A series of 2-substituted 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives were synthesized as inhibitors of D-amino acid oxidase (DAAO). Many compounds in this series were found to be potent DAAO inhibitors, with IC50 values in the double-digit nanomolar range. The 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione pharmacophore appears metabolically resistant to O-glucuronidation unlike other structurally related DAAO inhibitors. Among them, 6-hydroxy-2-(naphthalen-1-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione 11h was found to be selective over a number of targets and orally available in mice. Furthermore, oral coadministration of D-serine with 11h enhanced the plasma levels of D-serine in mice compared to the oral administration of D-serine alone, demonstrating its ability to serve as a pharmacoenhancer of D-serine.
A series of 2-substituted 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives were synthesized as inhibitors of D-amino acid oxidase (DAAO). Many compounds in this series were found to be potent DAAO inhibitors, with IC50 values in the double-digit nanomolar range. The 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione pharmacophore appears metabolically resistant to O-glucuronidation unlike other structurally related DAAO inhibitors. Among them, 6-hydroxy-2-(naphthalen-1-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione 11h was found to be selective over a number of targets and orally available in mice. Furthermore, oral coadministration of D-serine with 11h enhanced the plasma levels of D-serine in mice compared to the oral administration of D-serine alone, demonstrating its ability to serve as a pharmacoenhancer of D-serine.
d-Serine,
a full agonist at the glycine modulatory site
of the N-methyl-d-aspartate (NMDA) receptor,
has been actively explored as a potential therapeutic agent for the
treatment of schizophrenia.[1] Several clinical
studies with oral d-serine have shown promising results for
the treatment of not only positive symptoms but also negative symptoms
which have not responded well to existing drugs.[1] Further clinical development of d-serine, however,
could be hampered by the high doses of d-serine (60–120
mg/kg) required for the optimal efficacy[2] since d-serine was reported to be nephrotoxic in rats at
high doses.[3]d-Amino acid oxidase
(DAAO, EC 1.4.3.3) is a flavoenzyme that catalyzes the oxidation of d-amino acids including d-serine to the corresponding
α-keto acids. In mammals, DAAO is present in kidneys, liver,
and brain. Because the highest DAAO activity is found in the kidneys,[4] a substantial amount of orally administered d-serine is metabolized in the kidneys, contributing to its
rapid clearance. Indeed, pharmacokinetics studies of d-serine
in mice lacking DAAO unmasked the predominant role of DAAO in the
plasma clearance of d-serine.[5] Furthermore, d-serine-induced nephrotoxicity is most likely
due to the production of hydrogen peroxide as a byproduct of DAAO-mediated
oxidation because it does not occur in mutant rats lacking DAAO activity.[6] These findings collectively suggest that inhibition
of DAAO would exert dual beneficial effects on d-serine therapy:
(i) enhancement of d-serine bioavailability and (ii) attenuation
of d-serine induced nephrotoxicity.In the past decade,
concurrent with the functional studies of DAAO,[7] substantial strides have been made in identifying
potent and selective DAAO inhibitors of broad structural diversity
(Figure ).[8,9] Carboxylate-based DAAO inhibitors 1(10) and 2(11) presumably
originated from a prototype DAAO inhibitor, benzoic acid, have evolved
into a new class of compounds containing carboxylate bioisosteres
such as 3(12) and 4.[13] Crystal structure of 4 bound to DAAO revealed that the α-hydroxycarbonyl moiety of 4 retains the key interactions seen between the DAAO active
site and carboxylate-based inhibitors.[13] Subsequently, a variety of other heterocyclic frameworks bearing
an α-hydroxycarbonyl moiety were explored in the pursuit of
new scaffolds for potent DAAO inhibitors including 5(14) and 6.[15] Structural insights gained from DAAO in complex with imino-DOPA[16] led to the discovery of the latest DAAO inhibitors
possessing an additional substituent that extends to the secondary
binding site adjacent to the active site of DAAO. For example, our
group exploited this secondary binding site using kojic acid derivatives
such as compound 7 substituted at its 2-methyl group.[17] Similarly, Hondo et al. reported potent DAAO
inhibitors 8a–b based on a 4-hydroxypyridazin-3(2H)-one scaffold with a phenethyl group extending to the
secondary binding site.[18] The secondary
binding site was also exploited by carboxylate-based DAAO inhibitors
such as 9(19) and 10.[20]
Figure 1
Representative inhibitors of DAAO.
Representative inhibitors of DAAO.Recently, the pharmacokinetics
profile of several representative
DAAO inhibitors has been systematically compared in mice and rats.[21] Although most of the DAAO inhibitors were reported
to show negligible distribution to the brain, the poor CNS permeability
does not pose a major concern for our objective of increasing exposure
to orally administered d-serine by inhibition of peripheral
DAAO. To our surprise, however, carboxylate-based DAAO inhibitors
exhibited superior oral bioavailability relative to those containing
a carboxylate bioisostere. Subsequent investigation revealed that
most of these carboxylate bioisosteres are subject to glucuronidation
in liver microsomes, presumably contributing to their poor oral bioavailability.[22]As a part of our continuous effort to
identify new orally available
DAAO inhibitors, we have searched for a pharmacophore providing not
only potent DAAO inhibition but also considerable resistance to glucuronidation.
During the course of screening a library of FDA-approved small molecule
drugs, ceftriaxone (Figure ) was identified as a moderate DAAO inhibitor with an IC50 value of 10 μM. Although ceftriaxone does not offer
a particularly attractive molecular template for developing SAR studies,
the 3-mercapto-2-methyl-1,2-dihydro-1,2,4-triazine-5,6-dione portion
of ceftriaxone has caught our attention as its tautomer, 6-hydroxy-3-mercapto-2-methyl-1,2,4-triazin-5(2H)-one, has a structural feature common to other DAAO inhibitors.
Interestingly, 6-hydroxy-2-methyl-1,2,4-triazine-3,5(2H,4H)-dione 11a, one of the degradation
products of ceftriaxone in aqueous solution,[23] inhibited DAAO with an IC50 value of 2.8 μM, prompting
us to further examine the potential of 2-substituted 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives as new DAAO inhibitors.
Here we present a new class of DAAO inhibitors based on the 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione scaffold. With appropriate
substitutions at the 2-position, some 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives exhibited good
potency as well as improved metabolic stability. One of these derivatives
was also tested for its oral bioavailability as well as effects on d-serine plasma levels following oral coadministration with d-serine in mice.
Figure 2
Ceftriaxone and compound 11a.
Ceftriaxone and compound 11a.
Chemistry
The majority of 2-substituted
6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives were synthesized
using 6-bromo-1,2,4-triazine-3,5(2H,4H)-dione 12(24) as a starting
material (Scheme ).
Alkylation of the 2-position of 12 with alkyl halides
mediated by bis(trimethylsilyl)acetamide (BSA)[25] gave 2-substituted derivatives 13a–z. Subsequently, the 6-bromo group of 13a–z was replaced by the benzyloxy group by treating with benzyl
alcohol in the presence of potassium carbonate to give 14a–z. The final products 11a–z were obtained by removing the benzyl group of 14a–z by either catalytic hydrogenation or boron
tribromide. Anisole derivatives 11u–w are further converted into the corresponding phenolic derivatives 15u–w by boron tribromide.
Scheme 1
Synthesis
of 11a–z and 15u–w
Reagents and conditions: (a)
RI or RBr, BSA, acetonitrile, 82 °C; (b) K2CO3, BnOH, 150 °C; (c) H2, Pd/C, MeOH, rt; (d)
BBr3, dichloromethane, rt.
Synthesis
of 11a–z and 15u–w
Reagents and conditions: (a)
RI or RBr, BSA, acetonitrile, 82 °C; (b) K2CO3, BnOH, 150 °C; (c) H2, Pd/C, MeOH, rt; (d)
BBr3, dichloromethane, rt.Some
derivatives were synthesized using 6-bromo-4-[(phenylmethoxy)methyl]-1,2,4-triazine-3,5(2H,4H)-dione 16(26) as a starting material (Scheme ). Mitsunobu reaction of 16 and
alcohols with the aid of DIAD and PPh3 provided the 2-substituted
derivatives 17a–f. Reaction with
benzyl alcohol gave the benzyloxy protected intermediates 18a–f, which were subsequently converted into the
desired products 19a–f by either
catalytic hydrogenation or boron tribromide. Synthesis of ketone derivative 22 began with base-mediated coupling of 2-bromoacetophenone
to 16. The product 20 was converted into 21 by treating with benzyl alcohol. The removal of benzyl
and BOM groups from 21 by boron tribromide afforded 22. Synthetic intermediate 21 was also transformed
to the hydroxyl derivative 23. Synthesis of 2-phenyl
derivative 26 involves Chan–Lam coupling of 16 to phenylboronic acid to give 24. Subsequent
benzyloxylation and deprotection by BBr3 provided 26.
Reagents and conditions: (a)
ROH, Ph3P, DIAD, THF, 66 °C; (b) BnOH, NaH, DMF, 0
°C; (c) H2, Pd/C, MeOH, rt; (d) BBr3, dichloromethane,
rt; (e) BrCH2COPh, NaH, DMF, rt; (f) (i) H2,
[Rh(COD)Cl]2, Et3N, MeOH-EtOAc, rt, (ii) BBr3, dichloromethane, rt; (g) phenylboronic acid, pyridine, copper(II)
acetate, dichloromethane, rt.As shown in Scheme , we synthesized
two structurally close analogues of 11e, where its carboxylate
bioisostere moiety is slightly modified.
3-Thioxo derivative 30 was prepared using (2-phenylethyl)hydrazine 27 as a starting material. Reaction of 27 with
ammonium thiocyanate provided hydrazinecarbothioamide 28,[27] which was subsequently treated with
methyl chlorooxoacetate to give 29.[28] Cyclization of 29 in the presence of DBU afforded
the desired product 30.[28] Uracil
derivative 32 was prepared by BSA-mediated alkylation
of 5-hydroxypyrimidine-2,4(1H,3H)-dione 31 with phenethyl iodide.
