Inhibitors of nicotinamide: N -methyltransferase designed to mimic the methylation reaction transition state

Article abstract of DOI:10.1039/c7ob01357d

Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyses the methylation of nicotinamide to form N′-methylnicotinamide. Both NNMT and its methylated product have recently been linked to a variety of diseases, suggesting a role for the enzyme as a therapeutic target beyond its previously ascribed metabolic function in detoxification. We here describe the systematic development of NNMT inhibitors derived from the structures of the substrates involved in the methylation reaction. By covalently linking fragments of the NNMT substrates a diverse library of bisubstrate-like compounds was prepared. The ability of these compounds to inhibit NNMT was evaluated providing valuable insights into the structural tolerances of the enzyme active site. These studies led to the identification of new NNMT inhibitors that mimic the transition state of the methylation reaction and inhibit the enzyme with activity on par with established methyltransferase inhibitors.

Full text of DOI:10.1039/c7ob01357d

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Biomol. Chem., 2017, DOI: 10.1039/C7OB01357D.
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DOI: 10.1039/C7OB01357D
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Inhibitors of nicotinamide N-methyltransferase designed to mimic
the methylation reaction transition state
Received 00vth January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
Matthijs J. van Haren , Rebecca Taig , Jilles Kuppens , Javier Sastre Toraño , Ed E. Moret, Richard
b
c
c
a
B. Parsons, Davide Sartini , Monica Emanuelli and Nathaniel I. Martin *
DOI: 10.1039/x0xx00000x
Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyses the methylation of nicotinamide to form N’-
methylnicotinamide. Both NNMT and its methylated product have recently been linked to a variety of diseases, suggesting
a role for the enzyme as a therapeutic target beyond its previously ascribed metabolic function in detoxification. We here
describe the systematic development of NNMT inhibitors derived from the structures of the substrates involved in the
methylation reaction. By covalently linking fragments of the NNMT substrates a diverse library of bisubstrate-like
compounds was prepared. The ability of these compounds to inhibit NNMT was evaluated providing valuable insights into
the structural tolerances of the enzyme active site. These studies led to the identification of new NNMT inhibitors that
mimic the transition state of the methylation reaction and inhibit the enzyme with activity on par with established
methyltransferase inhibitors.
www.rsc.org/
Introduction
Nicotinamide N-methyltransferase (NNMT) is an enzyme that
transfers methyl group from S-adenosyl-L-methionine
AdoMet) to nicotinamide (NA) forming N’-methyl-
nicotinamide (MNA) and S-adenosyl-L-homocysteine (AdoHcy)
Fig. 1). The enzyme has gained much interest in the last
a
(
(
decade because of its involvement in a variety of diseases.
Whereas 30 years ago, the enzyme was thought to function
exclusively in detoxification and metabolic pathways, it has
now become clear that the enzyme has far more complex roles
in both healthy and disease states. In many types of cancer,
NNMT is overexpressed and it has the potential as a biomarker
Figure 1. Methylation of nicotinamide (NA) by NNMT using S-adenosyl-L-methionine
AdoMet) as the methyl donor, forming N’-methylnicotinamide (MNA) and S-adenosyl-
(
L-homocysteine (AdoHcy)
1
–4
transition state mimic approach. The recently reported NNMT
crystal structure reveals the interactions of the substrates with
for gastric, oral, renal and lung cancers. In this regard, the
overexpression of NNMT has recently been shown to cause
5
AdoMet depletion in cancer cells. Other studies have
1
5
the active site residues (Fig. 2). The active site can be roughly
divided into three binding pockets: one for the adenosine
group (A), one for the amino acid moiety (B) and one for
nicotinamide unit (C). In designing the inhibitors here
described we focused on the structural elements of the
substrates implicated in the transition state of the methylation
reaction.
suggested a role for NNMT as a cytoprotective agent in
6
Parkinson’s disease while other findings point to a potential
–8
9
link between MNA and lifespan regulation. In addition, the
upregulation of NNMT is observed in a range of metabolic
1
0–12
disorders including diabetes and obesity.
Despite its
implication in many disease states, very few inhibitors of
1
3,14
By linking fragments of the NNMT substrates known to
interact with the different active site pockets, bisubstrate-like
inhibitors were obtained and their inhibitory activity
evaluated. The first series of inhibitors examined were simple
N-alkylated nicotinamide analogues. In a second series, we
coupled the adenosine moiety to the nicotinamide ring via a
range of spacers. In a third approach, we omitted the
adenosine group and instead coupled nicotinamide to an
amino acid moiety again with a variety of spacers. Finally, we
also synthesized a “trivalent” compound comprising the
NNMT have been described to date.
Therefore, we set out
to develop inhibitors of NNMT based on a bi-substrate
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NNMT inhibitors, we found that N-alkylation of nicotinamide
with any group bigger than a methyl group - as present in MNA
-
lead to inactive compounds. While MNA was found to have
an IC50 value of 24.6 ± 3.2 µM (Table 1), none of the other
alkylated compounds exhibited NNMT inhibition at 250 µM.
The next series of NNMT inhibitors pursued were based on
a bisubstrate strategy. This approach is similar to that recently
described by our group in preparing inhibitors of protein
1
7
arginine N-methyltransferases . To this end, an adenosine
moiety was coupled to a nicotinamide mimicking group via a
series of different linkers (Scheme 2). Unlike the positively
charged N-alkylated compounds prepared in the first series
(Scheme 1), the bisubstrates were prepared as neutral
compounds wherein the pyridine of nicotinamide is replaced
with a benzene core. In examining the NNMT transition state
model (Fig. 2), it appears that the spacing between the ribose
group of the adenosine moiety and the nitrogen of
nicotinamide is 3 atoms. We therefore synthesized three-atom
spaced conjugates of adenosine and benzamide via N-, O- and
Figure 2. Schematic overview of the methylation reaction transition state of NNMT
showing the structures of substrates AdoMet in blue and nicotinamide in red and their
interactions with the active site residues. The active site can be divided into three S-linkages resulting in compounds
9
-
11
was prepared via reductive
with methyl 3-formylbenzoate,
.
binding pockets: adenosine pocket A; amino acid pocket B and nicotinamide pocket C.
Intermediate compound
amination of free amine
6
3
nicotinamide, adenosine and amino acid moieties with the aim followed by microwave-assisted amidation of the methyl ester
of simultaneously interacting with all three binding pockets. To using concentrated ammonium hydroxide in methanol. A final
assess NNMT inhibition, we used an assay recently developed deprotection step using TFA and water and subsequent
1
6
in our group. This assay employs Ultra High Performance purification by preparative HPLC gave compound
9. For the
(
UHP) Hydrophilic Liquid Interaction Chromatography (HILIC) preparation of thioether bisubstrate analogue 10
,
Quadrupole Time-Of-Flight Mass Spectrometry (QTOF-MS) to intermediate compound
rapidly and efficiently measure NNMT activity. Using this deprotection of S-acetylated thiol
7
was first prepared via a one-pot
followed by direct
4
method, we previously determined the IC50 values for the alkylation with methyl-3-bromomethylbenzoate. Subsequent
general methyltransferase inhibitors sinefungin and AdoHcy amidation, deprotection, and purification yielded compound
and here demonstrate that it can be used for the evaluation of 10. For the synthesis of oxy-ether linked conjugate 11 the
new NNMT inhibitors. In the sections below we describe the exocyclic adenine amine was dibenzoylated to avoid
synthesis of the different NNMT inhibitors designed and overalkylation by methyl-3-bromomethylbenzoate under the
discuss their inhibitory activity.
strongly basic conditions necessary for ether formation.
Following O-alkylation, intermediate was amidated followed
by deprotection and purification to yield 11. Somewhat
surprisingly, bisubstrate analogues 11 showed very little
8
Results and Discussion
9
-
NNMT inhibition at a concentration of 250 µM. This lack of
inhibition may be explained by both the omission of the
pyridine nitrogen as well as the absence of interactions with
the amino acid binding pocket. This explanation is supported
by the significantly reduced enzymatic activity previously
The inhibitors described in the present study all contain a
nicotinamide mimic and were designed to approximate the
NNMT transition state. The different inhibitors here explored
each build upon the nicotinamide mimic which was elaborated
to include structural elements of the adenosine and/or amino
acid moieties of the AdoMet cofactor. The NNMT inhibition of
all compounds prepared was first assessed at a fixed
concentration of 250 µM. In cases where at least 50%
inhibition was detected at this concentration full inhibition
curves were measured in triplicate to determine the
corresponding IC values.
1
5
reported for the Y20A mutant of NNMT. As seen in the
5
0
The first set of compounds studied were prepared directly
from nicotinamide, which was N-alkylated with ethyl, propyl,
isopropyl, allyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl and benzyl-groups (Scheme 1). For the synthesis of
compounds 2a-i, we developed a convenient microwave-
assisted alkylation procedure followed by straightforward Scheme 1. Synthesis of alkylated nicotinamides 2a-i via microwave-assisted coupling of
alkyl bromides or iodides. Reagents and conditions: a) alkyl iodide or alkyl bromide,
purification of the alkylated products via precipitation. When
3
CH CN, µW, 140°C, 1.5h, 29-92%.
testing the positively charged N-alkylated nicotinamides as
2
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Scheme 2. Synthesis of the adenosine-nicotinamide conjugates 9-11. Reagents and
conditions: a) methyl 3-formylbenzoate, Na(OAc) BH, 4A molsieves, DCE, rt, 16h, 60%;
b) methyl 3-bromomethylbenzoate, NaOCH , MeOH, Ar(g), -20°C to rt, 16h, 75%; c)
methyl 3-bromomethylbenzoate, NaH 55% dispersion in mineral oil, DMF, rt, 16h, 32%;
d) 25% NH (aq), MeOH, µW, 130°C, 3h; e) TFA, DCM, rt, 1h.
