Structure-Based Drug Design of Bisubstrate Inhibitors of Phenylethanolamine N-Methyltransferase Possessing Low Nanomolar Affinity at Both Substrate Binding Domains1

Article abstract of DOI:10.1021/acs.jmedchem.0c01475

The enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) catalyzes the final step in the biosynthesis of epinephrine and is a potential drug target, primarily for the control of hypertension. Unfortunately, many potent PNMT inhibitors also po

Full text of DOI:10.1021/acs.jmedchem.0c01475

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Structure-Based Drug Design of Bisubstrate Inhibitors of
Phenylethanolamine N‑Methyltransferase Possessing Low
Nanomolar Affinity at Both Substrate Binding Domains1
Jian Lu, Aaron G. Bart, Qian Wu, Kevin R. Criscione, Michael J. McLeish,* Emily E. Scott,*
and Gary L. Grunewald*
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ABSTRACT: The enzyme phenylethanolamine N-methyltransferase (PNMT, EC
2.1.1.28) catalyzes the final step in the biosynthesis of epinephrine and is a
potential drug target, primarily for the control of hypertension. Unfortunately,
many potent PNMT inhibitors also possess significant affinity for the a2-
adrenoceptor, which complicates the interpretation of their pharmacology. A
bisubstrate analogue approach offers the potential for development of highly
selective inhibitors of PNMT. This paper documents the design, synthesis, and
evaluation of such analogues, several of which were found to possess human
PNMT (hPNMT) inhibitory potency <5 nM versus AdoMet. Site-directed mutagenesis studies were consistent with bisubstrate
binding. Two of these compounds (19 and 29) were co-crystallized with hPNMT and the resulting structures revealed both
compounds bound as predicted, simultaneously occupying both substrate binding domains. This bisubstrate inhibitor approach has
resulted in one of the most potent (20) and selective (vs the a2‑adrenoceptor) inhibitors of hPNMT yet reported.
INTRODUCTION
■
The enzyme phenylethanolamine N-methyltransferase
(PNMT, EC 2.1.1.28) is found primarily in the adrenal glands
and is also located in the brain, heart, lung, and retina.1−4
Therein, it catalyzes the biosynthetic transformation (Figure 1)
As a bisubstrate enzyme, PNMT catalyzes the transfer of a
methyl group from S-adenosyl-L-methionine (AdoMet, 3) to
the amino group of 1. Inhibitors of this reaction fall into two
general classes. One class consists of compounds that compete
with the binding of the methyl-acceptor substrate (such as 1)
and a number of these have been investigated, including
hydrophobic ethanolamine alternate substrate inhibitors6−8
along with benzylamines and phenethylamines.9−11 Com-
pounds based on structural modification of conformationally
constrained benzylamines, such as in the tetrahydroisoquino-
line12−16 (e.g., 6a, 6b) or tetrahydrobenzazepine17,18 (e.g., 7)
ring systems (Figure 2) have been extensively studied for their
ability to inhibit PNMT in the central nervous system as a new
approach for the control of hypertension.19−22 However, most
of these compounds show significant affinity for the α2-
Figure 1. Biosynthesis of epinephrine.
of norepinephrine (1) into epinephrine (2).5 The enzyme is
Received: August 24, 2020
not selective for 1 as its methyl-acceptor substrate and
Axelrod5 identified a variety of phenylethanolamines, such as
5 (phenylethanolamine, PEA), that could also function as
methyl-acceptor substrates and, as a result, the enzyme was
named PNMT.
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Figure 2. Some Inhibitors of PNMT.
adrenoceptor, which complicates the interpretation of their
pharmacology.23
We have investigated numerous compounds in this class in
order to improve inhibitory potency at the PNMT active
site,24,25 CNS penetration,26,27 and selectivity versus the α2-
adrenoceptor.28
Figure 3. Inhibitors of COMT.
A second class of inhibitors of PNMT consists of
compounds that compete with the binding of the methyl-
donor (3), which include the co-substrate product (4) and its
analogues.29−33 However, inhibitors of this class show
significantly diminished inhibitory potency at PNMT,
compared to those that compete with the binding of 1, and
also show poor selectivity for PNMT, as they inhibit other
methyltransferases that utilize 3 as a co-substrate.34
Figure 4. THIQs used in the bisubstrate inhibitor design.
A bisubstrate inhibitor, based on a combination of elements
of these two inhibitor classes, could be used to achieve
selectivity over competing binding domains. Previous studies
indicated that the transfer of the methyl group from 3 to 1
occurs via a SN2 process, suggesting that the methyl transfer
reaction proceeds through a transition state that includes both
substrates bound in the active site of the enzyme.35−37 This
suggested that it would be possible to develop a bisubstrate
analogue inhibitor of PNMT containing structural features that
could simultaneously inhibit the binding of both substrates. A
bisubstrate inhibitor should possess high binding affinity for
the enzyme because of an entropic advantage of reduced
molecularity and an additive binding contribution from each of
the molecules it mimics.38 Also, the combination of the
analogues of both substrates in the same molecule should make
it less likely to be recognized by other pharmacologically
relevant sites and, for example, provide selectivity for PNMT
versus the α2-adrenoceptor. Thus, the goal of this project is to
design, synthesize, and evaluate potent and selective
bisubstrate inhibitors of PNMT to explore the potential of
the bisubstrate inhibitor approach for future inhibitor design.
The bisubstrate analogue approach has been applied to
develop inhibitors for other AdoMet-dependent methyltrans-
ferases.39−48 Most relevant among these is the 2.6 Å resolution
crystal structure of a bisubstrate inhibitor (8, Figure 3)
complexed with catechol O-methyltransferase (COMT) that
showed good inhibitory potency (IC50 = 9 nM),49 as compared
Figure 5. Active site cavity58 of the hPNMT-3 + 11 complex57 (gray)
[PDB: 2G70, Chain B] showing co-substrate 3 and inhibitor 11.
Labeled amino acids have been shown by site directed mutagenesis to
interact with the substrate or co-substrate.51,54 Carbon is shown in
white, nitrogen in blue, oxygen in red, and sulfur in yellow. Hydrogens
are not shown for clarity.
to entacapone (9), a clinically used inhibitor of COMT (IC50
=
14 nM).50
idea that a bisubstrate inhibitor of hPNMT could be created by
connecting a ligand binding in the PEA domain, such as 11,16
with an analogue of 3 using a suitable linker moiety.
Bisubstrate Inhibitor Design. From our earlier work with
the Martin group at the University of Queensland, we reported
the X-ray crystal structures of a number of ligands bound to
the active site of the enzyme,51−56 including the human PNMT
(hPNMT)-3 + 11 ternary complex.57 [see Figure 4 for 1,2,3,4-
tetrahydroisoquinoline (THIQ) structures.]
Examination of the active site in this crystal structure
showed that the two substrate binding domains, designated the
AdoMet domain and the PEA domain, are connected by an
unobstructed channel (Figure 5). This strongly supports the
In the hPNMT-3 + 11 complex, the C-3 atom of 11 is
approximately 5.8 Å from the sulfur atom of 3 and is very close
to the channel that connects the two binding domains in the
enzyme active site. Modeling studies showed that a four-atom
linker could meet the distance requirement between the C-3
atom of 11 and the sulfur atom of 3. To allow for synthetic
diversity, two linker types were considered. A four-atom amine
linker (e.g., 17, n = 2, Table 1), consisted of an ethyl-
B
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Table 1. In Vitro Activities of Bisubstrate Analogue Inhibitors of hPNMT
ab
,
ac
,
ac
,
d
e
PEA
PEA
cmpd
n
R
PNMT KAi doMet (nM)
PNMT K (nM)
PNMT K (nM)
α2 Ki (nM)
selectivity
is
ii
f
f
f
f
f
f
f
4
11
13
14
16
14,000 1000
g
f
h
f
f
f
47
5
19,000 1000
250,000 20,000
1,500 100
f
f
gi
,
j
31,000 3000
1400 200
58,000 14,000
i
2
28,000 2000
37,000 12,000
1,100,000 100,000
39
C-3 Attachment Amine Linker Compounds
115 18 220 52
17
18
19
20
21
22
23
24
25
2
2
2
3
3
3
4
5
6
H
Cl
NO2
H
Cl
NO2
H
H
38
2
15,000 2000
15,000 1000
64,000 6000
11,000 1000
13,000 1000
33,000 3000
7100 500
390
k
k
k
k
k
k
k
k
3.2 0.5
3.8 0.2
2.1 0.2
3.3 0.3
3.5 0.5
3.9 0.3
5.9 0.7
8.8 2.5
9.4 4.0
4700
17,000
5200
3900
9400
1800
82
k
k
k
7.8 1.8
k
10
15
3
4
6.0 3.0
k
k
12
13
6
4
k
k
6.0 0.9
k
k
15
4
42 18
57
18
6
1
100 12
66 10
120 33
145 67
4700 400
5000 500
H
280
C-3 Attachment Amide Linker Compounds
l
f
f
f
26
27
28
29
30
2
2
2
3
3
H
Cl
NO2
H
NO2
810 50
1300 20
94 11
3500 1560
9%
5%
8%
l
71
86
46
70
4
3
2
4
520 290
270 39
180 40
174 16
l
160 20
81
7
130,000 10,000
l
2800
f
220 30
7%
C-4 Attachment Amine Linker Compounds
31
32
2
3
H
H
5100 200
4300 200
12,600 2100
8400 890
17,800 3600
21,000 5250
52,000 5000
15,000 1000
10
3.5
a
b
Recombinant human enzyme was used, unless otherwise noted. Data were obtained by varying concentrations of the inhibitor and 3 at a fixed
c
concentration (100 μM) of 5. KAi doMet values were obtained by fitting initial rate data to a competitive inhibition model. Data were obtained by
varying concentrations of the inhibitor and 5 at a fixed concentration (5 μM) of 3. The slope and intercept inhibition constants, K and K
PEA
is
PEA
ii
d
respectively, were obtained by fitting initial rate data to a mixed (noncompetitive) inhibition model. In vitro activities for the inhibition of
e
f
g
h
[3H]clonidine binding to the α2-adrenoceptor. α2 Ki/KAi doMet. Data have not been determined. Reference 16. Previously reported data were for
i
j
k
l
bovine PNMT. Data are for bovine enzyme. Reference 24. Calculated using the Tight Binding routine in SigmaPlot (ref 83). Percent inhibition
of [3H]-clonidine binding at 100 μM. The Ki value was not calculated because of the compound showing poor solubility under assay conditions at
concentrations ≥1000 μM.
aminomethyl moiety [−(CH2)2NHCH2−], which could be
synthesized via reductive amination using an amine and an
aldehyde (Scheme 3). The corresponding amide linker (e.g.,
26, n = 2, Table 1), consisted of an N-ethylaminocarbonyl
moiety [−(CH2)2NHC(O)−], which could be synthesized
via the coupling of an amine and an acid (Scheme 6).
from the crystal structure of the hPNMT-3 + 11 complex,
using AutoDock59 and Sybyl.60 As expected, these studies
showed that molecules of this type could fit the active site, with
their THIQ and adenosyl moieties mimicking the binding of
the corresponding inhibitor and co-substrate analogues. For
example, the N-6 atom of the adenine ring in the AutoDock
model of 17 is 3.5 Å from the carboxylate of D158, which
suggested the possibility of a weak hydrogen bond interaction
Docking studies were conducted on the proposed
compounds 17 and 26, based on the hPNMT active site
C
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a
(3.7 Å in the crystal structure of hPNMT-3 + 11). The
aliphatic ring nitrogen of the THIQ moiety in the model of 26
was placed within 3.2 Å of the E219 carboxylate, which is in
the range of a hydrogen bond interaction61 (2.9 Å in hPNMT-
3 + 11). While these molecular modeling studies suggested
that a linker with a length of four atoms would be a good fit for
the hPNMT active site, we also proposed amine linker
compounds 20 and 23−25 (5−8 atom length linkers,
respectively, Table 1) and five-atom amide linker 29 to
explore the effects of an extension of the linker.
Scheme 1
Previous structure−activity relationship (SAR) studies
revealed that electron-withdrawing groups on the 7-position
of the THIQ ring (e.g., Cl and NO2) could increase the affinity
of these compounds for hPNMT.15,62 The chlorine and nitro
groups were chosen to represent an electron-withdrawing
lipophilic or hydrophilic substituent, respectively. Thus,
compounds 18, 19, 21, 22, 27, 28, and 30 (Table 1) were
proposed for synthesis.
The crystal structure of the hPNMT-3 + 11 complex also
showed that the C-4 atom of 11 is approximately 5.3 Å from
the sulfur atom of 3 and close to the channel between the two
ligands, although it is not as in line with the channel as is C-3.
Amine linker compounds 31 (four-atom linker) and 32 (five-
atom) were proposed to investigate the effect a change in the
orientation and directionality of the THIQ (10) moiety would
have on inhibitor potency.
a
Reagents and conditions: (a) H2NCH2CH2SH·HCl, NaOEt (98%)
and (b) HCO2H/H2O (4:1) (90%).
For the synthesis of inhibitors 17−32, N-Boc-protected
THIQ-3-carboxylic acids 39, 40, or 41 were used. Compound
39 was synthesized as shown (Scheme 2), 40 was synthesized
by modification of the literature procedure,75 and 41 was
commercially available.
a
Scheme 2
Examination of the X-ray crystal structures of hPNMT with
the AdoMet (3) bound,57,63 compared to structures with the
AdoHcy (4) bound,64,65 suggested that the positively charged
sulfonium species in 3 plays a minimal, if any, role in its
binding. Thus, because of the potential instability of the
sulfonium center,66−68 only a neutral sulfur atom was included
in the design of these inhibitors. For the syntheses of the target
compounds in this study, the proposed linker moiety would
replace the amino acid portion of 4 in order to retain a neutral
sulfur atom in the molecule. A comparison of the inhibitory
potencies of 16 and 4 would provide direct evidence as to
whether this structural simplification was justified.
a
Reagents and conditions: (a) ClCO2Et, pyridine (99%); (b)
(CH2O)n, HOAc/H2SO4 (69%); (c) 6 N HCl (48%); and (d)
(Boc)2O, Et3N (56%).
hPNMT uses an ordered binding mechanism,36 with 3
binding first, and it possesses an enclosed active site.51 It has
been hypothesized that PNMT must undergo a conformational
change before a substrate can bind in the PEA domain,51 which
may be triggered by the binding of 3 in a similar fashion as for
the mechanism proposed for COMT.69 In PNMT, this change
is hypothesized to expose the active site to the PEA domain
ligand.36 Because, by this mechanism, 3 must bind first, it is
not known if this conformational change will occur when using
the bisubstrate compounds.
Most of the compounds in this study conform to Lipinski’s
rules.70 For example, 20 has a clog P of 0.78, nine rotatable
bonds, five hydrogen bond donors, ten hydrogen bond
acceptors, and a molecular weight of 485, with a total polar
surface area71 of 140.72
Chemistry. Because the objective of this study was to
explore the optimal nature and length of the linkage for
bisubstrate inhibition, compounds of interest were prepared as
diastereomeric mixtures based on the unresolved nature of the
C-3 or C-4 attachment points on the THIQ moiety. Key
intermediate 34 was synthesized by reacting commercially
available adenosine analogue 33 with 2-aminoethylthiol
hydrochloride (Scheme 1).73,74 Deprotection of 34 with
formic acid/water (4:1) yielded 16.
The synthesis of 39 began by reacting commercially
available ester 35 with ethyl chloroformate to form carbamate
36, followed by a Pictet−Spengler reaction to yield 37.76
Treatment of 37 with HCl (6 N) at reflux resulted in the
formation of acid hydrochloride 38,77 which was then treated
with di-tert-butyl dicarbonate [(Boc)2O] to yield 39.
Inhibitors 17−19 were synthesized as shown in Scheme 3.
Weinreb amides 42−44 were formed by the treatment of 41,
39, or 40, respectively, with dimethylhydroxylamine hydro-
chloride and benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophospate (BOP). Reduction of 42
or 43 with LiAlH4 in anhydrous diethyl ether at room
temperature afforded aldehydes 45 or 46,78 while reduction of
44 with Cp2Zr(H)Cl (Schwartz reagent) gave aldehyde 47.79
Reductive aminations of 45−47 with amine 34 and NaBH4
yielded 48−50, followed by deprotection of the THIQ amine
and the 1,2-dihydroxyl groups to yield final products 17−19.
The syntheses of amines 61−64 are shown in Scheme 4.
Subjection of commercially available adenosine analogue 51 to
a Mitsunobu reaction with thioacetic acid provided thioester
5280 followed by treatment with bromides 53−56 and sodium
methoxide afforded 57−60,80 which were then hydrolyzed
with hydrazine to form amines 61−64.
D
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a
a
Scheme 3
Scheme 5
a
Reagents and conditions: (a) 61, 62, 63, or 64, NaBH4 (36−68%)
and (b) TFA/H2O (9:1) (42−75%).
a
Reagents and conditions: (a) N,O-dimethylhydroxylamine HCl salt,
Reduction of commercially available 76 with LiAlH4 gave
amino alcohol 77 (Scheme 7). Selective protection of the
amino group with di-tert-butyl dicarbonate followed by Swern
oxidation afforded aldehyde 79. Reductive amination with
amines 34 or 61 formed 80 or 81, respectively, which were
deprotected with TFA/H2O (9:1) to afford target products 31
or 32.
Biochemistry. hPNMT and its variants, all with a C-
terminal hexahistidine tag, were expressed in Escherichia coli
and purified as described previously.26 Enzyme activity was
monitored using a radiochemical assay,26,52 modified when
appropriate to account for the high binding affinity of some
inhibitors.82 Assays were carried out using four concentrations
of 3 or 5 as the variable substrate and three concentrations of
BOP, Et3N (62−83%); (b) for 42 or 43: LiAlH4 (77−88%); (c) for
44: Cp2Zr(H)Cl (56%); (d) 34, NaBH4 (48−57%); and (e) TFA/
H2O (9:1) (59−65%).
Reductive aminations of aldehydes 45−47 and the
appropriate amine 61−64 with NaBH4, yielding 65−70
followed by deprotection with TFA/H2O (9:1) afforded
inhibitors 20−25 (Scheme 5).