The inhibitory potency of the synthesized compounds were determined
using recombinant humanDAAO as previously reported.[12] In vitro DAAO inhibitory data are summarized in Tables and 2. As shown in Table , 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives containing a variety of substituents
at the 2-position displayed varying degrees of inhibitory potency
against DAAO. All compounds but 11c and 26 were found to be potent DAAO inhibitors, with IC50 values
in the low-micromolar to nanomolar range. Within the compounds containing
an aromatic ring, phenethyl derivative 11e and naphthalen-1-ylmethyl
derivative 11h exhibited the most potent inhibitory activity,
with IC50 values of 70 and 50 nM, respectively. Incorporation
of a heterocyclic ring (compounds 19a–d) did not result in improved potency. We conducted inhibitory kinetic
studies of compound 11h at various concentrations of d-serine to determine its mode of inhibition. As shown in Figure , a double reciprocal
plot of DAAO activity versus the d-serine concentrations
produced a pattern indicative of competitive inhibition with a Ki value of 60 nM. Furthermore, escalation of
FAD concentration from 10 to 100 μM did not affect IC50 value for 11h, providing additional supporting evidence
that compound 11h competes with d-serine for
the substrate-binding site distinct from the FAD-binding site of DAAO.
Table 1
Inhibition of Human DAAO by 6-Hydroxy-1,2,4-triazine-3,5(2H,4H)-dione Derivatives
Values are mean ± SD of at
least four experiments.
Table 2
Inhibition of Human DAAO by Analogues
of 11e
compd
R2
R3
R4
X1
X2
Y
Z
IC50 (μM)a
11e
H
H
H
H
H
O
N
0.07 ± 0.01
11j
Cl
H
H
H
H
O
N
0.10 ± 0.05
11k
H
Cl
H
H
H
O
N
0.06 ± 0.02
11l
H
H
Cl
H
H
O
N
0.04 ± 0.01
11m
F
H
H
H
H
O
N
0.08 ± 0.03
11n
H
F
H
H
H
O
N
0.06 ± 0.02
11o
H
H
F
H
H
O
N
0.05 ± 0.02
11p
CH3
H
H
H
H
O
N
0.08 ± 0.03
11q
H
CH3
H
H
H
O
N
0.07 ± 0.02
11r
H
H
CH3
H
H
O
N
0.09 ± 0.02
11s
H
CF3
H
H
H
O
N
0.10 ± 0.02
11t
H
H
CF3
H
H
O
N
0.08 ± 0.04
11u
OCH3
H
H
H
H
O
N
0.27 ± 0.07
11v
H
OCH3
H
H
H
O
N
0.12 ± 0.03
11w
H
H
OCH3
H
H
O
N
0.12 ± 0.03
15u
OH
H
H
H
H
O
N
0.09 ± 0.04
15v
H
OH
H
H
H
O
N
0.16 ± 0.05
15w
H
H
OH
H
H
O
N
0.11 ± 0.03
11x
H
OPh
H
H
H
O
N
0.24 ± 0.05
11y
H
H
OPh
H
H
O
N
0.08 ± 0.02
11z
H
H
Ph
H
H
O
N
0.05 ± 0.02
19e
H
H
H
CH3
H
O
N
3.3 ± 0.6
19f
H
H
H
F
F
O
N
0.40
22
H
H
H
O
O
O
N
0.85 ± 0.13
23
H
H
H
OH
H
O
N
0.47 ± 0.23
30
H
H
H
H
H
S
N
0.05 ± 0.01
32
H
H
H
H
H
O
CH
0.08 ± 0.01
Values are mean ± SD of at
least four experiments except for compound 19f (n = 2).
Figure 3
Double-reciprocal plot
of the oxidation of d-serine by
human DAAO in the presence of compound 11h. The straight
lines represent the least-squares fit of the data obtained by plotting
the reciprocal of the initial rate of change in the absorbance at
411 nm versus the reciprocal of the d-serine concentrations
(mM).
Values are mean ± SD of at
least four experiments.Values are mean ± SD of at
least four experiments except for compound 19f (n = 2).Double-reciprocal plot
of the oxidation of d-serine by
humanDAAO in the presence of compound 11h. The straight
lines represent the least-squares fit of the data obtained by plotting
the reciprocal of the initial rate of change in the absorbance at
411 nm versus the reciprocal of the d-serine concentrations
(mM).The potent inhibitory activity
of 11e prompted us
to conduct more focused SAR studies using 11e as a molecular
template (Table ).
A variety of substitutions were examined at the terminal phenyl group
of 11e (compounds 11j–z and 15u–w). Overall, there was
no significant change in inhibitory potency, with IC50 values
ranging from 40 to 270 nM. Analogues containing a large substituent
such as compounds 11x–z retained
IC50 values in the nanomolar range. Para substitutions
were particularly well tolerated, as demonstrated by phenoxy substituted
derivative 11y and phenyl substituted 11z with IC50 values of 80 and 50 nM, respectively. These
substituents are presumably oriented toward the entrance of the DAAO
active site, possessing a high degree of steric tolerance. In contrast,
a notable decrease in potency was seen in compound 19e containing a branched methyl group at the linker connecting the
phenyl and the 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione moieties. Other modifications at the linker (compounds 19f, 22, and 23) were not as disruptive
as the methyl group of 19e although these compounds still
showed somewhat lower potency compared to 11e. Compounds 30 and 32 possessing slightly modified carboxylate
bioisosteres retained potent DAAO inhibitory activity with IC50 values 50 and 80 nM, respectively, nearly identical to that
of 11e.As illustrated in Figure , proposed binding mode of 11e (white) to the
active site of DAAO is superposed onto the crystal structure of DAAO
in complex with 8a (cyan).[18] In this model, the α-hydroxycarbonyl moiety forms key hydrogen
bonds with Tyr228 and Arg283. It is conceivable that the 4-position
nitrogen group of 11e forms a hydrogen bond with the
backbone carbonyl of Gly313 in a similar manner as the 2-nitrogen
atom of 4-hydroxypyridazin-3(2H)-one moiety in 8a. This explains the relatively high inhibitory potency of
DAAO inhibitors with a 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione scaffold compared to the kojic acid based
compounds[17] represented by 7, which lacks a nitrogen atom in the core ring. In contrast, the
nitrogen atom at the 1-position of 11e does not seem
to be involved in the interaction with the active site of DAAO. This
is consistent with the similar inhibitory potency of the uracil derivative 32, in which the corresponding position is replaced by a carbon
atom. 3-Thioxo derivative 30 was also as potent as 11e, presumably due to the preservation of all of the key
interactions involved in binding of 11e to DAAO. The
secondary binding pocket occupied by the 2-phenylethyl group of 11e appears quite spacious and capable of accommodating larger
substituents owing to the movement of Tyr224 away from the active
site. This is in a good agreement with the high inhibitory potency
retained by compounds with bulky substituents such as compounds 11x–z.
Figure 4
Proposed binding mode of 11e (white) to the active
site of DAAO (3W4K). Key residues and FAD are shown in green and yellow, respectively.
Hydrogen-bonding interactions between 11h and the key
residues are shown as gray dashed lines. Compound 8a (cyan)
of 3W4K is also
shown for comparison.
Proposed binding mode of 11e (white) to the active
site of DAAO (3W4K). Key residues and FAD are shown in green and yellow, respectively.
Hydrogen-bonding interactions between 11h and the key
residues are shown as gray dashed lines. Compound 8a (cyan)
of 3W4K is also
shown for comparison.Table summarizes
in vitro metabolic stability of selected compounds in mouse liver
microsomes. As mentioned earlier, many of the DAAO inhibitors containing
carboxylate bioisosteres are known to be subject to a varying degree
of glucuronidation in liver microsomes.[22] For example, a kojic acid based DAAO inhibitor 7 is
extensively glucuronidated in mouse liver microsomes in the presence
of UDPGA, while compound 8b with the 4-hydroxypyridazin-3(2H)-one scaffold shows some resistance to glucuronidation.
6-Hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives 11e and 11h were found
to be completely resistant to glucuronidation in mouse liver microsomes.
3-Thioxo derivative 30 was also metabolically stable
against glucuronidation. In sharp contrast, uracil-based DAAO inhibitor 32 was fully glucuronidated within 60 min of incubation. This
trend appears consistent with our previous findings from other DAAO
inhibitors, showing that increased topological polar surface area
(tPSA) of the carboxylate bioisostere moiety contributes to higher
resistance to glucuronidation.[22] Compounds 11e and 11h were also stable in mouse liver microsomes
in the presence of NADPH, a cofactor for CYP-dependent oxidation.
Table 3
Metabolic Stability of DAAO Inhibitors
in Mouse Liver Microsomes
% remaining
after 60 min incubation
compd
tPSA (Å2)a
w/UDPGA
w/NADPH
7
47
5
NDb
8b
62
59
92
11e
82
≥99
≥99
11h
82
≥99
≥99
30
97
≥99
87
32
70
≤1
NDb
Topological surface area of the
carboxylate bioisostere moiety.
Not determined.
Topological surface area of the
carboxylate bioisostere moiety.Not determined.Further
pharmacological characterization was conducted with compound 11h. No evidence of mutagenicity was observed in the Ames
test using Salmonella typhimurium strain
(TA100) in the presence or absence of metabolic activation by S9 mixture.
Compound 11h was also tested in a panel of relevant in
vitro assays (Table ) including hERG channels, various sites of the NMDA receptors, and
another class of flavoenzymes, monoamine oxidases A and B (MAO-A and
MAO-B). Compound 11h showed no significant activity in
any of these assays at the highest tested concentrations.
Table 4
In Vitro Pharmacological Characterization
of 11h
assay
results
hERG channel
no inhibition
at concentrations up to 25 μM
NMDA receptor (agonist)
≤5% inhibition at 10 μM
NMDA receptor (glycine)
22% inhibition at 10 μM
NMDA receptor (phencyclidine)
≤5% inhibition at 10 μM
NMDA receptor (MK801)
≤5% inhibition at 10 μM
glycine receptor (strychnine sensitive)
≤5% inhibition at 10 μM
MAO-A
≤5% inhibition at 10 μM
MAO-B
≤5% inhibition at 10 μM
In vivo pharmacokinetic studies of 11h were carried
out in CD1mice by intravenous and oral administration (30 mg/kg).
The plasma pharmacokinetic parameters are summarized in Table , along with those for 8b for direct comparison. The plasma clearance of 11h in mice was 10.2 mL/min/kg with a terminal half-life of 0.9 h following
intravenous administration. Oral absorption of 11h was
rapid, and the plasma peak concentration was generally observed within
the first hour of oral administration. As predicted from the metabolic
stability data (Table ), the oral bioavailability of 11h (F = 79%) is significantly higher than that of 8b (F = 31%) despite the increased polarity. The improvement
in oral bioavailability of 11h over 8b clearly
illustrates the significant impact of glucuronidation on the pharmacokinetics
of DAAO inhibitors. Following oral administration, compound 11h showed negligible brain penetration with a brain to plasma
ratio of 0.01 in mice. Thus, compound 11h is unlikely
to increase the brain levels of endogenous d-serine by inhibiting
DAAO expressed in the brain. Given the favorable plasma exposure,
however, compound 11h should be capable of inhibiting
peripheral DAAO and minimize the metabolism of orally taken d-serine.
