
5
-Oxo-5-(tritylamino)pentanoic acid (13). To 2.80 g of KOH dissolved in 50 ml of ethanol
was added N-tritylglutarimide 11 (1.00 g, 2.8 mmol) and the mixture was refluxed for 48 hours.
The mixture was then concentrated to dryness and redissolved in H O. Acidification of the basic
2
solution with conc. HCl to pH=2 and filtration of the product gave compound 13 as a white
1
powder (0.96 g, yield 91%). H NMR (400 MHz, CD
3
OD) δ 7.30 – 7.17 (m, 15H), 2.37 (t, J =
1
3
7
3
.4 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H), 1.79-1.87 (m 2H). C NMR (101 MHz, CD OD) δ 175.5,
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Page 23 of 65
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
1
73.3, 144.6, 128.6, 127.3, 127.2, 126.7, 126.3, 35.2, 32.6, 20.7. HRMS (ESI): calculated for
+
C
24
H
23NO
3
[M+Na] 396.1576, found 396.1573.
5-Hydroxy-N-tritylpentanamide (15). To a solution of 13 (2.60 g, 6.96 mmol) in dry THF
60 mL) cooled to 0°C was added NaBH(OAc) (0.28 g, 7.3 mmol). The solution was stirred
(1.1 mL, 8.8 mmol) was added dropwise. The
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
(
3
2 3 2
until evolution of H stopped, and BF .OEt
reaction was stirred at room temperature for 4 hours. The reaction was quenched by adding 50
mL H O at 0°C. The mixture was extracted with EtOAc and the combined organic layers were
washed with sat. aq. Na CO , brine and dried over Na SO . The crude product was purified by
2
2
3
2
4
column chromatography (100% EtOAc) to give compound 15 as a white powder (1.60 g, 64%
1
yield). H NMR (400 MHz, CDCl
3
) δ 7.22 – 6.74 (m, 15H), 6.36 (br, 1H), 3.29 – 3.19 (br, 2H),
13
2
1
3
.01 (t, J = 7.2 Hz, 2H), 1.46 – 1.36 (m, 2H), 1.24 (m, 2H). C NMR (101 MHz, CDCl ) δ
71.9, 144.7, 128.6, 127.9, 127.0, 62.0, 37.0, 32.0, 21.4. HRMS (ESI): calculated for
+
C
24
H
25NO
2
[M+Na] 382.1783, found 382.1783.
5
-Oxo-N-tritylpentanamide (17). 5-hydroxy-N-tritylpentanamide 15 (1.30 g, 3.6 mmol) and PDC
Cl and stirred for 2 hours under N
atmosphere at room temperature. The mixture was filtered and the organic layer was washed with
brine, dried over anhydrous Na SO and concentrated under reduced pressure. The crude product was
purified by column chromatography (100% CH Cl ) to give compound 17 as an off-white powder
) δ 9.71 (s, 1H), 7.36 – 7.10 (m, 15H), 6.59 (s, 1H),
(2.00 g, 5.4 mmol) were dissolved in 50 mL of dry CH
2
2
2
2
4
2
2
1
(0.84 g, 65% yield). H NMR (400 MHz, CDCl
3
13
2.44 (t, J = 7.0 Hz, 2H), 2.32 (t, J = 7.2 Hz, 2H), 1.97 – 1.88 (m, 2H). C NMR (101 MHz, CDCl
3
) δ
202.0, 170.8, 144.6, 128.6, 127.9, 127.0, 70.5, 42.9, 36.1, 17.9. HRMS (ESI): calculated for
+
C
24 2
H23NO [M+Na] 380.1626, found 380.1629.