3
3
3
NNMT crystal structure (Fig. 2), the active site Tyr20 residue
coordinates both the nicotinamide moiety and the carbonyl of
the AdoMet amino acid group.
To separately assess the role of the AdoMet amino acid
group, bisubstrate inhibitors comprised of a nicotinamide
mimic linked to a carboxylic acid or amino acid moiety were
next prepared (Schemes 3-5). A range of different linkers was
used in preparing these conjugates. Based on the transition
Scheme 3. A) Synthesis of alkynes 12-17 via Sonogashira cross-coupling and
subsequent reduction of the alkynes to alkenes 24 and 25 and alkanes 18-23. B)
state model, the spacing between the carboxylic acid unit and Synthesis of alkyne 27 and its corresponding alkane 28. Reagents and conditions: a)
the aromatic ring of the nicotinamide unit is 6 atoms. We Pd(PPh
3
)
2
Cl
2
, CuI, Et
3
N, THF, 50°C, 4h, 27-89%; b) 10% Pd/C, H , MeOH, rt, 1h, 62-88%;
2
c) Lindlar catalyst, H
TFA, DCM, rt, 1h.
2
, MeOH, rt, 3h, 85%; d) Cp*RuCl(cod), H , DCM, rt, 16h, 60%; e)
2
therefore prepared analogues containing 5-, 6-, and 7-atom
linkers to investigate the optimal spacing for inhibitor design.
To examine the binding requirements for interaction with the
NNMT amino acid binding pocket, compounds bearing a
terminal carboxylic acid, a primary amide, or an amino acid
compounds 24-28 is rather surprising given their structural
similarity to the active analogues.
In addition to the carbon linked compounds, we also
prepared analogues containing hetero-atom spacers consisting
of either nitrogen or sulphur (Schemes 4 and 5). Synthesis of
the amine linked conjugate 35 was achieved via a reductive
amination approach similar to that used for the synthesis of
moiety were prepared. In preparing compounds 18
-25
(Scheme 3A) a Sonogashira cross-coupling reaction was used
to couple fragment pairs. Conveniently, we found that the
cross-coupling reaction did not require protecting groups on
either the amide or carboxyl functionalities which significantly
simplified access to the target compounds. Furthermore,
formation of the intermediate alkyne provided the opportunity
to also investigate the effect of altered rigidity and geometry
compound
9. As illustrated in Scheme 4, the protected
diaminobutyric acid building block 33 was coupled with
methyl-3-formylbenzoate to yield intermediate 34 which was
subsequently deprotected and amidated using the same
microwave-assisted procedure described for the preparation
in the linkers. Reduction of the alkyne intermediates 12
using standard hydrogenation conditions led to clean
formation of the fully saturated species 18 23. Partial
-17
of
9-11. Preparation of the thioether spaced conjugate 41
-
(Scheme 5) required access to thioacetate 38 which was
reduction was also investigated to provide both the E- and Z-
generated via an unusual but effective method using
19
potassium thioacetate absorbed on silica gel. Deprotection of
alkene variants derived from the 6-carbon spaced compound
14. To this end the Z-alkene 24 was prepared using the Lindlar
38 by treatment with potassium carbonate was followed by
20
coupling with bromide 36 to form protected thioether 40
catalyst while the E-alkene 25 was accessed using
.
1
8
Cp*RuCl(cod) as a catalyst. In both cases, the E/Z ratio was
greater than 95% and the desired products were well
separable from both the alkyne starting material and any over-
reduced alkane side-product. Compounds 27 and 28 were
synthesized in similar fashion starting from Boc-protected
homo-propargylglycine (Scheme 3B). The most active
compounds in this series were the 6- and 7-carbon spaced
Final compound 41 was obtained using the general procedures
for deprotection and amidation.
To complete the library of bisubstrate-based inhibitors, the
ternary compound 45 was also designed with the aim of
simultaneously interacting with all three binding pockets of the
NNMT active site (Scheme 6). To this end the previously
prepared compound
6
(Scheme 2) was coupled to aldehyde
via reductive amination to give 44 which was
subsequently deprotected and amidated to yield 45. In
performing the NNMT inhibition assays number of
compounds 14
,
16,
20, and 22 containing a terminal carboxylic
21
4
3
,
acid (see Table 1). The exception is compound 23, which
contains a terminal amide. The lack of inhibitory activity for
a
compounds known to inhibit the enzyme were also evaluated
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Scheme 4. Synthesis of compound 35. Reagents and conditions: a) 1. Ethyl
chloroformate, N-methyl morpholine, THF, -10°C to -5°C, 15 min 2. NaBH , water, 5°C,
.5 h, 95%; b) MsCl, DiPEA, DCE, 0°C, 30 min, 66%; c) NaN , DMF, rt, 16h, 72%; d) Pd/C,
, EtOH, rt, 1h, 89%; e) methyl-3-formylbenzoate, Na(OAc) BH, DCE, rt, 16h, 37%; f)
TFA, DCM, rt, 1h; g) 25% NH (aq), MeOH, µW, 130°C, 3h.
4
3
3
H
2
3
3
Scheme 6. Synthesis of compound 45. Reagents and conditions: a) O,N-dimethyl-
hydroxylamine.HCl, BOP, Et
3
N, DCM, rt, 2h, 90%; b) DiBAL-H in hexanes (1M), THF, -
7
3
8°C, 87%, c) 43, Na(OAc)
3
BH, DCE, rt, 16h, 53%; d) 25% NH (aq), MeOH, µW, 130°C,
3
as reference compounds (Fig. 3). As summarized in Table 1, the
general methyltransferase inhibitors AdoHcy (IC50 75.4 ± 6.3
µM) and sinefungin (17.0 ± 3.4 µM) display clear inhibition of
NNMT. Also included was the NNMT product inhibitor MNA
h; e) TFA, DCM, rt, 1h.
Interestingly, compounds
was linked to benzamide unit designed to mimic
nicotinamide, showed no significant inhibition. Rather, linking
of the benzamide unit to a simple carboxylate as in 14 16 20
, and 23 (amide not carboxylate) gave much better
9-11 wherein the adenosine moiety
a
(IC50 24.6 ± 3.2 µM). Given that MNA itself is the analyte
detected in the standard NNMT activity assay, the alternative
NNMT substrate 4-methyl-nicotinamide (4MeNA) was in this
case used. In this regard, we also evaluated 4-methyl-N’-
methyl-nicotinamide (4MeMNA) as an additional NNMT
product inhibitor (IC50 95.9 ± 14.1 µM), using the standard
assay conditions. We also studied the ability of the
polyaromatic compound norharmane to inhibit NNMT.
Previous studies revealed norharmane to be a weak substrate
,
,
,
2
2
inhibition and provide key insights into the preferred spacing
and rigidity of the linker used to connect the respective
bisubstrate-mimicking functionalities. The results indicate that
the optimal spacing is 6-7 atoms. Furthermore, trivalent
compound 45, designed to bind in all three NNMT binding
for NNMT with a K
M
value of 90 µM, significantly lower than
200 µM), but with a very
that of the native substrate NA (K
M
2
2
low turnover rate. We were therefore interested in assessing
whether such a strong binding, slowly converted NNMT
substrate might in fact act as an inhibitor by occupying the
1
6,22
NNMT active site.
Indeed, it was found that norharmane
is
inhibits NNMT with an IC50 of 115.3 ± 20.6 µM. The low K
M
offset by a diminished turnover rate giving norharmane the
ability to occupy the NNMT active site and effectively block the
methylation of nicotinamide.
Figure 3. Structures of reference compounds AdoHcy, sinefungin, N’-methyl-
nicotinamide (MNA) and 4-methyl-N’-methyl-nicotinamide (4MeMNA) and alternative
Among the newly synthesized compounds tested, only NNMT substrate norharmane.
those containing structural elements derived from both the
a
Table 1. IC50 values for NNMT inhibitors evaluated.
nicotinamide unit and the amino acid moiety of AdoMet
resulted in appreciable inhibition (>50% at 250 µM).
Compound
sinefungin
MNA
IC50 in µM
17.0 ± 3.4
24.6 ± 3.2
75.4 ± 6.3
95.9 ± 14.1
115.3 ± 20.6
> 250
Compound
IC50 in µM
> 250
18
19
20
21
22
23
24
25
26
27
28
35
41
45
> 250
AdoHcy
69.0 ± 14.8
> 250
4
MeMNA
norharmane
148.1 ± 36.3
30.8 ± 3.6
> 250
2
a-i
9
> 250
1
0
1
2
3
4
5
6
> 250
> 250
1
> 250
> 250
1
1
1
1
1
> 250
> 250
> 250
> 250
189.7 ± 30.0
> 250
> 250
> 250
57.8 ± 4.2
> 250
29.2 ± 4.0
Scheme 5. Synthesis of compound 41. Reagents and conditions: a) CBr
7%, b) SiO :AcSK, toluene, rt, 1h, 91%; c) K CO , MeOH, rt, 2h, 93%; d) 36, K
rt, 2h, 60%; e) TFA, DCM, rt, 1h; f) 25% NH (aq), MeOH, µW, 130°C, 3h.
4
, PPh
3
, DCM,
2
2
2
3
2
CO
3
, DMF,
17
a
3
Assays performed in triplicate on at least six different inhibitor concentrations.
Standard errors reported.
4
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To this end it is beneficial that the three main structural
elements found in 45 can be modified independently and with
relative ease. Once more potent NNMT inhibitors have been
identified, their selectivity and activity in cell-based assays will
be assessed. These findings will be reported in due course.