Table 5
Mouse Pharmacokinetics of 8b and 11h (30 mg/kg)
route
parameter
8ba
11h
iv
CL (mL/min/kg)
38.7
10.2
Vd (L/kg)
0.5
0.8
T1/2 (h)
0.2
0.9
AUClast (μg·h/mL)
12.7b
16.3
po
Tmax (h)
0.25
0.08
Cmax (μg/mL)
6.6
13.8
AUClast (μg·h/mL)
3.9
12.9
brain to plasma ratio
NDc
0.01
po/iv
F (%)
31
79
Intravenous administration
of 8b was conducted at 10 mg/kg.
The value is dose-normalized to
30 mg/kg.
Not determined.
Intravenous administration
of 8b was conducted at 10 mg/kg.The value is dose-normalized to
30 mg/kg.Not determined.To determine the effects of 11h on d-serine
plasma levels, mice (n = 3 per time point) were dosed
with 11h (30 mg/kg, po) along with d-serine
(30 mg/kg, po) simultaneously. As shown in Figure , compound 11h showed no ability
to enhance plasma Cmax of d-serine.
However, its plasma clearance was substantially reduced for a sustained
period of time, resulting in the significant increase in d-serine AUC0–6h (96.9 μg*h/mL) compared to
that of d-serine alone treatment (43.3 μg·h/mL).
The curve of d-serine concentration versus time in mice coadministered
with 11h is nearly identical to that of DAAO knockout
mice treated with oral d-serine.[5] The lack of enhancement in d-serine plasma Cmax in mice by coadministration with 11h is
in a good agreement with the negligible levels of DAAO found in mouse
liver as opposed to kidneys,[29] where DAAO
is abundant and most likely contributes to the plasma clearance of d-serine. Thus, the primary site of action for 11h in mice appears DAAO in kidneys, which explains its significant
effects on plasma clearance of d-serine but not on plasma Cmax value. Given the previously reported cross-species
variation in IC50 values between human and rat forms of
DAAO,[13] caution needs to be taken in establishing
the PK/PD relationship in mice as we have not measured inhibitory
potency of 11h in mouseDAAO. It is worth noting, however,
that 4-hydroxypyridazin-3(2H)-one derivatives represented
by 8b showed little difference in inhibitory potency
between human and mouseDAAO.[18] Given their
structural similarity to the 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione derivatives, we anticipate
comparative inhibitory potencies of 11h against human
and mouse forms of DAAO.
Figure 5
Effects of compound 11h (30 mg/kg,
po) on plasma pharmacokinetics
of oral d-serine (30 mg/kg) in mice.
Effects of compound 11h (30 mg/kg,
po) on plasma pharmacokinetics
of oral d-serine (30 mg/kg) in mice.
Conclusions
In the past decade, substantial efforts have
been made by multiple
groups to develop DAAO inhibitors of potential therapeutic value.
These efforts have not only provided structurally diverse DAAO inhibitors
but also contributed to the cumulative knowledge in this area, including
the use of carboxylate bioisosteres, exploitation of the secondary
binding pocket, and the strategy to minimize glucuronidation. We took
full advantage of the prior knowledge in guiding our efforts to develop
a new class of DAAO inhibitors based on the 6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione pharmacophore. The inhibitory
potency of these compounds was dependent on the variation of the substituents
at the 2-position. Many compounds containing an arylalkyl group at
this position showed potent inhibitory activity against DAAO. Among
these inhibitors, compound 11h exhibited good metabolic
stability in liver microsomes and selectivity over other relevant
targets. Compound 11h was also found to be orally available
and enhance plasma levels of coadministered d-serine by reducing
its clearance. The ability of compound 11h to sustain
high plasma levels of d-serine should be particularly attractive
for the treatment of chronic psychiatric disorders such as schizophrenia.
Experimental Section
General
All solvents
were reagent grade or HPLC grade.
Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. All reactions were
performed under nitrogen. Melting points were obtained on a Mel-Temp
apparatus and are uncorrected. 1H NMR spectra were recorded
at 400 MHz. 13C NMR spectra were recorded at 100 MHz. The
HPLC solvent system consisted of distilled water and acetonitrile,
both containing 0.1% formic acid. Preparative HPLC purification was
performed on an Agilent 1200 series HPLC system equipped with an Agilent
G1315DDAD detector using a Phenomenex Luna 5 μm C18 (2) column
(21.2 mm × 250 mm, 5 μm). Analytical HPLC was performed
on an Agilent 1200 series HPLC system equipped with an Agilent G1315DDAD detector (detection at 220 nm) and an Agilent 6120 quadrupole
MS detector. Unless otherwise specified, the analytical HPLC conditions
involve a gradient of 20% acetonitrile/80% water for 0.5 min followed
by an increase to 85% acetonitrile/15% water over 4 min and continuation
of 85% acetonitrile/15% water for 3.5 min with a Luna C18 column (2.1
mm × 50 mm, 3.5 μm) at a flow rate of 0.75 mL/min. All
final compounds tested were confirmed to be of ≥95% purity
by the HPLC methods described above. The model of compound 11h bound to humanDAAO (Figure ) was generated using AutoDock Vina.[30]
To a solution of 5-bromo-6-azauracil 12 (0.30 g, 1.6
mmol) in acetonitrile (5 mL) was added N,O-bis(trimethylsilyl)acetamide (4.0 mmol,
1.0 mL). The mixture was heated at 82 °C for 3 h, after which
methyl iodide (0.12 mL, 1.9 mmol) was added. After 24 h, more methyl
iodide (0.5 equiv) was added and the mixture was stirred for additional
24 h. The reaction mixture was concentrated, and the resulting residue
was dissolved in dichloromethane, washed with water and brine, dried
over Na2SO4, and concentrated to give 0.11 g
of 13a as a black solid (33% crude yield). This material
was used in the next step without further purification. 1H NMR (DMSO-d6) δ 3.44 (s, 3H),
12.50 (s, 1H).
A mixture
of 13a (0.10 g, 0.49 mmol) and K2CO3 (0.14 g, 1.0 mmol) in benzyl alcohol (1.0 mL) was heated overnight
at 150 °C. The reaction mixture was partitioned between aqueous
10% KHSO4 and EtOAc. The organic layer was dried over Na2SO4 and concentrated. The residual material was
purified using a Biotage Isolera One flash purification system with
a silica gel flash cartridge (EtOAc/hexanes) to give 0.070 g of 14a as a white solid (61% yield). 1H NMR (DMSO-d6) δ 3.36 (s, 3H), 5.12 (s, 2H), 7.37–7.46
(m, 5H), 12.16 (s, 1H).
To a solution of 14a (0.060 g, 0.26 mmol) in methanol
(5 mL) was added one
spatula tip of 10% Pd/C. The mixture was shaken under hydrogen (30
psi) for 1 h. The catalyst was removed by filtration, and the filtrate
was concentrated to give 0.035 g of 11a as a beige powder
(94% yield); mp 259 °C. 1H NMR (DMSO-d6) δ 3.27 (s, 3H), 11.11 (s, 1H), 12.03 (s, 1H).
LCMS (5% acetonitrile/95% water for 0.5 min followed by an increase
to 40% acetonitrile/60% water over 4 min and continuation of 40% acetonitrile/60%
water for 3.5 min): retention time 5.05 min, m/z 144 [M + H]+.
Compound 13b was prepared as described for the preparation
of 13a, except 1-iodo-3-methylbutane (1.6 equiv) was
used in place of methyl
iodide. The crude product was purified by trituration with cold diethyl
ether; yellow solid (27% yield). 1H NMR (DMSO-d6) δ 0.89 ((d, J = 6.3 Hz, 6H),
1.50 (m, 2H), 1.60 (m, 1H), 3.82 (t, J = 7.3 Hz,
2H), 12.48 (s, 1H).
Compound 13c was prepared as described for the preparation
of 13a, except 1-iodo-3,3-dimethylbutane (3.3 equiv)
was used
in place of methyl iodide; clear oil (19% yield). 1H NMR
(DMSO-d6) δ 0.92 (s, 9H), 1.53 (m,
2H), 3.82 (m, 2H), 12.50 (s, 1H).
Compound 11c was prepared from 14c as described
for the preparation of 11a, except the material was purified
by trituration with EtOAc/hexanes; white solid (77% yield); mp 219
°C. 1H NMR (DMSO-d6) δ
0.91 (m, 9H), 1.50 (m, 2H), 3.67 (m, 2H), 11.64 (s, 1H), 12.02 (s,
1H). LCMS: retention time 2.45 min, m/z 214 [M + H]+.
Compound 13d was prepared as described for the preparation
of 13a, except benzyl bromide (1.2 equiv) was used in
place of methyl iodide.
The crude product was purified by trituration with cold diethyl ether;
light-tan solid (71% yield). 1H NMR (DMSO-d6) δ 5.02 (s, 2H), 7.29–7.38 (m, 5H), 12.59
(s, 1H).
Compound 14d was prepared from 13d as described for the
preparation of 14a, except the product was purified by
trituration with EtOAc/hexanes; white solid (70% yield). 1H NMR (DMSO-d6): δ 4.91 (s, 2H),
5.11 (s, 2H), 7.29–7.40 (m, 10H), 12.24 (s, 1H).
Compound 11d was prepared from 14d as described
for the preparation
of 11a, except the hydrogenation was performed overnight
under atmospheric pressure of hydrogen; light-pink powder (81% yield);
mp 242 °C. 1H NMR (DMSO-d6) δ 4.85 (s, 2H), 7.26–7.36 (m, 5H), 11.72 (bs, 1H),
11.94 (bs, 2H). LCMS (5% acetonitrile/95% water for 0.5 min followed
by an increase to 40% acetonitrile/60% water over 4 min and continuation
of 40% acetonitrile/60% water for 3.5 min): retention time 2.84 min, m/z 220 [M + H]+.
Compound 13e was prepared as described for the preparation
of 13a, except phenethyl iodide (2.5 equiv) was used
in place of methyl
iodide. The crude product was purified by trituration with cold diethyl
ether; tan solid (64% yield). 1H NMR (DMSO-d6): δ 2.94 (t, J = 7.5 Hz, 2H),
4.04 (t, J = 7.6 Hz, 2H), 7.22 (m, 3H), 7.28 (m,
2H), 12.53 (s, 1H).
