6-Hydroxy-1,2,4-triazine-3,5(2H,4H)-dione Derivatives as Novel d -Amino Acid Oxidase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b00482

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.

Full text of DOI:10.1021/acs.jmedchem.5b00482

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Article
6-Hydroxy-1,2,4-triazine-3,5(2H,4H)-dione Derivatives
as Novel D-Amino Acid Oxidase Inhibitors
Niyada Hin, Bridget Duvall, Dana Ferraris, Jesse Alt, Ajit G Thomas, Rana Rais, Camilo
Rojas, Ying Wu, Krystyna M Wozniak, Barbara S. Slusher, and Takashi Tsukamoto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00482 • Publication Date (Web): 26 Aug 2015
Downloaded from http://pubs.acs.org on September 1, 2015
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ꢀHydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione Derivatives
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as Novel DꢀAmino Acid Oxidase Inhibitors
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Niyada Hin, Bridget Duvall, Dana Ferraris, Jesse Alt, Ajit G. Thomas, Rana Rais, Camilo
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,§
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†,‡
,†,‡
Rojas, Ying Wu, Krystyna M. Wozniak, Barbara S. Slusher, Takashi Tsukamoto*
†
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§
Brain Science Institute, Department of Neurology, and Department of Molecular and Comparative
Pathobiology, Johns Hopkins University, Baltimore, MD 21205, USA
CORRESPONDING AUTHOR *Phone: (410) 614ꢀ0982. Fax: (410) 614ꢀ0659. Eꢀmail:
ttsukamoto@jhmi.edu.
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ABSTRACT: A series of 2ꢀsubstituted 6ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione derivatives were
synthesized as inhibitors of Dꢀamino acid oxidase (DAAO). Many compounds in this series were found
to be potent DAAO inhibitors with IC values in the doubleꢀdigit nanomolar range. The 6ꢀhydroxyꢀ
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,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione pharmacophore appears metabolically resistant to Oꢀglucuronidation
unlike other structurally related DAAO inhibitors. Among them, 6ꢀhydroxyꢀ2ꢀ(naphthalenꢀ1ꢀylmethyl)ꢀ
,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione 11h was found to be selective over a number of targets and orally
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available in mice. Furthermore, oral coꢀadministration of Dꢀserine with 11h enhanced the plasma levels
of Dꢀserine in mice compared to the oral administration of Dꢀserine alone, demonstrating its ability to
serve as a pharmacoenhancer of Dꢀserine.
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INTRODUCTION
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Dꢀserine, a full agonist at the glycine modulatory site of the Nꢀmethylꢀdꢀaspartate (NMDA) receptor,
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has been actively explored as a potential therapeutic agent for the treatment of schizophrenia. Several
clinical studies with oral Dꢀserine have shown promising results for the treatment of not only positive
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symptoms, but also negative symptoms, which have not responded well to existing drugs. Further
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clinical development of Dꢀserine, however, could be hampered by the high doses of Dꢀserine (60ꢀ120
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mg/kg) required for the optimal efficacy, since Dꢀserine was reported to be nephrotoxic in rats at high
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doses. DꢀAmino acid oxidase (DAAO, EC 1.4.3.3) is a flavoenzyme that catalyzes the oxidation of Dꢀ
amino acids including Dꢀserine to the corresponding αꢀketo acids. In mammals, DAAO is present in
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kidneys, liver, and brain. Since the highest DAAO activity is found in the kidneys, a substantial amount
of orally administered Dꢀserine is metabolized in the kidneys, contributing to its rapid clearance. Indeed,
pharmacokinetics studies of Dꢀserine in mice lacking DAAO unmasked the predominant role of DAAO
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in the plasma clearance of Dꢀserine. Furthermore, Dꢀserineꢀinduced nephrotoxicity is most likely due to
the production of hydrogen peroxide as a byproduct of DAAOꢀmediated oxidation since it does not
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occur in mutant rats lacking DAAO activity. These findings collectively suggest that inhibition of
DAAO would exert dual beneficial effects on Dꢀserine therapy; (i) enhancement of Dꢀserine
bioavailability and (ii) attenuation of Dꢀserine induced nephrotoxicity.
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In the past decade, concurrent with the functional studies of DAAO, substantial strides have been
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made in identifying potent and selective DAAO inhibitors of broad structural diversity (Figure 1).
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Carboxylateꢀbased DAAO inhibitors 1 and
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presumably originated from a prototype DAAO
inhibitor, benzoic acid, have evolved into a new class of compounds containing carboxylate bioisosteres
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such as 3 and 4. Crystal structure of 4 bound to DAAO revealed that the αꢀhydroxycarbonyl moiety
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of 4 retains the key interactions seen between the DAAO active site and carboxylateꢀbased inhibitors.
Subsequently, a variety of other heterocyclic frameworks bearing an αꢀhydroxycarbonyl moiety were
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explored in the pursuit of new scaffolds for potent DAAO inhibitors including 5 and 6. Structural
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insights gained from DAAO in complex with iminoꢀDOPA led to the discovery of the latest DAAO
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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
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derivatives such as compound 7 substituted at its 2ꢀmethyl group. Similarly, Hondo et al. reported
potent DAAO inhibitors 8a-b based on a 4ꢀhydroxypyridazinꢀ3(2H)ꢀone scaffold with a phenethyl
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group extending to the secondary binding site. The secondary binding site was also exploited by
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carboxylateꢀbased DAAO inhibitors such as 9 and 10.
Recently, the pharmacokinetics profile of several representative DAAO inhibitors has been
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systematically compared in mice and rats. 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 Dꢀserine by inhibition of peripheral DAAO.
To our surprise, however, carboxylateꢀbased 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
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contributing to their poor oral bioavailability.
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 FDAꢀapproved small molecule drugs,
ceftriaxone (Figure 2) 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 3ꢀmercaptoꢀ2ꢀmethylꢀ1,2ꢀdihydroꢀ1,2,4ꢀtriazineꢀ5,6ꢀdione portion of ceftriaxone has caught
our attention as its tautomer, 6ꢀhydroxyꢀ3ꢀmercaptoꢀ2ꢀmethylꢀ1,2,4ꢀtriazinꢀ5(2H)ꢀone, has a structural
feature common to other DAAO inhibitors. Interestingly, 6ꢀhydroxyꢀ2ꢀmethylꢀ1,2,4ꢀtriazineꢀ
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,5(2H,4H)ꢀdione 11a, one of the degradation products of ceftriaxone in aqueous solution, inhibited
DAAO with an IC value of 2.8 ꢁM, prompting us to further examine the potential of 2ꢀsubstituted 6ꢀ
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hydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione derivatives as new DAAO inhibitors. Here we present a new
class of DAAO inhibitors based on the 6ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione scaffold. With
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appropriate substitutions at the 2ꢀposition, some 6ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(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 Dꢀserine plasma levels following oral coꢀadministration
with Dꢀserine in mice.
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CHEMISTRY
The majority of 2ꢀsubstituted 6ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione derivatives were synthesized
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using 6ꢀbromoꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione 12 as a starting material (Scheme 1). Alkylation of the
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ꢀposition of 12 with alkyl halides mediated by bis(trimethylsilyl)acetamide (BSA) gave 5ꢀsubstituted
derivatives 13a-z. Subsequently, the 6ꢀbromo 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
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1a-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.
Some derivatives were synthesized using 6ꢀbromoꢀ4ꢀ[(phenylmethoxy)methyl]ꢀ1,2,4ꢀtriazineꢀ
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,5(2H,4H)ꢀdione 16 as a starting material (Scheme 2). Mitsunobu reaction of 16 and alcohols with the
aid of DIAD and PPh provided the 5ꢀsubstituted derivatives 17a-f. Reaction with benzyl alcohol gave
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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 baseꢀmediated coupling of 2ꢀbromoacetophenone to 16. The product 20 was converted into
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1 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 2ꢀphenyl derivative 26 involves ChanꢀLam coupling of 16 to phenylboronic acid to give
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4. Subsequent benzyloxylation and deprotection by BBr provided 26.