Experimental
General methods
All reagents employed were of American Chemical Society
(
ACS) grade or finer and were used without further purification
1
unless otherwise stated. For compound characterization
H
NMR spectra were recorded at 400 MHz with chemical shifts
Figure 4. Proposed hydrogen bond network after modelling and minimization of
compound 45 (yellow) in the NNMT active site (aliphatic hydrogens omitted for clarity). reported in parts per million (ppm) downfield relative to
2
tetramethylsilane (TMS), H O (δ 4.79), methanol (δ 3.31) or
1
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 the
number of protons. Where appropriate, the multiplicity is
pockets, inhibits NNMT with an IC50 of 29.2 ± 4.0 µM, similar to
the inhibitory activity of reference inhibitors sinefungin and
MNA.
Modelling studies performed with compound 45 show
interactions in the enzyme’s active site similar to those
expected based upon the transition state model (Fig. 4). The
docking analysis reveals that the spacing of the compound
allows binding in all three binding pockets as hypothesized.
Although the NA pyridine ring is replaced by a benzene ring,
the pi-pi-stacking with tyrosine residue Y204 remains, which
appears to be sufficient to compensate for the loss of
interaction with the nitrogen in the pyridine ring of NA. All
other interactions that were found in the crystal structure of
NNMT in complex with nicotinamide and AdoHcy, shown in
Figure 2, are visible in the model as represented in Figure 4 as
well.
13
preceded by br, indicating that the signal was broad. C NMR
spectra were recorded at 101 MHz with chemical shifts
reported relative to CDCl
3
(δ 77.16), methanol (δ 49.00) or
DMSO (δ 39.52). The C NMR spectra of the compounds
recorded in D O could not be referenced. High-resolution mass
spectrometry (HRMS) analysis was performed using a Q-TOF
1
3
2
1
7
23
20
24
instrument. Compounds 3-5 , 30-33 , 36 and 42-43 were
synthesized according to previously described procedures. All
known compounds prepared 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 50%
3
CH CN (0.1% TFA) over 30 minutes at a flow rate of 1.0 ml
−1
min with UV detection at 214 nm and 254 nm.
Conclusions
We here describe the first systematic bisubstrate approach
towards the discovery of inhibitors of NNMT. While a number
of partial bisubstrate analogues were explored, our results
indicate that the best mimic of the NNMT transition state is
the trivalent compound 45. The absence of inhibitory activity
for the alkylated nicotinamides 2a-i and adenosine-
nicotinamide conjugates 9-11 are somewhat surprising. While
MNA shows an IC50 of 24.6 µM, N’-ethylnicotinamide has an
IC50 of >250 µM as do analogues bearing larger alkyl groups.
Furthermore, the simplified bisubstrate approach that we
previously applied in generating potent inhibitors of the
General procedure alkylated nicotinamides
Nicotinamide (500 mg, 1.0 eq, 4.0 mmol) was dissolved in 10
3
mL dry CH CN, followed by the addition of R-I or R-Br (2.2 eq, 9
mmol). The tube was then sealed and reacted in a microwave
for 90 minutes at 140°C. If necessary, the product was
precipitated with dry diethyl ether. The precipitated product
was filtered off and washed with dry diethyl ether.
1
-Ethyl-3-carbamoyl-pyridinium iodide 2a
Synthesized according to the general procedure (92% yield). H
NMR (400 MHz, D O) δ 9.38 (s, 1H), 9.09 (d, J = 6.1 Hz, 1H),
8.92 (d, J = 8.2 Hz, 1H), 8.24 (t, J = 7.2 Hz, 1H), 4.78 (d, J = 7.4
1
2
1
7
protein arginine methyltransferases was not successful here
as compounds 9-11 displayed essentially no NNMT inhibition.
While the set of compounds here described is not exhaustive,
the results do suggest that binding in at least the nicotinamide
and amino acid binding pockets is necessary for significant
inhibition of the enzyme. An additional finding of interest is
the inhibitory activity of norharmane, a known NNMT
1
3
Hz, 2H), 1.71 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, D
2
O) δ
1
65.9, 146.2, 144.0, 143.8, 133.9, 128.4, 58.0, 15.6. HRMS
+
(
ESI): calculated for C
-Propyl-3-carbamoyl-pyridinium bromide 2b
Synthesized according to the general procedure (697 mg,
8 11 2
H N O 151.0866, found 151.0867.
1
1
7
1
7
1%). H NMR (400 MHz, D
2
O) δ 9.36 (s, 1H), 9.07 (d, J = 6.0 Hz,
H), 8.93 (d, J = 8.1 Hz, 1H), 8.24 (t, J = 7.2 Hz, 1H), 4.70 (t, J =
3
substrate with
a low turnover rate. In this regard,
1
.3 Hz, 2H), 2.18 – 2.04 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H).
C
incorporation of a norharmane mimic in future inhibitor
designs may prove useful. Future plans are aimed at further
optimizing compound 45 in an attempt to enhance inhibition.
NMR (101 MHz, D
2
O) δ 165.84, 146.47, 144.20, 143.82, 133.83,
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DOI: 10.1039/C7OB01357D
ARTICLE
Organic and Biomolecular Chemistry
+
1
28.33, 63.86, 24.12, 9.63. HRMS (ESI): calculated for HRMS (ESI): calculated for C13
H
13
N
2
O
213.1022, found
+
C
9
H
13
N
2
O 165.1022, found 165.1020.
-Butyl-3-carbamoyl-pyridinium bromide 2c
Synthesized according to the general procedure (671 mg, 2,2-dimethyltetrahydrofuro[3,4-d][1,3]-dioxol-4-yl)methyl)-
213.1022.
1
Methyl 3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-
1
6
1
7
7
1
3%). H NMR (400 MHz, D
H), 8.93 (d, J = 8.2 Hz, 1H), 8.23 (t, J = 7.2 Hz, 1H), 4.73 (t, J = Amine
.5 Hz, 2H), 2.12 – 2.00 (m, 2H), 1.49 – 1.34 (m, 2H), 0.98 (t, J = dry DCE and methyl 3-formylbenzoate (255 mg, 1.55 mmol, 1.0
2
O) δ 9.35 (s, 1H), 9.07 (d, J = 6.1 Hz, amino)methyl)benzoate (6)
3
(500 mg, 1.63 mmol, 1.05 eq) was dissolved in 35 mL
1
3
.4 Hz, 3H). C NMR (101 MHz, D
2
O) δ 165.85, 146.43, 144.18, eq) was added along with crushed 4Å molsieves. The mixture
43.75, 133.84, 128.30, 62.30, 32.49, 18.68, 12.60. HRMS (ESI): was stirred for 5 hours at room temperature under N
2
+
calculated for C10
-Allyl-3-carbamoyl-pyridinium bromide 2d
Synthesized according to the general procedure (800 mg, at room temperature under N
H
15
N
2
O 179.1179, found 179.1175.
atmosphere before Na(OAc)
was added in small portions. The mixture was stirred overnight
atmosphere. Conversion was
) δ 9.53 (s, 1H), 9.22 (d, J = monitored by TLC (DCM:MeOH 8:2). The solvent was
.5 Hz, 1H), 9.02 (d, J = 7.9 Hz, 1H), 8.65 (s, 1H), 8.31 (t, J = 6.8 evaporated, redissolved in 150 mL DCE and filtered. The
and the
) δ 162.7, aqueous layer extracted with DCM. The organic layer was
46.4, 144.8, 143.7, 133.7, 131.4, 128.0, 122.4, 62.6. HRMS dried with Na SO , filtered and concentrated yielding (537
mg, 75%). H NMR (400 MHz, CDCl ): δ 8.12 (s, 1H), 7.96 (s,
1H), 7.92 (d, J = 7.7 Hz, 1H), 7.87 (s, 1H), 7.49 (d, J = 7.5 Hz,
Synthesized according to the general procedure (534 mg, 1H), 7.37 (t, J = 7.6 Hz, 1H), 6.03 – 5.89 (m, 3H), 5.49 (dd, J =
3
BH (461 mg, 2.18 mmol, 1.4 eq)
1
2
1
80%). H NMR (400 MHz, DMSO-d
6
5
Hz, 1H), 8.18 (s, 1H), 6.34 – 6.13 (m, 1H), 5.58 – 5.43 (m, 2H), organic layer was washed with saturated NaHCO
3
1
3
5.38 (d, J = 5.7 Hz, 2H). C NMR (101 MHz, DMSO-d
6
1
2
4
6
+
1
(ESI): calculated for C
9
H
11
N
2
O 163.0866, found 163.0863.
3
1-Isopropyl-3-carbamoyl-pyridinium iodide 2e
1
45%). H NMR (400 MHz, D
2
O) δ 9.37 (s, 1H), 9.14 (d, J = 5.9 Hz, 6.3, 3.3 Hz, 1H), 5.08 (dd, J = 6.3, 3.0 Hz, 1H), 4.44 – 4.36 (m,
1
H), 8.91 (d, J = 7.8 Hz, 1H), 8.23 (t, J = 7.0 Hz, 1H), 5.19 – 5.07 1H), 3.90 (s, 3H), 3.88 – 3.78 (m, 2H), 3.00 – 2.82 (m, 2H), 2.35
1
m, 1H), 1.76 (s, 3H), 1.74 (s, 3H). C NMR (101 MHz, DMSO- (s, 1H), 1.62 (s, 3H), 1.39 (s, 3H). C NMR (101 MHz, CDCl
3
13
(
3
): δ
d
6
) δ 162.8, 144.6, 143.6, 142.9, 134.0, 128.1, 64.8, 22.3. HRMS 167.2, 155.8, 153.2, 149.5, 140.6, 140.1, 132.7, 130.4, 129.3,
+
(
ESI): calculated for C
-Cyclopropylmethyl-3-carbamoyl-pyridinium bromide 2f
Synthesized according to the general procedure (335 mg,
9
H
13
N
2
O 165.1022, found 165.1016.
128.6, 128.4, 120.6, 114.6, 91.2, 85.6, 83.4, 82.4, 53.5, 52.2,
50.8, 27.5, 25.5.