Compound 14e was prepared from 13e as described
for the
preparation of 14a, except the product was purified by
trituration with EtOAc/hexanes; beige solid (74% yield). 1H NMR (DMSO-d6): δ 2.91 (t, J = 7.2 Hz, 2H), 5.05 (s, 2H), 3.96 (t, J = 7.2 Hz, 2H), 7.15 (m, 2H), 7.20 (m, 1H), 7.26–7.31 (m,
2H), 7.36–7.43 (m, 5H), 12.13 (s, 1H).
Compound 11e was prepared from 14e as described
for the
preparation of 11a, except a mixture of methanol and
acetic acid (3:1) was used as a solvent and that the hydrogenation
was performed at 50 psi for 1.5 h; light-pink powder (60% yield);
mp 200 °C. 1H NMR (DMSO-d6) δ 2.90 (t, J = 7.8 Hz, 2H), 3.87 (t, J = 7.6 Hz, 2H), 7.20 (m, 3H), 7.29 (m, 2H), 11.71 (s, 1H),
12.03 (s, 1H). LCMS: retention time 1.50 min, m/z 234 [M + H]+.
Compound 13f was prepared as described for the preparation
of 13a, except (3-iodopropyl)benzene (1.6 equiv) was
used in
place of methyl iodide; white solid (43% yield). 1H NMR
(DMSO-d6): δ 1.94 (m, 2H), 2.63
(t, J = 7.7 Hz, 2H), 3.83 (t, J =
6.9 Hz, 2H), 7.15–7.22 (m, 3H), 7.25–7.29 (m, 2H), 12.45
(s, 1H).
Compound 11f was prepared from 14f as described
for the
preparation of 11a, except a mixture of methanol and
acetic acid (3:1) was used as a solvent; light-pink solid (85% yield);
mp 153 °C. 1H NMR (DMSO-d6) δ 1.89 (m, 2H), 2.60 (t, J = 7.6 Hz, 2H),
3.67 (t, J = 6.9 Hz, 2H), 7.20 (m, 3H), 7.25 (m,
2H), 11.65 (bs, 1H), 11.93 (bs, 1H). LCMS: retention time 1.87 min, m/z 248 [M + H]+.
Compound 13g was prepared as described for the preparation
of 13a, except 2-(bromomethyl)naphthalene (1.2 equiv)
was used in place of methyl iodide. The reaction was heated overnight.
The crude product was purified by trituration with cold diethyl ether;
tan solid (56% yield). 1H NMR (DMSO-d6) δ 5.20 (s, 2H), 7.46 (dd, J = 1.5,
8.3 Hz, 1H), 7.51 (m, 2H), 7.22 (m, 1H), 7.86–7.92 (m, 4H),
12.62 (s, 1H).
To a solution of compound 14g (0.26 g, 0.72 mmol) in
dichloromethane (7.0 mL) was slowly added a 1.0 M solution of BBr3 (1.4 mL, 1.4 mmol) at room temperature. The reaction was
stirred for 1 h, after which 2 additional equiv of BBr3 were added. Stirring continued at rt for an additional hour, and
the reaction was quenched by the addition of water. The compound was
extracted with EtOAc. The organic layer was washed with brine, dried
over Na2SO4, and concentrated. The residual
material was purified by preparative HPLC (20% acetonitrile/80% water
for 5 min followed by an increase to 70% acetonitrile/30% water over
35 min and an increase to 100% acetonitrile over 10 min; flow rate
25 mL/min) to give 0.057 g of 11g as a white fluffy solid
(29% yield); mp 264 °C. 1H NMR (DMSO-d6) δ 5.03 (s, 2H), 7.44 (dd, J =
1.3, 8.3 Hz, 1H), 7.49 (m, 2H), 7.80 (s, 1H), 7.88 (m, 3H), 12.07
(bs, 2H). LCMS (20% acetonitrile/80% water for 0.25 min followed by
an increase to 85% acetonitrile/15% water over 1.5 min and continuation
of 85% acetonitrile/15% water for 2.25 min; flow rate 1.25 mL/min):
retention time 1.45 min, m/z 270
[M + H]+.
Compound 13h was prepared as described for the preparation
of 13a, except 1-(bromomethyl)-naphthalene (1.2 equiv)
was used in place of methyl iodide. The crude product was purified
by trituration with cold diethyl ether; orange solid (65% yield). 1H NMR (DMSO-d6) δ 5.49 (s,
2H), 7.49 (m, 2H), 7.59 (m, 2H), 7.91 (m, 1H), 7.98 (m, 1H), 8.14
(d, J = 8.1 Hz, 1H), 12.64 (br s, 1H).
Compound 11h was prepared from 14h as described
for the preparation of 11a, with the exception that a
mixture of methanol/ethyl acetate/acetic acid (1:1:0.1) was used as
a solvent. The hydrogenation was performed at 20 psi for 3 h; white
powder (90% yield); mp 260 °C (dec). 1H NMR (DMSO-d6) δ 5.33 (s, 2H), 7.41 (d, J = 7.1 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.57 (m,
2H), 7.88 (d, J = 8.1 Hz, 1H), 7.96 (m, 1H), 8.16
(d, J = 7.5 Hz, 1H), 11.68 (s, 1H), 12.20 (s, 1H).
LCMS (20% acetonitrile/80% water for 0.25 min followed by an increase
to 85% acetonitrile/15% water over 1.5 min and continuation of 85%
acetonitrile/15% water for 2.25 min; flow rate 1.25 mL/min): retention
time 1.43 min, m/z 270 [M + H]+. The corresponding sodium salt was used for all in vivo studies
involving compound 11h. The salt was prepared by dissolving 11h in 1.0 equiv of a 0.103 N volumetric solution of NaOH,
followed by the lyophilization.
Compound 13j was prepared as described for the preparation
of 13a, except 3-chloro-4-(2-iodoethyl)benzene (2.0 equiv)
was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; yellow solid (28% yield). 1H NMR (DMSO-d6) δ 3.07 (t, J = 7.1 Hz, 2H), 4.07 (t, J = 7.1 Hz, 2H),
7.26–7.29 (m, 2H), 7.34 (m, 1H), 7.41 (m, 1H), 12.54 (s, 1H).
Compound 11j was prepared from 14j as described
for the preparation of 11g, with the exception that 2
equiv of BBr3 were used and that the crude product was
purified by trituration with EtOAc/hexanes; white powder (39% yield);
mp 211 °C. 1H NMR (DMSO-d6) δ 3.04 (t, J = 7.1 Hz, 2H), 3.91 (t, J = 7.1 Hz, 2H), 7.24–7.29 (m, 3H), 7.41 (m, 1H),
11.66 (s, 1H), 12.04 (s, 1H). LCMS: retention time 2.90 min, m/z 268 [M + H]+.
Compound 13k was prepared as described for the preparation
of 13a, except 3-chloro-4-(2-iodoethyl)benzene (2.0 equiv)
was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; tan solid (73% yield). 1H NMR (DMSO-d6) δ 2.95 (t, J = 7.2 Hz, 2H), 4.06 (t, J = 7.3 Hz, 2H),
7.18 (m, 1H), 7.27–7.34 (m, 3H), 12.53 (s, 1H).
Compound 11k was prepared from 14k as described
for the preparation of 11g, with the exception that 2
equiv of BBr3 were added at rt and that the reaction was
stirred at rt for 1.5 h. The compound was purified by trituration
with EtOAc/hexanes; beige powder (54% yield); mp 219 °C. 1H NMR (DMSO-d6) δ 2.92 (t, J = 7.1 Hz, 2H), 3.90 (t, J = 7.2 Hz, 2H),
7.14 (m, 1H), 7.26–7.33 (m, 3H), 11.69 (s, 1H), 12.04 (s, 1H).
LCMS: retention time 2.91 min, m/z 268 [M + H]+.
Compound 13l was prepared as described for the preparation
of 13a, except 1-chloro-4-(2-iodoethyl)benzene (1.6 equiv)
was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; yellow solid (42% yield). 1H NMR (DMSO-d6) δ 2.94 (t, J = 7.3 Hz, 2H), 4.03 (t, J = 7.3 Hz, 2H),
7.25 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 12.52 (s, 1H).
Compound 11l was prepared from 14l as described
for the preparation of 11g, with the exception that only
2 equiv of BBr3 were used and that the compound was purified
by trituration with EtOAc/hexanes;vwhite solid (52% yield); mp 225
°C. 1H NMR (DMSO-d6) δ
2.90 (t, J = 7.2 Hz, 2H), 3.87 (t, J = 7.3 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 11.69 (s, 1H), 12.03 (s, 1H). LCMS (20%
acetonitrile/80% water for 0.25 min followed by an increase to 85%
acetonitrile/15% water over 1.5 min and continuation of 85% acetonitrile/15%
water for 2.25 min; flow rate 1.25 mL/min): retention time 1.39 min, m/z 268 [M + H]+.
Compound 13m was prepared as described for the preparation
of 13a, except 2-fluoro-4-(2-iodoethyl)benzene (2.0 equiv)
was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; yellow solid (34% yield). 1H NMR (DMSO-d6) δ 2.98 (t, J = 7.2 Hz, 2H), 4.05 (t, J = 7.2 Hz, 2H),
7.15 (m, 2H), 7.29 (m, 2H), 12.55 (s, 1H).
Compound 14m was prepared from 13m as described
for the preparation of 14a, with the exception that the
reaction was heated for 3 days and that the product was purified by
trituration with EtOAc/hexanes; white powder (65% yield). 1H NMR (DMSO-d6): δ 2.96 (t, J = 6.8 Hz, 2H), 3.97 (t, J = 6.6 Hz, 2H),
4.95 (s, 2H), 7.11 (m, 2H), 7.17 (m, 1H), 7.24 (m, 1H), 7.36–7.41
(m, 5H), 12.17 (s, 1H).
Compound 11m was prepared from 14m as described
for the preparation of 11a, with the exception that a
mixture of methanol and ethyl acetate (1:1) was used as the solvent
and that the hydrogenation was performed at 30 psi for 1.5 h; white
solid (83% yield); mp 194 °C. 1H NMR (DMSO-d6) δ 2.95 (t, J = 7.2
Hz, 2H), 3.88 (t, J = 7.2 Hz, 2H), 7.12 (m, 2H),
7.26 (m, 2H), 11.68 (bs, 1H), 11.99 (bs, 1H). LCMS: retention time
1.30 min, m/z 252 [M + H]+.
Compound 13n was prepared as described for the preparation of 13a, except 3-fluoro-4-(2-iodoethyl)benzene (2.0 equiv) was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; yellow solid (66% yield). 1H NMR (DMSO-d6) δ 2.96 (t, J = 7.3 Hz, 2H), 4.06 (t, J = 7.3 Hz, 2H),
7.05 (m, 2H), 7.22 (m, 1H), 7.33 (m, 1H), 12.53 (s, 1H).