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As shown in Scheme 3, we synthesized two structurally close analogs of 11e, where its carboxylate
bioisostere moiety is slightly modified. 3ꢀThioxo derivative 30 was prepared using (2ꢀ
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phenylethyl)hydrazine 27 as a starting material. Reaction of 27 with ammonium thiocyanate provided
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hydrazinecarbothioamide 28, which was subsequently treated with methyl chlorooxoacetate to give
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29. Cyclization of 29 in the presence of DBU afforded the desired product 30. Uracil derivative 32
was prepared by BSAꢀmediated alkylation of 5ꢀhydroxypyrimidineꢀ2,4(1H,3H)ꢀdione 31 with phenethyl
iodide.
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RESULTS AND DISCUSSION
The inhibitory potency of the synthesized compounds were determined using recombinant human
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DAAO as previously reported. In vitro DAAO inhibitory data are summarized in Tables 1 and 2. As
shown in Table 1, 6ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione derivatives containing a variety of
substituents at the 2ꢀposition displayed varying degrees of inhibitory potency against DAAO. All
compounds but 11c and 26 were found to be potent DAAO inhibitors with IC values in the lowꢀ
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micromolar to nanomolar range. Within the compounds containing an aromatic ring, phenethyl
derivative 11e and naphthalenꢀ1ꢀylmethyl derivative 11h exhibited the most potent inhibitory activity
with IC values of 70 nM and 50 nM, respectively. Incorporation of a heterocyclic ring (compounds
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9a-d) did not result in improved potency. We conducted inhibitory kinetic studies of compound 11h at
various concentrations of Dꢀserine to determine its mode of inhibition. As shown in Figure 3, a double
reciprocal plot of DAAO activity versus the Dꢀserine concentrations produced a pattern indicative of
competitive inhibition with a K value of 60 nM. Furthermore, escalation of FAD concentration from 10
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to 100 µM did not affect IC value for 11h, providing additional supporting evidence that compound
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1h competes with Dꢀserine for the substrateꢀbinding site distinct from the FADꢀbinding site of DAAO.
The potent inhibitory activity of 11e prompted us to conduct more focused SAR studies using 11e as a
molecular template (Table 2). A variety of substitutions were examined at the terminal phenyl group of
1e (compounds 11j-z and 15u-w). Overall, there was no significant change in inhibitory potency with
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IC values ranging from 40 to 270 nM. Analogs containing a large substituent such as compounds 11x-
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z retained IC values in the nanomolar range. Para substitutions were particularly well tolerated as
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and 50 nM, respectively. These substituents are presumably oriented towards 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
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ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione moieties. Other modifications at the linker (compounds 19f,
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somewhat lower potency compared to 11e. Compounds 30 and 32 possessing slightly modified
carboxylate bioisosteres retained potent DAAO inhibitory activity with IC values 50 nM and 80 nM,
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respectively, nearly identical to that of 11e.
As illustrated in Figure 4, proposed binding mode of 11e (white) to the active site of DAAO is
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superposed onto the crystal structure of DAAO in complex with 8a (cyan). In this model, the αꢀ
hydroxycarbonyl moiety forms key hydrogen bonds with Tyr228 and Arg283. It is conceivable that the
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ꢀposition nitrogen group of 11e forms a hydrogen bond with the backbone carbonyl of Gly313 in a
similar manner as the 2ꢀnitrogen atom of 4ꢀhydroxypyridazinꢀ3(2H)ꢀone moiety in 8a. This explains the
relatively high inhibitory potency of DAAO inhibitors with a 6ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀ
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dione scaffold compared to the kojic acid based compounds represented by 7 which lacks a nitrogen
atom in the core ring. In contrast, the nitrogen atom at the 1ꢀposition 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. 3ꢀ
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 2ꢀ
phenylethyl group of 11e appears quite spacious and capable of accommodating larger substituents
owing to the movement of Tyr224 away from the activeꢀsite. This is in a good agreement with the high
inhibitory potency retained by compounds with bulky substituents such as compounds 11x-z.
Table 3 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
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subject to a varying degree of glucuronidation in liver microsomes. For example, a kojic acidꢀbased
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DAAO inhibitor 7 is extensively glucuronidated in mouse liver microsomes in the presence of UDPGA,
while compound 8b with the 4ꢀhydroxypyridazinꢀ3(2H)ꢀone scaffold shows some resistance to
glucuronidation. 6ꢀHydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione derivatives 11e and 11h were found to be
completely resistant to glucuronidation in mouse liver microsomes. 3ꢀThioxo derivative 30 was also
metabolically stable against glucuronidation. In sharp contrast, uracilꢀbased 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
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0
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the carboxylate bioisostere moiety contributes to higher resistance to glucuronidation. Compounds 11e
and 11h were also stable in mouse liver microsomes in the presence of NADPH, a cofactor for CYPꢀ
dependent oxidation.
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 4) including hERG channels, various sites of the NMDA receptors, and
another class of flavoenzymes, monoamine oxidases A and B (MAOꢀA and MAOꢀB). Compound 11h
showed no significant activity in any of these assays at the highest tested concentrations.
In vivo pharmacokinetic studies of 11h were carried out in CD1 mice by intravenous and oral
administration (30 mg/kg). The plasma pharmacokinetic parameters are summarized in Table 5 along
with those for 8b for direct comparison. The plasma clearance of 11h in mice was 10.2 mL/min/kg with
a terminal halfꢀlife 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 3), 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
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Journal of Medicinal Chemistry
increase the brain levels of endogenous Dꢀserine 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 Dꢀserine.
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To determine the effects of 11h on Dꢀserine plasma levels, mice (n = 3 per time point) were dosed
with 11h (30 mg/kg, po) along with Dꢀserine (30 mg/kg, po) simultaneously. As shown in Figure 5,
compound 11h showed no ability to enhance plasma Cmax of Dꢀserine. However, its plasma clearance
was substantially reduced for a sustained period of time, resulting in the significant increase in Dꢀserine
AUC0ꢀ6h (96.9 µg*h/mL) compared to that of Dꢀserine alone treatment (43.3 µg*h/mL). The curve of Dꢀ
serine concentration versus time in mice coꢀadministered with 11h is nearly identical to that of DAAO
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knockout mice treated with oral Dꢀserine. The lack of enhancement in Dꢀserine plasma Cmax in mice
by coꢀadministration with 11h is in a good agreement with the negligible levels of DAAO found in
29
mouse liver as opposed to kidneys, where DAAO is abundant and most likely contributes to the
plasma clearance of Dꢀserine. Thus, the primary site of action for 11h in mice appears DAAO in
kidneys, which explains its significant effects on plasma clearance of Dꢀserine but not on plasma Cmax
value. Given the previously reported crossꢀspecies variation in IC values between human and rat forms
5
0
1
3
of DAAO, caution needs to be taken in establishing the PK/PD relationship in mice as we have not
measured inhibitory potency of 11h in mouse DAAO. It is worth noting, however, that 4ꢀ
hydroxypyridazinꢀ3(2H)ꢀone derivatives represented by 8b showed little difference in inhibitory
1
8
potency between human and mouse DAAO. Given their structural similarity to the 6ꢀhydroxyꢀ1,2,4ꢀ
triazineꢀ3,5(2H,4H)ꢀdione derivatives, we anticipate comparative inhibitory potencies of 11h against
human and mouse forms of DAAO.
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.
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We took full advantage of the prior knowledge in guiding our efforts to develop a new class of DAAO
inhibitors based on the 6ꢀhydroxyꢀ1,2,4ꢀtriazineꢀ3,5(2H,4H)ꢀdione pharmacophore. The inhibitory
potency of these compounds was dependent on the variation of the substituents at the 2ꢀposition. 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 coꢀadministered Dꢀserine by reducing its clearance. The ability of compound
11h to sustain constant levels of Dꢀserine should be particularly attractive for the treatment of chronic
psychiatric disorders such as schizophrenia.