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Journal of Medicinal Chemistry
Page 24 of 65
1
2
3
4
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
3
-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]-
dioxol-4-yl)methyl)amino)methyl)-N-tritylbenzamide (29). 3-Formyl-N-tritylbenzamide 6
(1.22 g, 3.12 mmol), 2’-3’-O-isopropylidene-6-aminomethyl-adenosine (1.00 g, 3.43 mmol) and
acetic acid (0.45 mL, 8 mmol) were dissolved in 1,2-dichloroethane (DCE, 50 mL) and stirred at
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
room temperature under a N
added and the reaction mixture was stirred overnight at room temperature. The reaction was
quenched by adding 1 N NaOH solution (50 mL), and the product was extracted with CH Cl
The combined organic layers were washed with brine and dried over Na SO . The solvent was
2 3
atmosphere. After 3 hours, NaBH(OAc) (1.09 g, 5.15 mmol) was
2
2
.
2
4
evaporated and the crude product was purified by column chromatography (10% MeOH in CH
2
Cl
2
)
1
to give compound 29 as a white powder (1.25 g, 59% yield). H NMR (400 MHz, DMSO-d
6
) δ
8
.89 (s, 1H), 8.34 (s, 1H), 8.06 (s, 1H), 7.79 (s, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.43 (d, J = 7.7 Hz,
H), 7.39 – 7.24 (m, 15H), 7.20 (m, 3H), 6.09 (d, J = 3.1 Hz, 1H), 5.76 (s, 1H), 5.46 (M, 1H),
1
5.00 (m, 1H), 4.28 – 4.23 (m, 1H), 3.73 (s, 2H), 2.75 - 2.66 (m, 2H), 1.54 (s, 3H), 1.31 (s, 3H).
1
3
6
C NMR (101 MHz, DMSO-d ) δ 166.9, 156.5, 153.1, 149.3, 145.3, 140.4, 135.6, 128.9, 128.3,
1
27.9, 127.6, 126.7, 126.5, 119.7, 113.7, 89.7, 85.3, 83.1, 82.6, 69.9, 55.3, 53.0, 50.8, 27.5, 25.7.
+
HRMS (ESI): calculated for C40
H
39
N
7
O
4
[M+Na] 704.2961, found 704.2975.
Methyl-3-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d]
1,3]dioxol-4-yl)methyl)amino)benzoate (30). Following the procedure described for compound
[
2
9, coupling methyl 3-formylbenzoate 8 (0.51 g, 3.12 mmol) and 2’-3’-O-isopropylidene-6-
aminomethyl-adenosine (1.00 g, 3.43 mmol) afforded compound 30 as a white powder (0.92 g,
1
6
5% yield). H NMR (400 MHz, CDCl
3
) δ 8.08 (s, 1H), 7.92 (s, 1H), 7.90 – 7.83 (m, 2H), 7.44
(
d, J = 7.6 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 6.37 (d, J = 5.7 Hz, 2H), 5.95 (d, J = 3.1 Hz, 1H),
5.45 (m, 1H), 5.04 (m, 1H), 4.40 – 4.34 (m, 1H), 3.86 (s, 3H), 3.79 (s, 2H), 2.90-2.83 (m, 2H),
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Page 25 of 65
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
1
.58 (s, 3H), 1.35 (s, 3H). C NMR (101 MHz, CDCl
3
) δ 167.1, 155.8, 155.8, 153.0, 149.2,
1
40.4, 140.4, 139.8, 132.6, 132.6, 130.1, 129.1, 129.1, 128.4, 128.4, 128.2, 120.2, 114.4, 91.0,
85.5, 83.2, 83.2, 82.2, 82.2, 53.3, 52.1, 52.1, 50.6, 27.3, 27.2, 25.4, 25.3. HRMS (ESI): calculated
+
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
for C22
H
26
N
6
O
5
[M+H] 455.2043, found 455.2050.