Compound 14n was prepared from 13n as described
for the preparation of 14a, except the product was purified
by trituration with EtOAc/hexanes; tan solid (78% yield). 1H NMR (DMSO-d6) δ 2.94 (t, J = 6.8 Hz, 2H), 3.98 (t, J = 6.9 Hz, 2H),
5.06 (s, 2H), 6.96–7.05 (m, 3H), 7.29 (m, 1H), 7.36–7.43
(m, 5H), 12.13 (s, 1H).
Compound 11n was prepared from 14n as described
for the preparation of 11a, with the exception that a
mixture of methanol and ethyl acetate (1:1) was used as a solvent
and that the hydrogenation was performed overnight at 30 psi; white
solid (79% yield); mp 197 °C. 1H NMR (DMSO-d6) δ 2.93 (t, J = 7.3
Hz, 2H), 3.89 (t, J = 7.2 Hz, 2H), 7.03 (m, 3H),
7.31 (m, 1H), 11.89 (bs, 2H). LCMS: retention time 2.10 min, m/z 252 [M + H]+.
Compound 13o was prepared as described for the preparation
of 13a, except 1-fluoro-4-(2-iodoethyl)benzene (2.0 equiv)
was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; yellow solid (42% yield). 1H NMR (DMSO-d6) δ 2.93 (t, J = 7.1 Hz, 2H), 4.03 (t, J = 7.3 Hz, 2H),
7.11 (m, 2H), 7.26 (m, 2H), 12.52 (s, 1H).
Compound 11o was prepared from 14o as described
for the preparation of 11a, except the hydrogenation
was performed under an atmospheric pressure of hydrogen for 3 h. The
product was purified by trituration with EtOAc/hexanes; light-pink
powder (73% yield); mp 193 °C. 1H NMR (DMSO-d6) δ 2.89 (t, J = 7.3
Hz, 2H), 3.87 (t, J = 7.3 Hz, 2H), 7.10 (m, 2H),
7.22 (m, 2H), 11.72 (bs, 1H), 11.97 (bs, 1H). LCMS: retention time
1.95 min, m/z 252 [M + H]+.
Compound 13p was prepared as described for the preparation of 13a, except 3-methyl-4-(2-iodoethyl)benzene (2.0 equiv) was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; tan solid (52% yield). 1H NMR (DMSO-d6) δ 2.31 (s, 3H),
2.94 (m, 2H), 3.98 (m, 2H), 7.12 (m, 3H), 7.15 (m, 1H), 12.54 (s,
1H).
Compound 11p was prepared from 14p as described
for the preparation of 11a, with the exception that a
mixture of methanol and ethyl acetate (1:1) was used as a solvent
and the hydrogenation was performed at 30 psi for 2 h; gray solid
(71% yield); mp 207 °C. 1H NMR (DMSO-d6) δ 2.31 (s, 3H), 2.89 (t, J =
7.7 Hz, 2H), 3.81 (t, J = 7.7 Hz, 2H), 7.10 (m, 3H),
7.14 (m, 1H), 11.93 (bs, 2H). LCMS: retention time 2.42 min, m/z 248 [M + H]+.
Compound 13q was prepared as described for the preparation
of 13a, except 3-methyl-4-(2-iodoethyl)benzene (2.0 equiv)
was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; tan solid (66% yield). 1H NMR (DMSO-d6) δ 2.27 (s, 3H),
2.89 (t, J = 7.6 Hz, 2H), 4.01 (t, J = 7.6 Hz, 2H), 7.02 (m, 3H), 7.18 (t, J = 7.3 Hz,
1H), 12.50 (s, 1H).
Compound 11q was prepared from 14q as described
for the preparation of 11a, with the exception that a
mixture of methanol and ethyl acetate (1:1) was used as a solvent
and that the hydrogenation was performed overnight at 30 psi; white
powder (82% yield); mp 210 °C. 1H NMR (DMSO-d6) δ 2.27 (s, 3H), 2.86 (t, J = 7.3 Hz, 2H), 3.85 (t, J = 7.2 Hz, 2H), 6.99 (m,
3H), 7.18 (t, J = 7.8 Hz, 2H), 11.71 (s, 1H), 12.01
(s, 1H). LCMS: retention time 2.53 min, m/z 248 [M + H]+.
Compound 13r was prepared as described for the preparation
of 13a, except 4-methyl-4-(2-iodoethyl)benzene (2.0 equiv)
was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; tan solid (63% yield). 1H NMR (DMSO-d6) δ 2.26 (s, 3H),
2.89 (t, J = 7.5 Hz, 2H), 4.00 (m, 2H), 7.10 (s,
4H), 12.52 (s, 1H).
Compound 13s was prepared as described for the preparation
of 13a, except 1-(2-iodoethyl)-3-(trifluoromethyl)benzene
(2.0 equiv) was used in place of methyl iodide. The crude product
was purified by trituration with cold diethyl ether; yellow solid
(48%). 1H NMR (DMSO-d6) δ
3.06 (t, J = 7.2 Hz, 2H), 4.10 (t, J = 7.1 Hz, 2H), 7.55 (m, 4H), 12.53 (s, 1H).
Compound 13t was prepared as described for the preparation
of 13a, except 1-(2-iodoethyl)-4-(trifluoromethyl)benzene
(2.0 equiv) was used in place of methyl iodide. The crude product
was purified using a Biotage Isolera One flash purification system
with a silica gel flash cartridge (EtOAc/hexanes); yellow solid (69%
yield). 1H NMR (DMSO-d6) δ
3.04 (t, J = 7.2 Hz, 2H), 4.08 (t, J = 7.1 Hz, 2H), 7.46 (d, J = 7.1 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 12.53 (s, 1H).
Compound 14t was prepared from 13t as described
for the preparation of 14a, except the product was purified
by trituration with EtOAc/hexanes; white solid (71% yield). 1H NMR (DMSO-d6) δ 3.01 (t, J = 6.7 Hz, 2H), 4.01 (t, J = 6.8 Hz, 2H),
5.02 (s, 2H), 7.38–7.42 (m, 7H), 7.62 (d, J = 8.1 Hz, 2H), 12.13 (s, 1H).
Compound 11t was prepared from 14t as described
for the preparation of 11a, with the exception that a
mixture of methanol and EtOAc (1:1) was used as a solvent and that
the hydrogenation was performed overnight at 30 psi; gray solid (83%
yield); mp 222 °C. 1H NMR (DMSO-d6) δ 3.01 (t, J = 7.1 Hz, 2H),
3.92 (t, J = 7.1 Hz, 2H), 7.42 (d, J = 7.8 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 11.71
(s, 1H), 12.02 (s, 1H). LCMS: retention time 2.99 min, m/z 302 [M + H]+.
Compound 13u was prepared as described for the preparation
of 13a, with the exception that 1-(2-iodoethyl)-2-methoxybenzene
(2.0 equiv) was used in place of methyl iodide. The crude product
was purified by trituration with cold diethyl ether; tan solid (57%
yield). 1H NMR (DMSO-d6) δ
2.91 (t, J = 6.9 Hz, 2H), 3.73 (s, 3H), 4.01 (t, J = 7.0 Hz, 2H), 6.87 (dt, J = 1.0, 7.6
Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 7.11 (m, 1H),
7.21 (m, 1H), 12.52 (s, 1H).
Compound 11u was prepared from 14u as described
for the preparation of 11a, with the exception that a
mixture of methanol and EtOAc (1:1) was used as a solvent and that
the hydrogenation was performed overnight under atmospheric pressure
of hydrogen; yellow powder (83% yield); mp 166 °C. 1H NMR (DMSO-d6) δ 2.88 (t, J = 7.2 Hz, 2H), 3.75 (s, 3H), 3.84 (t, J = 7.2 Hz, 2H), 6.85 (dt, J = 1.0, 7.3 Hz, 1H),
6.93 (d, J = 7.8 Hz, 1H), 7.07 (dt, J = 1.5, 7.3 Hz, 1H), 7.20 (dt, J = 1.8, 8.1 Hz,
1H), 11.63 (s, 1H), 12.0 (s, 1H). LCMS: retention time 2.03 min, m/z 264 [M + H]+.
Compound 15u was prepared from 11u as described
for the preparation of 11g, with the exception that 3
equiv of BBr3 were used and that the reaction was stirred
for 1.5 h at rt; off-white solid (68% yield); mp 205 °C. 1H NMR (DMSO-d6) δ 2.83 (t, J = 7.3 Hz, 2H), 3.83 (t, J = 7.3 Hz, 2H),
6.68 (dt, J = 1.3, 7.6 Hz, 1H), 6.75 (d, J = 7.8 Hz, 1H), 6.97–7.03 (m, 2H), 9.38 (s, 1H),
11.63 (bs, 1H), 11.97 (bs, 1H). LCMS: retention time 0.70 min, m/z 250 [M + H]+.
Compound 13v was prepared as described for the preparation
of 13a, except 1-(2-iodoethyl)-3-methoxybenzene (2.0
equiv) was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; tan solid (36% yield). 1H NMR (DMSO-d6) δ 2.91 (t, J = 7.5 Hz, 2H), 3.73 (s, 3H), 4.04 (m, 2H), 6.77 (m, 1H),
6.79 (m, 2H), 7.21 (t, J = 7.7 Hz, 1H), 12.53 (s,
1H).
Compound 11v was prepared from 14v as described
for the preparation of 11a, with the exception that a
mixture of methanol and EtOAc (1:1) was used as a solvent and that
the hydrogenation was performed overnight at 30 psi; off-white solid
(63% yield); mp 224 °C. 1H NMR (DMSO-d6) δ 2.88 (t, J = 7.6 Hz, 2H),
3.72 (s, 3H), 3.87 (t, J = 7.6 Hz, 2H), 6.75–6.78
(m, 3H), 7.20 (t, J = 8.0 Hz, 1H), 11.76 (bs, 1H),
11.99 (bs, 1H), 11.97 (bs, 1H). LCMS: retention time 1.80 min, m/z 264 [M + H]+.
Compound 13w was prepared as described for the preparation
of 13a, except 1-(2-iodoethyl)-4-methoxybenzene (2.0
equiv) was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; tan solid (58% yield). 1H NMR (DMSO-d6) δ 2.87 (t, J = 7.5 Hz, 2H), 3.71 (s, 3H), 3.99 (m, 2H), 6.84 (d, J = 8.6 Hz, 2H), 7.12 (m, 2H), 12.52 (s, 1H).