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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
1
under nitrogen. Melting points were obtained on a MelꢀTemp apparatus and are uncorrected. H NMR
1
3
spectra were recorded at 400 MHz. C 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
G1315D DAD detector using a Phenomenex Luna 5 ꢁm C18 (2) column (21.2 mm × 250 mm, 5 ꢁm).
Analytical HPLC was performed on an Agilent 1200 Series HPLC system equipped with an Agilent
G1315D DAD 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 ꢁm) 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 human DAAO (Figure 4) was generated using
3
0
AutoDock Vina.
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6
-Bromo-2-methyl-1,2,4-triazine-3,5(2H,4H)-dione (13a). To a solution of 5ꢀbromoꢀ6ꢀazauracil 12
1
2
(0.30 g, 1.6 mmol) in acetonitrile (5 mL) was added N,Oꢀbis(trimethylsilyl)acetamide (4.0 mmol, 1.0
3
4
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7
8
9
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6
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
2
4 h. The reaction mixture was concentrated and the resulting residue was dissolved in
0
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9
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dichloromethane, washed with water and brine, dried over Na SO , and concentrated to give 0.11 g of
2
4
1
3a as a black solid (33% crude yield). This material was used in the next step without further
1
purification: H NMR (DMSOꢀd ) δ 3.44 (s, 3H), 12.50 (s, 1H).
6
6
-(Benzyloxy)-2-methyl-1,2,4-triazine-3,5(2H,4H)-dione (14a). A mixture of 13a (0.10 g, 0.49
mmol) and K CO (0.14 g, 1.0 mmol) in benzyl alcohol (1.0 mL) was heated overnight at 150 °C. The
2
3
reaction mixture was partitioned between aqueous 10% KHSO and EtOAc. The organic layer was dried
4
over Na SO and concentrated. The residual material was purified using a Biotage Isolera One flash
2
4
purification system with a silica gel flash cartridge (EtOAc/hexanes) to give 0.070 g of 14a as a white
1
solid (61% yield): H NMR (DMSOꢀd ) δ 3.36 (s, 3H), 5.12 (s, 2H), 7.37ꢀ7.46 (m, 5H), 12.16 (s, 1H).
6
6
-Hydroxy-2-methyl-1,2,4-triazine-3,5(2H,4H)-dione (11a). 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
1
give 0.035 g of 11a as a beige powder (94% yield): mp 259 °C; H NMR (DMSOꢀd ) δ 3.27 (s, 3H),
6
1
4
1.11 (s, 1H), 12.03 (s, 1H). LCMS (5% acetonitrile/95% water for 0.5 min followed by an increase to
0% 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] .
6
-Bromo-2-isopentyl-1,2,4-triazine-3,5(2H,4H)-dione (13b). Compound 13b was prepared as
described for the preparation of 13a except 1ꢀiodoꢀ3ꢀmethylbutane (1.6 equiv.) was used in place of
methyl iodide. The crude product was purified by trituration with cold diethyl ether: yellow solid (27%
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1
yield); H NMR (DMSOꢀd ) δ 0.89 ( (d, J = 6.3 Hz, 6H), 1.50 (m, 2H), 1.60 (m, 1H), 3.82 (t, J = 7.3
6
1
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4
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9
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Hz, 2H), 12.48 (s, 1H).
6
-(Benzyloxy)-2-isopentyl-1,2,4-triazine-3,5(2H,4H)-dione (14b). Compound 14b was prepared
1
from 13b as described for the preparation of 14a: clear oil (36% yield); H NMR (DMSOꢀd ) δ 0.87 (d,
6
0
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2
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J = 6.3 Hz, 6H), 1.49 (m, 2H), 1.54 (m, 1H), 3.73 (t, J = 7.1 Hz, 2H), 5.14 (s, 2H), 7.36ꢀ7.45 (m, 5H),
1
2.14 (s, 1H).
6
-Hydroxy-2-isopentyl-1,2,4-triazine-3,5(2H,4H)-dione (11b). Compound 11b was prepared from
1
1
4b as described for the preparation of 11a: grey solid (quantitative yield); mp 171 °C; H NMR
(
DMSOꢀd ) δ 0.88 (d, J = 6.3 Hz, 6H), 1.47 (m, 2H), 1.54 (m, 1H), 3.66 (t, J = 7.3 Hz, 2H), 11.65 (s,
6
+
1H), 11.94 (s, 1H). LCMS: retention time 1.04 min, m/z 200 [M + H] .
6
-Bromo-2-(3,3-dimethylbutyl)-1,2,4-triazine-3,5(2H,4H)-dione (13c). Compound 13c was
prepared as described for the preparation of 13a except 1ꢀiodoꢀ3,3ꢀdimethylbutane (3.3 equiv.) was used
1
in place of methyl iodide: clear oil (19% yield); H NMR (DMSOꢀd ) δ 0.92 (s, 9H), 1.53 (m, 2H), 3.82
6
(m, 2H), 12.50 (s, 1H).
6-(Benzyloxy)-2-(3,3-dimethylbutyl)-1,2,4-triazine-3,5(2H,4H)-dione (14c). Compound 14c was
1
prepared from 13c as described for the preparation of 14a: clear oil (80% yield); H NMR (DMSOꢀd ) δ
6
0
.90 (s, 9H), 1.48 (m, 2H), 3.72 (m, 2H), 5.15 (s, 2H), 7.35ꢀ7.45 (m, 5H), 12.15 (s, 1H).
2-(3,3-Dimethylbutyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11c). Compound 11c was
prepared from 14c as described for the preparation of 11a except the material was purified by trituration
1
with EtOAc/hexanes: white solid (77% yield); mp 219 °C; H NMR (DMSOꢀd ) δ 0.91 (m, 9H), 1.50
6
+
(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] .
2
-Benzyl-6-bromo-1,2,4-triazine-3,5(2H,4H)-dione (13d). 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);
1
H NMR (DMSOꢀd ) δ 5.02 (s, 2H), 7.29ꢀ7.38 (m, 5H), 12.59 (s, 1H).
6
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2
-Benzyl-6-(benzyloxy)-1,2,4-triazine-3,5(2H,4H)-dione (14d). Compound 14d was prepared from
1
2
1
3d as described for the preparation of 14a except the product was purified by trituration with
3
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1
EtOAc/hexanes: white solid (70% yield); H NMR (DMSOꢀd ): δ 4.91 (s, 2H), 5.11 (s, 2H), 7.29ꢀ7.40
6
(m, 10H), 12.24 (s, 1H).
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2-Benzyl-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11d). Compound 11d was prepared from 14d
as described for the preparation of 11a except the hydrogenation was performed overnight under
1
atmospheric pressure of hydrogen: light pink powder (81% yield); mp 242 °C; H NMR (DMSOꢀd ) δ
6
4.85 (s, 2H), 7.26ꢀ7.36 (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]
.
6
-Bromo-2-phenethyl-1,2,4-triazine-3,5(2H,4H)-dione (13e). Compound 13e was prepared as
described for the preparation of 13a except phenethyl iodide (2.5 equiv.) was used in place of methyl
1
iodide. The crude product was purified by trituration with cold diethyl ether: tan solid (64% yield); H
NMR (DMSOꢀd ): 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
6
(s, 1H).
6-(Benzyloxy)-2-phenethyl-1,2,4-triazine-3,5(2H,4H)-dione (14e). Compound 14e was prepared
from 13e as described for the preparation of 14a except the product was purified by trituration with
1
EtOAc/hexanes: beige solid (74% yield); H NMR (DMSOꢀd ): δ 2.91 (t, J = 7.2 Hz, 2H), 5.05 (s, 2H),
6
3
.96 (t, J = 7.2 Hz, 2H), 7.15 (m, 2H), 7.20 (m, 1H), 7.26ꢀ7.31 (m, 2H), 7.36ꢀ7.43 (m, 5H), 12.13 (s,
H).