Compounds 26−30 were synthesized (Scheme 6) by
coupling 41, 39, or 40 with 34 or 61 to yield compounds
71−75.81 Treatment of 71 with formic acid/water (4:1)
yielded 26. Deprotection of 72−75 with trifluoroacetic acid
[TFA/H2O (9:1)] yielded final products 27−30.
a
Scheme 4
a
Reagents and conditions: (a) thioacetic acid, DIAD, PPh3 (100%); (b) NaOMe (64−85%); and (c) NH2NH2 (81−94%).
E
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a
Scheme 6
RESULTS AND DISCUSSION
■
The results of the kinetic studies are shown in Table 1.
hPNMT has been shown to function via an ordered, sequential
mechanism, wherein 3 binds first and facilitates the binding of
the methyl-acceptor substrate.36 Initial rate data for each
inhibitor were fit to equations for competitive-, noncompeti-
tive-, and mixed-type inhibition using the enzyme kinetics
module in SigmaPlot.83 The bisubstrate inhibitors all displayed
competitive inhibition versus AdoMet and noncompetitive
inhibition versus PEA (Table 1). This pattern is consistent
with a steady-state ordered mechanism, in which AdoMet
binds first.85 In light of that observation, the data in Table 1 for
the bisubstrate inhibitors were calculated using a competitive
AdoMet
binding model versus 3 (K
) and a mixed (non-
is
86
PEA
PEA
competitive) model versus 5 (K
and K ). It should be
is
ii
noted that the inhibition patterns were determined by
measuring the initial rates at varying concentrations of one
reactant and a fixed concentration of the second reactant.
Accordingly, the values in Table 1 should be treated as
apparent Ki values. For AdoMet, appKis = Ki.86 For PEA,
AdoMet
appKis = Ki(1 + [AdoMet]/K
) and appKii = Ki(1 +
ia
[AdoMet]/KAmdoMet).86 For 3, the fixed concentration was 5
μM, while for 5, the concentration was 100 μM. Using ITC,
36
AdoMet
PEA
is
K
was found to be 4.6 μM, thus appK should be ∼2
ia
AdoMet
× K
(i.e., 2 × Ki). Given the constraints of the assay, it
is
a
Reagents and conditions: (a) 34 or 61, EDCI, HOBT, N-
was gratifying to see that, in general, the Ki values determined
from the competitive pattern versus AdoMet were broadly
similar to those obtained from the mixed (noncompetitive)
pattern versus PEA (Table 1).
methylmorpholine (68−98%); (b) HCO2H/H2O (4:1) for 71; and
(c) TFA/H2O (9:1) for 72−75 (40−66%).
A comparison of the inhibitory potency of 4 (AdoHcy) with
that of 16 (the adenosyl fragment used in these bisubstrate
inhibitors) shows only a two-fold increase in potency with the
inclusion of the amino acid side chain, indicating that the
presence of the homocysteine side chain is not required for
binding.
All of the compounds with an amine linker were more
potent as inhibitors of hPNMT than their corresponding
amide analogues. For example, the best compounds in each of
the two linker types were 20 (KAi doMet = 2.1 nM) for an amine
linker and 29 (KAi doMet = 46 nM) for an amide linker. Each of
these compounds possess a five-atom length linker (Table 1, n
= 3), with hydrogen as the 7-substituent on the THIQ moiety.
The potency difference between the corresponding com-
pounds (comparing amine vs amide linker compounds: 17 vs
the inhibitor. Kinetic constants were obtained by fitting the
initial rate data using the Enzyme Kinetics Module in
SigmaPlot.83 At a minimum, assays were run in duplicate
and the average result presented.
α2-Adrenergic receptor binding assays were performed using
cortex obtained from male Sprague Dawley rats.84 [3H]-
clonidine was used as the radioligand to define the specific
binding and phentolamine was used to define the nonspecific
binding. Clonidine was used as the ligand to define α2-
adrenergic binding affinity for a direct comparison with
previous studies. At a minimum, assays were run in duplicate
and the average result presented.
a
Scheme 7
a
Reagents and conditions: (a) LiAlH4 (88%); (b) NaHCO3, (Boc)2O (88%); (c) oxalyl dichloride, DMSO, DIEA (71%); (d) 34 or 61, NaBH4
(54−60%); and (e) TFA/H2O (9:1) (74−86%).
F
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26, 18 vs 27, 19 vs 28, 20 vs 29, and 22 vs 30) was
approximately 20-fold (Table 1) in all cases. These results are
consistent with 3-substituted-THIQ 14 (aminomethyl) show-
ing over 20-fold greater potency (Table 1) as an inhibitor of
hPNMT than 13 (aminocarbonyl).
domains, site-directed mutagenesis was used to probe whether
19, one of the most potent inhibitors in this study, was binding
at both hPNMT substrate domains. The amino acid residues
mutated in these studies were those with established
interactions with either inhibitor 11 or co-substrate 3 based
on the hPNMT-3 + 11 structure (Figure 5).54 Both residues
E219 and K57 were mutated to alanine (E219A and K57A,
respectively). As mentioned earlier, E219 forms a hydrogen
bond with the aliphatic isoquinoline amine of tetrahydroiso-
quinoline-type inhibitors in the PEA binding domain. This
structure also showed that K57 is another important amino
acid residue in the PEA binding domain that forms a hydrogen
bond with the oxygen of the nitro moiety in 11.53 Because of
their considerable distances from the AdoMet domain, E219
and K57 were expected to have little effect on inhibitors
binding only in the AdoMet domain. In contrast, residues
D158 and C183 lie in the AdoMet domain and mutations of
these residues were expected to have little effect on the binding
of ligands binding in the PEA domain. D158 forms a hydrogen
bond with the N-6 atom of the adenine ring of 3,51 while C183
also forms a part of the binding domain for 3,51 being located
under the plane of the adenine ring system. Both were mutated
to alanine (D158A and C183A, respectively).
As expected, the introduction of an electron-withdrawing
group to the 7-position of the THIQ fragment increased the
hPNMT inhibitory potency of compounds possessing a four-
atom (n = 2) length linker. A comparison of both amine linker
compounds 18 (Cl) and 19 (NO2) compared to 17 (H) and
amide linker compounds 27 (Cl) and 28 (NO2) compared to
26 (H) showed an approximately 10-fold increase in potency.
However, this was not observed with compounds possessing a
five-atom length linker, with both amine linker compounds 21
(Cl) or 22 (NO2) compared to 20 (H) and amide linker 30
(NO2) compared to 29 (H), with at least a 40% reduction in
hPNMT inhibitory potency (Table 1). This may be due to the
increased length of the linker, such that the THIQ portion for
compounds possessing a five-atom length linker, would sit
further into the hydrophobic pocket formed by V53, M258,
V269, and V272 in the hPNMT active site,52 resulting in the
five-atom length amine linker 20 and its amide linker analogue
29, which possess a hydrogen as the 7-substituent on the
THIQ fragment, being the most potent compounds of their
respective series. Because of this possible positioning, adding a
7-substituent to the aromatic ring of the THIQ moiety of 20 or
29 could result in unfavorable steric interactions that would
offset the positive contribution of the electron-withdrawing
THIQ 7-substituent.
The effect of inhibitors 12 (THIQ fragment binding only in
the PEA domain), 16 (adenosyl fragment binding only in the
AdoMet domain), and 19 (bisubstrate inhibitor) on the
inhibition of mutant hPNMTs were compared to the wild-type
enzyme, as shown in Table 2. As expected, both the K57A or
Table 2. Relative Activities of Inhibitors 12, 16, and 19, at
Wild-Type (WT) and Mutant hPNMTs
Compounds possessing a six- (23), seven- (24), or eight-
atom (25) amine linker were prepared to explore the effect of
an extension of the linker. Compared to its five-atom linker
analogue (20, KAi doMet = 2.1 nM), the six-atom linker (23) was
slightly less potent (KAi doMet = 3.9 nM) and similar in potency
to compounds 21 or 22, wherein substitutions at the 7-position
of the THIQ moiety were incorporated (KAi doMet = 3.3 or 3.5
nM, respectively). When the linker was increased to seven
atoms, potency was further reduced as demonstrated for 24
(KAi doMet = 57 nM), potentially due to the negative steric
interactions or increasing entropic demand. In contrast, the
eight-atom linker analogue (25) showed a slight increase in
potency (KAi doMet = 18 nM) compared to 24, possibly resulting
from conformational adjustments by the enzyme. Similar
conformational adjustment had previously been shown to
reveal a cryptic binding site in hPNMT.55
Compounds 31 and 32 (four- and five-atom length amine
linkers, respectively) were synthesized to explore the effect of a
different orientation of the THIQ moiety, with the linker being
attached at the C-4 of THIQ (10). Both of these compounds
showed reduced hPNMT inhibitory potency of ca. 4−5 μM
compared to the equivalent C-3 attachment analogues, and
thus, this series was not investigated further.
As mentioned previously, a bisubstrate inhibitor should
show at least an additive increase in inhibitory potency over its
component fragments38 and this is indeed the case for all of
the bisubstrate compounds in this study. For example, the
increase in potency for 20 compared to 16 is greater than
∼13,000-fold and ∼4800-fold more potent when compared to
10 (KiPEA = 10 μM).87
Mutagenesis Studies. Because a number of the
compounds were very effective inhibitors of hPNMT and
docking suggested binding would be compatible with
simultaneous occupancy of both the AdoMet and PEA binding
a
relative potency
hPNMT variant
12
16
19
wild-type
K57A
E219A
D158A
C183A
1.0
1.0
0.46
1.8
35
7.3
1.0
10
69
15
22
18
43
8.1
4.9
a
Inhibitory potency relative to wild-type (WT) hPNMT for 12 and
19: Ki/Ki(WT); for 16: IC50/IC50(WT). Compound 5 was used as the
methyl-acceptor substrate.
E219A mutants had a larger impact on the binding of THIQ-
type inhibitor 12, which binds only in the PEA domain, than
the D158A or C183A mutants did, while the D158A and
C183A mutants showed a much larger impact on the hPNMT
inhibitory potency of 16, an inhibitor binding only in the
AdoMet domain, than the K57A or E219A mutants did.
Notably, all four mutants showed a strong effect on the
binding of inhibitor 19, which suggests that 19 interacts with
all four residues and thus occupies both the AdoMet and PEA
binding domains.
X-ray Structures of Bisubstrate Inhibitors in the
hPNMT Active Site. To confirm the general binding mode
of these bisubstrate inhibitors and to elucidate the details of
their interactions with hPNMT, crystals of 19 or 29 co-
crystallized with hPNMT were obtained. The structures of
these complexes were determined at 1.95 Å resolution for
hPNMT-1988 (Figure 6a) and at 2.20 Å for hPNMT-2989
(Figure 6b). Consistent with previous studies,51−53 the
asymmetric unit for both complexes contained two hPNMT
molecules which showed a root mean square deviation
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Figure 6. Active site cavity58 (gray) from the hPNMT-3 + 1157 crystal structure [PDB: 2G70]. Amino acids used in the site-directed mutagenesis
investigations are labeled. A molecule of Tris was identified in both hPNMT:bisubstrate inhibitor complexes. Atom colors are described in Figure 5.
The figures show (a) potent bisubstrate inhibitor 19 that contains a four-atom amine linker [PDB: 4MIK, Chain A] and (b) bisubstrate inhibitor
29 [PDB: 4MQ4, Chain A] that contains a five-atom length amide linker.
Figure 7. Active site cavity58 (shown in gray) based on the hPNMT-3 + 1157 crystal structure [PDB: 2G70]. Amino acids used in the site-directed
mutagenesis investigations are labeled. The molecule of Tris in the active site is shown for completeness. Atom colors are described in Figure 5.
The Figures show (a) superimposition58 of the X-ray structures of (3 + 11) (red) and 19 (blue) [PDB: 2G70, Chain B and 4MIK, Chain A,
respectively] and (b) superimposition58 of the X-ray structures of 19 (blue) and 29 (green) [PDB: 4MIK, Chain A and 4MQ4, Chain A,
respectively].
(rmsd)58 of 0.195 and 0.241 Å over all atoms of the hPNMT-
19 and hPNMT-29 complexes, respectively. By comparison,
the two molecules in the hPNMT-3 + 11 asymmetric unit57
showed an rmsd value of 0.307 Å. It was immediately apparent
that the presence of the bisubstrate inhibitor had little effect on
the overall conformation of hPNMT. This was confirmed by
the superposition of the monomers of the bisubstrate
complexes on those of the hPNMT-3 + 11 complex, which
provided average rmsd values (over all atoms) of 0.329 Å for
hPNMT-19 (Figure 7a) and 0.347 Å for hPNMT-29.
In both structures, the electron density for some of the linker
atoms was weak, implying flexibility, but clearly the main
portions of these bisubstrate inhibitors occupy both substrate
binding domains as predicted. The electron density was weaker
and the B-factors higher for the linker atoms of 19, which
incorporates a flexible amine linker, than for the corresponding
atoms of 29 and its more rigid amide linker (Table 3).
Compound 19, which possesses a four-atom length (n = 2)
linker, is one of the most potent bisubstrate inhibitors in this
study (Figure 6a).
(KAi doMet = 2.1 nM), which could provide insights into the
reasons why 20 was such a potent compound. However, 29
was less potent as an inhibitor of hPNMT (KAi doMet = 46 nM)
than 19 (KAi doMet = 3.8 nM).
Comparison of the crystal structure of hPNMT-19 with
hPNMT-3 + 11 showed considerable similarity between how
these compounds fit in the hPNMT active site (Figure 7a). In
the AdoMet binding domain, the closest distance between
D158 and the N-6 of the adenine ring is 2.9 Å in the hPNMT-
19 structure, which is shorter than the corresponding distance
in the hPNMT-3 + 11 structure (3.7 Å). This results from a
slight torsion in the terminal atoms of the D158 side chain,
rather than a change in the position of the adenine ring, and
results in a stronger hydrogen bond to 19 as compared to 3.
The position of C183, also in the AdoMet binding domain, is
unchanged. The sugar ring is positioned similarly in both
structures, conserving interactions between its hydroxyls and
D101. Although the sugar C-5 is positioned slightly differently
in the two structures, the position of the sulfur atom is highly
conserved. In the PEA binding domain, the THIQ ring of 19 is
shifted slightly toward E219 compared to the equivalent
position of 11 in the hPNMT-3 + 11 structure. As a result, the
closest distance between an E219 carbonyl oxygen and the
Compound 29 (Figure 6b) is the most potent amide linker
compound in this study and shares the five-atom linker length
(n = 3) with the most potent amine linker compound 20
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in contact with the E219 side chain carboxylate of PNMT at
distances of 2.7 and 3.3 Å. These observations indicate no
significant preference for binding of either of the C-3
stereoisomers. In contrast, the cocrystal structures of both 19
and 11 revealed that the THIQ nitrogen served as the
hydrogen donor contact to E219. The cocrystal structure with
19 showed only the C-3 (S)-stereoisomer, which is consistent
with the importance of the THIQ nitrogen-E219 contact
(Figure 5a).
The aromatic rings of the THIQ moieties in 19 and 29 lie in
the same plane, being sandwiched between the sidechains of
F182 and N39, as observed previously.51 Because the longer
linker of 29 seats the THIQ moiety deeper into the PEA
binding domain, the aromatic ring of the THIQ ring system
occupies approximately the same area of space as that occupied
by the nitro substituent of 19 (Figure 7b).
Table 3. X-ray Data Collection, Refinement, and Validation
Statistics
hPNMT-19
hPNMT-29
Data Collection
space group
P43212
P43212
cell dimensions: a, b, c (Å) 94.66, 94.66, 188.46
94.15, 94.15, 188.16
a
resolution (Å)
38.62−1.95
38.43−2.2
(2.06−1.95)
(2.32−2.2)
a
total reflections
unique reflections
1,613,327 (222,007)
63,094 (8974)
25.6 (24.7)
0.015 (0.426)
31.1 (1.7)
277,262 (40,988)
43,470 (6217)
6.4 (6.6)
0.023 (0.371)
20.5 (2.0)
a
a
redundancy
a
Rpim
a
Mn(I/sd)
a
completeness (%)
99.8 (98.7)
99.8 (99.8)
Refinement
resolution (Å)
no. Reflections
38.62−1.95
62,657
38.43−2.2
43,250
Unlike 19, which possesses a nitro group, 29 does not have a
substituent on its THIQ ring that can interact with K57 and
the C-7 of its THIQ ring is only 3.5 Å from the isopropyl
carbons of V53 (Figure 6b), which, along with M258, V269,
and V272, line the end of the PEA binding domain to form a
hydrophobic surface.52 The THIQ moiety of the five-atom
linker compounds is seated deeper into this hydrophobic
surface (Figure 6b), which may be responsible for the
increased inhibitory potency of 20 and 29 as compared to
their four-atom linker analogues (20: KAi doMet = 2.1 nM vs 17:
KAi doMet = 38 nM and 29: KiAdoMet = 46 nM vs 26: KiAdoMet = 810
nM, respectively). Addition of a chloro- or nitro-substituent to
the C-7 of the THIQ moiety in the five-atom linker
compounds was expected to grant a similar increase in
inhibitory potency as it did in the case of the four-atom linker
compounds, but unfavorable steric interactions with V53 may
be responsible for the observed reduction in inhibitory potency
of these compounds. For example, compare 22 (NO2, KiAdoMet
= 3.5 nM) to its equivalent hydrogen-substituted compound
(20, KAi doMet = 2.1 nM) or 30 (NO2 , KAi doMet = 70 nM) to 29
(H, KAi doMet = 46 nM). Decreasing the distance between the
THIQ moiety and V53 by increasing the length of the linker to
six or seven atoms, as in 23 (KAi doMet = 3.9 nM) or 24 (KAi doMet
= 57 nM), respectively, could be responsible for their
decreased inhibitory potency by increasing the possibility of
steric clash with V53.