Compound 11w was prepared from 14w as described
for the preparation of 11a, with the exception that a
mixture of methanol and EtOAc (1:1) was used as a solvent and that
the hydrogenation was performed at 50 psi for 3 h; off-white solid
(62% yield); mp 244 °C. 1H NMR (DMSO-d6) δ 2.83 (t, J = 7.5 Hz, 2H),
3.71 (s, 3H), 3.82 (t, J = 7.5 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.6 Hz, 2H),
11.74 (bs, 1H), 11.98 (bs, 1H). LCMS: retention time 1.75 min, m/z 264 [M + H]+.
Compound 15w was prepared from 11w as described
for the preparation of 11g: light-pink solid (54% yield);
mp >280 °C (decomp). 1H NMR (DMSO-d6) δ 2.78 (t, J = 7.6 Hz, 2H),
3.80 (t, J = 7.6 Hz, 2H), 6.65 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 8.3 Hz, 2H), 9.22 (s,
1H), 11.66 (s, 1H), 12.01 (s, 1H). LCMS (5% acetonitrile/95% water
for 0.5 min followed by an increase to 40% acetonitrile/60% water
over 4 min and continuation of 40% acetonitrile/60% water for 3.5
min): retention time 3.20 min, m/z 250 [M + H]+.
Compound 11x was prepared from 14x as described
for the preparation of 11a; tan solid (78% yield); mp
150–153 °C. 1H NMR (DMSO-d6): δ 2.89 (t, J = 7.2 Hz, 2H),
3.86 (t, J = 7.2 Hz, 2H), 6.79 (m, 1H), 6.83–6.86
(m, 1H), 6.95–6.98 (m, 3H), 7.10–7.14 (m, 1H), 7.28–7.40
(m, 3H), 11.86 (bs, 1H). LCMS (20% acetonitrile/80% water for 0.25
min followed by an increase to 85% acetonitrile/15% water over 1.5
min and continuation of 85% acetonitrile/15% water for 2.25 min; flow
rate 1.25 mL/min): retention time 1.78 min, m/z 326 [M + H]+.
Compound 13y was prepared as described for the preparation
of 13a, except 1-(2-iodoethyl)-4-phenoxybenzene (2.0
equiv) was
used in place of methyl iodide; tan solid (24% yield). 1H NMR (DMSO-d6): δ 2.93 (m, 2H),
4.04 (m, 2H), 6.93–6.98 (m, 4H), 7.10–7.14 (m, 1H),
7.21–7.25 (m, 2H), 7.36–7.40 (m, 2H), 12.54 (s, 1H).
Compound 11y was prepared from 14y as described
for the preparation of 11g; white solid (69% yield);
mp 207–209 °C. 1H NMR (DMSO-d6): δ 2.90 (t, J = 7.2 Hz, 2H),
3.88 (t, J = 7.3 Hz, 2H), 6.92–6.98 (m, 4H),
7.10–7.14 (m, 1H), 7.21 (m, 2H), 7.35–7.40 (m, 2H),
11.69 (bs, 1H), 12.04 (bs, 1H). LCMS (20% acetonitrile/80% water for
0.25 min followed by an increase to 85% acetonitrile/15% water over
1.5 min and continuation of 85% acetonitrile/15% water for 2.25 min;
flow rate 1.25 mL/min): retention time 1.80 min, m/z 326 [M + H]+.
Compound 13z was prepared as described for the preparation
of 13a, except 4-(2-iodoethyl)biphenyl (2.0 equiv) was
used in place of methyl iodide. The crude product was purified by
trituration with cold diethyl ether; tan solid (59% yield). 1H NMR (DMSO-d6) δ 2.99 (t, J = 7.5 Hz, 2H), 4.08 (t, J = 7.6 Hz, 2H),
7.31–7.37 (m, 3H), 7.45 (t, J = 7.6 Hz, 2H),
7.59–7.65 (m, 4H), 12.55 (s, 1H).
Compound 11z was prepared from 14z as described
for the preparation of 11a, with the exception that a
mixture of methanol and EtOAc (2:1) was used as a solvent and that
the hydrogenation was performed overnight at 30 psi; white powder
(89% yield); mp 254 °C. 1H NMR (DMSO-d6) δ 2.95 (t, J = 7.6 Hz, 2H),
3.92 (t, J = 7.5 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.35 (t, J = 6.8 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H),
7.63 (d, J = 7.1 Hz, 2H), 11.71 (s, 1H), 12.06 (s,
1H). LCMS: retention time 3.37 min, m/z 310 [M + H]+.
To a solution of 16 (0.30 g, 0.96 mmol), 2-(pyridin-2-yl)ethanol
(0.12 g, 1.0 mmol), and triphenylphosphine (0.30 g, 1.1 mmol) in THF
(5 mL) was added dropwise diisopropyl azodicarboxylate (0.23 mL, 1.2
mmol) at 0 °C. The mixture was stirred at 0 °C for 10 min,
then heated at 66 °C for 6.5 h. The reaction was concentrated
and the residual oil was purified using a Biotage Isolera One flash
purification system with a silica gel flash cartridge (EtOAc/hexanes)
to give 0.34 g of 17a as a clear oil (85% yield). 1H NMR (CDCl3) δ 3.22 (t, J = 7.1 Hz, 2H), 4.37 (t, J = 7.6 Hz, 2H), 4.68 (s,
2H), 5.49 (s, 2H), 7.12–7.19 (m, 2H), 7.29–7.34 (mt,
5H), 7.61 (dt, J = 2.0, 7.8 Hz, 1H), 8.51 (d, J = 4.8 Hz, 1H).
To a solution of benzyl alcohol (0.13 mL, 1.2 mmol) in DMF (3 mL)
was added 60% w/w sodium hydride (60% dispersion in mineral oil, 0.049
g, 1.2 mmol) at 0 °C. The mixture was stirred for 10 min, and
a solution of 17a (0.34 g, 0.81 mmol) in DMF (5 mL) was
added via syringe. The reaction was stirred at 0 °C for 30 min
and quenched by the addition of water and EtOAc. The organic layer
was dried over Na2SO4 and concentrated. The
residual oil was purified using a Biotage Isolera One flash purification
system with a silica gel flash cartridge (EtOAc/hexanes) to give 0.18
g of 18a as an oil (50% yield). 1H NMR (CDCl3) δ 3.17 (t, J = 7.1 Hz, 2H) 4.27 (t, J = 5.6 Hz, 2H), 4.67 (s, 2H), 5.07 (s, 2H), 5.47 (s, 2H),
7.12 (m, 2H), 7.29–7.42 (m, 10H), 7.59 (dt, J = 2.0, 7.8 Hz, 1H), 8.54 (d, J = 5.1 Hz, 1H).
To a solution
of 18a (0.18 g, 0.40 mmol) in dichloromethane
(8 mL) was added a 1.0 M solution of BBr3 in dichloromethane
(0.80 mL, 0.80 mmol) at rt. The reaction was stirred for 45 min, concentrated,
and purified by preparative HPLC (0% acetonitrile/100% water for 5
min followed by an increase to 20% acetonitrile/80% water over 35
min and an increase to 40% acetonitrile/60% water for 10 min; flow
rate 10 mL/min) to give 0.017 g of 19a as a white solid
(18% yield); mp 225 °C. 1H NMR (DMSO-d6) δ 3.17 (t, J = 5.8 Hz, 2H),
4.04 (t, J = 6.8 Hz, 2H), 7.59 (m, 2H), 8.07 (m,
1H), 8.63 (m, 1H), 11.64 (bs, 1H), 12.04 (s, 1H). LCMS (5% acetonitrile/95%
water for 0.5 min followed by an increase to 40% acetonitrile/60%
water over 4 min and continuation of 40% acetonitrile/60% water for
3.5 min; flow rate 0.25 mL/min; column Luna Plus C18, 2.1 mm ×
100 mm, 1.8 μm): retention time 1.06 min, m/z 235 [M + H]+.
Compound 18b was prepared from 17b as described
for the preparation of 18a, except the reaction was stirred
at 0 °C and warmed up to rt over a period of 4 h; clear oil (47%
yield). 1H NMR (CDCl3): δ 4.27 (t, J = 5.9 Hz, 2H), 4.43 (t, J = 5.9 Hz, 2H),
4.67 (s, 2H), 5.05 (s, 2H), 5.46 (s, 2H), 6.21 (t, J = 2.1 Hz, 1H), 7.20 (m, 1H), 7.28–7.41 (m, 10H), 7.51 (m,
1H).
To a solution of 19b (0.14 g, 0.32 mmol) in methanol
(5 mL) was added one spatula tip of 10% Pd/C. The mixture was hydrogenated
overnight under an atmospheric pressure of hydrogen and filtered through
a pad of Celite. The filtrate was concentrated, and the residual material
was triturated with EtOAc/MeOH to give 0.060 g of compound 19b as a white solid (83% yield); mp 275 °C (dec). 1H NMR (DMSO-d6) δ 4.01 (t, J = 6.2 Hz, 2H), 4.36 (t, J = 6.2 Hz, 2H),
6.20 (t, J = 2.1 Hz, 1H), 7.40 (d, J = 1.5 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 11.69
(s, 1H), 12.04 (s, 1H). LCMS (5% acetonitrile/95% water for 0.5 min
followed by an increase to 40% acetonitrile/60% water over 4 min and
continuation of 40% acetonitrile/60% water for 3.5 min; flow rate
0.25 mL/min; column Luna Plus C18, 2.1 mm × 100 mm, 1.8 μm):
retention time 1.32 min, m/z 224
[M + H]+.
Compound 17c was prepared as described for the preparation
of 17a, except 2-(1H-benzo[d]imidazol-1-yl)ethanol was used in place of 2-(pyridin-2-yl)ethanol
and that the compound was purified by trituration with EtOAc/hexanes;
beige solid (72% yield). 1H NMR (CDCl3) δ
4.38 (t, J = 6.6 Hz, 2H), 4.56 (t, J = 6.4 Hz, 2H), 4.61 (s, 2H), 5.42 (s, 2H), 7.28–7.34 (m,
7H), 7.39 (m, 1H), 7.80 (m, 1H), 7.91 (s, 1H).
Compound 18c was prepared from 17c as described
for the preparation of 18a, with the exception that the
reaction was stirred overnight at rt and that the compound was purified
by a silica gel flash chromatography (1% NH4OH/EtOAc,);
white solid (34% yield). 1H NMR (CDCl3) δ
4.25 (t, J = 5.9 Hz, 2H), 4.47 (t, J = 5.9 Hz, 2H), 4.60 (s, 2H), 4.75 (s, 2H), 5.40 (s, 2H), 7.27–7.36
(m, 13H), 7.71 (s, 1H), 7.78 (m, 1H).