1
6-Hydroxy-2-phenethyl-1,2,4-triazine-3,5(2H,4H)-dione (11e). Compound 11e was prepared from
4e as described for the preparation of 11a except a mixture of methanol and acetic acid (3:1) was used
1
as a solvent and that the hydrogenation was performed at 50 psi for 1.5 h: light pink powder (60%
1
yield); mp 200 °C; H NMR (DMSOꢀd ) δ 2.90 (t, J = 7.8 Hz, 2H), 3.87 (t, J = 7.6 Hz, 2H), 7.20 (m,
6
+
3
H), 7.29 (m, 2H), 11.71 (s, 1H), 12.03 (s, 1H). LCMS: retention time 1.50 min, m/z 234 [M + H] .
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-Bromo-2-(3-phenylpropyl)-1,2,4-triazine-3,5(2H,4H)-dione (13f). Compound 13f was prepared
1
2
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9
1
1
1
1
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as described for the preparation of 13a except (3ꢀiodopropyl)benzene (1.6 equiv.) was used in place of
1
methyl iodide: white solid (43% yield); H NMR (DMSOꢀd ): δ 1.94 (m, 2H), 2.63 (t, J = 7.7 Hz, 2H),
6
3
.83 (t, J = 6.9 Hz, 2H), 7.15ꢀ7.22 (m, 3H), 7.25ꢀ7.29 (m, 2H), 12.45 (s, 1H).
0
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6-(Benzyloxy)-2-(3-phenylpropyl)-1,2,4-triazine-3,5(2H,4H)-dione (14f). Compound 14f was
1
prepared from 13f as described for the preparation of 14a: clear oil (85% yield oil); H NMR (DMSOꢀ
d6): δ 1.93 (m, 2H), 2.60 (t, J = 7.7 Hz, 2H), 3.76 (t, J = 6.8 Hz, 2H), 5.13 (s, 2H), 7.18 (m, 3H), 7.24
(
m, 2H), 7.35ꢀ7.46 (m, 5H), 12.09 (s, 1H).
6
-Hydroxy-2-(3-phenylpropyl)-1,2,4-triazine-3,5(2H,4H)-dione (11f). Compound 11f was prepared
from 14f as described for the preparation of 11a except a mixture of methanol and acetic acid (3:1) was
1
used as a solvent: light pink solid (85% yield); mp 153 °C; H NMR (DMSOꢀd ) δ 1.89 (m, 2H), 2.60 (t,
6
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] .
6
-Bromo-2-(naphthalen-2-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13g). Compound 13g was
prepared as described for the preparation of 13a except 2ꢀ(bromomethyl)naphthalene (1.2 equiv.) was
used in place of methyl iodide. The reaction was heated overnight. The crude product was purified by
1
trituration with cold diethyl ether: tan solid (56% yield); H NMR (DMSOꢀd ) δ 5.20 (s, 2H), 7.46 (dd, J
6
= 1.5, 8.3 Hz, 1H), 7.51 (m, 2H), 7.22 (m, 1H), 7.86ꢀ7.92 (m, 4H), 12.62 (s, 1H).
6
-(Benzyloxy)-2-(naphthalen-2-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14g). Compound 14g
1
was prepared from 13g as described for the preparation of 14a: white solid foam (76% yield); H NMR
(DMSOꢀd ) δ 5.09 (s, 2H), 5.12 (s, 2H), 7.26ꢀ7.31 (m, 3H), 7.36 (m, 2H), 7.45 (dd, J = 1.8, 7.6 Hz, 1H),
6
7
.50ꢀ7.53 (m, 2H), 7.85ꢀ7.92 (m, 4H), 12.27 (s, 1H).
6-Hydroxy-2-(naphthalen-2-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11g). 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
BBr (1.4 mL, 1.4 mmol) at room temperature. The reaction was stirred for 1 h after which 2 additional
3
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Journal of Medicinal Chemistry
equiv. of BBr were added. Stirring continued at rt for an additional hour and the reaction was quenched
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5
5
5
5
5
5
5
5
6
by the addition of water. The compound was extracted with EtOAc. The organic layer was washed with
brine, dried over Na SO , and concentrated. The residual material was purified by preparative HPLC
2
4
(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
0
1
2
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4
5
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7
8
9
0
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7
8
9
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9
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9
0
1
white fluffy solid (29% yield): mp 264 °C; H NMR (DMSOꢀd ) δ 5.03 (s, 2H), 7.44 (dd, J = 1.3, 8.3
6
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
8
5% acetonitrile/15% water for 2.25 min; flow rate 1.25 mL/min): retention time 1.45 min, m/z 270 [M
+
+
H] .
6-Bromo-2-(naphthalen-1-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13h). Compound 13h was
prepared as described for the preparation of 13a except 1ꢀ(bromomethyl)ꢀnaphthalene (1.2 equiv.) was
used in place of methyl iodide. The crude product was purified by trituration with cold diethyl ether:
1
orange solid (65% yield); H NMR (DMSOꢀd ) δ 5.49 (s, 2H), 7.49 (m, 2H), 7.59 (m, 2H), 7.91 (m,
6
1
H), 7.98 (m, 1H), 8.14 (d, J = 8.1 Hz, 1H), 12.64 (br s, 1H).
-(Benzyloxy)-2-(naphthalen-1-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14h). Compound 14h
6
was prepared from 13h as described for the preparation of 14a except the product was purified by silica
1
gel column chromatography (30% EtOAc in hexanes): white solid (40% yield); H NMR (DMSOꢀd ) δ
6
5.04 (s, 2H), 5.39 (s, 2H), 7.31 (s, 5H), 7.49 (d, J = 4.8 Hz, 2H), 7.57 (m, 2H), 7.91 (t, J = 4.8 Hz, 1H),
7
.98 (m, 1H), 8.22 (d, J = 7.6 Hz, 1H), 12.29 (s, 1H).
-Hydroxy-2-(naphthalen-1-ylmethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11h). Compound 11h was
6
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
1
2
0 psi for 3 h: white powder (90% yield); mp 260 °C (dec); H NMR (DMSOꢀd ) δ 5.33 (s, 2H), 7.41
6
(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
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Journal of Medicinal Chemistry
Page 16 of 56
followed by an increase to 85% acetonitrile/15% water over 1.5 min and continuation of 85%
1
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7
8
9
1
1
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1
1
1
1
1
1
1
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2
2
2
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6
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.
0
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0
6
-Bromo-2-(2-(naphthalen-1-yl)ethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13i). Compound 13i was
prepared as described for the preparation of 13a except 1ꢀ(2ꢀiodoethyl)naphthalene (2.0 equiv.) was
used in place of methyl iodide. The crude product was purified by trituration with cold diethyl ether: tan
1
solid. (46% yield); H NMR (DMSOꢀd ) δ 3.41 (t, J = 7.5 Hz, 2H), 4.13 (t, J = 7.6 Hz, 2H), 7.43 (m,
6
2
H), 7.55 (m, 2H), 7.81 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 12.56 (s,
1
H).
6
-(Benzyloxy)-2-(2-(naphthalen-1-yl)ethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14i). Compound 14i
was prepared from 13i as described for the preparation of 14a except the product was purified by
1
trituration with EtOAc/hexanes: yellow solid (56% yield); H NMR (DMSOꢀd ) δ 3.38 (t, J = 6.8 Hz,
6
2
H), 4.06 (t, J = 7.1 Hz, 2H), 4.91 (s, 2H), 7.30 (m, 1H), 7.37ꢀ7.44 (m, 6H), 7.54 (m, 2H), 7.80 (d, J =
.1 Hz, 1H), 7.92 (d, J = 6.6 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H) 12.15 (s, 1H).
8
6
-Hydroxy-2-(2-(naphthalen-1-yl)ethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11i). Compound 11i was
prepared from 14i as described for the preparation of 11a except a mixture of methanol and acetic acid
1
(
2:1) was used as a solvent: light pink powder (90% yield); mp 300 °C (dec); H NMR (DMSOꢀd ) δ
6
3
.37 (t, J = 8.1 Hz, 2H), 3.93 (bs, 2H), 7.38 (bs, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H),
7.59 (t, J = 7.2 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 8.20 (bs, 1H), 11.95 (s, 1H),
+
1
2.28 (d, 1H). LCMS: retention time 2.99 min, m/z 284 [M + H] .