1
3-(((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-di-
1
63%). H NMR (400 MHz, DMSO-d
6
) δ 9.61 (s, 1H), 9.32 (d, J = hydroxytetrahydrofuran-2-yl)methyl)amino)-methyl)
5.0 Hz, 1H), 8.98 (d, J = 7.6 Hz, 1H), 8.63 (s, 1H), 8.28 (t, J = 6.5 benzamide (9)
Hz, 1H), 8.16 (s, 1H), 4.59 (d, J = 7.0 Hz, 2H), 1.94 – 1.23 (m, Compound
6
(128 mg, 0.27 mmol) was dissolved in 1 mL
) δ MeOH and 1 mL 25% NH (aq) and crimp sealed. The mixture
62.8, 146.1, 144.4, 143.6, 133.7, 127.9, 64.9, 12.0, 4.1. HRMS was reacted for 4 hours at 130°C in the microwave. The
1
3
1
H), 0.61 (s, 2H), 0.60 (s, 2H). C NMR (101 MHz, DMSO-d
6
3
1
+
(
ESI): calculated for C
-Cyclobutylmethyl-3-carbamoyl-pyridinium bromide 2g
Synthesized according to the general procedure (510 mg, concentrated and purified by preparative HPLC. H NMR (D
9
H
11
N
2
O 177.1022, found 177.1026.
mixture was concentrated and redissolved in 7 mL TFA:H
2
O
1
(5:2). After 45 minutes at room temperature, the mixture was
1
2
O):
2
7%). H NMR (400 MHz, D O) δ 9.28 (s, 1H), 9.01 (d, J = 6.1 Hz, δ 8.39 (s, 1H), 8.17 (s, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.78 (s, 1H),
1
4
1
7
2
1
H), 8.92 (d, J = 8.0 Hz, 1H), 8.21 (t, J = 6.8 Hz, 1H), 4.75 (d, J = 7.65 (d, J = 7.8 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 6.15 (d, J = 4.2
.6 Hz, 2H), 3.01 (dt, J = 15.0, 7.7 Hz, 1H), 2.17 – 2.05 (m, 3H), Hz, 1H), 4.90 – 4.77 (m, 1H+ H2O), 4.54 (t, J = 5.3 Hz, 1H), 4.49
1
3
13
2 2
O) δ 165.8, 146.2, – 4.32 (m, 4H), 3.64 – 3.37 (m, 3H). C NMR (101 MHz, D O) δ
.01 – 1.86 (m, 4H). C NMR (101 MHz, D
43.7, 141.9, 133.8, 128.3, 66.4, 35.8, 17.4. HRMS (ESI): 171.5, 148.1, 147.7, 133.7, 133.3, 130.8, 129.5, 128.9, 128.3,
+
calculated for C11
-Cyclohexylmethyl-3-carbamoyl-pyridinium bromide 2h
Synthesized according to the general procedure (342 mg,
H
15
N
2
O 191.1179, found 191.1181.
119.2, 90.1, 79.8, 73.3, 71.5, 50.4, 47.9. HRMS: calculated for
+
1
C
18
H
21
N
7
O
4
[M+H] 400.1728, found 400.1758.
Methyl 3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-
O) δ 9.29 (s, 1H), 9.01 (d, J = 5.2 Hz, 2,2-dimethyltetrahydrofuro[3,4-d][1,3]-dioxol-4-yl)methyl)
H), 8.93 (d, J = 8.3 Hz, 1H), 8.23 (t, J = 7.3 Hz, 1H), 4.57 (d, J = thio)methyl)benzoate (7)
.3 Hz, 2H), 2.12 – 1.98 (m, 1H), 1.83 – 1.54 (m, 4H), 1.32 – Thioacetate (546 mg, 1.5 mmol, 1.0 eq) was dissolved in 30
O) δ 165.8, 146.6, 143.8, mL dry deoxygenated methanol and methyl-3-bromomethyl-
41.8, 133.7, 128.2, 67.9, 39.0, 29.2, 25.4, 24.9. HRMS (ESI): benzoate (514 mg, 2.2 mmol, 1.5 eq) was added. The mixture
1
2
1
7
1
1
9%). H NMR (400 MHz, D
2
4
1
3
.04 (m, 6H). C NMR (101 MHz, D
2
+
calculated for C13
H
19
N
2
O 219.1492, found 219.1499.
was cooled to -20°C and freshly prepared sodium methoxide
(from 82 mg Na(s) in 3 mL MeOH) was added. The mixture was
1
-Benzyl-3-carbamoyl-pyridinium bromide 2i
Synthesized according to the general procedure (1063 mg, stirred overnight at room temperature under N
2
atmosphere.
) δ 9.73 (s, 1H), 9.38 (d, J = The solvent was evaporated, redissolved in chloroform and
.0 Hz, 1H), 9.01 (d, J = 8.1 Hz, 1H), 8.66 (s, 1H), 8.30 (t, J = 7.1 washed with water. The organic layer was dried, concentrated
1
89%). H NMR (400 MHz, DMSO-d
6
6
Hz, 1H), 8.20 (s, 1H), 7.64 – 7.58 (m, 2H), 7.47 – 7.38 (m, 3H), and purified by column chromatography (2.5% MeOH in DCM)
1
3
1
5
6 3
.98 (s, 2H). C NMR (101 MHz, DMSO- d ) δ 162.7, 146.4, yielding 7 (450 mg, 64%) H NMR (400 MHz, CDCl ): δ 8.27 (s,
144.8, 143.9, 134.0, 134.0, 129.4, 129.2, 129.1, 128.3, 63.4. 1H), 7.94 – 7.84 (m, 3H), 7.39 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6
Hz, 1H), 6.07 – 6.00 (m, 3H), 5.44 (dd, J = 6.4, 1.9 Hz, 1H), 4.99
6
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Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
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DOI: 10.1039/C7OB01357D
Organic and Biomolecular Chemistry
ARTICLE
(
dd, J = 6.3, 3.4 Hz, 1H), 4.33 (td, J = 6.6, 3.4 Hz, 1H), 3.89 (s, (5:2). After 45 minutes at room temperature, the mixture was
1
H), 3.72 (s, 2H), 2.71 (ddd, J = 43.7, 13.7, 6.7 Hz, 2H), 1.59 (s, concentrated and purified by preparative HPLC. H NMR (400
3
3
1
1
1
3
3 2
H), 1.37 (s, 3H). C NMR (101 MHz, CDCl ): δ 166.9, 155.8, MHz, D O): δ 8.47 (s, 1H), 8.36 (s, 1H), 7.79 (s, 1H), 7.74 (d, J =
53.2, 149.3, 140.0, 138.4, 133.4, 130.6, 130.0, 128.7, 128.5, 7.5 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 6.13
20.4, 114.6, 90.8, 86.8, 84.1, 83.9, 52.3, 36.3, 33.5, 27.2, 25.5. (d, J = 5.2 Hz, 1H), 5.00 – 4.83 (m, 2H), 4.76 (t, J = 5.3 Hz, 1H),
3
-(((((2S,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-di-
hydroxytetrahydrofuran-2-yl)methyl)thio)methyl)benzamide
10)
Compound
MeOH and 1 mL 25% NH
was reacted for 4 hours at 130°C in the microwave. The
mixture was concentrated and redissolved in 7 mL TFA:H
5:2). After 45 minutes at room temperature, the mixture was iodo-N-tritylbenzamide (490 mg, 1.0 mmol) and TEA (5 ml),
4.44 (t, J = 5.1 Hz, 1H), 4.29 (d, J = 30.9 Hz, 1H), 3.97 – 3.79 (m,
1
3
13
2H). C NMR (101 MHz, D
172.4, 133.1, 131.1, 129.2, 126.8, 126.1, 117.7, 114.8, 88.5,
(100 mg, 0.21 mmol) was dissolved in 1 mL 85.6, 74.1, 70.2, 61.1. HRMS: calculated for C18
2 2
O) δ C NMR (101 MHz, d o) δ
(
7
21 6 5
H N O
+
3
(aq) and crimp sealed. The mixture [M+Na] 423.1387, found 423.1411.
5-(3-Carbamoylphenyl)pent-4-ynoic acid (12)
To a suspension of 4-pentynoic acid (122 mg, 1.24 mmol), 3-
2
O
(
1
concentrated and purified by preparative HPLC. H NMR (400 Pd(PPh
3
)
2
Cl
2
(14 mg, 0.02 mmol) was added and stirred at
environment. After
MHz, CD OD): δ 8.43 (s, 1H), 8.33 (s, 1H), 7.71 (s, 1H), 7.68 (d, J room temperature for 5 minutes under N
3
2
=
7.7 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), addition of THF (2 ml) and Cu(I)I (8 mg, 0.04 mmol), the
.05 (d, J = 4.8 Hz, 1H), 4.78 (t, J = 5.1 Hz, 1H), 4.34 (t, J = 5.1 mixture was heated to 50°C for 4 hours. The solvent was
6
Hz, 1H), 4.24 (dt, J = 6.5, 4.8 Hz, 1H), 3.82 (d, J = 2.2 Hz, 2H), evaporated in vacuo, and the crude product was purified by
2
.92 (dd, J = 14.2, 4.8 Hz, 1H), 2.82 (dd, J = 14.2, 6.7 Hz, 1H). column chromatography (SiO
2
: DCM - 5% MeOH) affording the
OD) δ 172.7, 149.5, 140.1, 134.2, trityl-protected product (89 mg, 20%) as an orange powder.