Compound 19c was prepared from 18c as described
for the preparation of 19b; beige solid (18% yield);
mp 260 °C (dec). 1H NMR (DMSO-d6) δ 3.99 (t, J = 5.8 Hz, 2H), 4.50
(t, J = 5.8 Hz, 2H), 7.18 (m, 1H), 7.23 (m, 1H),
7.51 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H), 8.14 (s, 1H). LCMS (5% acetonitrile/95% water for
0.5 min followed by an increase to 40% acetonitrile/60% water over
4 min and continuation of 40% acetonitrile/60% water for 3.5 min;
flow rate 0.25 mL/min; column Luna Plus C18, 2.1 mm × 100 mm,
1.8 μm): retention time 3.26 min, m/z 274 [M + H]+.
Compound 18d was prepared from 17d as described
for the preparation of 18a, with the exception that the
reaction was stirred for 4 h at rt and that the compound was precipitated
from EtOAc; white solid (51% yield). 1H NMR (CDCl3) δ 4.28 (t, J = 5.3 Hz, 2H), 4.52 (s, 2H),
4.63 (t, J = 5.8 Hz, 2H), 4.66 (s, 2H), 5.43 (s,
2H), 6.42 (d, J = 3.5 Hz, 1H),), 7.02 (m, 2H), 7.22
(dd, J = 1.8, 7.1 Hz, 2H), 7.29–7.36 (m, 8H),
7.84 (dd, J = 1.5, 7.8 Hz, 1H), 8.22 (dd, J = 1.5, 4.8 Hz, 1H).
Compound 19d was prepared from 18d as described
for the preparation of 19a, with the exception that 4.0
equiv of BBr3 were used and that the reaction was stirred
for 2 h at rt. The crude material was purified by preparative HPLC
(5% acetonitrile/95% water for 5 min followed by an increase to 35%
acetonitrile/65% water over 35 min and an increase to 50% acetonitrile/50%
water for 10 min; flow rate 15 mL/min); beige powder (79% yield);
mp 259 °C. 1H NMR (DMSO-d6) δ 4.03 (t, J = 4.8 Hz, 2H), 4.51 (t, J = 5.1 Hz, 2H), 6.43 (d, J = 3.3 Hz, 1H),
7.05 (dd, J = 2.8, 7.6 Hz, 1H), 7.46 (d, J = 3.3 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H),
8.16 (d, J = 4.3 Hz, 1H), 11.89 (bs, 2H). LCMS (5%
acetonitrile/95% water for 0.5 min followed by an increase to 40%
acetonitrile/60% water over 4 min and continuation of 40% acetonitrile/60%
water for 3.5 min): retention time 0.45 min, m/z 274 [M + H]+.
Compound 19e was prepared from 18e as described
for the
preparation of 19b, with the exception that the product
was purified by trituration with EtOAc/hexanes; beige solid (12% yield);
mp 217 °C. 1H NMR (DMSO-d6) δ 1.19 (d, J = 7.1 Hz, 3H), 3.22 (m, 1H),
3.78 (d, J = 6.8 Hz, 2H), 7.22 (m, 3H), 7.28 (m,
2H), 11.65 (s, 1H), 11.99 (s, 1H). LCMS (20% acetonitrile/80% water
for 0.25 min followed by an increase to 85% acetonitrile/15% water
over 1.5 min and continuation of 85% acetonitrile/15% water for 2.25
min; flow rate 1.25 mL/min): retention time 0.93 min, m/z 248 [M + H]+.
Compound 17f was prepared as described for the preparation
of 17a, except 2,2-difluoro-2-phenylethanol was used
in place of 2-(pyridin-2-yl)ethanol: colorless oil (95% yield). 1H NMR (DMSO-d6) δ 4.53 (s,
2H), 4.66 (t, J = 13.9 Hz, 2H), 5.30 (s, 2H), 7.27–7.36
(m, 5H), 7.50 (m, 3H), 7.56 (m, 2H).
Compound 18f was prepared from 17f as described
for the preparation of 18a, with the exception that 1.2
equiv of benzyl alcohol and 1.2 equiv of sodium hydride were used
and that the reaction was stirred at rt for 2 h; white solid (59%
yield). 1H NMR (DMSO-d6) δ
4.49 (s, 2H), 4.56 (t, J = 12.8 Hz, 2H), 4.83 (s,
2H), 5.27 (s, 2H), 7.26–7.33 (m, 5H), 7.40 (m, 5H), 7.50–7.58
(m, 5H).
Compound 19f was prepared from 18f as described
for the preparation of 19b, with the exception that a
mixture of methanol, EtOAc, and acetic acid (1:1:0.1) was used as
solvent. A mixture of compound 19f and its N-hydroxylmethyl derivative was obtained as a beige solid. The residue
was redissolved in methanol and treated with a catalytic amount of
sodium carbonate and stirred over 2 days. The reaction mixture was
acidified with a 10% KHSO4 solution to pH 4, and the precipitate
was filtered and then washed thoroughly with water and with 10% EtOAc/hexanes
to give compound 19g as a white powder (36% yield); mp
245 °C. 1H NMR (DMSO-d6) δ 4.38 (t, J = 14.0 Hz, 2H), 7.50 (m, 5H),
11.88 (m, 1H), 12.17 (m, 1H). LCMS: retention time 2.25 min, m/z 270 [M + H]+.
To a suspension of NaH (0.14 g, 3.5 mmol) in DMF (2.5 mL) at rt was
slowly added a solution of 16 (1.0 g, 3.2 mmol) in DMF
(7 mL) via syringe. The mixture was stirred at rt for 1 h, after which
bromoacetophenone (0.70 g, 3.5 mmol) was added in one portion. The
reaction was stirred for 3.5 h. The reaction mixture was partitioned
between EtOAc and water. The organic layer was dried over Na2SO4 and concentrated. The residual material was purified
by a Biotage Isolera One flash purification system with a silica gel
flash cartridge (EtOAc/hexanes) to give 1.27 g of 20 as
a white solid (92% yield). 1H NMR (CDCl3) δ
4.72 (s, 2H), 5.40 (s, 2H), 5.54 (s, 2H), 7.31–7.39 (m, 5H),
7.54 (t, J = 7.6 Hz, 2H), 7.67 (m, 1H), 7.97 (m,
2H).
Compound 21 was prepared from 20 as described
for the preparation of 18a, with the exception that 2.2
equiv of benzyl alcohol and 2.2 equiv of sodium hydride were used;
yellow oil (45% yield). 1H NMR (CDCl3) δ
4.72 (s, 2H), 5.16 (s, 2H), 5.29 (s, 2H), 5.53 (s, 2H), 7.29–7.41
(m, 10H), 7.54 (t, J = 7.3 Hz, 2H), 7.67 (m, 1H),
7.99 (m, 2H).
Compound 22 was prepared from 21 as described
for the preparation of 19a, with the exception that 4.0
equiv of BBr3 were used and that the reaction was stirred
for 10 min. The crude material was purified by trituration with EtOAc/hexanes;
white solid (63% yield); mp 220 °C. 1H NMR (DMSO-d6) δ 5.27 (s, 2H), 7.58 (t, J = 7.8 Hz, 2H), 7.71 (t, J = 7.6 Hz, 1H), 8.02 (d, J = 7.3 Hz, 2H), 11.78 (bs, 1H), 12.30 (s, 1H). LCMS (20%
acetonitrile/80% water for 0.25 min followed by an increase to 85%
acetonitrile/15% water over 1.5 min and continuation of 85% acetonitrile/15%
water for 2.25 min; flow rate 1.25 mL/min): retention time 0.26 min, m/z 248 [M + H]+.
To a solution of 21 (0.17 g, 0.37 mmol) in a 2:1 mixture
of methanol and EtOAc (15 mL) were added a small spatula tip of chloro(1,5-cyclooctadiene)rhodium(I)
dimer and one drop of triethylamine. The mixture was stirred for 36
h under hydrogen (300 psi) using a mechanical stirrer. The reaction
was filtered and concentrated to give a brown oil, which was subsequently
dissolved in dichloromethane (5 mL) and treated with a 1.0 M solution
of BBr3 in dichloromethane (1.5 mL, 1.5 mmol) for 1.5 h.
The reaction was quenched by the addition of water. The mixture was
concentrated and purified by preparative HPLC (10% acetonitrile/90%
water for 5 min, followed by an increase to 50% acetonitrile/50% water
over 35 min and then an increase to 70% acetonitrile/30% water over
10 min; flow rate 15 mL/min) to give 0.024 g of 23 as
a beige solid (26% yield); mp 202 °C. 1H NMR (DMSO-d6) δ 3.66 (dd, J = 4.0,
13.1 Hz, 1H), 3.85 (dd, J = 9.1, 13.4 Hz, 1H), 4.91
(dd, J = 4.3, 9.4 Hz, 1H), 5.52 (bs, 1H), 7.27–7.34
(m, 5H), 11.66 (s, 1H), 12.03 (s, 1H). LCMS (5% acetonitrile/95% water
for 0.25 min followed by an increase to 40% acetonitrile/60% water
over 1.5 min and continuation of 40% acetonitrile/60% water for 2.25
min; flow rate 1.25 mL/min): retention time 1.61 min, m/z 250 [M + H]+.
To a solution of 16 (0.50 g, 1.6 mmol) in dichloromethane
(25 mL) were added pyridine (0.26 mL, 3.20 mmol), phenylboronic acid
(0.39 g, 3.20 mmol), and copper(II) acetate (0.44 g, 2.4 mmol). The
mixture was stirred at rt overnight and filtered through a pad of
Celite. The filtrate was concentrated, and the resulting residue was
partitioned between EtOAc and water. The organic layer was washed
with a 10% KHSO4 solution, dried over Na2SO4, and concentrated. The residue was purified by flash column
chromatography using EtOAc/hexanes as eluent to give 0.62 g of 24 as a white solid (quantitative yield). 1H NMR
(CDCl3) δ 4.77 (s, 2H), 5.61 (s, 2H), 7.30–7.37
(m, 5H), 7.42 (m, 1H), 7.46–7.49 (m, 4H).
Compound 26 was prepared from 25 as described
for the preparation
of 19a, with the exception that 4.0 equiv of BBr3 were used. After completion of the reaction, the reaction
mixture was concentrated and the resulting residue was partitioned
between EtOAc and water. The organic layer was washed with brine,
dried over Na2SO4, and concentrated. The crude
product was purified by trituration with EtOAc/hexanes; white powder
(33% yield); mp 239 °C. 1H NMR (DMSO-d6) δ 7.32 (t, J = 7.3 Hz, 1H),
7.44 (t, J = 7.8 Hz, 1H), 7.49 (m, 2H), 11.85 (bs,
1H), 12.23 (s, 1H). LCMS (20% acetonitrile/80% water for 0.25 min
followed by an increase to 85% acetonitrile/15% water over 1.5 min
and continuation of 85% acetonitrile/15% water for 2.25 min; flow
rate 1.25 mL/min): retention time 1.08 min, m/z 206 [M + H]+.