6
-Bromo-2-(2-chlorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13j). Compound 13j was
prepared as described for the preparation of 13a except 3ꢀchloroꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
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Page 17 of 56
Journal of Medicinal Chemistry
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ether: yellow solid (28% yield); H NMR (DMSOꢀd ) δ 3.07 (t, J = 7.1 Hz, 2H), 4.07 (t, J = 7.1 Hz,
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
2H), 7.26ꢀ7.29 (m, 2H), 7.34 (m, 1H), 7.41 (m, 1H), 12.54 (s, 1H).
6
-(Benzyloxy)-2-(2-chlorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14j). Compound 14j was
1
prepared from 13j as described for the preparation of 14a: white solid (85% yield); H NMR (DMSOꢀ
d6) δ 3.05 (t, J = 6.7 Hz, 2H), 4.00 (t, J = 6.7 Hz, 2H), 4.92 (s, 2H), 7.22ꢀ7.27 (m, 4H), 7.34ꢀ7.43 (m,
0
1
2
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8
9
0
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5
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7
8
9
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2
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4
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8
9
0
5
H), 12.15 (s, 1H).
-(2-Chlorophenethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11j). Compound 11j was
2
prepared from 14j as described for the preparation of 11g with the exception that 2 equiv. of BBr were
3
used and that the crude product was purified by trituration with EtOAc/hexanes: white powder (39%
1
yield); mp 211 °C; H NMR (DMSOꢀd ) δ 3.04 (t, J = 7.1 Hz, 2H), 3.91 (t, J = 7.1 Hz, 2H), 7.24ꢀ7.29
6
+
(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] .
6
-Bromo-2-(3-chlorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13k). Compound 13k was
prepared as described for the preparation of 13a except 3ꢀchloroꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv) was
used in place of methyl iodide. The crude product was purified by trituration with cold diethyl ether: tan
1
solid (73% yield); H NMR (DMSOꢀd ) δ 2.95 (t, J = 7.2 Hz, 2H), 4.06 (t, J = 7.3 Hz, 2H), 7.18 (m,
6
1
H), 7.27ꢀ7.34 (m, 3H), 12.53 (s, 1H).
-(Benzyloxy)-2-(3-chlorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14k). Compound 14k was
6
prepared from 13k as described for the preparation of 14a except the product was purified by trituration
1
with EtOAc/hexanes: beige powder (70% yield); H NMR (DMSOꢀd ) δ 2.93 (t, J = 6.9 Hz, 2H), 3.98
6
(t, J = 6.9 Hz, 2H), 5.05 (s, 2H), 7.09 (m, 1H), 7.25ꢀ7.31 (m, 3H), 7.36ꢀ7.43 (m, 5H), 12.13 (s, 1H).
2-(3-Chlorophenethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11k). Compound 11k was
prepared from 14k as described for the preparation of 11g with the exception that 2 equiv. of BBr were
3
added at rt and that the reaction was stirred at rt for 1.5 h. The compound was purified by trituration
1
with EtOAc/hexanes: beige powder (54% yield); mp 219 °C; H NMR (DMSOꢀd ) δ 2.92 (t, J = 7.1 Hz,
6
2
H), 3.90 (t, J = 7.2 Hz, 2H), 7.14 (m, 1H), 7.26ꢀ7.33 (m, 3H), 11.69 (s, 1H), 12.04 (s, 1H). LCMS:
+
retention time 2.91 min, m/z 268 [M + H] .
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6
-Bromo-2-(4-chlorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13l). Compound 13l was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
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2
2
2
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5
6
prepared as described for the preparation of 13a except 1ꢀchloroꢀ4ꢀ(2ꢀiodoethyl)benzene (1.6 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
1
ether: yellow solid (42% yield); H NMR (DMSOꢀd ) δ 2.94 (t, J = 7.3 Hz, 2H), 4.03 (t, J = 7.3 Hz,
6
2
H), 7.25 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 12.52 (s, 1H).
-(Benzyloxy)-2-(4-chlorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14l). Compound 14l was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
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7
8
9
0
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5
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7
8
9
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2
3
4
5
6
7
8
9
0
6
prepared from 13l as described for the preparation of 14a except the reaction was heated over three
1
days: white solid (72% yield); H NMR (DMSOꢀd ) δ 2.90 (t, J = 6.7 Hz, 2H), 3.96 (t, J = 6.9 Hz, 2H),
6
5
.05 (s, 2H), 7.15 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 7.36ꢀ7.43 (m, 5H), 12.12 (s, 1H).
-(4-Chlorophenethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11l). Compound 11l was
2
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: white solid (52%
1
yield); mp 225 °C; H NMR (DMSOꢀd ) δ 2.90 (t, J = 7.2 Hz, 2H), 3.87 (t, J = 7.3 Hz, 2H), 7.20 (d, J =
6
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
8
5% acetonitrile/15% water for 2.25 min; flow rate 1.25 mL/min): retention time 1.39 min, m/z 268 [M
+
+
H] .
6
-Bromo-2-(2-fluorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13m). Compound 13m was
prepared as described for the preparation of 13a except 2ꢀfluoroꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
1
ether: yellow solid (34% yield); H NMR (DMSOꢀd ) δ 2.98 (t, J = 7.2 Hz, 2H), 4.05 (t, J = 7.2 Hz,
6
2H), 7.15 (m, 2H), 7.29 (m, 2H), 12.55 (s, 1H).
6
-(Benzyloxy)-2-(2-fluorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14m). Compound 14m was
prepared from 13m as described for the preparation of 14a with the exception that the reaction was
heated for three days and that the product was purified by trituration with EtOAc/hexanes: white powder
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Page 19 of 56
Journal of Medicinal Chemistry
1
(
65% yield); H NMR (DMSOꢀd ): δ 2.96 (t, J = 6.8 Hz, 2H), 3.97 (t, J = 6.6 Hz, 2H), 4.95 (s, 2H), 7.11
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
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4
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5
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5
5
5
5
5
6
(
m, 2H), 7.17 (m, 1H), 7.24 (m, 1H), 7.36ꢀ7.41 (m, 5H), 12.17 (s, 1H).
2-(2-Fluorophenethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11m). 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
0
1
2
3
4
5
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7
8
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1
2
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1
2
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9
0
1
psi for 1.5 h: white solid (83% yield); mp 194 °C; H NMR (DMSOꢀd ) δ 2.95 (t, J = 7.2 Hz, 2H), 3.88
6
(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] .
6
-Bromo-2-(3-fluorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13n). Compound 13n was
prepared as described for the preparation of 13a except 3ꢀfluoroꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
1
ether: yellow solid (66% yield); H NMR (DMSOꢀd ) δ 2.96 (t, J = 7.3 Hz, 2H), 4.06 (t, J = 7.3 Hz,
6
2
H), 7.05 (m, 2H), 7.22 (m, 1H), 7.33 (m, 1H), 12.53 (s, 1H).
6-(Benzyloxy)-2-(3-fluorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14n). Compound 14n was
prepared from 13n as described for the preparation of 14a except the product was purified by trituration
1
with EtOAc/hexanes: tan solid (78% yield); H NMR (DMSOꢀd ) δ 2.94 (t, J = 6.8 Hz, 2H), 3.98 (t, J =
6
6
.9 Hz, 2H), 5.06 (s, 2H), 6.96ꢀ7.05 (m, 3H), 7.29 (m, 1H), 7.36ꢀ7.43 (m, 5H), 12.13 (s, 1H).
-(3-Fluorophenethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11n). Compound 11n was
2
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
1
psi: white solid (79% yield); mp 197 °C; H NMR (DMSOꢀd ) δ 2.93 (t, J = 7.3 Hz, 2H), 3.89 (t, J = 7.2
6
+
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] .
6
-Bromo-2-(4-fluorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13o). Compound 13o was
prepared as described for the preparation of 13a except 1ꢀfluoroꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
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Journal of Medicinal Chemistry
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1
ether: yellow solid (42% yield); H NMR (DMSOꢀd ) δ 2.93 (t, J = 7.1 Hz, 2H), 4.03 (t, J = 7.3 Hz,
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
H), 7.11 (m, 2H), 7.26 (m, 2H), 12.52 (s, 1H).