It is notable that both the hPNMT-19 and hPNMT-29
structures contained the electron density for another molecule
in the portion of the AdoMet binding domain that would
normally bind the homocysteine sidechain of AdoMet. A
survey of all components in the protein and crystallization
buffers revealed that this density was best fit as a molecule of
the buffer Tris (tris(hydroxymethyl)amino methane). As
shown in Figure 7a, two of the Tris oxygens are positioned
in exactly the same locations as the oxygens in the
homocysteine carboxylate, while the third hydroxyl group is
directed into approximately the same space as the
homocysteine amine group and the Tris amine is directed
into the space normally occupied by the homocysteine
sidechain. The density was strong in both structures. This is
consistent with Tris inhibition being observed in PNMT
assays, which prompted a switch to phosphate buffer, and may
suggest that hPNMT strongly prefers a compatible molecule in
the homocysteine-binding portion of the AdoMet binding
domain. This adventitious occupancy of the homocysteine-
binding area of the AdoMet binding domain by a molecule of
R
work/Rfree
0.1824/0.2295
0.1826/0.2207
residues modeled for
molecules
Wilson B factor
A: 26−280,
A: 17−280,
B: 16−280
B: 26−280
40.22
Number of Atoms/B Factor
8135/50.60
40/54.25
47.57
protein
8115/60.67
40/65.85
128/63.68
181/56.18
ligand (Tris)
ligand (inhibitor)
water
128/82.59
314/51
RMS Deviations
0.013
bond lengths (Å)
bond angles (deg)
0.013
1.443
1.493
Structure Analysis
Ramachandran (%)
98.85
1.15
0
most favored
allowed
disallowed
98.07
1.93
0
a
Highest resolution shell in parentheses.
ligand amine nitrogen was 2.6 Å, in the hPNMT-19 structure,
while the equivalent distance in hPNMT-3 + 11 was 2.9 Å.
Similarly, the distance between the THIQ nitro oxygen and the
K57 amine was shorter for hPNMT-19 (2.7 Å), compared to
the equivalent interaction in hPNMT-3 + 11 (3.4 Å). Again,
these shorter interaction distances for both K57 and E219 may
indicate an increase in the strength of the binding interactions
of 19 compared to 11.
Despite the fact that the linker in 29 is one atom longer than
for 19, an overlay of the hPNMT-19 and hPNMT-29
structures (Figure 7b) shows that they both bind in a similar
fashion, with a rmsd of 0.225 Å for the alignment of all atoms
of their respective hPNMT A chains.
In the AdoMet binding domain, the adenosyl moieties of the
two bisubstrate inhibitors superimpose well, with only 0.2 Å
differences between their respective adenosine N-6s, ribose
ring oxygens, and sulfurs, and no significant differences in the
positions of D158 or C183. While the five-atom amide linker
passes through the channel between the AdoMet and PEA
binding domains, its longer length and the different positions
of these atoms bury the THIQ moiety deeper into the PEA
binding domain. There are two possible orientations of the
THIQ moiety in the hPNMT-29 structure, with the plane of
the THIQ being flipped by 180°. The electron density does
not distinguish between them and, as a result, they were each
modeled with 50% occupancy. Regardless, for both orienta-
tions, the carbonyl oxygen of the linker amide appeared to be
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4.6 mm, particle diameter: 5 μm, Grace Vydac, Hesperia, CA.). The
HPLC system consisted of a Shimadzu SLC-10A VP system
controller, two LC-6AD pumps, and an SPD-M10A VP diode array
detector and monitored at wavelengths specified for each compound.
System A parameters consisted of the following: a linear gradient
solvent system of H2O (0.1% TFA)/CH3CN from 99/1 to 1/99 in 40
min, with a flow rate of 1 mL/min. System B parameters consisted of
the following: a linear gradient solvent system of H2O (0.1% TFA)/
MeOH from 99/1 to 1/99 in 40 min, with a flow rate of 1 mL/min.
AdoMet was obtained from Sigma-Aldrich (St. Louis, MO).
[methyl-3H]AdoMet and [3H]clonidine were obtained from Perki-
nElmer (Boston, MA). Stock inhibitor solutions (10 mM) were made
by dissolving the compound in H2O (for salts) or in 0.01 N HClO4
(for free amines).
Tris suggests that further improvements in the potency of
these bisubstrate inhibitors may be achievable.
CONCLUSIONS
■
A series of compounds was developed in this study as potential
bisubstrate inhibitors of hPNMT, based on the information
from the X-ray crystal structure of the hPNMT-3 + 11 ternary
complex, molecular modeling studies, and previous SAR
studies. Kinetic studies showed that many of these new
analogues were not only low nanomolar inhibitors of hPNMT
but also displayed only micromolar affinity for the α2-
adrenoceptor. This additional selectivity would be highly
advantageous for delineating the separate pharmacological
effects of PNMT inhibition and α2-adrenoceptor affinity. For
example, 19 showed a six-fold increase in hPNMT inhibitory
potency and a four-fold decrease in α2-adrenoceptor affinity as
compared to 11, its mono-substrate analogue, making 19 one
of the most potent (KiAdoMet = 3.8 nM) and selective (vs the α2-
adrenoceptor, Table 1) inhibitors of hPNMT yet reported.
While docking studies predicted the bisubstrate binding
nature of these new inhibitors and the site-directed muta-
genesis results were consistent with these compounds
occupying both binding domains, the binding mode of these
inhibitors was confirmed by the X-ray crystal structures of
hPNMT-19 and hPNMT-29.
Protein Expression and Purification. Expression vectors
containing genes for wt, K57A and E219A PNMT were available
from previous studies.26,54 The D158A and C183A variants were
obtained by mutagenesis of pET17PNMT26 using the QuikChange
site-directed mutagenesis kit (Agilent, Santa Clara, CA).
Hexahistidine-tagged hPNMT was expressed and purified as
described previously.26,54
Radiochemical Assay for hPNMT Inhibitory Potency. The
radiochemical assay used to measure the inhibitory potency of these
compounds against hPNMT was performed as described previously.82
Kinetic data were fit to equations for competitive- (eq 1) or
noncompetitive (mixed)-type (eq 2) inhibition using SigmaPlot.83
Vmax[S]
v =
[I]
The object of these studies was to determine whether co-
occupancy of both the PEA and AdoMet binding sites might
lead to further enhancement of hPNMT inhibitory potency
and selectivity. These compounds were successful at both of
these objectives. While the most potent compound (20) obeys
Lipinski’s rules, it is likely that further optimization of drug-like
properties will be required before it could be considered as a
clinical candidate.
[S] + Km 1 +
(
)
Kis
(1)
Vmax[S]
v =
[I]
Kis
[I]
Kii
Km 1 +
+ [S] 1 +
(
)
(
)
(2)
Where appropriate, data were fit using the tight-binding competitive
and mixed inhibition routines in the Enzyme Kinetics module of
SigmaPlot.
α2-Adrenoceptor Radioligand Binding Assay. Tissue samples
were prepared and the radioligand receptor binding assay was
performed according to the method of U’Prichard et al.84 All animal
use was conducted in accordance with institutional guidelines.
Molecular Modeling. Initial molecular modeling studies were
performed on a Silicon Graphics Octane workstation using Sybyl.60
Docking of inhibitors into the PNMT active site was performed using
AutoDock 3.0 (default settings).59 The initial ligand was overlayed
with the cocrystallized ligand and minimized with the Tripos force
field before it was docked into the active site of PNMT. Graphics for
Figures 5−7 were generated on an iMac i7 using PyMOL.58
Protein Crystallization, Data Collection, and Structure
Determination. Purified hPNMT was mixed with 19 (final
concentration of 240 μM) and concentrated to 35 mg/mL final
protein concentration. DTT was added to 10 μM. Crystals were
grown from 1 μL of the 35 mg/mL hPNMT-19 protein complex
mixed with 1 μL of well solution (0.1 M Na cacodylate pH 5.6
(Hampton Research, Aliso Viejo, CA), 0.17 M LiCl, and 9% (w/v)
PEG 6000 (Hampton Research)) equilibrated against 750 μL of this
same solution by the hanging drop vapor diffusion method.
Tetragonal rods appeared after 10 days of incubation at 17 °C,
growing from a background of heavy precipitation. Crystals were
immersed in cryoprotectant (well solution with 25% ethylene glycol)
and flash cooled in liquid nitrogen for storage until data collection.
Crystals of hPNMT-29 were grown in a similar manner with slight
modifications. hPNMT was concentrated to 40 mg/mL, and a 200
mM stock solution of 29 dissolved in 100% dimethyl sulfoxide
(DMSO) was added directly to the protein sample to a final
concentration of 800 μM. Crystals were grown with the same
crystallization reagents and conditions used for hPNMT-19. Crystals
with a tetragonal morphology grew after 4 days of incubation at 17
°C. Crystal harvesting and storage was performed as described
previously.
EXPERIMENTAL SECTION
■
The purity of all biologically tested compounds was determined to be
≥95% by combustion analysis (17−23, 26−27, 29, and 32) or high-
performance liquid chromatography (HPLC) (24, 25, 28, 30, and
31). See below for details. HPLC tracings are included in Supporting
Information.
All reagents and solvents used were of reagent grade or were
purified by standard methods before use. Melting points were
determined in open capillary tubes on a Thomas−Hoover melting
point apparatus calibrated with known compounds but are otherwise
uncorrected. All proton (1H NMR) and carbon (13C NMR) nuclear
magnetic resonance spectra were taken on a Bruker DRX-400 or a
Bruker AM-500 spectrometer. Multiplicity abbreviations are as
follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad; and
ex, exchangeable. Electron-impact mass spectra [MS (EI)], chemical-
ionization mass spectra [MS (CI)], and high-resolution mass spectra
(HRMS) were obtained on a Varian Atlas CH-5, a Ribermag R 10-10,
a VG ZAB, or an LCT Premier high-resolution instrument. The
intensity of each peak in the mass spectrum relative to the base peak is
reported in parentheses. Combustion analyses were performed by
Quantitative Technologies, Inc. (Whitehouse, New Jersey). Flash
chromatography was performed using silica gel 60 (230−400 mesh)
supplied by Universal Adsorbents, Atlanta, Georgia. Methanol
(MeOH) and ethanol (EtOH) were anhydrous unless stated
otherwise and were prepared by distillation over magnesium.
Anhydrous tetrahydrofuran (THF) and diethyl ether (Et2O) were
distilled from sodium-benzophenone ketyl. Hexanes refer to the
mixture of hexane isomers (bp 40−70 °C). All reactions that required
anhydrous conditions were performed under dried argon, and all
glassware was either oven dried or flame dried before use.
The purity of some final products was determined by HPLC using
a Vydac 218TP54 column (C18 reversed-phase, column size: 250 ×
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Data sets were collected from single crystals at the Stanford
Synchrotron Radiation Lightsource (Stanford, CA) Beamline 12-2 at
a wavelength of 1.03 Å. Data integration and scaling were carried out
employing the programs XDS90 and SCALA.91 The structures were
solved by molecular replacement using Phaser92 with the hPNMT-3-
fluoromethyl-7-thiomorpholinosulfonamide-THIQ/AdoMet structure
as a search model55 (Protein Data Bank code 2G72, molecule A)
using the entire resolution range to 2.0 Å. The solution for both
hPNMT inhibitor complexes (P432121 space group) had an
asymmetric unit consisting of two hPNMT molecules. Log likelihood
gain values were 6058 for hPNMT-19 and 8361 for hPNMT-29,
respectively. Iterative model building and refinement were carried out
using COOT93 and PHENIX.94 The statistics for data collection,
refinement, and validation are shown in Table 3. Structures were
deposited to the Protein Data Bank under accession codes 4MIK
(hPNMT-19) and 4MQ4 (hPNMT-29).
prepared in a similar manner as 17 but using 50 (150 mg, 0.23 mmol)
as the starting material to afford 19 (70 mg, 59%) as slightly yellow
foam: H NMR (500 MHz, CDCl3/CD3OD, 10:1): δ 8.33 (s, 1H),
1
8.24 (s, 1H), 8.01 (m, 2H), 7.34 (d, J = 8.2 Hz, 1H), 6.03 (d, J = 4.6
Hz, 1H), 4.79 (t, J = 5.0 Hz, 1H), 4.39 (t, J = 5.0 Hz, 1H), 4.26 (d, J =
5.2 Hz, 1H), 4.11 (m, 2H), 3.06−3.01 (m, 3H), 2.90−2.76 (m, 6H),
2.64 (m, 2H); 13C NMR (125.7 MHz, CDCl3/CD3OD, 10:1): δ
155.8, 152.5, 149.1, 146.1, 142.0, 139.9, 136.6, 129.9, 120.8, 120.7,
119.1, 88.8, 84.2, 73.4, 72.5, 52.9, 52.1, 48.1, 47.0, 33.7, 32.4, 31.8;
MS (CI) m/z (relative intensity): 517 (MH+, 40), 327 (20), 290
(25), 136 (80); HRMS (ESI+) m/z: calcd for C22H29N8O5S (MH+),
517.1982; obsd, 517.1961; Anal. Calcd for C22H28N8O5S·1/
2CF3COOH: C, 48.16; H, 5.01; N, 19.54. Found: C, 48.19; H,
5.21; N, 19.31.
5′-Deoxy-5′-{3-[(1,2,3,4-tetrahydro-isoquinolin-3-yl)-
methyl]amino-propyl}thio-adenosine (20). This compound was
prepared in a similar manner as 17 using the procedure outlined
previously, but using 65 (1.00 g, 1.60 mmol) as the starting material
to afford 20 (420 mg, 54%) as white foam: IR (KBr) 3424, 3181,
2925, 2827, 1650, 1593, 1470, 1327, 1127, 748 cm−1; 1H NMR (500
MHz, CD3OD): δ 8.26 (s, 1H), 8.16 (s, 1H), 7.12−7.03 (m, 4H),
5.99 (d, J = 4.9 Hz, 1H), 4.76 (t, J = 5.0 Hz, 1H), 4.32 (t, J = 4.9 Hz,
1H), 4.19 (m, 1H), 4.09 (s, 2H), 3.27 (m, 1H), 3.08−2.95 (m, 2H),
2.92−2.61 (m, 8H), 1.86 (quintet, J = 7.0 Hz, 2H); 13C NMR (125.7
MHz, CD3OD): δ 154.2, 150.9, 147.6, 138.3, 130.5, 130.4, 127.1,
125.0, 124.6, 124.1, 117.5, 87.0, 82.6, 71.8, 71.0, 49.8, 49.1, 46.8, 44.1,
32.2, 28.8, 27.7, 25.1; MS (CI) m/z (relative intensity): 486 (MH+,
45), 284 (20), 250 (40), 132 (80); HRMS (ES+) m/z: calcd for
C23H32N7O3S (MH+), 486.2287; obsd, 486.2265; Anal. Calcd for
C23H31N7O3S·1/2H2O: C, 55.85; H, 6.52; N, 19.82. Found: C, 55.72;
H, 6.45; N, 19.39.
5′-(2-Aminoethyl)thio-5′-deoxy-adenosine (16). Compound
34 (200 mg, 0.546 mmol) was dissolved in HCOOH/H2O (4:1, 10
mL). The solution was stirred at room temperature for 8 h and
Na2CO3 was added until the mixture was basic. The solvents were
removed under reduced pressure and the residue was purified by flash
chromatography (silica gel) with CHCl3/CH3OH/NH3·H2O
(10:1:0.1) as eluent to yield 16 as a white solid foam (160 mg,
1
90%): H NMR (400 MHz, CD3OD): δ 8.29 (s, 1H), 8.18 (s, 1H),
5.98 (d, J = 5.0 Hz, 1H), 4.77 (t, J = 5.2 Hz, 1H), 4.32 (t, J = 5.0 Hz,
1H), 4.17 (quartet, J = 4.6 Hz, 1H), 3.29 (m, 1H), 2.91 (m, 2H), 2.72
(m, 2H), 2.63 (m, 2H); 13C NMR (100 MHz, CD3OD): δ 156.3,
152.9, 149.6, 140.4, 119.5, 89.1, 84.7, 73.8, 73.0, 40.7, 35.6, 33.9; MS
(CI) m/z (relative intensity): 327 (MH+, 25), 199 (75), 136 (90);
HRMS (ESI+) m/z: calcd for C12H19N6O3S (MH+), 327.1239; obsd,
327.1211; Anal. Calcd for C12H18N6O3S·1/3(CHCl3·H2O): C, 39.80;
H, 5.15; N, 22.58. Found: C, 39.90; H, 5.00; N, 22.44.
5′-Deoxy-5′-{3-[(7-chloro-1,2,3,4-tetrahydroisoquinolin-3-
yl)methyl]amino-propyl}thio-adenosine (21). This compound
was prepared in a similar manner as 17 using the procedure outlined
previously, but using 66 (270 mg, 0.410 mmol) as the starting
material to afford 21 (130 mg, 61.0%) as white foam: 1H NMR (400
MHz, CD3OD): δ 8.32 (s, 1H), 8.21 (s, 1H), 7.10 (m, 3H), 5.99 (d, J
= 5.0 Hz, 1H), 4.78 (t, J = 5.0 Hz, 1H), 4.33 (t, J = 5.0 Hz, 1H), 4.23
(m, 1H), 3.97 (s, 2H), 2.95 (m, 3H), 2.79−2.45 (m, 8H), 1.79
(quintet, J = 7.1 Hz, 2H); 13C NMR (125.7 MHz, CD3OD): δ 155.9,
152.4, 149.2, 139.9, 136.8, 132.6, 131.0, 130.2, 125.9, 125.5, 119.1,
88.7, 84.3, 73.4, 72.5, 53.8, 52.6, 48.4, 48.1, 33.8, 31.9, 30.1, 28.8; MS
(CI) m/z (relative intensity): 520 (MH+, 45), 271 (20), 239 (15),
136 (80); HRMS (ESI+) m/z: calcd for C23H31ClN7O3S (MH+),
520.1898; obsd, 520.1892; Anal. Calcd for C23H30ClN7O3S·MeOH:
C, 52.21; H, 6.21; N, 17.76. Found: C, 52.36; H, 5.91; N, 17.55.
5′-Deoxy-5′-{3-[(7-nitro-1,2,3,4-tetrahydroisoquinolin-3-yl)-
methyl]amino-propyl}thio-adenosine (22). This compound was
prepared in a similar manner as 17 using the procedure outlined
previously, but using 67 (400 mg, 0.600 mmol) as the starting
material to afford 22 (210 mg, 66%) as slightly yellow foam: 1H NMR
(400 MHz, CDCl3/CD3OD, 10:1): δ 8.30 (s, 1H), 8.23 (s, 1H), 7.99
(m, 2H), 7.32 (d, J = 8.3 Hz, 1H), 6.02 (d, J = 4.6 Hz, 1H), 4.76 (t, J
= 4.6 Hz, 1H), 4.36 (t, J = 5.1 Hz, 1H), 4.27 (m, 1H), 4.12 (m, 2H),
2.98 (m, 4H), 2.71 (m, 7H), 1.82 (quintet, J = 7.1 Hz, 2H); 13C
NMR (100 MHz, CDCl3/CD3OD, 10:1): δ 156.2, 152.9, 149.6,
146.5, 142.6, 140.2, 137.1, 130.4, 121.4, 121.3, 119.6, 89.3, 84.4, 74.1,
72.9, 54.2, 52.7, 34.4, 33.2, 30.7, 29.3; MS (CI) m/z (relative
intensity): 531 (MH+, 40), 145 (20), 79 (70); HRMS (ESI+) m/z:
calcd for C23H31N8O5S (MH+), 531.2138; obsd, 531.2132; Anal.