1-Phenethylhydrazinecarbothioamide
(28)
To a suspension of 27 (5.00
g, 21.3 mmol) in ethanol
(50 mL) was added ammonium thiocyanate (1.60 g, 21.0 mmol) at rt.
The white suspension was heated at 78 °C for 50 h. The reaction
was cooled to rt, and the resultant solid was removed by filtration.
The filtrate was concentrated by approximately one-half, and the resultant
solid was removed by filtration. This was repeated four times. The
remaining filtrate was partitioned between EtOAc (50 mL) and satd
NaHCO3 (20 mL). The organic layer was dried over MgSO4 and concentrated. The crude material was purified by silica
gel column chromatography (80% EtOAc/hexanes w/0.1% NH4OH) to give 0.66 g of 28 as a white solid (16% yield). 1H NMR (DMSO-d6) δ 2.94 (m,
2H), 4.09 (m, 2H), 4.88 (s, 2H), 7.18–7.33 (m, 5H), 7.44 (br
s, 2H).
To a solution of 28 (0.66
g, 3.4 mmol) in THF (15 mL) was added a solution of methyl chlorooxoacetate
(0.41 g, 3.4 mmol) in THF (5 mL) over 2 min at rt. The mixture was
stirred for 14 h and concentrated to dryness. The residue was dissolved
in EtOAc (50 mL) and washed with satd NaHCO3 (25 mL). The
organic layer was dried over MgSO4 and concentrated. The
crude material was purified by silica gel column chromatography (5%
MeOH/CH2Cl2) to give 0.30 g of 29 as a white solid (31% yield). 1H NMR (CDCl3) δ 3.09 (t, J = 7.03 Hz, 2H), 3.88 (s, 3H),
4.32 (m, 2H), 6.03 (s, 2H), 7.25–7.37 (m, 5H), 8.22 (brs, 1H).
To a solution of 29 (0.30 g, 1.1 mmol) in THF (5 mL) was added dropwise a solution
of DBU (0.32 g, 2.1 mmol) in THF (3 mL) at rt. The mixture was stirred
at rt for 14 h and then heated to 50 °C for 30 min. The reaction
was concentrated to dryness. The residue was partitioned between EtOAc
and 5% KHSO4. The organic layer was separated, dried over
MgSO4, and concentrated. The crude material was purified
by silica gel chromatography (1% AcOH/EtOAc) to give 0.031 g of 30 as a yellow solid (11% yield); mp 190–230 °C. 1H NMR (CD3OD) δ 3.11 (m, 2H), 4.41 (m, 2H),
7.19–7.31 (m, 5H). LCMS: retention time 2.90 min, m/z 250 [M + H]+.
To a solution of 5-hydroxypyrimidine-2,4(1H,3H)-dione 31 (0.30 g, 2.3
mmol) in acetonitrile (15 mL) was added N,O-bis(trimethylsilyl)acetamide (2.0 mL, 8.2 mmol). The mixture
was heated at 82 °C for 3 h, after which phenethyl iodide (0.54
g, 2.3 mmol) was added via syringe. The reaction was heated for 48
h at the same temperature, and the reaction mixture was concentrated
in vacuo. The resulting residue was dissolved in dichloromethane,
and the organic solution was washed twice with water, dried over Na2SO4, and concentrated. The crude material was triturated
with cold diethyl ether and filtered to give 0.080 g of 32 as a tan solid (15% yield). 1H NMR (DMSO-d6): δ 2.86 (t, J = 8.0 Hz, 2H),
3.79 (t, J = 8.0 Hz, 2H), 7.10 (s, 1H), 7.22–7.31
(m, 5H), 8.53 (s, 1H), 11.36 (s, 1H). LCMS (20% acetonitrile/80% water
for 0.25 min followed by an increase to 85% acetonitrile/15% water
over 1.5 min and continuation of 85% acetonitrile/15% water for 2.25
min; flow rate 1.25 mL/min): retention time 0.45 min, m/z 233 [M + H]+.
Human DAAO
(hDAAO) Purification
HEK cells expressing
hDAAO were grown to confluence, harvested, washed, and resuspended
in ice-cold assay buffer (Tris buffer, 50 mM, pH 8.5) containing flavin
adenine dinucleotide (FAD, 100 μM) prior to sonication. The
resulting cell lysate was centrifuged at 16000g for
10 min at 4 °C, and the hDAAO was purified using an UNO Q6 ion-exchange
column pre-equilibrated in diluted assay buffer (10 mM, pH 8.5) on
a FPLC system (Bio-Rad; Hercules, CA). The hDAAO was eluted by a linear
gradient of 0–0.5 M NaCl, and the fractions with the highest
DAAO activity were combined and stored at −80 °C in 20%
glycerol until usage. The specific activity with respect to d-serine was determined to be 35.0 ± 0.2 μmol/min/mg, comparable
to that of purified protein previously reported.[31] The final concentration of hDAAO in the assay described
below was adjusted in such a manner that a rate of 2.0 mOD/min can
be achieved for the oxidation of d-serine (5 mM) in the absence
of a DAAO inhibitor.
In Vitro DAAO Asay
d-Serine
was purchased
from Bachem Biosciences Inc. (King of Prussia, PA), horseradish peroxidase
from Worthington Biochemical Corporation (Freehold, NJ), and o-phenylenediamine from Pierce Biotechnology, Inc. (Rockford,
IL). All other chemicals were obtained from Sigma-Aldrich (St. Louis,
MO). A reliable 96-well plate d-amino acid oxidase (DAAO)
assay was developed based on previously published methods.[32] Briefly, d-serine (5 mM) was oxidatively
deaminated by hDAAO in the presence of molecular oxygen and flavin
adenosine dinucleotide (FAD; 10 μM) to yield the corresponding
α-keto acid, ammonia, and hydrogen peroxide. The resulting hydrogen
peroxide was quantified using horseradish peroxidase (0.01 mg/mL)
and o-phenylenediamine (180 μg/mL), which turns
yellowish-brown upon oxidation. DAAO activity was correlated to the
rate formation of the colored product, i.e., rate of change of absorbance
at 411 nm. All reactions were carried out for 20 min at room temperature
in a 100 μL volume in Tris buffer (50 mM, pH 8.5). Additionally,
stock solutions and serial dilutions of potential DAAO inhibitors
were made in 20:80 DMSO:buffer with a final assay DMSO concentration
of 2%.
In Vitro Metabolic Stability Studies
The metabolic
stability was evaluated using mouse liver microsomes. For the cytochrome
P450 (CYP)-mediated metabolism, the reaction was carried out with
100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH
regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3
mM MgCl2, 0.4 U/mL glucose-6-phosphate dehydrogenase, 50
μM sodium citrate). Reactions, in duplicate, were initiated
by addition of the liver microsomes to the incubation mixture (compound
final concentration was 5 μM; 0.5 mg/mL microsomes). For the
glucuronidation reaction, a test compound were added to TRIS-HCl buffer
(50 mM, pH 7.5) with microsomes (0.5 mg/mL), along with MgCl2 (8 mM) and alamethicin (25 μg/mL), and preincubated at 37
°C. The reaction was initiated (in duplicate) with UDPGA (2 mM;
final concentration). Controls in the absence cofactors were carried
out to determine the specific cofactor-free degradation. After 60
min of incubation, aliquots of the mixture were removed and the reaction
quenched by addition of two times the volume of ice-cold acetonitrile
spiked with the internal standard. Compound disappearance was monitored
over time using a liquid chromatography and tandem mass spectrometry
(LC/MS/MS) method.
In Vivo Pharmacokinetics
All procedures
involving mice
were approved by and conformed to the guidelines of the Institutional
Animal Care Committee of the Johns Hopkins University. In vivo pharmacokinetics
studies were performed using male CD1mice (n = 3
for each time point for each group). Approximately 1 mL of whole blood
was collected from each animal by cardiac puncture into heparinized
microcentrifuge tubes, capped, gently inverted a few times, and stored
on wet ice until centrifugation (10 min at 800g,
4 °C). Thereafter, the top layer of each tube (∼400 μL
plasma) was aspirated via transfer pipet, dispensed into a clean nonheparinized
microcentrifuge tube, and stored at −80 °C until subsequent
analyses. Plasma levels of 8b and 11h were
analyzed using an Agilent 1290 HPLC system coupled to an Agilent 6520
QTOF-MS system. Plasma d-serine level analyses were carried
out using the previously reported method involving derivatization
with Marfey’s reagent,[33] followed
by the quantification of the derivatized d-serine using an
Agilent 1290 HPLC system coupled to an Agilent 6520 QTOF-MS system.
Authors: Jos H M Lange; Jennifer Venhorst; Maria J P van Dongen; Jurjen Frankena; Firas Bassissi; Natasja M W J de Bruin; Cathaline den Besten; Stephanie B A de Beer; Chris Oostenbrink; Natalia Markova; Chris G Kruse Journal: Eur J Med Chem Date: 2011-05-13 Impact factor: 6.514
Authors: James F Berry; Dana V Ferraris; Bridget Duvall; Niyada Hin; Rana Rais; Jesse Alt; Ajit G Thomas; Camilo Rojas; Kenji Hashimoto; Barbara S Slusher; Takashi Tsukamoto Journal: ACS Med Chem Lett Date: 2012-09-16 Impact factor: 4.345
Authors: Allen J Duplantier; Stacey L Becker; Michael J Bohanon; Kris A Borzilleri; Boris A Chrunyk; James T Downs; Lain-Yen Hu; Ayman El-Kattan; Larry C James; Shenping Liu; Jiemin Lu; Noha Maklad; Mahmoud N Mansour; Scot Mente; Mary A Piotrowski; Subas M Sakya; Susan Sheehan; Stefanus J Steyn; Christine A Strick; Victoria A Williams; Lei Zhang Journal: J Med Chem Date: 2009-06-11 Impact factor: 7.446
Authors: Niyada Hin; Bridget Duvall; James F Berry; Dana V Ferraris; Rana Rais; Jesse Alt; Camilo Rojas; Barbara S Slusher; Takashi Tsukamoto Journal: Bioorg Med Chem Lett Date: 2016-02-23 Impact factor: 2.823
Figure 1
Figure 2
Scheme 1
Scheme 2
Scheme 3
Figure 3
Figure 4
Figure 5