33.8, 129.9, 128.8, 127.1, 120.1, 89.9, 85.3, 74.6, 73.6, 67.7, The intermediate was dissolved in DCM (2 mL) and a mixture
1
3
C NMR (101 MHz, CD
3
1
3
+
6.8, 34.0. HRMS: calculated for C18
H
21
N
6
O
4
S [M+H] 417.1340, of TFA/TIPS/H
2
O (95:2.5:2.5, 5 mL) was added. After full
found 417.1366.
deprotection as determined via TLC (DCM / 5% MeOH), the
Methyl 3-((((3aR,4R,6R,6aR)-6-(6-(N-benzoylbenzamido)- mixture was concentrated and purified by preparative HPLC to
1
H-purin-9-yl)-2,2-dimethyltetrahydrofuro-[3,4-d][1,3]dioxol- yield compound 12 as a white powder (21 mg, 50%). H NMR
9
4-yl)methoxy)methyl)benzoate (8)
3
(400 MHz, CD OD) δ 7.88 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.53
The protected adenosine
added to a suspension of sodium hydride as dispersion in 2H), 2.51 (t, J = 7.3 Hz, 2H). C NMR (101 MHz, CD
mineral oil (103 mg, 2.14 mmol, 1.1 eq) dissolved in 15 mL dry 176.9, 171.5, 135.6, 135.3, 131.7, 129.6, 127.9, 125.5, 90.3,
5
(1.0 g, 1.94 mmol, 1.0 eq) was (d, J = 7.7 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 2.72 (t, J = 7.2 Hz,
1
3
3
OD) δ
+
DMF. After 30 minutes
bromomethylbenzoate (580 mg, 2.52 mmol, 1.3 eq) in DMF (2 218.0812, found 218.0804.
mL) was added slowly and the mixture was stirred overnight at 5-(3-Carbamoylphenyl)pent-4-ynoic amide (13)
room temperature. The reaction was quenched with saturated Following the procedure described for compound 12, coupling
NH Cl and the mixture was concentrated. The residue was 4-pentynoic amide (117 mg, 1.2 mmol) and 3-iodobenzamide
redissolved in EtoAc and washed with saturated NH Cl. The (247 mg, 1.0 mmol) afforded compound 13 (169 mg, 78%) as a
aqueous phase was extracted with EtoAc and the combined white powder. H NMR (400 MHz, DMSO-d
a
3
solution of methyl-3- 81.3, 35.5, 16.4. HRMS: calculated for C12H11NO [M+H] :
4
4
1
6
) δ 7.99 (s, 1H),
organic layers were dried over sodium sulfate, concentrated 7.83 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H),
and purified by column chromatography (2.5% MeOH in DCM) 7.41 – 7.31 (m, 2H), 6.85 (s, 1H), 2.59 (t, J = 7.3 Hz, 2H), 2.32 (t,
1 13
3 6
(450 mg, 64%). H NMR (400 MHz, CDCl ): δ 8.49 (s, J = 7.3 Hz, 2H). C NMR (101 MHz, DMSO-d ) δ 172.76, 167.48,
yielding
8
1
=
7
H), 8.09 (s, 1H), 7.96 (s, 1H), 7.92 (d, J = 7.5 Hz, 2H), 7.84 (d, J 134.99, 134.18, 130.59, 129.03, 127.47, 123.52, 91.10, 80.33,
+
7.7 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.4 Hz, 1H), 34.47, 15.41. HRMS: calculated for C12
.45 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.8 Hz, 2H), 7.31 (t, J = 7.7 217.0972, found 217.0991.
12 2 2
H N O [M+H] :
Hz, 1H), 7.28 – 7.21 (m, 1H), 7.14 (t, J = 7.6 Hz, 2H), 6.06 (d, J =
6-(3-Carbamoylphenyl)hex-5-ynoic acid (14)
2.3 Hz, 1H), 5.62 (d, J = 2.5 Hz, 2H), 5.45 (dd, J = 6.4, 2.3 Hz, Following the procedure described for compound 12, coupling
1
H), 5.10 (dd, J = 6.3, 3.3 Hz, 1H), 4.60 – 4.49 (m, 2H), 4.43 (dd, of 5-hexynoic acid (137 mg, 1.22 mmol) and 3-iodo-N-
1
3
J = 11.5, 5.3 Hz, 1H), 3.85 (s, 3H), 1.61 (s, 3H), 1.38 (s, 3H).
C
tritylbenzamide (490 mg, 1.0 mmol) afforded the trityl-
NMR (101 MHz, CDCl
3
): δ 172.1, 167.0, 166.1, 154.3, 152.2, protected intermediate (232 mg, 49 %) as a peach colored
52.0, 142.7, 138.0, 135.9, 133.4, 132.6, 131.1, 130.3, 129.7, powder. The deprotection and purification procedure on 0.2
29.6, 129.4, 129.3, 128.9, 128.7, 128.6, 128.6, 128.5, 128.0, mmol scale as described for compound 12 afforded final
1
1
1
1
27.0, 115.1, 91.1, 84.7, 83.9, 81.4, 64.0, 52.1, 51.2, 27.3, 25.5. compound 14 (21 mg, 42%). H NMR (400 MHz, CD
-(((((2S,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3,4-di- (s, 1H), 7.76 (d, J = 7.8, 1.7 Hz, 1H), 7.51 (d, J = 7.7, 1.6 Hz, 1H),
hydroxytetrahydrofuran-2-yl)methoxy)methyl)benzamide 7.38 (t, J = 1.8 Hz, 1H), 2.51 – 2.43 (m, 4H), 1.92 – 1.82 (m, 2H).
11)
Compound
MeOH and 1 mL 25% NH
was reacted for 4 hours at 130°C in the microwave. The found 232.0974.
3
OD) δ 7.86
3
1
3
(
C NMR (101 MHz, CD
3
OD) δ 175.46, 170.13, 134.18, 133.79,
8
(100 mg, 0.21 mmol) was dissolved in 1 mL 130.20, 128.17, 126.42, 124.21, 89.49, 79.97, 32.33, 23.68,
+
3
3
(aq) and crimp sealed. The mixture 17.98. HRMS: calculated for C13H13NO [M+H] : 232.0968,
mixture was concentrated and redissolved in 7 mL TFA:H
2
O
6-(3-Carbamoylphenyl)hex-5-ynoic amide (15)
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DOI: 10.1039/C7OB01357D
ARTICLE
Organic and Biomolecular Chemistry
1
Following the procedure described for compound 12, coupling compound 19 (69 mg, 67%) as a white powder. H NMR (400
5-hexynoic amide (133 mg, 1.2 mmol) and 3-iodobenzamide MHz, CD
3
OD) δ 7.71 (s, 1H), 7.68 (d, J = 6.8 Hz, 1H), 7.40 – 7.33
(
247 mg, 1.0 mmol) afforded compound 15 (144 mg, 63%) as a (m, 2H), 2.71 (t, J = 7.1 Hz, 2H), 2.24 (t, J = 7.2 Hz, 2H), 1.72 –
13
) δ 7.99 (s, 1H), 1.62 (m, 4H). C NMR (101 MHz, CD
1
white powder. H NMR (400 MHz, DMSO-d
6
3
OD) δ 179.1, 172.6,
7
–
2
.85 (s, 1H), 7.78 (d, J = 7.8 Hz,1H), 7.50 (d, J = 7.7 Hz, 1H), 7.41 144.1, 135.0, 133.1, 129.5, 128.6, 126.2, 36.4, 36.3, 32.0, 26.4.
+
16 2 2
7.36 (m, 2H), 7.28 (s, 1H), 6.73 (s, 1H), 2.41 (t, J = 7.0 Hz, 2H), HRMS: calculated for C12H N O [M+H] : 221.1285, found
1
.19 (t, J = 7.4 Hz, 2H), 1.76 – 1.68 (m, 2H). C NMR (101 MHz, 221.1294.
3
DMSO-d
6
) δ 174.03, 167.49, 134.97, 134.22, 130.59, 129.02,
6-(3-Carbamoylphenyl)hexanoic acid (20)
1
27.46, 123.61, 91.24, 80.74, 34.33, 24.57, 18.72. HRMS: Compound 14 (100 mg, 0.21 mmol) was reduced following the
+
[M+H] : 231.1128, found 231.1131.
calculated for C13
H
13
N
2
O
2
procedure described for compound 18 affording the trityl-
7-(3-Carbamoylphenyl)hept-6-ynoic acid (16)
protected alkane (88 mg, 88 %) as a white powder. The
Following the procedure described for compound 12, coupling deprotection and purification procedure as described for
-heptynoic acid (302.7 mg, 2.4 mmol) and 3-iodo-N- compound 12 afforded final compound 20 (15 mg, 29%
6
1
3
tritylbenzamide (980 mg, 2.0 mmol) afforded the trityl- isolated yield). H NMR (400 MHz, CD OD) δ 7.71 (s, 1H), 7.67
protected intermediate (712 mg, 72 %) as a yellow oil. The (d, J = 6.8 Hz, 1H), 7.40 – 7.33 (m, 2H), 2.69 (t, J = 7.6 Hz, 2H),
deprotection and purification procedure on 0.2 mmol scale as 2.28 (t, J = 7.4 Hz, 2H), 1.72 – 1.61 (m, 4H), 1.43 – 1.34 (m, 2H).
1
3
described for compound 12 afforded final compound 16 (15
C NMR (101 MHz, CD
OD) δ 7.84 (s, 133.1, 129.5, 128.6, 126.1, 49.6, 49.4, 49.2, 49.0, 49.0, 48.8,
H), 7.75 (d, J = 7.8 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.38 (t, J = 48.6, 48.4, 36.5, 34.8, 32.2, 29.7, 25.9. HRMS: calculated for
3
OD) δ 177.6, 172.7, 144.3, 134.9,
1
mg, 27% isolated yield). H NMR (400 MHz, CD
3
1
7
–
+
.8 Hz, 1H), 2.44 (t, J = 6.9 Hz, 2H), 2.34 (t, J = 7.2 Hz, 2H), 1.80
1
C
13
H17NO
3
[M+H] : 236.1281, found 236.1290.