tert-Butyl-3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)methyl)amino)methyl)benzoate (31). Following the procedure described for
compound 29, coupling tert-butyl 3-formylbenzoate 9 (0.64 g, 3.12 mmol) and 2’-3’-O-
isopropylidene-6-aminomethyl-adenosine (1.00 g, 3.43 mmol) afforded compound 31 as a white
1
powder (0.77 g, 50% yield). H NMR (400 MHz, CDCl
3
) δ 8.11 (s, 1H), 7.89 (s, 1H), 7.86-7.83
(m, 2H), 7.43 (d, J = 7.7 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 6.36 – 6.27 (m, 2H), 5.96 (d, J = 3.3
Hz, 1H), 5.46 (m, 1H), 5.04 (m, 1H), 4.38 (m, 1H), 3.80 (s, 2H), 2.94-2.81 (m, 2H), 1.58 (s, 3H),
1
3
1
3
.55 (s, 9H), 1.36 (s, 3H). C NMR (101 MHz, CDCl ) δ 165.7, 155.8, 155.8, 153.0, 149.3,
140.2, 139.8, 132.0, 132.0, 129.0, 128.2, 128.1, 120.3, 114.5, 91.0, 85.4, 83.2, 82.2, 80.9, 53.4,
+
5
0.6, 28.1, 27.3, 25.4. HRMS (ESI): calculated for C25
H
32
N
6
O
5
[M+H] 497.2512, found 497.2511.
9
-((3aR,4R,6R,6aR)-2,2-dimethyl-6-(((naphthalen-2-ylmethyl)amino)methyl)tetrahydrofuro-
[
3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine (32). Following the procedure described for
compound 29, coupling 2-naphthaldehyde (0.49 g, 3.12 mmol) and 2’-3’-O-isopropylidene-6-
aminomethyl-adenosine (1.00 g, 3.43 mmol) afforded compound 32 as a white powder (1.03 g,
1
7
4% yield). H NMR (400 MHz, CDCl
3
) δ 8.11 (s, 1H), 7.88 (s, 1H), 7.78 (m, 3H), 7.70 (s, 1H),
7.48 – 7.38 (m, 3H), 6.05 (s, 2H), 5.99 (d, J = 3.3 Hz, 1H), 5.48 (m, 1H), 5.06 (m, 1H), 4.45 –
13
4
.39 (m, 1H), 3.95 (s, 2H), 3.01 – 2.87 (m, 2H), 2.33 (br, 2H), 1.61 (s, 3H), 1.38 (s, 3H). C
3
NMR (101 MHz, CDCl ) δ 155.7, 153.0, 149.3, 139.9, 137.4, 133.3, 132.6, 128.0, 127.6 126.4,
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Journal of Medicinal Chemistry
Page 26 of 65
1
2
3
4
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
1
26.4, 126.0, 125.5, 120.3, 114.5, 91.0, 85.6, 83.3, 82.3, 53.9, 50.7, 27.3, 25.4. HRMS (ESI):
+
calculated for C24
H
26
N
6
O
3
[M+H] 447.2145, found 447.2167.
3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]-
dioxol-4-yl)methyl)(4-oxo-4-(tritylamino)butyl)amino)methyl)-N-tritylbenzamide (33). Oxo-
N-tritylbutanamide 16 (62 mg, 0.18 mmol), compound 29 (100 mg, 0.15 mmol) and AcOH (1
drop) were dissolved in 1,2-dichloroethane (DCE, 10 mL) and stirred at room temperature under
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 N
was stirred overnight at room temperature. The reaction was quenched by adding 1 N NaOH (10
mL), and the product was extracted with CH Cl . The combined organic layers were washed
with brine and dried over Na SO . The solvent was evaporated and the crude product was
2 4
atmosphere. After 3 hours, NaBH (49 mg, 0.23 mmol) was added and the reaction mixture
2
2
2
4
purified by column chromatography (10% MeOH in CH
2
2
Cl ) to give compound 33 as a white
1
powder (83 mg, 55% yield). H NMR (400 MHz, CDCl
3
) δ 8.15 (s, 1H), 7.69 (s, 1H), 7.67 (s,
1H), 7.53 (d, J = 7.1 Hz, 2H), 7.39 – 7.09 (m, 32H), 6.61 (s, 1H), 5.95 (d, J = 1.9 Hz, 1H), 5.65
(s, 2H), 5.36 (m, 1H), 4.89 (m, 1H), 4.40 – 4.34 (m, 1H), 3.56 (d, J = 3.4 Hz, 2H), 2.68 (d, J =
13
6
.8 Hz, 2H), 2.46 (m, 2H), 2.26 (m, 2H), 1.81 – 1.69 (m, 2H), 1.52 (s, 3H), 1.30 (s, 3H). C
NMR (101 MHz, CDCl
3
) δ 171.5, 166.7, 155.4, 152.9, 149.0, 144.8, 144.7, 140.0, 139.9, 135.3,
1
5
1
31.5, 128.8, 128.7, 128.0, 127.9, 127.0, 126.9, 125.3, 114.1, 90.8, 85.7, 83.8, 83.4, 70.7, 70.4,
+
8.6, 56.0, 53.5, 34.9, 27.0, 25.3, 22.7. HRMS (ESI): calculated for C63
009.4765, found 1009.4765.