6-(Benzyloxy)-2-(4-fluorophenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14o). Compound 14o was
1
prepared from 13o as described for the preparation of 14a: white solid (81% yield); H NMR (DMSOꢀ
d6) δ 2.90 (t, J = 6.8 Hz, 2H), 3.95 (t, J = 6.9 Hz, 2H), 5.07 (s, 2H), 7.09 (m, 2H), 7.17 (m, 2H), 7.36ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.43 (m, 5H), 12.12 (s, 1H).
-(4-Fluorophenethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11o). Compound 11o was
2
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
1
EtOAc/hexanes: light pink powder (73% yield); mp 193 °C; H NMR (DMSOꢀd ) δ 2.89 (t, J = 7.3 Hz,
6
2
H), 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] .
6-Bromo-2-(2-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13p). Compound 13p was
prepared as described for the preparation of 13a except 3ꢀmethylꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
1
ether: tan solid (52% yield); H NMR (DMSOꢀd ) δ 2.31 (s, 3H), 2.94 (m, 2H), 3.98 (m, 2H), 7.12 (m,
6
3
H), 7.15 (m, 1H), 12.54 (s, 1H).
6-(Benzyloxy)-2-(2-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14p). Compound 14p was
prepared from 13p as described for the preparation of 14a except the product was purified by trituration
1
with EtOAc/hexanes: beige powder (66% yield); H NMR (DMSOꢀd ) δ 2.30 (s, 3H), 2.90 (t, J = 7.5
6
Hz, 2H), 3.90 (t, J = 7.8 Hz, 2H), 5.07 (s, 2H), 7.03 (m, 1H), 7.09ꢀ7.12 (m, 2H), 7.14 (m, 1H), 7.36ꢀ7.45
(m, 5H), 12.16 (s, 1H).
6
-Hydroxy-2-(2-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11p). 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:
1
grey solid (71% yield); mp 207 °C; H NMR (DMSOꢀd ) δ 2.31 (s, 3H), 2.89 (t, J = 7.7 Hz, 2H), 3.81 (t,
6
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Journal of Medicinal Chemistry
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
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
[
M + H] .
6-Bromo-2-(3-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13q). Compound 13q was
prepared as described for the preparation of 13a except 3ꢀmethylꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ether: tan solid (66% yield): H NMR (DMSOꢀd ) δ 2.27 (s, 3H), 2.89 (t, J = 7.6 Hz, 2H), 4.01 (t, J =
6
7
.6 Hz, 2H), 7.02 (m, 3H), 7.18 (t, J = 7.3 Hz, 1H), 12.50 (s, 1H).
6-(Benzyloxy)-2-(3-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14q). Compound 14q was
prepared from 13q as described for the preparation of 14a except the product was purified by trituration
1
with EtOAc/hexanes: yellow powder (62% yield); H NMR (DMSOꢀd ) δ 2.25 (s, 3H), 2.87 (t, J = 6.8
6
Hz, 2H), 3.94 (t, J = 7.0 Hz, 2H), 5.07 (s, 2H), 6.98 (m, 3H), 7.16 (t, J = 7.6 Hz, 1H), 7.37ꢀ7.45 (m, 5H),
1
2.14 (s, 1H).
-Hydroxy-2-(3-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11q). Compound 11q was
6
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
1
psi: white powder (82% yield); mp 210 °C; H NMR (DMSOꢀd ) δ 2.27 (s, 3H), 2.86 (t, J = 7.3 Hz,
6
2
H), 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] .
6-Bromo-2-(4-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13r). Compound 13r was
prepared as described for the preparation of 13a except 4ꢀmethylꢀ4ꢀ(2ꢀiodoethyl)benzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
1
ether: tan solid (63% yield); H NMR (DMSOꢀd ) δ 2.26 (s, 3H), 2.89 (t, J = 7.5 Hz, 2H), 4.00 (m, 2H),
6
7
.10 (s, 4H), 12.52 (s, 1H).
6
-(Benzyloxy)-2-(4-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14r). Compound 14r was
1
prepared from 13r as described for the preparation of 14a: white foam (72% yield); H NMR (DMSOꢀ
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d6) δ 2.24 (s, 3H), 2.86 (t, J = 7.2 Hz, 2H), 3.93 (t, J = 7.1 Hz, 2H), 5.06 (s, 2H), 7.03 (m, 2H), 7.06 (m,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2H), 7.36ꢀ7.43 (m, 5H), 12.12 (s, 1H).
6
-Hydroxy-2-(4-methylphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11r). Compound 11r was
prepared from 14r as described for the preparation of 11a except the hydrogenation was performed at 30
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
psi for 2 h: white powder (54% yield); mp 219 °C; H NMR (DMSOꢀd ) δ 2.25 (s, 3H), 2.85 (t, J = 7.5
6
Hz, 2H), 3.84 (t, J = 7.6 Hz, 2H), 7.08 (q, J = 8.1, 11.4 Hz, 4H), 11.68 (s, 1H), 12.02 (s, 1H). LCMS:
+
retention time 2.49 min, m/z 248 [M + H] .
6-Bromo-2-(3-(trifluoromethyl)phenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13s). Compound 13s
was prepared as described for the preparation of 13a except 1ꢀ(2ꢀiodoethyl)ꢀ3ꢀ(trifluoromethyl)benzene
(2.0 equiv.) was used in place of methyl iodide. The crude product was purified by trituration with cold
1
diethyl ether: yellow solid (48%); H NMR (DMSOꢀd ) δ 3.06 (t, J = 7.2 Hz, 2H), 4.10 (t, J = 7.1 Hz,
6
2
H), 7.55 (m, 4H), 12.53 (s, 1H).
6-(Benzyloxy)-2-(3-(trifluoromethyl)phenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14s). Compound
1
1
4s was prepared from 13s as described for the preparation of 14a: white solid (85%); H NMR
(
DMSOꢀd ) δ 3.02 (t, J = 6.7 Hz, 2H), 4.01 (t, J = 6.8 Hz, 2H), 5.00 (s, 2H), 7.36 (m, 1H), 7.40 (m,
6
4
H), 7.50 (m, 4H), 12.13 (s, 1H).
-Hydroxy-2-(3-(trifluoromethyl)phenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11s). Compound
6
11s was prepared from 14s as described for the preparation of 11a: white powder (85% yield); mp 201
1
°
C; H NMR (DMSOꢀd ) δ 3.02 (t, J = 7.1 Hz, 2H), 3.93 (t, J = 7.2 Hz, 2H), 7.51 (m, 3H), 7.574 (m,
6
+
1
H), 11.70 (s, 1H), 12.03 (s, 1H). LCMS: retention time 3.23 min, m/z 302 [M + H] .
6-Bromo-2-(4-(trifluoromethyl)phenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13t). Compound 13t
was prepared as described for the preparation of 13a except 1ꢀ(2ꢀiodoethyl)ꢀ4ꢀ(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%
1
yield); H NMR (DMSOꢀd ) 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),
6
7
2
.64 (d, J = 8.1 Hz, 2H), 12.53 (s, 1H).
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Journal of Medicinal Chemistry
6
-(Benzyloxy)-2-(4-(trifluoromethyl)phenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14t). Compound
1
2
1
4t was prepared from 13t as described for the preparation of 14a except the product was purified by
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
trituration with EtOAc/hexanes: white solid (71% yield); H NMR (DMSOꢀd ) δ 3.01 (t, J = 6.7 Hz,
6
2
H), 4.01 (t, J = 6.8 Hz, 2H), 5.02 (s, 2H), 7.38ꢀ7.42 (m, 7H), 7.62 (d, J = 8.1 Hz, 2H), 12.13 (s, 1H).
6-Hydroxy-2-(4-(trifluoromethyl)phenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11t). Compound
1t was prepared from 14t as described for the preparation of 11a with the exception that a mixture of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
methanol and EtOAc (1:1) was used as a solvent and that the hydrogenation was performed overnight at
1
30 psi: gray solid (83% yield); mp 222 °C; H NMR (DMSOꢀd ) δ 3.01 (t, J = 7.1 Hz, 2H), 3.92 (t, J =
6
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] .