3
3
1.72 (m, 2H), 1.66 – 1.58 (m, 2H). C NMR (101 MHz, CD OD)
6-(3-Carbamoylphenyl)hexanoic amide (21)
δ 177.5, 171.7, 135.6, 135.1, 131.6, 129.6, 127.7, 125.7, 91.6, Compound 15 (96 mg, 0.42 mmol) was reduced following the
8
1.0, 34.4, 29.1, 25.3, 19.6. HRMS: calculated for C14
H
15NO
3
procedure described for compound 18 affording final
+
1
[
M+Na] : 268.0944, found 268.0941.
-(3-Carbamoylphenyl)hept-6-ynoic amide (17)
Following the procedure described for compound 12, coupling (m, 2H), 2.69 (d, J = 7.6 Hz, 2H), 2.20 (d, J = 7.4 Hz, 2H), 1.73 –
compound 21 (21 mg, 22%) as a white powder. H NMR (400
7
3
MHz, CD OD) δ 7.71 (s, 1H), 7.67 (d, J = 6.8 Hz, 1H), 7.39 – 7.33
1
3
5-hexynoic amide (150 mg, 1.2 mmol) and 3-iodo-N- 1.61 (m, 4H), 1.43 – 1.33 (m, 2H). C NMR (101 MHz, CD
3
OD) δ
tritylbenzamide (247 mg, 0.5 mmol) afforded the trityl- 179.2, 172.6, 144.3, 134.9, 133.1, 129.5, 128.7, 126.1, 36.5,
+
protected intermediate (218 mg, 89 %) as a yellow powder. 36.4, 32.2, 29.7, 26.6. HRMS: calculated for C13
The deprotection and purification procedure on 0.2 mmol 235.1441, found 235.1450.
18 2 2
H N O [M+H] :
scale as described for compound 12 afforded final compound
7-(3-Carbamoylphenyl)heptanoic acid (22)
1
7 (22 mg, 44% isolated yield). H NMR (400 MHz, CD OD) δ Compound 16 (100 mg, 0.21 mmol) was reduced following the
1
7
7
3
.88 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 7.7 Hz, 1H), procedure described for compound 18 affording the trityl-
.40 (t, J = 7.8 Hz, 1H), 2.47 (t, J = 6.9 Hz, 3H), 2.27 (t, J = 7.4 protected alkane (80 mg, 79 %) as a white powder. The
1
3
Hz, 2H), 1.85 – 1.76 (m, 3H), 1.69 – 1.61 (m, 3H). C NMR (101 deprotection and purification procedure as described for
MHz, CD OD) δ 178.9, 171.6, 135.6, 135.2, 131.6, 129.6, 127.8, compound 12 (0.1 mmol scale) afforded final compound 22
3
1
1
3
25.8, 91.6, 81.1, 36.0, 29.3, 26.1, 19.7. HRMS: calculated for (7.4 mg, 30% isolated yield). H NMR (400 MHz, CD OD) δ 7.71
+
[M+H] : 245.1258, found 245.1185.
C
14
H
16
N
2
O
2
(s, 1H), 7.68 (d, J = 6.5 Hz, 1H), 7.41 – 7.34 (m, 2H), 2.69 (t, J =
7.5 Hz, 2H), 2.28 (t, J = 7.3 Hz, 2H), 1.71 – 1.56 (m, 4H), 1.42 –
5-(3-Carbamoylphenyl)pentanoic acid (18)
1
3
To a solution of the trityl-protected intermediate of compound 1.35 (m, 4H). C NMR (101 MHz, CD
3
OD) δ 172.7, 144.5,
1
2
(90 mg, 0.19 mmol) in MeOH (5 mL), a suspension of Pd/C 134.9, 133.1, 129.5, 128.6, 126.1, 36.6, 35.0, 32.4, 30.0, 29.9,
+
(
10 %, 20 mg, excess) in MeOH (5 mL) was added. The mixture 26.0. HRMS: calculated for C14
was hydrogenated using an H -balloon for 2 hours. The 250.1439.
catalyst was removed by filtration over celite and the solvent 7-(3-Carbamoylphenyl)heptanoic amide (23)
was evaporated in vacuo, affording the trityl-protected alkane Compound 17 (90 mg, 0.19 mmol) was reduced following the
56 mg, 62 %) as a white powder. The deprotection and procedure described for compound 18 affording the trityl-
3
H19NO [M+H] : 250.1438, found
2
(
purification procedure as described for compound 12 afforded protected alkane (69 mg, 76%) as a white powder. The
1
final compound 18 (9 mg, 33% isolated yield). H NMR (400 deprotection and purification procedure as described for
3
MHz, CD OD) δ 7.69 (s, 1H), 7.66 (d, J = 6.7 Hz, 1H), 7.38 – 7.31 compound 12 afforded final compound 23 (12 mg, 36%
1
(
m, 2H), 2.67 (d, J = 7.0 Hz, 2H), 2.30 (d, J = 7.1 Hz, 2H), 1.70 – isolated yield). H NMR (400 MHz, DMSO-d
6
) δ 7.92 (s, 1H),
OD) δ 177.5, 172.6, 7.71 – 7.64 (m, 2H), 7.37 – 7.26 (m, 3H), 7.21 (s, 1H), 6.66 (s,
44.0, 134.9, 133.1, 129.5, 128.6, 126.2, 36.4, 34.7, 31.9, 25.6. 1H), 2.60 (tj, J = 7.5 Hz, 2H), 2.01 (t, J = 7.4 Hz, 2H), 1.63 – 1.52
1
3
1
.59 (m, 4H). C NMR (101 MHz, CD
3
1
+
13
HRMS: calculated for C12
H15NO
3
[M+H] : 222.1125, found (m, 2H), 1.51 – 1.42 (m, 2H), 1.34 – 1.20 (m, 4H). C NMR (101
MHz, DMSO-d ) δ 174.3, 168.0, 142.3, 134.2, 131.1, 128.1,
127.4, 124.8, 35.1, 35.0, 30.8, 28.5, 28.4, 25.0. HRMS:
2
22.1137.
-(3-Carbamoylphenyl)pentanoic amide (19)
Compound 13 (100 mg, 0.46 mmol) was reduced following the calculated for C14
procedure described for compound 18 affording final (Z)-6-(3-Carbamoylphenyl)hex-5-enoic acid (24)
6
5
+
20 2 2
H N O [M+H] : 249.1598, found 249.1603.
8
| Org. Biomol. Chem., 2017, 00, 1-11
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Page 9 of 13
Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C7OB01357D
Organic and Biomolecular Chemistry
ARTICLE
Alkyne 20 (70 mg, 0.30 mmol) was selectively reduced using 7.91 (s, 1H), 7.73 – 7.65 (m, 2H), 7.39 – 7.29 (m, 3H), 3.90 (t, J =
Lindlar catalyst (15 mg, 20% w/w) in MeOH (3.5 mL) under H 5.8 Hz, 1H), 2.62 (t, J = 7.6 Hz, 2H), 1.79 (tt, J = 14.0, 6.7 Hz,
atmosphere at room temperature. After 3 hours, the reaction 2H), 1.61 (p, J = 7.5 Hz, 2H), 1.51 – 1.26 (m, 2H). C NMR (101
was complete as seen by NMR. The catalyst was filtered off MHz, DMSO-d ) δ 171.1, 168.0, 141.9, 134.3, 131.2, 128.1,
over celite, the solvent evaporated and the crude product 127.4, 124.9, 51.9, 34.7, 30.3, 29.9, 24.0. HRMS: calculated for
2
1
3
6
+
purified by preparative HPLC affording (Z)-alkene 24 (60 mg,
1
C
13
H
18
N
2
O
3
[M+H] : 251.1390, found 251.1414.
0
6
.26 mmol, 85%). H NMR (400 MHz, DMSO-d ) δ 7.96 (s, 1H),
Methyl-(S)-3-(((4-(tert-butoxy)-3-((tert-butoxycarbonyl)
7
=
2
.77 – 7.70 (m, 2H), 7.46 – 7.40 (m, 2H), 7.35 (s, 1H), 6.47 (d, J amino)-4-oxobutyl)amino)methyl)benzoate (34)
11.7 Hz, 1H), 5.71 (dt, J = 11.7, 7.2 Hz, 1H), 2.31 (q, J = 6.8 Hz, To a solution of methyl 3-formylbenzoate (140 mg, 0.9 mmol)
1
3
H), 2.24 (t, J = 7.4 Hz, 2H), 1.65 (p, J = 7.5 Hz, 2H). C NMR and compound 33 (220 mg, 0.8 mmol) in dry DCE (15 ml),
) δ 167.8, 163.1, 137.0, 134.4, 132.7, crushed 4 molsieves were added. After 2 hours, Na(OAc) BH
31.1, 128.6, 128.2, 127.6, 125.7, 33.1, 27.5, 24.6. HRMS: (254 mg, 1.2 mmol) was added over 5 minutes. The mixture
(101 MHz, DMSO-d
6
Å
3
1
+
calculated for C13
H15NO
3
[M+Na] : 256.0944, found 256.0966.
was reacted overnight, quenched with saturated NaHCO
3
,
(E)-6-(3-Carbamoylphenyl)hex-5-enoic acid (25)
filtered and the aqueous phase extracted with DCM. The
Alkyne 20 (50 mg, 0.22 mmol) was selectively reduced using a organic layers were combined, washed with Brine, dried and
Cp*RuCl(cod) catalyst (4.1 mg, 5 mol-%) in DCM (3 mL) under concentrated. Purification via column chromatography
2
H atmosphere at room temperature. After overnight stirring, (gradient of 2.5 to 6% MeOH in DCM) afforded compound 34
1
3
NMR shows 60% conversion with >95% E-alkene. The catalyst (90 mg, 27 %) as a white powder. H NMR (400 MHz, CDCl ) δ
was filtered off over celite, the solvent evaporated and the 7.94 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.52 (d, J = 7.4 Hz, 1H),
crude product purified by preparative HPLC affording (E)- 7.36 (t, J = 7.6 Hz, 1H), 5.65 (d, J = 7.4 Hz, 1H), 4.34 – 4.10 (m,
1
alkene 25. H NMR (400 MHz, DMSO-d
6
) δ 7.97 (s, 1H), 7.90 (s, 1H), 3.88 (s, 3H), 3.78 (s, 2H), 2.68 (t, J = 6.6 Hz, 2H), 2.06 –
1
H), 7.71 (d, J = 7.0 Hz, 1H), 7.52 (d, J = 7.0 Hz, 1H), 7.44 – 7.26 1.69 (m, 2H), 1.40 (s, 18H). C NMR (101 MHz, CDCl
3
1
3
) δ 171.9,
(
(
m, 2H), 6.51 – 6.29 (m, 2H), 2.38 – 2.10 (m, 4H), 1.77 – 1.60 167.1, 155.7, 140.5, 132.9, 130.3, 129.3, 128.6, 128.4, 81.8,
1
3
m, 2H). C NMR (101 MHz, DMSO-d
6
) δ 174.3, 167.8, 137.3, 79.5, 53.6, 52.8, 52.1, 45.4, 32.6, 28.4, 28.0.