60 8 5
H N O [M+H]
3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)methyl)(5-oxo-5-(tritylamino)pentyl)amino)methyl)-N-tritylbenzamide
34). Following the procedure described for compound 33, coupling compound 29 (100 mg, 0.15
mmol) with 5-oxo-N-tritylpentanamide 17 (64 mg, 0.18 mmol) afforded compound 34 as a white
(
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Page 27 of 65
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
1
powder (88 mg, 57% yield). H NMR (400 MHz, CDCl
H), 7.52 (s, 1H), 7.41 – 7.13 (m, 32H), 6.62 (s, 1H), 5.96 (d, J = 1.8 Hz, 1H), 5.83 (br, 2H),
5.38 (m, 1H), 4.92 (m, 1H), 4.40 – 4.34 (m, 1H), 3.54 (s, 2H), 2.65 (d, J = 6.9 Hz, 2H), 2.46 –
3
) δ 8.16 (s, 1H), 7.67 (s, 2H), 7.57 (br,
1
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.38 (m, 2H), 2.13 (m, 2H), 1.56 (s, 3H), 1.42 – 1.33 (m, 2H), 1.30 (s, 3H). C NMR (101 MHz,
CDCl ) δ 171.6, 166.6, 155.5, 152.9, 149.1, 144.8, 144.8, 140.3, 140.0, 135.2, 131.6, 128.8,
28.7, 128.2, 128.0, 127.9, 127.5, 127.0, 127.0, 125.4, 114.1, 90.9, 85.9, 83.8, 83.4, 77.3, 70.7,
3
1
7
0.4, 58.6, 56.1, 53.9, 37.1, 27.1, 26.3, 25.4, 23.1. HRMS (ESI): calculated for
+
C
64
H
62
N
8
O
5
[M+H] 1023.4921, found 1023.4918.
tert-Butyl 4-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro-
[3,4-d][1,3]dioxol-4-yl)methyl)(3-(tritylcarbamoyl)benzyl)amino)butanoate (35). Following
the procedure described for compound 33, coupling tert-butyl 4-oxobutanoate 22 (29 mg, 0.18
mmol) and compound 29 (100 mg, 0.15 mmol) afforded compound 35 as a white powder (61 mg,
1
49% yield). H NMR (400 MHz, CDCl
3
) δ 8.16 (s, 1H), 7.70 (d, J = 5.8 Hz, 2H), 7.58 (d, J =
7
1
–
1
1
2
.6 Hz, 2H), 7.38 – 7.15 (m, 17H), 5.97 (d, J = 2.0 Hz, 3H), 5.36 (m, 1H), 4.93 (m, 1H), 4.35 (m,
H), 3.63 – 3.52 (m, 2H), 2.76 – 2.63 (m, 2H), 2.47 (t, J = 7.1 Hz, 2H), 2.23 – 2.12 (m, 2H), 1.75
1
3
3
1.65 (m, 2H), 1.55 (s, 3H), 1.36 (s, 9H), 1.29 (s, 3H). C NMR (101 MHz, CDCl ) δ 172.7,
66.7, 155.5, 152.9, 149.0, 144.8, 144.8, 140.0, 139.9, 131.6, 128.8, 128.7, 128.0, 128.0, 127.0,
25.5, 120.1, 90.8, 85.8, 83.9, 83.4, 80.1, 70.7, 61.9, 58.8, 55.9, 53.4, 33.0, 32.4, 28.0, 27.1, 25.3,
+
2.4. HRMS (ESI): calculated for C48
H
53
N
7
O
6
[M+H] 824.4136, found 824.4142.