Calcd for C23H30N8O5S·1/2CHCl3: C, 47.82; H, 5.21; N, 18.98.
Found: C, 47.85; H, 5.17; N, 18.82.
5′-Deoxy-5′-{2-[(1,2,3,4-tetrahydroisoquinolin-3-yl)-
methyl]amino-ethyl}thio-adenosine (17). A solution of 48 (500
mg, 0.820 mmol) in TFA/H2O (9:1, 9 mL) was stirred at room
temperature for 1 h and then the solution was evaporated under
vacuum. The residue was dissolved in MeOH (5 mL), Na2CO3
solution (sat, 5 mL) was added to the solution and the mixture was
stirred for 10 min. The solvent was evaporated under reduced
pressure. The resulting brown residue was purified by flash
chromatography (silica gel) eluting with CHCl3/MeOH/NH3OH/
EtOAc/hexane (10:1:0.1:0.1:0.1) to yield 17 (300 mg, 78%) as a
white foam: 1H NMR (500 MHz, CDCl3/CD3OD, 10:1): δ 8.30 (d, J
= 3.3 Hz, 1H), 8.21 (s, 1H), 7.10−7.02 (m, 4H), 6.02 (d, J = 4.7 Hz,
1H), 4.79 (t, J = 5.0 Hz, 1H), 4.36 (t, J = 5.0 Hz, 1H), 4.24 (d, J = 5.2
Hz, 1H), 3.98 (br s, 2H), 3.01 (m, 3H), 2.80−2.51 (m, 8H); 13C
NMR (125.7 MHz, CDCl3/CD3OD, 10:1): δ 155.9, 152.5, 149.2,
139.9, 134.4, 133.6, 128.7, 125.9, 125.7, 125.5, 119.1, 88.7, 84.3, 73.4,
72.6, 53.8, 52.9, 48.3, 48.2, 33.7, 32.4, 32.0; MS (CI) m/z (relative
intensity): 472 (MH+, 25), 284 (40), 189 (50), 79 (80); HRMS
(FAB+) m/z: calcd for C22H30N7O3S (MH+), 472.2131; obsd,
472.2152; Anal. Calcd for C22H29N7O3S·1/2CHCl3: C, 50.87; H,
5.60; N, 18.46. Found: C, 50.93; H, 5.51; N, 18.14.
5′-{2-[(7-Chloro-1,2,3,4-tetrahydroisoquinolin-3-yl)methyl]-
amino-ethyl}thio-5′-deoxy-adenosine (18). This compound was
prepared in a similar manner as 17 but using 49 (1.30 g, 2.01 mmol)
as the starting material to afford 18 (370 mg, 36%) as white foam: 1H
NMR (500 MHz, CD3OD): δ 8.32 (d, J = 0.8 Hz, 1H), 8.22 (s, 1H),
7.10−7.02 (m, 3H), 6.03 (d, J = 4.7 Hz, 1H), 4.80 (t, J = 5.0 Hz, 1H),
4.37 (t, J = 5.0 Hz, 1H), 4.24 (d, J = 5.3 Hz, 1H), 3.93 (m, 2H), 2.98
(m, 2H), 2.86 (m, 1H), 2.84−2.66 (m, 6H), 2.43 (m, 2H); 13C NMR
(125.7 MHz, CD3OD): δ 155.8, 152.5, 149.2, 139.9, 136.8, 132.6,
130.9, 130.2, 125.9, 125.5, 119.1, 88.7, 84.3, 73.4, 72.6, 53.3, 52.7,
48.4, 48.3, 33.7, 32.0, 31.8; MS (CI) m/z (relative intensity): 506
(MH+, 50), 257 (10), 166 (50), 136 (70); HRMS (FAB+) m/z: calcd
for C22H29ClN7O3S (MH+), 506.1741; obsd, 506.1735; Anal. Calcd
for C22H28ClN7O3S·2/3H2O: C, 51.01; H, 5.71; N, 18.93. Found: C,
51.09; H, 5.46; N, 18.61.
5′-Deoxy-5′-{4-[(1,2,3,4-tetrahydroisoquinolin-3-yl)-
methyl]amino-butyl}thio-adenosine (23). This compound was
prepared in a similar manner as 17 using the procedure outlined
previously, but using 68 (450 mg, 0.700 mmol) as the starting
1
material to afford 23 (240 mg, 68%) as white foam: H NMR (400
MHz, CDCl3/CD3OD, 10:1): δ 8.29 (s, 1H), 8.24 (s, 1H), 7.11 (m,
3H), 7.04 (m, 1H), 6.02 (s, 1H), 4.76 (m, 1H), 4.36 (m, 1H), 4.25
(m, 1H), 4.02 (s, 2H), 3.02 (m, 2H), 2.99 (m, 1H), 2.77 (m, 2H),
5′-Deoxy-5′-{2-[(7-nitro-1,2,3,4-tetrahydroisoquinolin-3-yl)-
methyl]amino-ethyl}thio-adenosine (19). This compound was
K
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Article
2.61 (m, 6H), 1.61 (m, 4H); 13C NMR (100 MHz, CDCl3/CD3OD,
10:1): δ 156.3, 152.9, 149.6, 140.2, 134.9, 134.1, 129.2, 126.5, 126.3,
126.1, 119.6, 89.2, 84.6, 74.1, 72.9, 54.6, 53.4, 49.5, 48.8, 34.3, 33.0,
32.9, 28.7, 27.5; MS (CI) m/z (relative intensity): 500 (MH+, 50),
251 (20), 164 (25), 136 (70); HRMS (ESI+) m/z: calcd for
C24H34N7O3S (MH+), 500.2444; obsd, 500.2439; Anal. Calcd for
C24H33N7O3S·1/3(CHCl3·H2O): C, 53.58; H, 6.28; N, 17.98. Found:
C, 53.75; H, 6.26; N, 17.71.
5′-Deoxy-5′-{5-[(1,2,3,4-tetrahydroisoquinolin-3-yl)-
methyl]amino-pentyl}thio-adenosine (24). This compound was
prepared in a similar manner as 17 using the procedure outlined
previously, but using 69 (0.75 g, 1.2 mmol) as the starting material to
afford 24 (0.44 g, 75%) as slightly yellow foam: 1H NMR (500 MHz,
CDCl3): δ 8.20 (s, 1H), 8.08 (s, 1H), 7.08 (br s, 2H, NH2), 7.00 (m,
4H), 6.02 (s, 1H), 4.72 (m, 1H), 4.34 (m, 1H), 4.29 (m, 1H), 4.00 (s,
2H), 3.02 (m, 1H), 2.87−2.48 (m, 10H), 1.47−1.28 (m, 6H); 13C
NMR (125.7 MHz, CDCl3): δ 155.9, 152.9, 149.4, 139.6, 135.3,
134.1, 129.4, 126.5, 126.3, 126.1, 119.8, 89.4, 84.4, 74.4, 73.0, 54.6,
53.2, 49.8, 48.0, 34.8, 33.2, 29.5, 29.2, 26.4; MS (ESI+) m/z (relative
intensity): 514 (MH+, 30); HRMS (ESI+) m/z: calcd for
C25H36N7O3S (MH+), 514.2600; obsd, 514.2614; HPLC analysis of
the diastereomeric product mixture observed a single peak at 11.2 min
(System A, 250 nm, purity 96%), and at 14.2 min (System B, 240 nm,
96%).
5′-Deoxy-5′-{6-[(1,2,3,4-tetrahydroisoquinolin-3-yl)-
methyl]amino-hexyl}thio-adenosine (25). This compound was
prepared in a similar manner as 17 using the procedure outlined
previously, but using 70 (0.70 g, 1.0 mmol) as the starting material to
afford 25 (0.24 g, 43%) as slightly yellow foam: 1H NMR (400 MHz,
CDCl3): δ 8.17 (s, 1H), 8.03 (s, 1H), 7.05 (br ex s, 2H, NH2), 7.00
(m, 4H), 6.02 (s, 1H), 4.69 (m, 1H), 4.27 (m, 2H), 3.97 (m, 2H),
2.97 (m, 1H), 2.86−2.45 (m, 10H), 1.40 (m, 4H), 1.20 (m, 4H); 13C
NMR (100 MHz, CDCl3): δ 156.0, 153.0, 149.4, 139.5, 135.5, 134.2,
129.4, 126.4, 126.3, 126.1, 119.8, 89.3, 84.2, 74.4, 73.0, 54.9, 53.4,
50.1, 48.1, 34.7, 33.2, 31.2, 29.8, 29.6, 28.7, 26.9; MS (ESI+) m/z
(relative intensity): 528 (MH+, 20), 405 (50); HRMS (ESI+) m/z:
calcd for C26H38N7O3S (MH+), 528.2757; obsd, 528.2759; HPLC
analysis of the diastereomeric product mixture observed a single peak
at 15.6 min (System A, 255 nm, purity 97%), and at 15.7 min (System
B, 250 nm, purity 99%).
CD3OD, 10:1): δ 8.45 (s, 1H), 8.43 (s, 1H), 7.31 (m, 1H), 7.26 (m,
2H), 6.21 (d, J = 4.6 Hz, 1H), 4.94 (t, J = 5.1 Hz, 1H), 4.54 (t, J = 4.9
Hz, 1H), 4.45 (quartet, J = 5.4 Hz, 1H), 4.18−4.13 (m, 2H), 3.72
(dd, J = 5.4, 10.2 Hz, 1H), 3.64 (t, J = 6.6 Hz, 2H), 3.23−3.18 (m,
2H), 3.16−3.00 (m, 2H), 2.94 (m, 2H); 13C NMR (125.7 MHz,
CDCl3/CD3OD, 10:1): δ 173.8, 155.8, 152.5, 149.1, 139.7, 137.0,
132.2, 131.4, 130.0, 126.2, 125.4, 119.3, 88.9, 84.0, 73.7, 72.4, 56.0,
46.2, 38.4, 33.8, 31.9, 30.5; MS (FAB+) m/z (relative intensity): 520
(MH+, 29), 329 (80), 176 (100), 154 (100); HRMS (FAB+) m/z:
calcd for C22H27ClN7O4S (MH+), 520.1534; obsd, 520.1531; Anal.
Calcd for C22H26ClN7O4S·2/3CHCl3: C, 45.40; H, 4.48; N, 16.35.
Found: C, 45.62; H, 4.53; N, 16.36.
5′-Deoxy-5′-[2-(7-nitro-1,2,3,4-tetrahydroisoquinoline-3-
carboxamido)ethyl]thio-adenosine (28). This compound was
prepared in a similar manner as 26 but using 73 (0.70 g, 1.0 mmol) as
1
the starting material to afford 28 (220 mg, 40%) as yellow foam: H
NMR (400 MHz, DMSO-d6): δ 8.37 (s, 1H), 8.15 (s, 1H), 8.10 (ex
m, 1H), 7.97 (m, 2H), 7.39 (d, J = 8.8 Hz, 1H), 7.31 (ex s, 1H), 5.89
(d, J = 5.8 Hz, 1H), 5.53−5.35 (ex m, 1H), 4.75 (t, J = 5.0 Hz, 1H),
4.14 (m, 1H), 4.05−3.91 (m, 2H), 4.01 (m, 1H), 3.46 (quartet, J =
4.7 Hz, 1H), 3.34 (br ex m, 2H), 3.26 (m, 2H), 3.06−2.83 (m, 4H),
2.61 (t, J = 6.6 Hz, 2H); 13C NMR (100 MHz, DMSO-d6): δ 172.9,
156.9, 153.5, 150.3, 146.3, 144.1, 140.7, 139.0, 131.1, 121.5, 121.4,
120.0, 88.1, 84.6, 73.4, 73.3, 55.9, 47.1, 39.1, 34.1, 32.1, 31.7; MS
(ESI+) m/z (relative intensity): 531 (MH+, 100), 396 (15), 266 (10);
HRMS (ESI+) m/z: calcd for C22H27N8O6S (MH+), 531.1774; obsd,
531.1782; HPLC analysis of the diastereomeric product mixture
observed a single peak at 11.1 min (System A, 289 nm, purity 95%),
and at 13.1 min (System B, 300 nm, purity 96%).
5′-Deoxy-5′-[3-(1,2,3,4-tetrahydroisoquinoline-3-carboxa-
mido)-propyl]thio-adenosine (29). This compound was prepared
in a similar manner as 26 using the procedures outlined previously,
but using 74 (0.70 g, 1.1 mmol) as the starting material to afford 29
(300 mg, 55%) as white foam: 1H NMR (500 MHz, CD3OD): δ 8.26
(s, 1H), 8.15 (s, 1H), 7.05 (m, 3H), 6.97 (t, J = 4.2 Hz, 1H), 5.96 (d,
J = 4.9 Hz, 1H), 4.73 (m, 1H), 4.30 (t, J = 5.0 Hz, 1H), 4.17 (q, J =
5.0 Hz, 1H), 3.92 (q, J = 10.3 Hz, 2H), 3.44 (dd, J = 4.7, 10.5 Hz,
1H), 3.25 (m, 2H), 2.90 (m, 4H), 2.54 (t, J = 7.1 Hz, 2H), 1.73
(quintet, J = 7.1 Hz, 2H); 13C NMR (125.7 MHz, CD3OD): δ 174.0,
155.8, 152.4, 149.2, 139.9, 134.7, 133.4, 128.5, 125.9, 125.8, 125.4,
119.1, 88.6, 84.2, 73.5, 72.5, 56.3, 46.4, 37.7, 33.8, 31.2, 29.6, 28.9;
MS (CI) m/z (relative intensity): 500 (MH+, 50), 219 (10), 136
(70); HRMS (ESI+) m/z: calcd for C23H30N7O4S (MH+), 500.2080;
obsd, 500.2082; Anal. Calcd for C23H29N7O4S·H2O: C, 53.37; H,
6.04; N, 18.94. Found: C, 53.29; H, 5.68; N, 18.66.
5′-Deoxy-5′-[3-(7-nitro-1,2,3,4-tetrahydroisoquinoline-3-
carboxamido)propyl]thio-adenosine (30). This compound was
prepared in a similar manner as 26 using the procedure outlined
previously, but using 75 (0.90 g, 1.3 mmol) as the starting material to
afford 30 (470 mg, 66%) as white foam: 1H NMR (400 MHz,
CDCl3/CD3OD, 10:1): δ 8.28 (s, 1H), 8.17 (s, 1H), 7.96 (d, J = 8.3
Hz, 1H), 7.94 (s, 1H), 7.29 (d, J = 8.3 Hz, 1H), 5.98 (d, J = 4.8 Hz,
1H), 4.73 (t, J = 5.0 Hz, 1H), 4.32 (t, J = 5.0 Hz, 1H), 4.21 (quartet, J
= 5.1 Hz, 1H), 4.08 (m, 2H), 3.53 (m, 1H), 3.29 (m, 2H), 3.06−2.87
(m, 4H), 2.59 (t, J = 7.2 Hz, 2H), 1.77 (quintet, J = 7.0 Hz, 2H); 13C
NMR (100 MHz, CDCl3/CD3OD, 10:1): δ 173.8, 156.3, 152.9,
149.6, 146.7, 142.2, 140.2, 137.3, 130.2, 121.2, 121.0, 119.6, 89.1,
84.6, 74.0, 72.9, 56.0, 46.7, 38.3, 34.3, 31.6, 30.1, 29.3; (FAB+) m/z
(relative intensity): 545 (MH+, 45), 460 (15), 307 (55), 242 (20),
154 (100), 136 (85); HRMS (FAB+) m/z: calcd for C23H29N8O6S
(MH+), 545.1931; obsd, 545.1912; HPLC analysis of the diastereo-
meric product mixture observed a single peak at 11.1 min (System A,
280 nm, purity 96%), and at 14.2 min (System B, 280 nm, purity
98%).
5′-Deoxy-5′-[2-(1,2,3,4-tetrahydro-isoquinoline-3-
carboxamido)ethyl]thio-adenosine (26). Compound 71 (4.40 g,
7.04 mmol) was dissolved in H2O/HCOOH (1:4, 20 mL). The
solution was stirred at the room temperature for 40 h. The H2O and
HCOOH were removed under reduced pressure, the residue was
mixed with CH3OH (10 mL), and NH3·H2O was added until pH =
12. The solvents were removed under reduced pressure and the
residue was purified by flash chromatography (silica gel) with CHCl3/
MeOH (10:1) as eluent to yield 26 as white foam (2.60 g, 76%): IR
(KBr) 3421, 2914, 1650, 1598, 1475, 1424, 1327, 1250, 1132, 748
1
cm−1; H NMR (400 MHz, CD3OD): δ 8.32 (s, 1H), 8.21 (s, 1H),
7.13−7.02 (m, 4H), 6.03 (d, J = 5.0 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H),
4.35 (t, J = 4.9 Hz, 1H), 4.25−4.21 (m, 1H), 4.04−3.94 (m, 2H),
3.53 (dd, J = 4.7, 10.5 Hz, 1H), 3.42 (m, 2H), 3.02−2.86 (m, 4H),
2.74−2.70 (m, 2H); 13C NMR (100 MHz, CD3OD): δ 174.4, 156.3,
152.9, 149.7, 140.4, 135.2, 133.8, 129.0, 126.5, 126.2, 125.9, 119.6,
89.1, 84.9, 73.9, 73.0, 56.7, 46.8, 38.9, 34.1, 32.1, 31.5; MS (CI) m/z
(relative intensity): 486 (MH+, 60), 237 (15), 164 (80), 132 (80);
HRMS (FAB+) m/z: calcd for C22H28N7O4S (MH+), 486.1923; obsd,
486.1926; Anal. Calcd for C22H27N7O4S·1/2H2O: C, 53.43; H, 5.71;
N, 19.82. Found: C, 53.19; H, 5.51; N, 19.52.