34.5, 131.0, 129.5, 128.7, 128.5, 126.0, 124.7, 33.1, 31.8, (S)-2-Amino-4-((3-carbamoylbenzyl)amino)butanoic acid
[M+H] : 234.1125, found (35)
Compound 34 (80 mg, 0.19 mmol) was first deprotected in
1
2
2
+
4.1. HRMS: calculated for C13
34.1143.
H15NO
3
(
S)-2-((Tert-butoxycarbonyl)amino)-6-(3-
carbamoylphenyl) hex-5-ynoic acid 26)
Following the procedure described for compound 12, coupling NH
TFA:DCM (1:2) for 1 hour at room temperature. The mixture
(
was concentrated, redissolved in 1 mL MeOH and 1 mL 25%
3
(aq) and crimp sealed. The mixture was reacted for 4
Boc-L-homopropargylglycine (114 mg, 0.5 mmol) and 3- hours at 130°C in the microwave. The mixture was
iodobenzamide (130 mg, 0.5 mmol) afforded compound 26 concentrated and purified by preparative HPLC affording
1
1
140 mg, 81%) as a white powder. H NMR (400 MHz, DMSO- compound 34 as a white powder. H NMR (400 MHz, DMSO-
(
d
=
–
9
1
2
6
) δ 8.02 (s, 1H), 7.88 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.52 (d, J
6
d ) δ 8.06 – 8.01 (m, 2H), 7.92 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.7
7.6 Hz, 1H), 7.47 – 7.37 (m, 2H), 7.14 (d, J = 7.9 Hz, 1H), 4.11 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.46 (s, 1H), 4.23 (s, 2H), 4.12 –
1
3.98 (m, 1H), 2.57 – 2.43 (m, 2H), 2.02 – 1.77 (m, 2H), 1.38 (s, 3.96 (m, 1H), 3.24 – 2.96 (m, 2H), 2.26 – 2.01 (m, 2H). C NMR
3
1
H). C NMR (101 MHz, DMSO-d
3
6 6
) δ 173.8, 167.1, 155.6, (101 MHz, DMSO-d ) δ 170.2, 167.4, 134.7, 132.6, 132.0,
34.6, 133.8, 130.2, 128.6, 127.1, 123.0, 90.1, 80.5, 78.1, 52.7, 129.5, 128.6, 127.7, 50.0, 49.7, 43.3, 26.5. HRMS: calculated
+
9.9, 28.2, 15.8.
S)-2-Amino-6-(3-carbamoylphenyl)hex-5-ynoic acid (27)
Compound 26 (70 mg, 0.2 mmol) was deprotected in TFA/DCM Following a procedure described in literature , methyl-3-
1:1, 4 mL) for 2 hours at room temperature. The mixture was bromomethylbenzoate (1.15 g, 5.0 mmol) was reacted with
for C12
17 3 3
H N O [M+H] : 252.1343, found 252.1360.
(
Methyl 3-((acetylthio)methyl)benzoate (38)
1
9
(
concentrated and the product purified by preparative HPLC silica-gel supported potassium thioacetate (7.5 g, 1 mmol/g,
1
affording compound 27 as a white powder. H NMR (400 MHz, 7.5 mmol) in toluene (20 mL). After 1 hour at room
6
DMSO-d ) δ 8.34 (br s, 2H), 8.02 (s, 1H), 7.90 (s, 1H), 7.84 (d, J temperature, the mixture was filtered and concentrated
=
7.8 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.49 – 7.40 (m, 2H), 4.03 yielding compound 38 as a slightly yellow oil (1.01 g, 91%). The
1
3
(t, J = 6.0 Hz, 1H), 2.74 – 2.53 (m, 2H), 2.19 – 1.97 (m, 2H).
C
compound had NMR spectra and mass spectra consistent with
2
5
) δ 170.6, 167.0, 134.6, 133.8, 130.3, the assigned structures .
NMR (101 MHz, DMSO-d
6
1
28.6, 127.2, 122.8, 89.0, 81.0, 51.2, 29.2, 15.1. HRMS: Methyl 3-(mercaptomethyl)benzoate (39)
+
[M+H] : 247.1077, found 247.1105.
26
Following a procedure described in literature , compound 38
calculated for C13
H
14
N
2
O
3
(S)-2-Amino-6-(3-carbamoylphenyl)hexanoic acid (28)
(224 mg, 1.0 mmol) was deacetylated using potassium
Compound 26 (100 mg, 0.46 mmol) was reduced following the carbonate (350 mg, 2.5 mmol) in methanol (15 mL). After
procedure described for compound 18. The intermediate was stirring for 2 hours at room temperature, the reaction was
deprotected in TFA/DCM (1:1, 4 mL) for 2 hours at room quenched using 0.1 M HCl to pH 2.5, diluted with water and
temperature. The mixture was concentrated and the product extracted with DCM. The organic phase was washed with
was purified by preparative HPLC affording compound 28 as a Brine, dried and concentrated yielding compound 39 (170 mg,
1
white powder. H NMR (400 MHz, DMSO-d
6
) δ 8.20 (s, 2H),
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB01357D
ARTICLE
Organic and Biomolecular Chemistry
9
3%). The compound had NMR spectra and mass spectra 90.9, 85.5, 83.9, 83.5, 81.8, 79.6, 58.8, 55.8, 52.9, 52.2, 50.7,
2
7
consistent with the assigned structures .
Methyl (S)-3-(((4-(tert-butoxy)-3-((tert-butoxycarbonyl)- [M+H] 712.3365, found 712.3371.
amino)-4-oxobutyl)thio)methyl)benzoate (40) (S)-2-Amino-4-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-
To a solution of thiol 39 (170 mg, 0.9 mmol) and bromide 36 3,4-dihydroxytetrahydrofuran-2-yl)methyl)(3-carbamoyl-
49 7 9
29.7, 28.5, 28.1, 27.3, 25.5. HRMS: calculated for C35H N O
+
(
270 mg, 0.8 mmol) in dry DMF (10 mL), potassium carbonate benzyl)-amino)butanoic acid (45)
350 mg, 2.5 mmol) was added. After 2 hours, at room Compound 44 (80 mg, 0.19 mmol) was first deprotected in
(
temperature, TLC shows completion and the mixture was TFA:DCM (1:2) for 1 hour at room temperature. The mixture
concentrated, redissolved in DCM and washed with water. The was concentrated, redissolved in 1 mL MeOH and 1 mL 25%
3
aqueous phase was extracted with DCM, the organic layers NH (aq) and crimp sealed. The mixture was reacted for 4
were combined, washed with Brine, dried and concentrated. hours at 130°C in the microwave. The mixture was
Purification via column chromatography (hexanes/ EtOAc 8:1) concentrated and purified by preparative HPLC affording
1
1
afforded compound 40 (245 mg, 60 %) as a clear oil. H NMR compound 45 as a white powder. H NMR (400 MHz, (D
400 MHz, CDCl ) δ 7.94 (s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.49 (d, 8.27 (s, 1H), 8.18 (s, 1H), 7.77 – 7.57 (m, 3H), 7.42 (t, J = 7.7 Hz,
J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 5.08 (d, J = 7.5 Hz, 1H), 1H), 6.10 (s, 1H), 4.66 (dd, J = 5.3, 2.9 Hz, 1H), 4.62 – 4.43 (m,
2
O): δ
(
3
4
.23 – 4.17 (m, 1H), 3.88 (s, 3H), 3.71 (s, 2H), 2.45 – 2.35 (m, 4H), 3.87 (dd, J = 9.2, 3.7 Hz, 1H), 3.73 – 3.61 (m, 3H), 2.58 –
1
3
13
2
H), 2.08 – 1.74 (m, 2H), 1.39 (s, 18H). C NMR (101 MHz, 2.38 (m, 1H), 2.38 – 2.23 (m, 1H). C NMR (101 MHz, D
) δ 171.3, 166.8, 155.3, 138.8, 133.4, 130.5, 129.9, 128.7, 171.7, 167.1, 155.6, 153.1, 149.3, 139.3, 133.6, 130.1, 130.0,
28.4, 82.1, 79.7, 53.4, 52.2, 35.9, 32.8, 28.3, 28.0, 27.2.
S)-2-Amino-4-((3-carbamoylbenzyl)thio)butanoic
2
O)
CDCl
3
δ
1
128.5, 128.4, 120.5, 114.5, 90.9, 85.5, 83.9, 83.5, 81.8, 79.6,
(
acid 58.8, 55.8, 52.9, 52.2, 50.7, 29.7, 28.5, 28.1, 27.3, 25.5. HRMS:
+
(41)
calculated for C22
H
28
N
8
O
6
[M+H] 501.2205; found 501.2223.