tert-Butyl-5-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro-
3,4-d][1,3]dioxol-4-yl)methyl)(3-(tritylcarbamoyl)benzyl)amino)pentanoate (36). Following
[
the procedure described for compound 33, coupling tert-butyl 5-oxopentanoate 23 (31 mg, 0.18
mmol) and compound 29 (100 mg, 0.15 mmol) afforded compound 36 as a white powder (67 mg,
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Page 28 of 65
1
2
3
4
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
1
5
3
3% yield). H NMR (400 MHz, CDCl ) δ 8.17 (s, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 7.8
Hz, 1H), 7.51 (s, 1H), 7.42 – 7.27 (m, 14H), 7.19 (t, J = 7.7 Hz, 1H), 5.98 (s, 1H), 5.72 (s, 2H),
5.38 (m, 1H), 4.94 (s, 1H), 4.40 – 4.33 (m, 1H), 3.57 (s, 2H), 2.70 – 2.65 (m, 2H), 2.47 (t, J = 7.0
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
Hz, 2H), 2.13 (t, J = 7.2 Hz, 2H), 1.57 (s, 3H), 1.52 – 1.45 (m, 2H), 1.41 (s, 9H), 1.32 (s, 3H).
13
3
C NMR (101 MHz, CDCl ) δ 172.9, 166.6, 155.4, 152.9, 149.1, 144.8, 140.2, 139.9, 135.2,
1
5
31.6, 128.7, 128.2, 128.0, 127.4, 127.0, 125.4, 120.2, 114.1, 90.8, 85.8, 83.9, 83.4, 80.0, 70.7,
8.7, 56.0, 54.0, 35.2, 28.1, 27.1, 26.3, 25.3, 22.7. HRMS (ESI): calculated for
+
C
49
H
55
N
7
O
5
[M+H] 838.4292, found 838.4298.
tert-Butyl-(S)-4-((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)(3-(tritylcarbamoyl)benzyl)amino)-2-
(tert-butoxycarbonyl)amino)butanoate (37). Following the procedure described for compound
3, coupling tert-butyl (R)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoate 27 (49 mg, 0.18
(
3
mmol) and compound 29 (100 mg, 0.15 mmol) afforded compound 37 as a white powder (94 mg,
1
6
7
1
7% yield). H NMR (400 MHz, CDCl
3
) δ 8.15 (s, 1H), 7.70 (m, 2H), 7.57 (d, J = 9.9 Hz, 2H),
.41 – 7.14 (m, 15H), 5.98 (s, 1H), 5.59 (s, 2H), 5.37 (m, 2H), 4.91 (s, 1H), 4.36 (s, 1H), 4.17 (s,
H), 3.62 (d, J = 13.8 Hz, 1H), 3.54 (d, J = 13.8 Hz, 1H), 2.76 – 2.48 (m, 4H), 1.99 (d, J = 6.2
1
3
Hz, 1H), 1.76 (br, 1H), 1.57 (s, 3H), 1.39 (m, 15H), 1.32 (s, 3H). C NMR (101 MHz, CDCl
3
) δ
1
1
71.6, 166.6, 155.4, 152.9, 149.0, 144.8, 140.0, 139.5, 135.4, 131.6, 128.8, 128.0, 127.0, 125.5,
14.2, 90.7, 85.6, 83.9, 83.4, 81.7, 79.4, 70.7, 58.9, 58.2, 55.9, 52.7, 50.9, 50.6, 36.5, 29.7, 28.3,
+
28.2, 28.0, 27.9, 27.1, 25.3. HRMS (ESI): calculated for C53
39.4784.