6
-Bromo-2-(2-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13u). Compound 13u was
prepared as described for the preparation of 13a with except of 1ꢀ(2ꢀiodoethyl)ꢀ2ꢀmethoxybenzene (2.0
equiv.) was used in place of methyl iodide. The crude product was purified by trituration with cold
1
diethyl ether: tan solid (57% yield); H NMR (DMSOꢀd ) δ 2.91 (t, J = 6.9 Hz, 2H), 3.73 (s, 3H), 4.01
6
(
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).
-(Benzyloxy)-2-(2-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14u). Compound 14u
6
was prepared from 13u as described for the preparation of 14a except the product was purified by
1
trituration with EtOAc/hexanes: white solid (63% yield); H NMR (DMSOꢀd ) δ 2.89 (t, J = 6.7 Hz,
6
2
H), 3.74 (s, 3H), 3.94 (t, J = 6.7 Hz, 2H), 4.89 (s, 2H), 6.84 (t, J = 7.3 Hz, 1H), 6.93 (d, J = 8.1 Hz,
H), 7.02 (dd, J = 1.8, 7.3 Hz, 1H), 7.20 (m, 1H), 7.35ꢀ7.41 (m, 5H), 12.12 (s, 1H).
1
6
-Hydroxy-2-(2-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11u). 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
1
atmospheric pressure of hydrogen: yellow powder (83% yield); mp 166 °C; H NMR (DMSOꢀd ) δ 2.88
6
(
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
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Journal of Medicinal Chemistry
Page 24 of 56
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).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
LCMS: retention time 2.03 min, m/z 264 [M + H] .
6-Hydroxy-2-(2-hydroxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (15u). Compound 15u was
prepared from 11u as described for the preparation of 11g with the exception that 3 equiv. of BBr were
3
1
used and that the reaction was stirred for 1.5 h at rt: offꢀwhite solid (68% yield); mp 205 °C; H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
DMSOꢀd ) 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
6
=
7.8 Hz, 1H), 6.97ꢀ7.03 (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] .
6
-Bromo-2-(3-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13v). Compound 13v was
prepared as described for the preparation of 13a except 1ꢀ(2ꢀiodoethyl)ꢀ3ꢀmethoxybenzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
1
ether: tan solid (36% yield); H NMR (DMSOꢀd ) δ 2.91 (t, J = 7.5 Hz, 2H), 3.73 (s, 3H), 4.04 (m, 2H),
6
6.77 (m, 1H), 6.79 (m, 2H), 7.21 (t, J = 7.7 Hz, 1H), 12.53 (s, 1H).
6
-(Benzyloxy)-2-(3-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14v). Compound 14v was
prepared from 13v as described for the preparation of 14a except the product was purified by trituration
1
with EtOAc/hexanes: white solid (64% yield); H NMR (DMSOꢀd ) δ 2.89 (t, J = 7.1 Hz, 2H), 3.70 (s,
6
3
H), 3.96 (t, J = 7.2 Hz, 2H), 5.06 (s, 2H), 6.72ꢀ6.78 (m, 3H), 7.19 (t, J = 7.8 Hz, 1H), 7.36ꢀ7.44 (m,
5H), 12.14 (s, 1H).
-Hydroxy-2-(3-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11v). Compound 11v was
6
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:
1
offꢀwhite solid (63% yield); mp 224 °C; H NMR (DMSOꢀd ) δ 2.88 (t, J = 7.6 Hz, 2H), 3.72 (s, 3H),
6
3
.87 (t, J = 7.6 Hz, 2H), 6.75ꢀ6.78 (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] .
6
-Hydroxy-2-(3-hydroxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (15v). Compound 15v was
1
prepared from 11v as described for the preparation of 11g: white solid (53% yield); mp 232 °C; H
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Journal of Medicinal Chemistry
NMR (DMSOꢀd ) δ 2.80 (t, J = 7.2 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 6.58 (m ,3H), 7.07 (t, J = 7.7 Hz,
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
H), 9.32 (s, 1H), 11.69 (s, 1H), 12.04 (s, 1H). LCMS: retention time 0.37 min, m/z 250 [M + H] .
6-Bromo-2-(4-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13w). Compound 13w was
prepared as described for the preparation of 13a except 1ꢀ(2ꢀiodoethyl)ꢀ4ꢀmethoxybenzene (2.0 equiv.)
was used in place of methyl iodide. The crude product was purified by trituration with cold diethyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ether: tan solid (58% yield); H NMR (DMSOꢀd ) δ 2.87 (t, J = 7.5 Hz, 2H), 3.71 (s, 3H), 3.99 (m, 2H),
6
6
.84 (d, J = 8.6 Hz, 2H), 7.12 (m, 2H), 12.52 (s, 1H).
6-(Benzyloxy)-2-(4-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14w). Compound 14w
was prepared from 13w as described for the preparation of 14a except the product was purified by
1
trituration with EtOAc/hexanes: white solid (70% yield); H NMR (DMSOꢀd ) δ 2.84 (t, J = 7.1 Hz,
6
2
H), 3.69 (s, 3H), 3.91 (t, J = 7.1 Hz, 2H), 5.06 (s, 2H), 6.82 (t, J = 8.8 Hz, 2H), 7.05 (t, J = 8.6 Hz, 2H),
.17 (m, 2H), 7.36ꢀ7.44 (m, 5H), 12.12 (s, 1H).
7
6-Hydroxy-2-(4-methoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11w). 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: offꢀ
1
white solid (62% yield); mp 244 °C; H NMR (DMSOꢀd ) δ 2.83 (t, J = 7.5 Hz, 2H), 3.71 (s, 3H), 3.82
6
(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]
6-Hydroxy-2-(4-hydroxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (15w). Compound 15w was
prepared from 11w as described for the preparation of 11g: light pink solid (54% yield); mp > 280 °C
1
(
decomp); H NMR (DMSOꢀd ) δ 2.78 (t, J = 7.6 Hz, 2H), 3.80 (t, J = 7.6 Hz, 2H), 6.65 (d, J = 8.3 Hz,
6
2
H), 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] .
6
-Bromo-2-(3-phenoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13x). Compound 13x was
prepared as described for the preparation of 13a except 1ꢀ(2ꢀiodoethyl)ꢀ3ꢀphenoxybenzene (2.0 equiv.)
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1
was used in place of methyl iodide. Yield: 21% (white solid). H NMR (DMSOꢀd ): δ 2.93 (t, J = 7.1
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hz, 2H), 4.05 (t, J = 7.1 Hz, 2H), 6.85ꢀ6.67 (m, 2H), 6.95ꢀ7.01 (m, 3H), 7.10ꢀ7.15 (m, 1H), 7.29ꢀ7.39
(m, 3H), 12.53 (s, 1H).
6
-(Benzyloxy)-2-(3-phenoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14x). Compound 14x was
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
prepared from 13x as described for the preparation of 14a: white solid (52% yield); H NMR (DMSOꢀ
d6): δ 2.88 (t, J = 6.5 Hz, 2H), 3.96 (t, J = 6.5 Hz, 2H), 5.03 (s, 2H), 6.75 (m, 1H), 6.84ꢀ6.86 (m, 1H),
6
.91ꢀ6.96 (m, 3H), 7.09ꢀ7.13 (m, 1H), 7.28ꢀ7.39 (m, 8H), 12.14, (bs, 1H).
6
-Hydroxy-2-(3-phenoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11x). Compound 11x was
1
prepared from 14x as described for the preparation of 11a: tan solid (78% yield); mp 150ꢀ153 °C; H
NMR (DMSOꢀd ): δ 2.89 (t, J = 7.2 Hz, 2H), 3.86 (t, J = 7.2 Hz, 2H), 6.79 (m, 1H), 6.83ꢀ6.86 (m, 1H),
6
6
.95ꢀ6.98 (m, 3H), 7.10ꢀ7.14 (m, 1H), 7.28ꢀ7.40 (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] .