Compound 40 (100 mg, 0.23 mmol) was first deprotected in
TFA:DCM (1:2) for 1 hour at room temperature. The mixture Enzymatic activity assay
was concentrated, redissolved in 1 mL MeOH and 1 mL 25%
The enzyme was cloned, expressed and purified as previously
15
described. Purity of the enzyme was confirmed using
NH
3
(aq) and crimp sealed. The mixture was reacted for 4
hours at 130°C in the microwave. The mixture was
SDS−PAGE with Coomassie blue staining (see supplemental
figure S1) and NNMT identity was confirmed using SDS−PAGE
and Western blotting. Catalytic activity of recombinant protein
concentrated and purified by preparative HPLC affording
1
compound 41 as a white powder. H NMR (400 MHz, D
2
O) δ
7
7
–
1
3
2
.77 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 7.8 Hz, 2H),
15
was evaluated as previously reported with 1 unit of enzyme
.49 (t, J = 7.7 Hz, 1H), 4.09 (t, J = 6.4 Hz, 1H), 3.86 (s, 2H), 2.66
activity representing the formation of 1 nmol of MNA per hour
of incubation at 37 °C. The homogeneous recombinant NNMT
specific activity was 10260 units/mg of protein at a protein
concentration of 1.1 mg/mL. NNMT was used at a final
concentration of 150 nM in a mixture containing 50 mM Tris
Buffer (pH 8.6) and 1 mM DTT. The compounds were
incubated with the enzyme at room temperature for 10
minutes before initiating the reaction with a mixture of NA and
1
2.55 (m, 2H), 2.29 – 2.04 (m, 2H). C NMR (101 MHz, D
3
2
O) δ
72.6, 171.9, 138.8, 133.0, 132.8, 129.1, 127.7, 126.2, 51.9,
+
4.5, 29.3, 25.8. HRMS: calculated for C12
69.0954, found 269.0955.
16 2 3
H N O [M+H] :
Methyl 3-(((((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-
,2-dimethyltetrahydrofuro[3,4-d][1,3]-dioxol-4-yl)methyl)-
2
(
(S)-4-(tert-butoxy)-3-((tert-butoxycarbonyl)amino)-4-oxo-
butyl)-amino)-methyl)benzoate (44)
To a solution of compound (200 mg, 0.44 mmol) and
aldehyde 43 (126 mg, 0.46 mmol) in dry DCE (10 ml), crushed
AdoMet at their K
The concentration of inhibitor ranged from 1 µM to 1 mM
DMSO was kept below 5% final concentration). The formation
M
values of 200 µM and 8.5 µM respectively.
6
(
4
Å
molsieves were added. After 2 hours, Na(OAc)
.62 mmol) was added over 5 minutes. The mixture was
, filtered
3
BH (130 mg,
of MNA was measured after 30 minutes at 37°C. The reaction
was quenched by addition of 15 µL sample to 70 µL
acetonitrile containing 5 µM deutero-methylated nicotinamide
as internal standard. The enzymatic activity assays were
performed using UHP-HILIC-MS as previously described with
0
reacted overnight, quenched with saturated NaHCO
3
and the aqueous phase was extracted with DCM. The organic
layers were combined, washed with Brine, dried and
concentrated. Purification via column chromatography
16
minor modifications. UHP-HILIC-MS analysis was performed
(
gradient of 2.5 to 7.5% MeOH in DCM) afforded compound 44
using a 1290 Infinity UHPLC system (Agilent Technologies,
Waldbronn, BW, Germany) consisting of a binary pump, an
1
(
156 mg, 53 %) as a white powder. H NMR (400 MHz, CDCl
.20 (s, 1H), 7.89-7.87 (m, 2H), 7.83 (br s, 1H), 7.49 (d, J = 7.7
Hz, 1H), 7.30 (t, J = 7.9 Hz, 1H), 6.02 (br s, 1H), 5.73 (br s, 1H),
.41-5.36 (br m, 2H), 4.90 (dd, J = 3.5, 6.4 Hz, 2H), 4.38-4.34
3
) δ
8
autosampler and
a
temperature controlled column
department at 65°C with a Waters (Milford, MA, USA) acquity
UPLC BEH amide 1.7 µm 3.0x100 mm HILIC column coupled to
a Q-TOF II mass spectrometer with an electrospray ionization
source and liquid chromatography sprayer from Bruker
Daltonics (Bremen, HB, Germany), operated in positive mode.
The analytes were isocratically eluted using 20% water
5
(
m, 1H), 4.17-4.16 (m, 1H), 3.91 (s, 3H), 3.70 (d, J = 13.7 Hz, 1H
, 3.54 (dd, J = 13.7 Hz, 1H), 2.81-2.76 (m, 2H), 2.70-2.61 (m,
)
2
1
1
1
H), 2.55-2.48 (m, 2H), 2.01-1.97 (m, 2H), 1.80-1.76 (m, 1H),
.60 (s, 3H), 1.43 (s, 9H), 1.40 (s, 9H), 1.37 (s, 3H), 1.26 (br s,
3
H). C NMR (101 MHz, CDCl
3
) δ 171.7, 167.1, 155.6, 153.1,
4
containing 300 µM formic acid and 550 µM NH OH (pH 9.2)
49.3, 139.3, 133.6, 130.1, 130.0, 128.5, 128.4, 120.5, 114.5,
1
0 | Org. Biomol. Chem., 2017, 00, 1-11
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Organic and Biomolecular Chemistry
Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
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DOI: 10.1039/C7OB01357D
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and 80% acetonitrile with a flow-rate of 0.6 mL/min and a run-
time of 2.6 minutes. The injection volume was 10 µL and the
spectra sample rate was 0.8 Hz. For the analysis of the
inhibitory activity of MNA, 4MeNA was used as a substrate at
5
6
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124.
1
6
M
its K concentration of 400 µM. The other parameters in the
assay were kept the same. The enzymatic activity was
determined by measurement of the formation of MNA. The
results were normalized with the “no inhibitor” control set at
7
8
Z. H. Milani, D. B. Ramsden and R. B. Parsons, J. Biochem.
Mol. Toxicol., 2013, 27, 451–456.
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K. Schmeisser, J. Mansfeld, D. Kuhlow, S. Weimer, S.
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100% activity. The “no inhibitor” controls were independently
performed for each IC50 determination. The measured
activities were plotted as a function of inhibitor concentration.
The “no inhibitor” control was plotted at 0.1 µM, 10-fold lower
than the lowest concentration measured. Plotting the “no
inhibitor” control at 0.01 µM or 0.001 µM had no effect on the
curve fit or the IC50 data generated.
9
The data was fitted using non-linear regression analysis of the
Sigmoidal dose-response curve generated using normalized 10
data and a variable slope following equation 1;
R. Giuliante, D. Sartini, T. Bacchetti, R. Rocchetti, I. Klöting,
C. Polidori, G. Ferretti and M. Emanuelli, Metab. Syndr.
Relat. Disord., 2015, 13, 165–170.
ꢀ
ꢁꢁ
1
1
D. Kraus, Q. Yang, D. Kong, A. S. Banks, L. Zhang, J. T.
Rodgers, E. Pirinen, T. C. Pulinilkunnil, F. Gong, Y. Wang, Y.
Cen, A. A. Sauve, J. M. Asara, O. D. Peroni, B. P. Monia, S.
Bhanot, L. Alhonen, P. Puigserver and B. B. Kahn, Nature,
Y= (ꢀꢂꢀꢁ(
Equation [1]
(ꢃꢄꢅꢆꢇꢈꢉꢊꢋ)∗ꢌꢍꢎꢎꢏꢎꢄꢐꢑ))
)
where Y = percent activity, X = the logarithmic concentration
of the compound and Hill Slope = slope factor or Hill
coefficient. The IC50 value was determined by the
concentration resulting in a half-maximal percent activity.
Values are reported along with standard errors of the mean
2
014, 508, 258–262.
1
1
2
3
M. Liu, L. Li, J. Chu, B. Zhu, Q. Zhang, X. Yin, W. Jiang, G.
Dai, W. Ju, Z. Wang, Q. Yang and Z. Fang, J. Clin. Endocrinol.
Metab., 2015, 100, 3112–3117.
(S.E.M., calculated using the symmetrical CI function in
B. D. Horning, R. M. Suciu, D. A. Ghadiri, O. A. Ulanovskaya,
M. L. Matthews, K. M. Lum, K. M. Backus, S. J. Brown, H.
Rosen and B. F. Cravatt, J. Am. Chem. Soc., 2016, 138,
Graphpad Prism 6) indicating the precision of the mean values
obtained.
1
3335–13343.
Modelling studies
1
4
H. Neelakantan, H. Wang, V. Vance, J. D. Hommel, S. F.
McHardy and S. J. Watowich, J. Med. Chem., 2017, DOI:
acs.jmedchem.7b00389.
2
Compound 45 was built in Yasara version 16.9.23 , based on
8
the AdoHcy and NA structure in complex with NNMT (PDB-ID:
1
5
ROD). The model was energy minimized with the steepest
3
1
1
5
6
Y. Peng, D. Sartini, V. Pozzi, D. Wilk, M. Emanuelli and V. C.
Yee, Biochemistry, 2011, 50, 7800–7808.
2
9
descent method using the Amber14 forcefield , before
analysing the hydrogen bond interactions with Yasara (Fig. 4).
M. J. van Haren, J. Sastre Toraño, D. Sartini, M. Emanuelli,
R. B. Parsons and N. I. Martin, Biochemistry, 2016, 55,
5
307–5315.
Acknowledgements
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Financial support provided by Utrecht University and the
Netherlands Organization for Scientific Research (VIDI grant to
NIM).
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Organic and Biomolecular Chemistry
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TABLE OF CONTENTS GRAPHIC
NNMT Active Site
+
Bisubstrate Inhibitor
NNMT inhibitors designed to simultaneously occupy the different substrate binding pockets of the
enzyme active site reveal key structural features required for potent inhibition.
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