-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)methyl)(isopropyl)amino)methyl)-N-tritylbenzamide (38). Following the
H
61
N
8
O
8
[M+H] 939.4749, found
9
3
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7
8
9
procedure described for compound 33, coupling 5 mL dry acetone (large excess) and compound
1
2
9 (100 mg, 0.15 mmol) afforded compound 38 as a white powder (68 mg, 63% yield). H NMR
(400 MHz, CDCl
3
) δ 8.22 (s, 1H), 7.75 (s, 1H), 7.60 (d, J = 10.4 Hz, 2H), 7.49 – 7.40 (m, 2H),
.33 – 7.21 (m, 15H), 5.91 (s, 1H), 5.34 (m, 2H), 4.92 – 4.87 (m, 1H), 4.23 (d, J = 3.2 Hz, 1H),
.57 (s, 2H), 2.92 – 2.83 (m, 1H), 2.76 – 2.68 (m, 1H), 2.59 (m, 1H), 1.49 (s, 3H), 1.25 (s, 3H),
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
7
3
1
1
9
1
3
3
.01 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.7, 155.7,
52.9, 149.0, 144.8, 141.4, 139.9, 135.2, 131.4, 128.7, 128.3, 128.0, 127.1, 125.4, 120.2, 114.0,
0.9, 86.5, 83.7, 83.3, 70.7, 54.4, 51.8, 50.5, 27.1, 25.4, 18.7, 17.2. HRMS (ESI): calculated for
+
C
43
H
45
N
7
O
4
[M+H] 724.3611, found 724.3618.
Methyl
3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro-
[3,4-d][1,3]dioxol-4-yl)methyl)(4-oxo-4-(tritylamino)butyl)amino)methyl)benzoate
(39).
Following the procedure described for compound 33, coupling 4-oxo-N-tritylbutanamide 16 (62
mg, 0.18 mmol) and compound 30 (68 mg, 0.15 mmol) afforded compound 39 as a white powder
1
(
63 mg, 54% yield). H NMR (400 MHz, CDCl
3
) δ 8.17 (s, 1H), 7.90 (s, 1H), 7.84 – 7.79 (m,
1
H), 7.77 (s, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.31 – 7.08 (m, 17H), 6.60 (s, 1H), 5.97 (d, J = 2.2
Hz, 1H), 5.67 (s, 2H), 5.34 (m, 1H), 4.86 (m, 1H), 4.34 (m, 1H), 3.85 (s, 3H), 3.57 (m, 2H), 2.74
–
3
1
2.62 (m, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.31-2.16 (m, 2H), 1.75 (m, 2H), 1.53 (s, 3H), 1.32 (s,
13
3
H). C NMR (101 MHz, CDCl ) δ 171.5, 167.0, 155.4, 153.0, 149.1, 144.7, 139.9, 139.6,
33.3, 129.9, 129.7, 128.7, 128.2, 128.1, 127.9, 127.0, 126.9, 120.2, 114.2, 90.8, 85.4, 83.7, 83.4,
70.4, 58.5, 55.7, 53.6, 52.1, 34.9, 27.0, 25.2, 22.7. HRMS (ESI): calculated for
+
C
45
H
48
N
7
O
6
[M+H] 782.3666, found 782.3666.
Methyl
3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro-
[3,4-d][1,3]dioxol-4-yl)methyl)(5-oxo-5-(tritylamino)pentyl)amino)methyl)benzoate
(40).
ACS Paragon Plus Environment
29