5′-[2-(7-Chloro-1,2,3,4-tetrahydro-isoquinoline-3-
carboxamido)ethyl]thio-5′-deoxy-adenosine (27). A solution of
72 (200 mg, 0.300 mmol) in TFA/H2O (9:1, 3 mL) was stirred at
room temperature for 2 h, and the solvents were evaporated under the
vacuum. The residue was dissolved in MeOH (5 mL), and NaOH
(10%) was added until pH = 12. The solvents were removed under
reduced pressure. The resulting brown residue was purified by flash
chromatography (silica gel) eluting with CHCl3/MeOH (20:1) to
5′-Deoxy-5′-{2-[(1,2,3,4-tetrahydroisoquinolin-4-yl)-
methyl]amino-ethyl}thio-adenosine (31). A solution of 80
(0.930 g, 1.49 mmol) in TFA/H2O (9:1, 18 mL) was stirred at
room temperature for 1 h and then the solution was evaporated under
vacuum. The residue was dissolved in MeOH (20 mL). Na2CO3
solution (sat, 7 mL) was added to the solution and the mixture was
1
yield 27 (80 mg, 51%) as white foam: H NMR (500 MHz, CDCl3/
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stirred for 10 min. The solvent was evaporated under reduced
pressure. The resulting brown residue was purified by flash
chromatography (silica gel) eluting with CHCl3/MeOH/NH4OH/
EtOAc/hexanes (10:1:0.1:0.1:0.1) to yield 31 (0.60 g, 86%) as white
( )-Diethyl 7-Chloro-3,4-dihydro-2,3(1H)-isoquinolinedi-
carboxylate (37). To a solution of carbamate 36 (2.30 g, 7.69
mmol) in AcOH (9 mL)/H2SO4 (3 mL) was added paraformalde-
hyde (240 mg). After stirring overnight, the mixture was poured into
ice water (50 mL). The resulting solution was extracted with EtOAc
(3 × 20 mL). The combined organic extracts were washed with
sodium bicarbonate (sat, 30 mL) and brine (30 mL) and dried over
anhydrous Na2SO4. The solvent was removed under reduced pressure
1
foam: H NMR (500 MHz, CD3OD): δ 8.27 (s, 1H), 8.20 (s, 1H),
7.64−7.36 (m, 1H), 7.31 (m, 2H), 7.15 (dd, J = 7.6, 25.4 Hz, 1H),
5.98 (m, 1H), 4.79 (m, 1H), 4.31 (m, 1H), 4.20 (m, 2H), 4.14 (m,
1H), 3.57 (m, 1H), 3.34−3.19 (m, 2H), 3.14 (m, 2H), 2.93−2.58 (m,
6H); 13C NMR (125.7 MHz, CD3OD): δ 155.9, 152.5, 149.1, 140.0,
137.1, 130.9, 128.4, 128.2, 127.9, 125.9, 119.1, 88.9, 84.3, 73.1, 72.7,
66.6, 58.4, 54.0, 52.1, 45.6, 33.8, 30.7; MS (CI) m/z (relative
intensity): 472 (MH+, 60), 339 (25), 204 (30), 144 (80), 115 (40);
HRMS (FAB+) m/z: calcd for C22H30N7O3S (MH+), 472.2112; obsd,
472.2112; HPLC analysis of the diastereomeric product mixture
observed two partially resolved peaks at 9.3 and 9.7 min for System A
(251 nm, combined purity 95%), and at 10.7 and 10.9 min for System
B (250 nm, combined purity 99%).
5′-Deoxy-5′-{3-[(1,2,3,4-tetrahydroisoquinolin-4-yl)-
methyl]amino-propyl}thio-adenosine (32). This compound was
prepared in a similar manner as 31 using the procedures outlined
previously but using 81 (1.10 g, 1.76 mmol) as the starting material to
afford 32 (0.65 g, 74%) as white foam: 1H NMR (500 MHz,
CD3OD): δ 8.30 (s, 1H), 8.19 (s, 1H), 7.45−7.15 (m, 1H), 7.17 (m,
2H), 7.00 (m, 1H), 5.99 (d, J = 5.0 Hz, 1H), 4.78 (t, J = 5.0 Hz, 1H),
4.33 (t, J = 5.0 Hz, 1H), 4.20 (q, J = 5.0 Hz, 1H), 3.90 (d, J = 10.0 Hz,
2H), 3.15−3.02 (m, 2H), 2.96−2.79 (m, 5H), 2.63−2.54 (m, 4H),
1.76−1.67 (m, 2H); 13C NMR (125.7 MHz, CD3OD): δ 157.4,
154.0, 150.8, 141.5, 137.6, 136.8, 129.9, 127.6, 127.4, 127.3, 120.6,
90.2, 85.8, 74.9, 74.0, 70.1, 59.7, 55.3, 47.2, 37.9, 35.4, 31.8, 30.3; MS
(CI) m/z (relative intensity): 486 (MH+, 60), 237 (20), 136 (80);
HRMS (ESI+) m/z: calcd for C23H32N7O3S (MH+), 486.2287; obsd,
486.2292; Anal. Calcd for C23H31N7O3S·1/2CHCl3: C, 51.76; H,
5.82; N, 17.98. Found: C, 52.08; H, 5.92; N, 17.75.
1
to afford 37 as colorless oil (1.66 g, 69%): H NMR (500 MHz,
CDCl3): δ 7.02−6.95 (m, 3H), 5.05−4.83 (m, 1H), 4.63−4.42 (m,
2H), 4.14−3.92 (m, 4H), 3.14−2.96 (m, 2H), 1.24−1.13 (m, 3H),
1.01 (t, J = 7.1 Hz, 3H); 13C NMR (125.7 MHz, CDCl3): δ (170.8,
170.6), (155.9, 155.3), (134.9, 134.3), (132.2, 132.1), (130.2, 130.1),
(129.3, 128.6), (126.7, 126.6), (126.0, 125.9), (62.1, 61.8), 61.0,
(53.3, 52.6), (43.9, 43.8), (30.8, 30.5), (14.5, 14.4), 13.9; MS (EI) m/
z (relative intensity): 311 (M+, 60), 238 (100), 164 (40), 103 (25);
HRMS (ESI+) m/z: calcd for C15H18ClNO4 (M+) 311.0924; obsd,
311.0918.
(
)-7-Chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
Acid Hydrochloride (38·HCl). A solution of 37 (500 mg, 1.61
mmol) in HCl (6 N, 20 mL) was heated at reflux for 40 h. The
reaction mixture was cooled to room temperature, Et2O (20 mL) was
added, and the mixture was filtered to afford 38·HCl (490 mg, 48%)
1
as a white solid: mp ≥ 285 °C; H NMR (500 MHz, DMSO-d6): δ
7.41 (s, 1H), 7.35−7.30 (m, 2H), 4.40 (dd, J = 4.9, 11.0 Hz, 1H),
4.33 (m, 2H), 3.34−3.10 (m, 2H); 13C NMR (125.7 MHz, DMSO-
d6): δ 170.0, 131.6, 131.2, 131.0, 130.3, 127.8, 126.6, 53.2, 43.7, 27.9;
MS (CI) m/z (relative intensity): 212 (MH+, 90), 166 (75); HRMS
(FAB+) m/z: calcd for C10H11ClNO2 (MH+), 212.0478; obsd,
212.0464.
(
)-7-Chloro-3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinoline-3-carboxylic Acid (39). A mixture of 38·
HCl (200 mg, 0.810 mmol) and CH2Cl2 (5 mL) was cooled to 0 °C
and Et3N (326 mg, 3.20 mmol) was added slowly. A solution of
(Boc)2O (352 mg, 1.61 mmol) in CH2Cl2 (1 mL) was then added
over 10 min. The mixture was stirred at room temperature overnight.
Aqueous citric acid (sat, 6 mL) was added and the mixture was stirred
for 5 min. The organic phase was washed with brine (2 × 5 mL) and
dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure and the residue was purified by flash chromatog-
raphy (silica gel) eluting with CHCl3/MeOH (20:1) to yield 39 as a
5′-(2-Aminoethyl)thio-5′-deoxy-2′,3′-O-(1-methylethyli-
dene)-adenosine (34). Sodium (2.80 g, 123 mmol) was dissolved in
EtOH (200 mL) at 0 °C, and the mixture was stirred for 0.5 h. 2-
Aminoethylthiol·HCl salt (7.00 g, 61.6 mmol) was added slowly into
the sodium ethoxide solution at 0 °C and the mixture was allowed to
warm up to room temperature, and then stirred for 20 min.
Compound 33 (10.0 g, 30.6 mmol) was added and the mixture was
refluxed for 8 h. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography (silica gel) eluting
with CHCl3/MeOH/NH4OH (10:1:0.1) to yield 34 as yellowish
1
white solid (140 mg, 56%): mp 103−105 °C; H NMR (400 MHz,
CDCl3): δ 7.14−7.02 (m, 3H), 5.15−4.78 (m, 1H), 4.67−4.41 (m,
2H), 3.24−3.06 (m, 2H), 1.51, 1.42 (two peaks, 9H); 13C NMR (100
MHz, CDCl3): δ (177.4, 176.9), (155.9, 155.2), (135.8, 134.8),
(132.9, 132.8), (130.7, 130.5), (130.3, 129.7), (127.5, 127.3), (126.8,
126.6), 81.7, (54.2, 52.5), (44.6, 44.0), (31.2, 30.8), 28.8, 28.6, 28.4;
MS (CI) m/z (relative intensity): 312 (MH+, 60), 212 (80), 166
(80), 84 (25); HRMS (FAB+) m/z: calcd for C15H18ClNO4 (M+)
311.0924; obsd, 311.0888.
1
foam (11.0 g, 98%): H NMR (400 MHz, CD3OD): δ 8.29 (s, 1H),
8.24 (s, 1H), 6.20 (d, J = 2.3 Hz, 1H), 5.53 (dd, J = 2.4, 6.4 Hz, 1H),
5.08 (dd, J = 3.0, 6.4 Hz, 1H), 4.34 (m, 1H), 2.81 (m, 2H), 2.70 (m,
2H), 2.60 (m, 2H), 1.60 (s, 3H), 1.21 (s, 3H); 13C NMR (100 MHz,
CD3OD): δ 156.4, 153.0, 149.2, 140.9, 119.6, 114.5, 90.7, 87.2, 84.2,
84.1, 40.7, 35.4, 33.9, 26.4, 24.5; MS (CI) m/z (relative intensity):
367 (MH+, 70), 232 (35), 164 (75), 136 (80), 44 (45); HRMS
(FAB+) m/z: calcd for C15H23N6O3S (MH+), 367.1552; obsd,
367.1571.
(
)-3,4-Dihydro-2(1H)-(1,1-dimethylethoxy)carbonyl-7-
nitro-isoquinoline-3-carboxylic Acid (40). THIQ-3-carboxylic
acid hydrochloride (15·HCl, 2.00 g, 9.36 mmol) was dissolved in
concentrated H2SO4 (8 mL) at 0 °C. KNO3 (1.04 g, 10.3 mmol) was
added in small portions over 30 min. The reaction mixture was stirred
at room temperature for 8 h and then was poured onto ice (200 g).
NH3·H2O was added to the mixture until pH = 12. The water was
removed under reduced pressure to yield a yellow residue. The
residue was mixed with CH2Cl2 (100 mL) and the mixture was cooled
to 0 °C. Et3N (1.40 g, 13.8 mmol) was added slowly. A solution of
(Boc)2O (3.06 g, 14.0 mmol) in CH2Cl2 (10 mL) was then added
over 10 min. The mixture was warmed to room temperature and
stirred overnight. Aqueous citric acid (sat, 20 mL) was added and the
mixture was stirred for 5 min. The organic phase was washed with
brine (2 × 40 mL) and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure and the residue was purified by
chromatography on a silica gel column eluting with CHCl3/MeOH
(30:1) to yield yellow foam (1.70 g), which was purified by the
reported procedures to afford regiopure 40.75
(
)-4-Chloro-N-(ethoxycarbonyl)-phenylalanine, Ethyl
Ester (36).95 4-Chlorophenylalanine ethyl ester hydrochloride 35
(2.00 g, 7.57 mmol) was mixed with pyridine (2.27 g, 28.8 mmol) and
CHCl3 (100 mL) at 0 °C. Ethyl chloroformate (0.990 g, 9.08 mmol)
was added dropwise and the suspension was warmed to room
temperature. The mixture was stirred for 8 h. Ice water (80 mL) was
added and the mixture was stirred for 10 min. The phases were
separated, and the aqueous phase was extracted with CHCl3 (2 × 40
mL). The organic phases were combined, washed with HCl (3 N, 3 ×
30 mL) and brine (2 × 30 mL), and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure to afford 36 as
colorless oil (2.25 g, 99%): 1H NMR (400 MHz, CDCl3): δ 7.26 (d, J
= 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 5.32 (br s, 1H), 4.58 (m,
1H), 4.17−4.05 (m, 4H), 3.18−2.99 (m, 2H), 1.23 (m, 6H); 13C
NMR (100 MHz, CDCl3): δ 171.8, 156.3, 134.9, 133.3, 131.1, 128.9,
61.9, 61.5, 55.0, 38.0, 14.9, 14.6; MS (FAB+) m/z (relative intensity):
300 (MH+, 75), 226 (100), 154 (30); HRMS (ESI+) m/z: calcd for
C14H19ClNO4 (MH+), 300.1003; obsd, 300.1003.
M
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Journal of Medicinal Chemistry
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Article
( )-1,1-Dimethylethyl 3,4-Dihydro-3-(N-methoxy-N-meth-
ylcarbamoyl)-isoquinoline-2(1H)-carboxylate (42).78 Weinreb
amide 42 was prepared by following the literature procedure.16 BOP
(3.67 g, 8.30 mmol) was added to a stirred solution of 41 (2.30 g,
8.30 mmol) and Et3N (1.16 mL, 8.30 mmol) in CH2Cl2 (70 mL).
After five minutes, N,N-dimethylhydroxylamine hydrochloride (972
mg, 9.96 mmol) and Et3N (1.34 mL, 9.96 mmol) were added to the
solution. The reaction was stirred for 24 h at room temperature. The
mixture was diluted with CH2Cl2 (70 mL), washed with HCl (3 N, 2
× 50 mL), aqueous NaHCO3 (sat, 50 mL), and brine (50 mL), and
dried over anhydrous Na2SO4. The solvent was evaporated under
reduced pressure and the resulting crude product was purified by
chromatography on a silica gel column eluting with EtOAc/hexanes
(1:2) to yield 42 (2.21 g, 83%) as colorless crystals: mp 128−130 °C;
1H NMR (400 MHz, CDCl3): δ 7.20−7.14 (m, 4H), 5.24−4.47 (m,
peaks, 1H), 7.21−7.11 (m, 4H), 4.74−4.57 (m, 3H), 3.12−3.10 (m,
2H), 1.55−1.47 (m, 9H).78,96
(
)-1,1-Dimethylethyl 7-Chloro-3,4-dihydro-3-formyl-iso-
quinoline-2(1H)-carboxylate (46). This compound was prepared
in a similar manner as 45 but using 43 (1.70 g, 4.79 mmol) as the
starting material to afford 46 (1.25 g, 88%) as colorless oil: IR (KBr)
3436, 3058, 2971, 2925, 2802, 2704, 2249, 1736, 1690, 1598, 1490,
1
1388, 1250, 1158, 1024, 866, 814, 732 cm−1; H NMR (500 MHz,
CDCl3): δ 9.53, 9.49 (two peaks, 1H), 7.18−7.12 (m, 3H), 4.87−4.49
(m, 3H), 3.22−3.04 (m, 2H), 1.55, 1.48 (two peaks, 9H); 13C NMR
(100 MHz, CDCl3): δ (201.1, 200.5), 155.0, (136.1, 135.1), (133.2,
133.0), (131.1, 130.7), (130.3, 129.6), (127.9, 127.6), (126.9, 126.7),
(81.9, 81.7), (60.6, 59.3), (45.0, 44.4), (29.0, 28.0), (28.8, 28.7); MS
(ESI+) m/z (relative intensity): 296 (MH+, 100), 240 (35), 196 (30);
HRMS (ESI+) m/z: calcd for C15H19ClNO3 (MH+), 296.1053; obsd,
296.1039.
3H), 3.85, 3.78 (two peaks, 3H), 3.19, 3.16 (two peaks, 3H), 3.08−
2.97 (m, 2H), 1.52, 1.46 (two peaks, 9H); 13C NMR (100 MHz,
CDCl3): δ (173.4, 172.9), (155.7, 155.1), (135.9, 134.9), (133.4,
132.5), (128.3, 127.7), (127.4, 127.2), (127.1, 127.0), (126.4, 126.2),
(80.8, 80.6), (61.9, 61.7), (53.2, 51.1), (45.8, 44.9), (32.8, 32.5),
(31.6, 31.4), 28.9.
( )-1,1-Dimethylethyl 3,4-Dihydro-3-formyl-7-nitro-isoqui-
noline-2(1H)-carboxylate (47). Weinreb amide 44 (300 mg, 0.820
mmol) was mixed with Cp2Zr(H)Cl (Schwartz reagent) (0.630 g,
2.47 mmol) and THF (15 mL). The mixture was stirred at room
temperature for 1 h. Water (0.1 mL) was added to the reaction and
the solvent was removed under reduced pressure. The residue was
purified by chromatography on a silica gel column eluting with
hexanes/EtOAc (4:1) to yield aldehyde 47 (140 mg, 56%) as slightly
yellow oil: IR (KBr) 3012, 2976, 2919, 2858, 1737, 1701, 1521, 1393,
1352, 1163, 892, 856, 738 cm−1; 1H NMR (500 MHz, CDCl3): δ 9.55
(d, J = 12.0 Hz, 1H), 8.07 (m, 2H), 7.38 (d, J = 8.5 Hz, 1H), 4.99−
( )-1,1-Dimethylethyl 7-Chloro-3,4-dihydro-3-(N-methoxy-
N-methylcarbamoyl)-isoquinoline-2(1H)-carboxylate (43).
This compound was synthesized using the same procedure as 42
except 39 (1.90 g, 6.11 mmol) was used as the starting material to
afford 43 (1.77 g, 82%) as a white solid: mp 175−177 °C; IR (KBr)
3011, 2975, 2929, 1690, 1670, 1486, 1393, 1168, 1004, 861, 763
1
4.61 (m, 3H), 3.38−3.16 (m, 2H), 1.56, 1.50 (two peaks, 9H); H
1
cm−1; H NMR (500 MHz, CDCl3): δ 7.15−7.05 (m, 3H), 5.28−
NMR (500 MHz, CDCl3): δ (199.7, 199.3), (155.1, 154.7), (146.9,
146.8), (139.7, 139.5), 134.3, (129.6, 129.0), (122.3, 122.0), 121.5,
(81.8, 81.7), (59.6, 58.2), (44.7, 44.0), (28.8, 27.9), 28.2; MS (ESI+)
m/z (% relative intensity): 307 (MH+, 95), 294 (50), 265 (80), 189
(45); HRMS (ESI+) m/z: calcd for C15H19N2O5 (MH+), 307.1294;
obsd, 307.1288.
4.47 (m, 3H), 3.84−3.68 (m, 3H), 3.15−2.97 (m, 5H), 1.51, 1.39
(two peaks, 9H); 13C NMR (125.7 MHz, CDCl3): δ (172.5, 172.1),
(155.1, 154.4), (136.8, 135.9), (132.3, 132.1), (131.0, 130.3), (129.3,
128.7), (126.9, 126.7), (126.0, 125.8), (80.6, 80.4), (61.4, 61.2),
(52.2, 50.1), (45.0, 44.2), (32.3, 32.0), (30.6, 30.4), 28.4; MS (FAB+)
m/z (relative intensity): 355 (MH+, 50), 255 (100), 154 (75); HRMS
(FAB+) m/z: calcd for C17H24ClN2O4 (MH+), 355.1425; obsd,
355.1321.
5′-Deoxy-5′-{2-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-3-yl]methyl}amino-ethyl}thio-2′,3′-O-(1-
methylethylidene)-adenosine (48). A mixture of aldehyde 45
(1.20 g, 4.56 mmol), amine 34 (1.90 g, 5.19 mmol), and molecular
sieves (4 Å, 2.0 g) in THF (60 mL) was stirred at room temperature
for 3 h. The mixture was filtered and the solvent was removed under
reduced pressure. The residue was dissolved in ethanol (60 mL), and
sodium borohydride (240 mg, 6.30 mmol) was added to the solution
over 1.5 h. The mixture was stirred for 12 h at room temperature and
water (0.3 mL) was added. The solvent was removed under reduced
pressure and the residue was purified by chromatography on a silica
gel column eluting with CHCl3/MeOH (30:1) to yield 48 (1.34 g,
( )-1,1-Dimethylethyl 3,4-Dihydro-3-(N-methoxy-N-meth-
ylcarbamoyl)-7-nitro-isoquinoline-2(1H)-carboxylate (44).
BOP (2.33 g, 5.28 mmol) was added to a stirred solution of crude
40 (1.70 g, 5.28 mmol) and Et3N (0.74 mL, 5.3 mmol) in CH2Cl2
(60 mL). After five minutes, N-dimethylhydroxylamine hydrochloride
(0.46 g, 6.3 mmol) and Et3N (0.88 mL, 6.3 mmol) were added to the
solution. The mixture was stirred for 24 h at room temperature. The
mixture was then diluted with CH2Cl2 (20 mL) and washed with HCl
(3 N, 2 × 20 mL), aqueous NaHCO3 (sat, 30 mL), and brine (30
mL), and dried over anhydrous Na2SO4. The solvent was evaporated
under reduced pressure and the resulting crude product was purified
by chromatography on a silica gel column eluting with EtOAc/
hexanes (1:5) to yield amide 44 (1.20 g, 35% from 15) as slightly
1
48%) as white foam: H NMR (500 MHz, CDCl3): δ 8.34 (s, 1H),
7.92 (s, 1H), 7.18−7.10 (m, 4H), 6.08 (s, 1H), 5.96 (br s, 2H), 5.51
(dd, J = 1.8, 6.4 Hz, 1H), 5.05 (dd, J = 2.6, 6.2 Hz, 1H), 4.73 (m,
1H), 4.59−4.36 (m, 2H), 4.23 (m, 1H), 3.06−2.82 (m, 2H), 2.79−
2.43 (m, 8H), 1.62 (s, 3H), 1.49 (s, 9H), 1.40 (s, 3H); 13C NMR
(125.7 MHz, CDCl3): δ 155.6, 155.3, 153.1, 149.1, 140.0, 132.9,
129.1, 126.5, 126.1, 120.2, 114.4, 90.8, 86.8, 83.9, 83.7, 79.9, 50.2,
(49.9, 48.4), 48.2, (43.3, 42.5), 34.1, 32.8, 30.9, 28.5, 27.0, 25.3; MS
(CI) m/z (relative intensity): 612 (MH+, 45), 512 (50), 379 (40),
132 (70); HRMS (FAB+) m/z: calcd for C30H42N7O5S (MH+),
612.2968; obsd, 612.2944.
1
yellow crystals: mp 174−175 °C; H NMR (400 MHz, CDCl3): δ
8.04 (br s, 2H), 7.30 (m, 1H), 5.42−5.07 (m, 1H), 4.85 (m, 2H),
3.86, 3.81 (two peaks, 3H), 3.30−3.10 (m, 5H), 1.57, 1.49 (two
peaks, 9H); 13C NMR (100 MHz, CDCl3): δ (172.4, 172.1), (155.3,
154.7), 147.1, (140.5, 139.9), (136.8, 136.0), (129.6, 129.0), (122.4,
122.1), (121.7, 121.6), (81.4, 81.3), (62.0, 61.8), (51.9, 49.9), (45.6,
44.7), (32.7, 32.4), 31.6, 28.8; MS (ESI+) m/z (relative intensity):
366 (MH+, 62), 266 (60), 177 (65); HRMS (ESI+) m/z: calcd for
C17H24N3O6 (MH+), 366.1665; obsd, 366.1662.
5′-{2-{[7-Chloro-3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-3-yl]methyl}amino-ethyl}thio-5′-deoxy-
2′,3′-O-(1-methylethylidene)-adenosine (49). This compound
was prepared in a similar manner as 48 but using aldehyde 46 (1.20 g,
4.05 mmol) as the starting material to afford 49 (1.50 g, 57%) as
( )-1,1-Dimethylethyl 3,4-Dihydro-3-formyl-isoquinoline-
2(1H)-carboxylate (45). To a stirred solution of 42 (160 mg,
0.500 mmol) in Et2O (6 mL), LiAlH4 (24 mg, 0.63 mmol) was added
at 0 °C. The reaction was stirred for 1 h at room temperature and the
reaction mixture was then hydrolyzed with a solution of KHSO4 (120
mg, 0.880 mmol) in water (2.5 mL). The aqueous phase was
separated and extracted with Et2O (3 × 5 mL). The organic phases
were combined, washed with HCl (3 N, 10 mL), NaHCO3 (sat, 10
mL), and brine (10 mL) and then dried over anhydrous Na2SO4.
After solvent evaporation, aldehyde 45 (100 mg, 77%) was obtained
1
white foam: H NMR (500 MHz, CDCl3): δ 8.27 (s, 1H), 7.89 (s,
1H), 7.06−6.97 (m, 3H), 6.70 (br s, 2H), 6.05 (s, 1H), 5.48 (d, J =
6.4 Hz, 1H), 5.02 (dd, J = 2.5, 5.8 Hz, 1H), 4.67 (m, 1H), 4.53−4.32
(m, 2H), 4.11 (m, 1H), 2.93−2.77 (m, 2H), 2.72−2.34 (m, 8H), 1.56
(s, 3H), 1.43 (s, 9H), 1.35 (s, 3H); 13C NMR (125.7 MHz, CDCl3):
δ 156.0, 155.0, 153.0, 149.0, 139.8, 134.7, 131.6, 131.3, 130.4, 126.6,
126.0, 120.1, 114.2, 90.7, 86.8, 83.9, 83.7, 80.0, 49.9, 49.5, 48.1, (42.8,
42.1), 34.0, 32.8, 30.3, 28.5, 27.0, 25.2; MS (CI) m/z (relative
1
in pure form as oil: H NMR (500 MHz, CDCl3): δ 9.52, 9.48 (two
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01475
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intensity): 646 (MH+, 50), 379 (50), 244 (25), 164 (40), 136 (60);
HRMS (FAB+) m/z (MH+), calcd for C30H41ClN7O5S (MH+),
646.2578; obsd, 646.2572.
7.4 Hz, 2H), 1.65 (m, 2H), 1.62 (s, 3H), 1.47 (m, 2H), 1.41 (s, 3H),
1.30 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 168.9, 156.3, 153.5,
149.5, 140.4, 134.2, 132.5, 123.6, 120.6, 114.7, 91.3, 87.5, 84.4, 84.3,
38.3, 34.6, 32.8, 29.8, 28.8, 28.6, 27.5, 26.8, 25.7; MS (ESI+) m/z
(relative intensity): 553 (MH+, 80), 418 (30); HRMS (ESI+) m/z:
calcd for C27H33N6O5S (MH+), 553.2233; obsd, 553.2228.
5′-Deoxy-5′-{2-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-7-nitro-isoquinolin-3-yl]methyl}amino-ethyl}thio-
2′,3′-O-(1-methylethylidene)-adenosine (50). This compound
was prepared in a similar manner as 48 but using aldehyde 47 (140
mg, 0.460 mmol) as the starting material to afford 50 (150 mg, 50%)
as slightly yellow foam: 1H NMR (500 MHz, CDCl3): δ 8.35 (s, 1H),
8.04 (d, J = 8.3 Hz, 1H), 8.02 (s, 1H), 7.94 (s, 1H), 7.31 (d, J = 8.3
Hz, 1H), 6.09 (s, 1H), 5.73 (br s, 2H, NH2), 5.53 (dd, J = 2.0, 6.4 Hz,
1H), 5.07 (t, J = 2.9 Hz, 1H), 5.03−4.20 (m, 4H), 3.08−2.94 (m,
2H), 2.87−2.44 (m, 8H), 1.62 (s, 3H), 1.51 (s, 9H), 1.41 (s, 3H);
13C NMR (125.7 MHz, CDCl3): δ 155.7, 155.1, 153.1, 149.2, 146.5,
141.0, 140.1, 134.6, 130.2, 121.7, 121.4, 120.2, 114.5, 90.9, 86.9, 84.0,
83.8, 80.6, 53.5, 50.1, 48.1, (42.8, 42.1), 34.1, 32.8, 31.3, 28.5, 27.1,
25.3; MS (ESI+) m/z (% relative intensity): 657 (MH+, 100), 557
(60), 422 (40), 301 (55), 191 (60); HRMS (ESI+) m/z: calcd for
C30H41N8O7S (MH+), 657.2819; obsd, 657.2802.
5′-(3-Aminopropyl)thio-5′-deoxy-2′,3′-O-(1-methylethyli-
dene)-adenosine (61).29,73,97 Phthalimide 57 (3.20 g, 6.27 mmol)
was mixed with ethanol (60 mL) and heated to 50 °C until 57 was
completely dissolved. Hydrazine (610 mg, 18.8 mmol) was added
dropwise and the reaction mixture was stirred at 80 °C for 3.5 h. The
solid was filtered off and washed with MeOH (20 mL). The filtrates
were combined together. The solvent was removed under reduced
pressure, and the residue was purified by chromatography on a silica
gel column with CHCl3/MeOH/NH4OH (10:1:0.1) to yield 61
1
(2.08 g, 87%) as colorless oil: H NMR (400 MHz, CDCl3): δ 8.33
(s, 1H), 8.02 (s, 1H), 7.51 (br s, 2H, NH2), 6.17 (s, 1H), 5.58 (d, J =
6.4 Hz, 1H), 5.08 (m, 1H), 4.41 (m, 1H), 2.81 (m, 2H), 2.70 (t, J =
6.8 Hz, 2H), 2.65 (m, 2H), 1.65 (br s, 2H), 1.161 (s, 3H), 1.46 (m,
2H), 1.40 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 156.7, 153.2,
149.2, 140.1, 120.3, 114.4, 91.0, 87.1, 84.2, 84.1, 40.9, 34.5, 33.3, 30.1,
27.2, 25.6; MS (CI) m/z (relative intensity): 381 (MH+, 50), 3222
(50), 290 (25), 186 (80), 164 (25), 136 (50); HRMS (FAB+) m/z:
calcd for C16H25N6O3S (MH+), 381.1709; obsd, 381.1713.
Compounds 52 and 57 were synthesized by following the literature
procedures.80
5′-Deoxy-5′-[4-(1,3-dioxoisoindolin-2-yl)butyl]thio-2′,3′-O-
(1-methylethylidene)-adenosine (58). Protected thionucleoside
52 (3.60 g, 10.0 mmol) and N-(4-bromobutyl)phthalimide (54) (4.23
g, 15.0 mmol) were added to oxygen-free absolute MeOH (150 mL)
under argon. The suspension was cooled to −20 °C. NaOMe (1.20 g,
22.2 mmol, in 10 mL MeOH) solution was freshly prepared and
slowly added into the suspension. The mixture was then allowed to
warm up to room temperature and was stirred for 12 h. The solvent
was removed under reduced pressure and the residue was mixed with
H2O/CHCl3 (1:1, 100 mL). The water layer was extracted with
CHCl3 (3 × 30 mL). The chloroform phases were combined together
and dried with anhydrous Na2SO4. The solvent was removed under
reduced pressure, and the residue was purified by flash chromatog-
raphy (silica gel) eluting with CH2Cl2/MeOH (10:1) to yield 58 (4.4
5′-(4-Aminobutyl)thio-5′-dexoy-2′,3′-O-(1-methylethyli-
dene)-adenosine (62). This compound was prepared in a similar
manner as 61 using the procedure outlined previously, but using
phthalimide 58 (1.50 g, 2.86 mmol) as the starting material to afford
1
62 (1.10 g, 98%) as colorless oil: H NMR (400 MHz, CD3OD): δ
8.26 (s, 1H), 8.21 (s, 1H), 6.17 (d, J = 2.2 Hz, 1H), 5.54 (dd, J = 2.2,
6.3 Hz, 1H), 5.05 (dd, J = 2.9, 6.3 Hz, 1H), 4.33 (m, 1H), 2.76 (d, J =
6.9 Hz, 2H), 2.54 (t, J = 6.9 Hz, 2H), 2.45 (t, J = 6.4 Hz, 2H), 1.57 (s,
3H), 1.44 (m, 4H), 1.37 (s, 3H); 13C NMR (100 MHz, CD3OD): δ
156.4, 153.0, 149.2, 140.9, 119.6, 114.3, 90.8, 87.5, 84.2, 84.1, 41.0,
34.1, 32.1, 31.8, 27.0, 26.4, 24.5; MS (CI) m/z (relative intensity):
395 (MH+, 30), 203 (10), 96 (60); HRMS (FAB+) m/z: calcd for
C17H27N6O3S (MH+), 395.1865; obsd, 395.1877.
5′-(5-Aminopentyl)thio-5′-dexoy-2′,3′-O-(1-methylethyli-
dene)-adenosine (63). This compound was prepared in a similar
manner as 61 using the procedure outlined previously, but using
phthalimide 59 (3.2 g, 5.9 mmol) as the starting material to afford 63
(2.3 g, 94%) as colorless oil: 1H NMR (500 MHz, CDCl3): δ 8.35 (s,
1H), 7.98 (s, 1H), 6.30 (br s, 2H, NH2), 6.09 (d, J = 1.7 Hz, 1H),
5.55 (dd, J = 1.7, 6.3 Hz, 1H), 5.06 (dd, J = 2.9, 6.3 Hz, 1H), 4.40 (m,
1H), 3.15 (br s, 2H, NH2), 2.79 (m, 2H), 2.71 (t, J = 7.1 Hz, 2H),
2.44 (t, J = 7.4 Hz, 2H), 1.61 (s, 3H), 1.45 (m, 4H), 1.40 (s, 3H),
1.32 (m, 2H); 13C NMR (125.7 MHz, CDCl3): δ 155.7, 153.0, 149.1,
140.1, 120.2, 114.3, 90.9, 87.2, 83.9, 83.8, 41.4, 34.2, 32.4, 32.0, 29.2,
27.0, 25.8, 25.3; MS (FAB+) m/z (relative intensity): 409 (MH+,
100), 274 (55), 215 (65), 185 (95), 136 (78); HRMS (FAB+) m/z:
calcd for C18H29N6O3S (MH+), 409.2022; obsd, 409.1997.
1
g, 82%) as a white solid: mp 174−176 °C; H NMR (500 MHz,
CDCl3): δ 8.35 (s, 1H), 7.95 (s, 1H), 7.83 (m, 2H), 7.70 (m, 2H),
6.20 (br s, 2H, NH2), 6.09 (d, J = 2.1 Hz, 1H), 5.52 (dd, J = 2.1, 6.4
Hz, 1H), 5.07 (m, 1H), 4.38 (m, 1H), 3.65 (t, J = 7.0 Hz, 2H), 2.83−
2.77 (m, 2H), 2.54 (t, J = 7.0 Hz, 2H), 1.73 (m, 2H), 1.61 (s, 3H),
1.58 (m, 2H), 1.40 (s, 3H); 13C NMR (125.7 MHz, CDCl3): δ 168.3,
155.6, 153.0, 149.1, 139.9, 133.8, 132.0, 123.1, 120.2, 114.3, 90.8,
86.8, 83.9, 83.7, 37.3, 34.2, 31.9, 27.5, 27.0, 26.7, 25.3; MS (CI) m/z
(relative intensity): 525 (MH+, 80), 234 (50), 202 (20), 164 (60);
HRMS (FAB+) m/z: calcd for C25H29N6O5S (MH+), 525.1920; obsd,
525.1929.
5′-Deoxy-5′-[5-(1,3-dioxoisoindolin-2-yl)pentyl]thio-2′,3′-
O-(1-methylethylidene)-adenosine (59). This compound was
prepared in a similar manner as 58 using the procedure outlined
previously, but using phthalimide 55 (3.6 g, 10 mmol) as the starting
1
materials to afford 59 (3.41 g, 64%) as white foam: H NMR (500
MHz, CDCl3): δ 8.35 (s, 1H), 7.93 (s, 1H), 7.83 (m, 2H), 7.70 (m,
2H), 6.09 (d, J = 2.0 Hz, 1H), 5.99 (br s, 2H, NH2), 5.54 (dd, J = 2.0,
6.3 Hz, 1H), 5.06 (dd, J = 3.0, 6.3 Hz, 1H), 4.39 (m, 1H), 3.65 (t, J =
7.0 Hz, 2H), 2.83−2.77 (m, 2H), 2.46 (t, J = 7.0 Hz, 2H), 1.64 (m,
2H), 1.61 (s, 3H), 1.52 (m, 2H), 1.40 (s, 3H), 1.36 (m, 2H); 13C
NMR (125.7 MHz, CDCl3): δ 168.3, 155.6, 153.1, 149.2, 140.0,
133.8, 132.1, 123.1, 120.3, 114.3, 90.8, 87.0, 83.9, 83.8, 37.7, 34.2,
32.3, 29.0, 28.0, 27.0, 25.9, 25.3; MS (FAB+) m/z (relative intensity):
539 (MH+, 70), 307 (60), 154 (100); HRMS (FAB+) m/z: calcd for
C26H31N6O5S (MH+), 539.2077; obsd, 539.2077.
5′-(6-Aminohexyl)thio-5′-dexoy-2′,3′-O-(1-methylethyli-
dene)-adenosine (64). This compound was prepared in a similar
manner as 61 using the procedure outlined previously, but using
phthalimide 60 (5.4 g, 9.8 mmol) as the starting material to afford 64
1
(4.1 g, 99%) as colorless oil: H NMR (400 MHz, CDCl3/CD3OD
10:1): δ 8.33 (s, 1H), 7.97 (s, 1H), 6.82 (br ex s, 2H, NH2), 6.11 (s,
1H), 5.53 (d, J = 6.3 Hz, 1H), 5.08 (m, 1H), 4.40 (m, 1H), 2.84 (m,
2H), 2.66 (t, J = 7.0 Hz, 2H), 2.47 (t, J = 7.3 Hz, 2H), 2.37 (br ex s,
2H, NH2), 1.62 (s, 3H), 1.50 (m, 2H), 1.41 (s, 3H), 1.39 (m, 2H),
1.27 (m, 4H); 13C NMR (100 MHz, CDCl3/CD3OD 10:1): δ 156.5,
153.1, 149.0, 140.1, 120.2, 114.2, 90.8, 87.2, 84.0, 83.9, 41.9, 34.3,
33.4, 32.5, 29.6, 28.6, 27.1, 26.5, 25.4; MS (ESI+) m/z (relative
intensity): 423 (MH+, 50), 288 (100), 230 (50); HRMS (ESI+) m/z:
calcd for C19H31N6O3S (MH+), 423.2178; obsd, 423.2153.
5′-Deoxy-5′-{3-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-3-yl]methyl}amino-propyl}thio-2′,3′-O-
(1-methylethylidene)-adenosine (65). This compound was
prepared in a similar manner as 48 using the procedure outlined
previously, but using aldehyde 45 (1.53 g, 5.85 mmol) and amine 61
5′-Deoxy-5′-[6-(1,3-dioxoisoindolin-2-yl)hexyl]thio-2′,3′-O-
(1-methylethylidene)-adenosine (60). This compound was
prepared in a similar manner as 58 using the procedure outlined
previously, but using phthalimide 56 (5.00 g, 16.1 mmol) as the
starting material to afford 60 (5.40 g, 71%) as a white solid: mp 85−
1
87 °C; H NMR (400 MHz, CDCl3): δ 8.33 (s, 1H), 7.96 (s, 1H),
7.83 (m, 2H), 7.70 (m, 2H), 6.48 (br s, 2H, NH2), 6.11 (d, J = 2.1
Hz, 1H), 5.54 (dd, J = 2.1, 6.4 Hz, 1H), 5.07 (dd, J = 3.1, 6.4 Hz,
1H), 4.40 (m, 1H), 3.66 (t, J = 7.3 Hz, 2H), 2.80 (m, 2H), 2.46 (t, J =
O
https://dx.doi.org/10.1021/acs.jmedchem.0c01475
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
1
(2.00 g, 5.26 mmol) to afford 65 (1.30 g, 36%) as white foam: H
NMR (400 MHz, CDCl3): δ 8.25 (s, 1H), 7.90 (s, 1H), 7.06 (m, 4H),
6.97 (br s, 2H, NH2), 6.05 (d, J = 1.6 Hz, 1H), 5.48 (dd, J = 1.6, 6.3
Hz, 1H), 5.01 (m, 1H), 4.74−4.14 (m, 4H), 2.98−2.73 (m, 2H), 2.70
(m, 2H), 2.57−2.46 (m, 4H), 2.39−2.34 (m, 2H), 1.58 (m, 2H), 1.54
(s, 3H), 1.42 (s, 9H), 1.33 (s, 3H); 13C NMR (125.7 MHz, CDCl3):
δ 156.1, 155.2, 153.0, 148.9, 139.7, 132.8, 129.0, 128.8, 126.4, 126.0,
120.1, 114.1, 90.7, 86.7, 83.9, 83.7, 79.7, 50.4, (49.6, 48.4), 48.0,
(43.1, 42.4), 34.2, 30.9, 30.1, 29.8, 28.4, 27.0, 25.2; MS (ESI+) m/z
(relative intensity): 626 (MH+, 100), 526 (75), 391 (20), 313 (50);
HRMS (ESI+) m/z: calcd for C31H44N7O5S (MH+), 626.3125; obsd,
626.3122.
5′-Deoxy-5′-{3-{[7-chloro-3,4-dihydro-2(1H)-(1,1-
dimethylethoxy)carbonyl-isoquinolin-3-yl]methyl}amino-
propyl}thio-2′,3′-O-(1-methylethylidene)-adenosine (66). This
compound was prepared in a similar manner as 48 using the
procedure outlined previously, but using aldehyde 46 (450 mg, 1.52
mmol) and amine 61 (600 mg, 1.58 mmol) as the starting materials to
afford 66 (0.68 g, 68%) as white foam: 1H NMR (500 MHz, CDCl3):
δ 8.28 (s, 1H), 7.91 (s, 1H), 7.06−6.97 (m, 3H), 6.63 (br s, 2H,
NH2), 6.06 (d, J = 1.6 Hz, 1H), 5.50 (d, J = 6.2 Hz, 1H), 5.02 (dd, J =
2.9, 6.2 Hz, 1H), 4.72 (m, 1H), 4.54−4.13 (m, 2H), 4.34 (m, 1H),
2.93−2.76 (m, 2H), 2.70 (m, 2H), 2.57−2.36 (m, 6H), 1.56 (m, 5H),
1.44 (s, 9H), 1.35 (s, 3H); 13C NMR (125.7 MHz, CDCl3): δ 155.9,
155.1, 153.0, 149.0, 139.8, 134.7, 131.6, 131.3, 130.3, 126.6, 125.9,
120.1, 114.2, 90.8, 86.7, 83.9, 83.7, 80.0, 50.3, 49.5, 48.1, (42.8, 42.1),
34.3, 30.4, 30.2, 29.8, 28.4, 27.0, 25.2; MS (FAB−) m/z (relative
intensity): 660 (MH+, 68), 560 (20), 307 (90), 289 (55), 154 (100),
136 (70); HRMS (ESI+) m/z: calcd for C31H43ClN7O5S (MH+),
660.2735; obsd, 660.2759.
43%) as white foam: H NMR (500 MHz, CDCl3): δ 8.33 (s, 1H),
7.94 (s, 1H), 7.16 (m, 4H), 6.13 (br s, 2H, NH2), 6.09 (s, 1H), 5.54
(m, 1H), 5.06 (dd, J = 3.0, 6.3 Hz, 1H), 4.82 (m, 1H), 4.70−4.24 (m,
2H), 4.40 (m, 1H), 3.07−2.83 (m, 2H), 2.77 (m, 2H), 2.59−2.49 (m,
6H), 1.62 (s, 3H), 1.49 (s, 9H), 1.45 (m, 4H), 1.40 (s, 3H), 1.36 (m,
2H); 13C NMR (125.7 MHz, CDCl3): δ 155.7, 155.5, 153.2, 149.2,
140.0, 132.9, 129.2, 129.1, 126.6, 126.2, 126.1, 120.2, 114.3, 90.9,
87.1, 84.0, 83.9, 80.0, 50.8, (49.6, 48.6), 49.4, (43.4, 42.5), 34.3, 32.5,
31.2, 29.6, 29.4, 28.5, 27.1, 26.4, 25.3; MS (ESI+) m/z (relative
intensity): 654 (MH+, 45), 554 (20), 327 (100); HRMS (ESI+) m/z:
calcd for C33H48N7O5S (MH+), 654.3438; obsd, 654.3446.
5′-Deoxy-5′-{6-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-3-yl]methyl}amino-hexyl}thio-2′,3′-O-
(1-methylethylidene)-adenosine (70). This compound was
prepared in a similar manner as 48 using the procedures outlined
previously, but using aldehyde 45 (0.60 g, 2.3 mmol) and amine 64
(0.97 g, 2.3 mmol) as the starting materials to afford 70 (0.90 g, 59%)
as white foam: 1H NMR (400 MHz, CDCl3): δ 8.34 (s, 1H), 7.94 (s,
1H), 7.14 (m, 4H), 6.35 (br s, 2H, NH2), 6.09 (d, J = 2.0 Hz, 1H),
5.54 (dd, J = 2.0, 6.3 Hz, 1H), 5.07 (dd, J = 3.0, 6.3 Hz, 1H), 4.90−
4.45 (m, 2H), 4.40 (m, 1H), 4.23 (m, 1H), 3.10−2.85 (m, 2H), 2.79
(m, 2H), 2.64−2.44 (m, 6H), 1.62 (s, 3H), 1.50 (s, 9H), 1.45 (m,
2H), 1.40 (s, 3H), 1.34 (m, 2H), 1.25 (m, 4H); 13C NMR (100 MHz,
CDCl3): δ 156.3, 155.7, 153.5, 149.6, 140.4, 133.4, 133.3, 129.6,
129.5, 127.0, 126.5, 120.7, 114.8, 91.3, 87.4, 84.4, 84.3, 80.3, 51.1,
49.9, 48.6, (43.7, 42.9), 34.7, 33.0, 31.6, 30.4, 29.9, 29.1, 28.9, 27.5,
27.2, 25.7; MS (ESI+) m/z (relative intensity): 668 (MH+, 100), 568
(30); HRMS (ESI+) m/z: calcd for C34H50N7O5S (MH+), 668.3594;
obsd, 668.3593.
5′-Deoxy-5′-{2-[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinoline-3-carboxamido]ethyl}thio-2′,3′-O-(1-
methylethylidene)-adenosine (71). To a stirred mixture of acid
41 (2.50 g, 9.02 mmol), amine 34 (3.30 g, 9.02 mmol), and HOBt·
H2O (1.22 g, 9.02 mmol) in DMF (25 mL) was added EDCI·HCl
(1.73 g, 9.02 mmol) and then N-methylmorpholine (1.0 mL, 9.0
mmol) under argon at 0 °C. The reaction mixture was warmed to
room temperature and stirred for 12 h. The solvent was removed
under reduced pressure and the residue was purified by flash
chromatography (silica gel) eluting with CHCl3/MeOH (40:1) to
yield 71 (5.50 g, 98%) as white foam: 1H NMR (500 MHz, CD3OD):
δ 8.26 (s, 1H), 8.23 (s, 1H), 7.15 (m, 4H), 6.17 (s, 1H), 5.49 (m,
1H), 5.03 (br s, 1H), 4.60−4.46 (m, 3H), 4.30 (br s, 1H), 3.21−3.08
(m, 4H), 2.76 (m, 2H), 2.46−2.40 (m, 2H), 1.58 (s, 3H), 1.51, 1.42
(two peaks, 9H), 1.38 (s, 3H); 13C NMR (100 MHz, CD3OD): δ
(173.8, 173.1), 156.3, (156.1, 156.4), 152.9, 149.2, 140.9, (134.9,
134.2), (133.9, 133.5), (128.0, 127.5), (127.4, 127.2), 126.9, 126.2,
119.6, 114.5, 90.6, 86.9, 84.2, 84.1, 81.1, (56.9, 55.3), (45.4, 44.5),
(38.9, 38.8), 33.9, (32.5, 31.9), 31.5, 27.8, 26.5, 24.7; MS (FAB+) m/z
(relative intensity): 626 (MH+, 50), 524 (15), 136 (100); HRMS
(FAB+) m/z: calcd for C30H40N7O6S (MH+), 626.2761; obsd,
626.2763.
5′-Deoxy-5′-{3-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-7-nitro-isoquinolin-3-yl]methyl}amino-propyl}thio-
2′,3′-O-(1-methylethylidene)-adenosine (67). This compound
was prepared in a similar manner as 48 using the procedure outlined
previously, but using aldehyde 47 (500 mg, 1.63 mmol) and amine 61
(500 mg, 1.32 mmol) as the starting materials to afford 67 (420 mg,
1
48%) as yellow foam: H NMR (500 MHz, CDCl3): δ 8.36 (s, 1H),
8.03 (m, 2H), 7.94 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 6.09 (d, J = 1.4
Hz, 1H), 5.72 (br s, 2H, NH2), 5.55 (dd, J = 1.7, 6.3 Hz, 1H), 5.06
(dd, J = 3.0, 6.3 Hz, 1H), 4.88 (m, 1H), 4.56−4.28 (m, 2H), 4.39 (m,
1H), 3.07−2.96 (m, 2H), 2.77 (m, 2H), 2.62−2.44 (m, 6H), 1.64 (m,
2H), 1.63 (s, 3H), 1.51 (s, 9H), 1.41 (s, 3H); 13C NMR (125.7 MHz,
CDCl3): δ 155.5, 155.0, 153.0, 149.2, 146.5, 140.9, 140.0, 134.7,
130.0, 121.6, 121.3, 120.3, 114.3, 90.9, 86.9, 83.9, 83.8, 80.4, 50.5,
49.4, 48.1, 42.5, 34.4, 31.2, 30.3, 29.8, 28.4, 27.0, 25.3; MS (CI) m/z
(relative intensity): 671 (MH+, 75), 571 (30), 240 (75); HRMS
(ESI+) m/z: calcd for C31H43N8O7S (MH+), 671.2975; obsd,
671.2987.
5′-Deoxy-5′-{4-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-3-yl]methyl}amino-butyl}thio-2′,3′-O-(1-
methylethylidene)-adenosine (68). This compound was prepared
in a similar manner as 48 using the procedure outlined previously, but
using aldehyde 45 (440 mg, 1.69 mmol) and amine 62 (0.670 g, 1.69
mmol) as the starting materials to afford 68 (0.52 g, 48%) as white
5′-{2-[7-Chloro-3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinoline-3-carboxamido]ethyl}thio-5′-deoxy-
2′,3′-O-(1-methylethylidene)-adenosine (72). This compound
was prepared in a similar manner as 71 using the procedures outlined
previously but using 39 (170 mg, 0.550 mmol) as the starting material
to afford 72 (300 mg, 83%) as white foam: IR (KBr): 3334, 3191,
2981, 2930, 1660, 1593, 1486, 1383, 1209, 1163, 1096, 871, 738
1
foam: H NMR (400 MHz, CDCl3): δ 8.35 (s, 1H), 7.94 (s, 1H),
7.13 (m, 4H), 6.09 (d, J = 2.0 Hz, 1H), 5.96 (br s, 2H, NH2), 5.53
(dd, J = 2.0, 6.3 Hz, 1H), 5.06 (dd, J = 3.0, 6.3 Hz, 1H), 4.82 (m,
1H), 4.71−4.23 (m, 2H), 4.40 (m, 1H), 3.07−2.83 (m, 2H), 2.77 (m,
2H), 2.57 (m, 6H), 1.62 (s, 3H), 1.50 (s, 9H), 1.45 (m, 4H), 1.41 (s,
3H); 13C NMR (100 MHz, CDCl3): δ 155.7, 155.4, 153.0, 149.1,
139.9, 132.9, 130.8, 129.0, 128.8, 126.5, 126.1, 120.2, 114.3, 90.8,
86.8, 83.9, 83.8, 79.9, 50.6, (49.6, 48.5), 48.9, (43.3, 42.4), 34.2, 32.4,
31.1, 29.0, 28.4, 27.1, 27.0, 25.3; MS (CI) m/z (relative intensity):
640 (MH+, 30), 407 (20), 292 (15), 136 (60); HRMS (ESI+) m/z:
calcd for C32H46N7O5S (MH+), 640.3281; obsd, 640.3292.
5′-Deoxy-5′-{5-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-3-yl]methyl}amino-pentyl}thio-2′,3′-O-
(1-methylethylidene)-adenosine (69). This compound was
prepared in a similar manner as 48 using the procedure outlined
previously, but using aldehyde 45 (0.760 g, 2.91 mmol) and amine 63
(1.18 g, 2.91 mmol) as the starting materials to afford 69 (0.81 g,
1
cm−1; H NMR (500 MHz, CD3OD): δ 8.26 (s, 1H), 8.23 (s, 1H),
7.16−7.09 (m, 3H), 6.17 (s, 1H), 5.49 (m, 1H), 5.03 (br s, 1H),
4.58−4.46 (m, 3H), 4.30 (br s, 1H), 3.21−3.05 (m, 4H), 2.77 (m,
2H), 2.49 (m, 2H), 1.58 (s, 3H), 1.50, 1.42 (two peaks, 9H), 1.37 (s,
3H); 13C NMR (125.7 MHz, CD3OD): δ (172.9, 172.3), 155.9,
(155.4, 155.0), 152.6, 148.7, 140.3, (136.4, 135.7), (132.2, 131.9),
(129.1, 128.6), (128.5, 127.8), (126.9, 126.7), 125.7, 119.1, 114.0,
90.2, 86.5, 83.7, 83.6, 80.7, (56.0, 54.4), (44.6, 43.7), (38.4, 38.3),
33.4, (31.4, 30.9), 31.1, 27.3, 26.0, 24.2; MS (CI) m/z (relative
intensity): 660 (M+, 25), 560 (20), 324 (25), 292 (60), 136 (80), 120
(90); HRMS (FAB+) m/z: calcd for C30H39ClN7O6S (MH+),
660.2371; obsd, 660.2366.
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5′-Deoxy-5′-{2-[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-7-nitro-isoquinoline-3-carboxamido]ethyl}thio-
2′,3′-O-(1-methylethylidene)-adenosine (73). This compound
was prepared in a similar manner as 71 using the procedures outlined
previously, but using 40 (500 mg, 1.55 mmol) as the starting material
128.9, 126.5, 126.3, 126.1, 68.0, 47.9, 47.0, 38.6; MS (CI) m/z
(relative intensity): 163 (M+, 20), 162 (50), 130 (75), 105 (98), 91
(45); HRMS (FAB+) m/z: calcd for C10H14NO (MH+), 164.1075;
obsd, 164.1057.
(
)-1,1-Dimethylethyl 3,4-Dihydro-4-hydroxymethyl-iso-
1
to afford 73 (0.86 g, 83%) as white foam: H NMR (500 MHz,
quinoline-2(1H)-carboxylate (78). To a stirred solution of 77
(70 mg, 0.43 mmol) in MeOH (6.6 mL) was added NaHCO3 (253
mg, 3.01 mmol). (Boc)2O (281 mg, 1.29 mmol) was then added and
the mixture was stirred at room temperature overnight. The solvent
was removed under reduced pressure and the residue was mixed with
water (10 mL) and then extracted with CH2Cl2 (3 × 10 mL). The
organic layers were combined together, washed with brine (20 mL),
and dried with anhydrous Na2SO4. The solvent was removed and the
residue was purified by column chromatography (silica gel) eluting
with hexanes/EtOAc (4:1) to provide 78 (100 mg, 88%) as colorless
oil: 1H NMR (500 MHz, CDCl3): δ 4.92 (d, J = 17.0 Hz, 1H), 4.51−
4.34 (m, 2H), 3.65 (m, 2H), 3.40 (br s, 1H), 3.10−3.00 (m, 2H),
1.51 (s, 9H); 13C NMR (125.7 MHz, CDCl3): δ 156.2, 135.0, 133.3,
129.4, 126.8, 126.4, 126.0, 80.5, 64.1, (46.6, 45.4), (42.2, 41.5), 41.0,
28.3; MS (CI) m/z (relative intensity): 264 (MH+, 50), 233 (18), 208
(40), 162 (90), 130 (40); HRMS (FAB+) m/z: calcd for C15H22NO3
(MH+), 264.1600; obsd, 264.1592.
CDCl3): δ 8.30 (s, 1H), 8.00 (m, 2H), 7.95 (s, 1H), 7.32 (m, 1H),
6.74−6.39 (br, 2H), 6.07 (br s, 1H), 5.49 (br s, 1H), 5.01 (br s, 1H),
4.76−4.48 (m, 3H), 4.30 (br s, 1H), 3.29 (m, 2H), 3.05 (m, 2H),
2.72 (m, 2H), 2.49 (m, 2H), 1.59 (s, 3H), 1.56 (s, 9H), 1.39 (s, 3H);
13C NMR (125.7 MHz, CDCl3): δ 170.9, 155.6, 155.0, 152.9, 149.0,
146.9, 141.3, 140.1, 134.4, 129.4, 122.1, 121.2, 120.1, 114.4, 90.7,
86.7, 83.9, 83.6, 81.8, 52.8, 44.5, 38.0, 33.7, 32.1, 30.7, 28.2, 26.9,
25.2; MS (ESI+) m/z (% relative intensity) 671 (MH+, 100), 571
(10), 480 (5), 436 (10), 292 (10); HRMS (ESI+) m/z: calcd for
C30H39N8O8S (MH+), 671.2612; obsd, 671.2614.
5′-Deoxy-5′-{3-[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinoline-3-carboxamido]propyl}thio-2′,3′-O-(1-
methylethylidene)-adenosine (74). To a stirred mixture of acid
41 (0.50 g, 1.8 mmol), amine 61 (0.69 g, 1.81 mmol), and HOBt
(244 mg, 1.81 mmol) in DMF (12 mL) was added EDCI·HCl (347
mg, 1.81 mmol), followed by N-methylmorpholine (183 mg, 1.81
mmol) under argon at 0 °C. The reaction mixture was warmed to
room temperature and stirred for 12 h. The solvent was removed
under reduced pressure and the residue was purified by flash
chromatography (silica gel) eluting with CHCl3/MeOH (40:1) to
yield 74 (0.78 g, 68%) as white foam: 1H NMR (400 MHz, CDCl3): δ
8.25 (s, 1H), 7.89 (s, 1H), 7.11 (m, 4H), 6.73 (br s, 2H, NH2), 6.04
(s, 1H), 6.03 (br s, 1H), 5.48 (m, 1H), 4.98 (m, 1H), 4.77 (m, 1H),
4.59−4.40 (m, 2H), 4.28 (m, 1H), 3.13 (m, 2H), 3.05 (m, 2H), 2.68
(m, 2H), 2.13 (m, 2H), 1.54 (s, 3H), 1.49 (m, 2H), 1.39 (s, 9H), 1.34
(s, 3H); 13C NMR (100 MHz, CDCl3): δ (172.4, 171.7), 156.4,
(156.1, 155.6), 153.4, 149.4, 140.3, 134.3, 133.6, 128.6, 128.0, 127.0,
126.6, 120.5, 114.7, 91.3, 87.1, 84.4, 84.2, 81.5, (57.1, 54.9), (45.6,
45.0), 38.4, 34.6, (32.6, 31.2), 29.8, 29.3, 28.8, 27.5, 25.7; MS (CI)
m/z (relative intensity): 640 (MH+, 75), 292 (20), 219 (30), 132
(75); HRMS (ESI+) m/z: calcd for C31H42N7O6S (MH+), 640.2917;
obsd, 640.2934.
( )-1,1-Dimethylethyl 3,4-Dihydro-4-formyl-isoquinoline-
2(1H)-carboxylate (79). To a solution of oxalyl chloride (135 mg,
1.06 mmol) in CH2Cl2 (3 mL) was added dimethyl sulfoxide (0.14
mL, 1.9 mmol) at −78 °C. The mixture was then stirred for 30 min. A
solution of alcohol 78 (100 mg, 0.380 mmol) in CH2Cl2 (4 mL) was
added dropwise to the reaction at −78 °C and the reaction mixture
was then stirred for another 1.5 h. N,N-Diisopropylethylamine (0.60
mL) was added slowly to the reaction and the mixture was warmed up
to room temperature. The solution was poured into 1 M HCl solution
(20 mL). The organic layer was washed with 1 M HCl (10 mL),
NaHCO3 (sat, 10 mL), and brine (10 mL) and then dried with
anhydrous Na2SO4. The solvent was removed under reduced pressure
and the residue was purified by column chromatography (hexanes/
EtOAc 6:1) to provide aldehyde 79 (70 mg, 71%) as colorless oil: 1H
NMR (500 MHz, CDCl3): δ 9.73 (s, 1H), 7.30−7.20 (m, 4H), 4.90−
4.50 (m, 2H), 4.34 (m, 1H), 3.60−3.30 (m, 2H), 1.50 (s, 9H); 13C
NMR (125.7 MHz, CDCl3): δ 199.6, 154.4, 134.1, 129.7, 128.9,
127.8, 127.0, 126.8, 80.5, 51.6, (45.8, 45.0), (41.4, 40.2), 28.1; MS
(ESI+) m/z (relative intensity): 262 (MH+, 100), 206 (88), 188 (40),
145 (40), 117 (32); HRMS (FAB+) m/z: calcd for C15H20NO3
(MH+), 262.1443; obsd, 262.1421.
5′-Deoxy-5′-{3-[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-7-nitro-isoquinoline-3-carboxamido]propyl}thio-
2′,3′-O-(1-methylethylidene)-adenosine (75). This compound
was prepared in a similar manner as 74 using the procedure outlined
previously for 74, but using acid 40 (500 mg, 1.55 mmol) as the
1
starting material to afford 75 (1.0 g, 94%) as white foam: H NMR
5′-Deoxy-5′-{2-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-4-yl]methyl}amino-ethyl}thio-2′,3′-O-(1-
methylethylidene)-adenosine (80). A mixture of aldehyde 79 (70
mg, 0.27 mmol), amine 34 (110 mg, 0.300 mmol), molecular sieves
(4 Å, 110 mg), and THF (6 mL) was stirred at room temperature for
3 h. The mixture was filtered and the solvent was removed under
reduced pressure. The residue was combined with ethanol (6 mL)
and sodium borohydride (15 mg, 0.40 mmol) was added over 1.5 h.
The mixture was stirred for 12 h at room temperature and water (0.1
mL) was added. The solvent was removed under reduced pressure
and the residue was purified by chromatography on a silica gel column
in CHCl3/MeOH (50:1) to yield 80 (90 mg, 54%) as white foam: 1H
NMR (400 MHz, CDCl3): δ 8.32 (s, 1H), 7.95 (br s, 1H), 7.60−7.23
(m, 1H), 7.16 (m, 2H), 7.08 (m, 1H), 6.56 (ex m, 2H), 6.09 (m, 1H),
5.51 (m, 1H), 5.08 (m, 1H), 4.82 (m, 1H), 4.42 (m, 1H), 4.29 (m,
2H), 3.15 (m, 1H), 2.89−2.66 (m, 10H), 1.54 (s, 3H), 1.48 (s, 9H),
1.40 (s, 3H); 13C NMR (125.7 MHz, CDCl3): δ 155.6, 155.0, 153.0,
149.1, 139.9, (136.8, 136.2), 133.0, 128.9, 126.8, 126.3, 126.0, 120.2,
114.3, 90.8, 86.8, 84.0, 83.7, 79.8, 52.5, 48.7, (46.1, 45.2), (43.0,
41.8), (39.1, 38.7), 34.2, 32.8, 28.4, 27.0, 25.3; MS (ESI+) m/z
(relative intensity): 612 (MH+, 100), 512 (40), 377 (10); HRMS
(ESI+) m/z: calcd for C30H42N7O5S (MH+), 612.2968; obsd,
612.2957.
(400 MHz, CDCl3): δ 8.22 (s, 1H), 7.93 (m, 2H), 7.91 (s, 1H), 7.27
(d, J = 6.7 Hz, 1H), 6.80 (br s, 2H, NH2), 6.68 (br s, 1H), 6.04 (s,
1H), 5.46 (dd, J = 1.4, 6.1 Hz, 1H), 4.96 (dd, J = 2.6, 6.1 Hz, 1H),
4.66 (m, 2H), 4.42 (m, 1H), 4.26 (m, 1H), 3.34−3.09 (m, 2H), 3.08
(m, 2H), 2.65 (m, 2H), 2.28 (m, 2H), 1.52 (s, 3H), 1.46 (m, 2H),
1.42 (s, 9H), 1.32 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 171.0,
156.4, (156.0, 155.2), 153.4, 149.4, 146.8, 141.9, 140.4, (135.7,
134.7), 129.7, 122.9, 121.5, 120.5, 114.7, 91.2, 87.1, 84.3, 84.2, 82.1,
(56.0, 53.5), (45.0, 44.5), 38.7, 34.6, (32.2, 30.9), 30.0, 29.3, 28.7,
27.4, 25.7; (ESI+) m/z (relative intensity): 685 (MH+, 100), 306
(15), 262 (25); HRMS (ESI+) m/z: calcd for C31H41N8O8S (MH+),
685.2768; obsd, 685.2760.
( )-4-Hydroxymethyl-1,2,3,4-tetrahydroisoquinoline (77).
Lactam 76 (200 mg, 0.980 mmol) was dissolved in THF (20 mL).
LiAlH4 (111 mg, 2.93 mmol) was added slowly into the solution at 0
°C. The reaction mixture was warmed to room temperature, after
which it was heated at reflux for 8 h. The reaction mixture was cooled
to 0 °C. H2O (0.1 mL), 15% NaOH (0.1 mL) and then H2O (3.3
mL) were added slowly to the mixture. The mixture was stirred at
room temperature for 30 min and then filtered through a Celite pad.
The filtrate was concentrated and the resulting residue was purified by
flash chromatography (silica gel) eluting with CHCl3/MeOH/
1
NH4OH (5:1:0.1) to yield 77 (140 mg, 88%) as a yellow oil: H
5′-Deoxy-5′-{3-{[3,4-dihydro-2(1H)-(1,1-dimethylethoxy)-
carbonyl-isoquinolin-4-yl]methyl}amino-propyl}thio-2′,3′-O-
(1-methylethylidene)-adenosine (81). This compound was
prepared in a similar manner as 80 using the procedures outlined
NMR (500 MHz, CDCl3): δ 7.19 (m, 3H), 7.04 (d, J = 7.2 Hz, 1H),
4.05 (ex s, 2H), 3.98 (br s, 2H), 3.96−3.82 (m, 2H), 3.42−3.09 (m,
2H), 2.80 (m, 1H); 13C NMR (125.7 MHz, CDCl3): δ 135.5, 135.3,
Q
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Article
previously but using aldehyde 79 (1.30 g, 5.00 mmol) and amine 61
(2.40 g, 6.30 mmol) as the starting materials to afford 81 (1.87 g,
60%) as white foam: H NMR (500 MHz, CDCl3): δ 8.36 (s, 1H),
This work was supported by NIH grant HL34193 “Synthesis of
Inhibitors of Epinephrine Biosynthesis”. Structural work by
A.G.B. was partially supported by a K-INBRE Undergraduate
Scholarship funded by NIH P20 GM103418 and startup funds
from the University of Kansas (to E. E. S). We thank David
VanderVelde and Sarah Neuenswander of the University of
Kansas Nuclear Magnetic Resonance Laboratory for their
assistance. A 500 MHz NMR spectrometer was partially
funded by the National Science Foundation Grant CHE-
9977422. We also thank Gerald Lushington of the University
of Kansas Molecular Graphics and Modeling Laboratory and
Todd Williams of the University of Kansas Mass Spectrometry
Laboratory for their assistance. Crystallization employed
facilities of the University of Kansas Protein Structure
Laboratory (PSL). The PSL was partially supported by NIH
through the KU COBRE Center for Protein Structure and
Function (NIH RR17708). X-ray diffraction data were
collected at the Stanford Synchrotron Radiation Lightsource.
Use of the Stanford Synchrotron Radiation Lightsource, SLAC
National Accelerator Laboratory, is supported by the U.S.
Department of Energy, Office of Science, Office of Basic
Energy Sciences under Contract no. DE-AC02-76SF00515.
The SSRL Structural Molecular Biology Program is supported
by the DOE Office of Biological and Environmental Research,
and by the National Institutes of Health, National Institute of
General Medical Sciences.
1
7.95 (s, 1H), 7.18 (m, 3H), 7.10 (m, 1H), 6.09 (s, 1H), 5.82 (br s,
2H, NH2), 5.53 (d, J = 6.1 Hz, 1H), 5.07 (m, 1H), 4.85 (m, 1H), 4.42
(m, 1H), 4.32 (m, 2H), 3.20−2.90 (m, 2H), 2.84 (m, 2H), 2.80 (m,
1H), 2.70 (m, 4H), 2.60 (m, 2H), 1.73 (t, J = 6.6 Hz, 2H), 1.62 (s,
3H), 1.49 (s, 9H), 1.42 (s, 3H); 13C NMR (125.7 MHz, CDCl3): δ
155.6, 155.0, 153.1, 149.2, 139.9, 136.0, 133.2, 128.8, 126.7, 126.5,
126.3, 120.2, 114.3, 90.9, 86.8, 84.0, 83.8, 79.8, 52.8, (48.8, 48.2),
(46.1, 45.3), (43.0, 41.7), (39.0, 38.6), 34.4, 30.3, (29.9, 29.7), 28.4,
27.0, 25.3; MS (CI) m/z (relative intensity): 626 (MH+, 75), 393
(20), 102 (50); (ESI+) m/z: calcd for C31H44N7O5S (MH+),
626.3125; obsd, 626.3108.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01475.
■
sı
HPLC tracings for 24, 25, 28, 30, and 31 (CSV)
Molecular formula strings for all new compounds (PDF)
Accession Codes
Atomic coordinates have been released prior to article
publication under PDB ID codes 4MIK (19) and 4MQ4 (29).
AUTHOR INFORMATION
Corresponding Authors
■
Michael J. McLeish − Department of Chemistry and Chemical
Biology, Purdue School of Science, IUPUI, Indianapolis, Indiana
46202, United States; orcid.org/0000-0002-9221-9119;
Email: mcleish@iupui.edu
ACKNOWLEDGMENTS
■
We thank Michael F. Rafferty for the helpful and detailed
comments on this manuscript. G.L.G. thanks all of the research
students he has had the pleasure of working with over the past
five decades. In particular, the work (and friendship) of his first
five students (Bharat V. Kamdar, Ann Marie Warner, Winton
Dennis Jones, Jr., Dale B. Mirth, and Allan H. Mizoguchi) were
especially important to a young assistant professor beginning a
research career and their work made that of later students
much easier. Molecular modeling studies by a later student
(James A. Monn), conducted long before anything about the
structure of hPNMT was known, led to the undertaking of the
project described in this manuscript. Early work on much
simpler approaches by postdoctoral research associates John R.
Ross, Akmal Bhatti and Meri Slavica were instrumental in the
ultimate success described in this paper.
Emily E. Scott − Department of Medicinal Chemistry, University
of Kansas, Lawrence, Kansas 66045, United States;
Email: scottee@med.umich.edu
Gary L. Grunewald − Department of Medicinal Chemistry,
University of Kansas, Lawrence, Kansas 66045, United States;
orcid.org/0000-0003-1297-7436; Email: ggrunewald@
ku.edu
Authors
Jian Lu − Department of Medicinal Chemistry, University of
Kansas, Lawrence, Kansas 66045, United States
Aaron G. Bart − Department of Medicinal Chemistry, University
of Kansas, Lawrence, Kansas 66045, United States
Qian Wu − Department of Chemistry and Chemical Biology,
Purdue School of Science, IUPUI, Indianapolis, Indiana 46202,
United States
Kevin R. Criscione − Department of Medicinal Chemistry,
University of Kansas, Lawrence, Kansas 66045, United States;
orcid.org/0000-0001-8429-3696
ABBREVIATIONS
■
AdoHcy, S-adenosyl-homocysteine; AdoMet, S-adenosyl-L-
methionine; hPNMT, human phenylethanolamine N-methyl-
transferase; PNMT, phenylethanolamine N-methyltransferase;
THIQ, 1,2,3,4-tetrahydroisoquinoline
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01475
REFERENCES
■
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NOTE ADDED IN PROOF
■
While the manuscript of this paper was under review, a similar
bisubstrate inhibitor design strategy for hPNMT was
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