6
-Bromo-2-(4-phenoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (13y). Compound 13y was
prepared as described for the preparation of 13a except 1ꢀ(2ꢀiodoethyl)ꢀ4ꢀphenoxybenzene (2.0 equiv.)
1
was used in place of methyl iodide: tan solid (24% yield); H NMR (DMSOꢀd ): δ 2.93 (m, 2H), 4.04
6
(m, 2H), 6.93ꢀ6.98 (m, 4H), 7.10ꢀ7.14 (m, 1H), 7.21ꢀ7.25 (m, 2H), 7.36ꢀ7.40 (m, 2H), 12.54 (s, 1H).
6
-(Benzyloxy)-2-(4-phenoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14y). Compound 14y was
1
prepared from 13y as described for the preparation of 14a: white solid (41% yield); H NMR (DMSOꢀ
d6): δ 2.90 (t, J = 7.0 Hz, 2H), 3.96 (t, J = 7.0 Hz, 2H), 5.08 (s, 2H), 6.91ꢀ6.96 (m, 4H), 7.09ꢀ7.12 (m,
1
H), 7.16ꢀ7.18 (m, 2H), 7.33ꢀ7.45 (m, 7H), 12.14 (s, 1H).
6-Hydroxy-2-(4-phenoxyphenethyl)-1,2,4-triazine-3,5(2H,4H)-dione (11y). Compound 11y was
1
prepared from 14y as described for the preparation of 11g: white solid (69% yield); mp 207ꢀ209 °C; H
NMR (DMSOꢀd ): δ 2.90 (t, J = 7.2 Hz, 2H), 3.88 (t, J = 7.3 Hz, 2H), 6.92ꢀ6.98 (m, 4H), 7.10ꢀ7.14 (m,
6
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H), 7.21 (m, 2H), 7.35ꢀ7.40 (m, 2H), 11.69 (bs, 1H), 12.04 (bs, 1H). LCMS (20% acetonitrile/80%
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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] .
2
-(2-(Biphenyl-4-yl)ethyl)-6-bromo-1,2,4-triazine-3,5(2H,4H)-dione (13z). Compound 13z was
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prepared as described for the preparation of 13a except 4ꢀ(2ꢀiodoethyl)biphenyl (2.0 equiv.) was used in
place of methyl iodide. The crude product was purified by trituration with cold diethyl ether: tan solid
1
(59% yield); H NMR (DMSOꢀd ) δ 2.99 (t, J = 7.5 Hz, 2H), 4.08 (t, J = 7.6 Hz, 2H), 7.31ꢀ7.37 (m,
6
3
H), 7.45 (t, J = 7.6 Hz, 2H), 7.59ꢀ7.65 (m, 4H), 12.55 (s, 1H).
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-(Benzyloxy)-2-(2-(biphenyl-4-yl)ethyl)-1,2,4-triazine-3,5(2H,4H)-dione (14z). Compound 14z
1
was prepared from 13z as described for the preparation of 14a: white solid (53% yield); H NMR
(DMSOꢀd ) δ 2.96 (t, J = 7.2 Hz, 2H), 4.00 (t, J = 7.1 Hz, 2H), 5.05 (s, 2H),7.25 (d, J = 8.1 Hz, 2H),
6
7.34ꢀ7.46 (m, 8H), 7.58ꢀ7.64 (m, 4H), 12.16 (s, 1H).
-(2-(Biphenyl-4-yl)ethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (11z). Compound 11z was
2
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:
1
white powder (89% yield); mp 254 °C; H NMR (DMSOꢀd ) δ 2.95 (t, J = 7.6 Hz, 2H), 3.92 (t, J = 7.5
6
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] .
4
-(Benzyloxymethyl)-6-bromo-2-(2-(pyridin-2-yl)ethyl)-1,2,4-triazine-3,5(2H,4H)-dione
(17a).
To a solution of 16 (0.30 g, 0.96 mmol), 2ꢀ(pyridinꢀ2ꢀyl)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
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7a as a clear oil (85% yield): H NMR (CDCl ) δ 3.22 (t, J = 7.1 Hz, 2H), 4.37 (t, J = 7.6 Hz, 2H), 4.68
3
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(
s, 2H), 5.49 (s, 2H), 7.12ꢀ7.19 (m, 2H), 7.29ꢀ7.34 (mt, 5H), 7.61 (dt, J = 2.0, 7.8 Hz, 1H), 8.51 (d, J =
4.8 Hz, 1H).
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-(Benzyloxy)-4-(benzyloxymethyl)-2-(2-(pyridin-2-yl)ethyl)-1,2,4-triazine-3,5(2H,4H)-dione
(18a). To a solution of benzyl alcohol (0.13 mL, 1.2 mmol) in DMF (3 mL) was added 60% w/w
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sodium hydride (60% dispersion in mineral oil, 0.049 g, 1.2 mmol) at 0 °C. The mixture was stirred for
0 min and a solution of 17a (0.34 g, 0.81 mmol) in DMF (5 mL) was added via syringe. The reaction
1
was stirred at 0 °C for 30 min and quenched by the addition of water and EtOAc. The organic layer was
dried over Na SO and concentrated. The residual oil was purified using a Biotage Isolera One flash
2
4
purification system with a silica gel flash cartridge (EtOAc/hexanes) to give 0.18 g of 18a as an oil
1
(50% yield): H NMR (CDCl ) δ 3.17 (t, J = 7.1 Hz, 2H) 4.27 (t, J = 5.6 Hz, 2H), 4.67 (s, 2H), 5.07 (s,
3
2
H), 5.47 (s, 2H), 7.12 (m, 2H), 7.29ꢀ7.42 (m, 10H), 7.59 (dt, J = 2.0, 7.8 Hz, 1H), 8.54 (d, J = 5.1 Hz,
1
H).
6-Hydroxy-2-(2-(pyridin-2-yl)ethyl)-1,2,4-triazine-3,5(2H,4H)-dione (19a). To a solution of 18a
(0.18 g, 0.40 mmol) in dichloromethane (8 mL) was added a 1.0 M solution of BBr in dichloromethane
3
(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
1
10 mL/min) to give 0.017 g of 19a as a white solid (18% yield): mp 225 °C; H NMR (DMSOꢀd ) δ
6
3
1
.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,
H), 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 ꢁm): retention time 1.06 min, m/z 235
+
[
M + H] .
2
-(2-(1H-Pyrazol-1-yl)ethyl)-4-(benzyloxymethyl)-6-bromo-1,2,4-triazine-3,5(2H,4H)-dione
(17b). Compound 17b was prepared as described for the preparation of 17a except 2ꢀ(1Hꢀpyrazolꢀ1ꢀ
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yl)ethanol was used in place of 2ꢀ(pyridinꢀ2ꢀyl)ethanol: clear oil (86% yield); H NMR (CDCl ) δ 4.36
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(
t, J = 5.9 Hz, 2H), 4.50 (t, J = 5.9 Hz, 2H), 4.67 (s, 2H), 5.47 (s, 2H), 6.24 (t, J = 2.1 Hz, 1H), 7.29ꢀ
7.34 (m, 5H), 7.36 (m, 1H), 7.46 (m, 1H).
-(2-(1H-Pyrazol-1-yl)ethyl)-6-(benzyloxy)-4-(benzyloxymethyl)-1,2,4-triazine-3,5(2H,4H)-dione
2
(18b). Compound 18b was prepared from 17b as described for the preparation of 18a except the
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reaction was stirred at 0 °C and warmed up to rt over a period of 4 h: clear oil (47% yield); H NMR
(CDCl ): 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
3
(t, J = 2.1 Hz, 1H), 7.20 (m, 1H), 7.28ꢀ7.41 (m, 10H), 7.51 (m, 1H).
-(2-(1H-Pyrazol-1-yl)ethyl)-6-hydroxy-1,2,4-triazine-3,5(2H,4H)-dione (19b). To a solution of
9b (0.14 g, 0.32 mmol) in methanol (5 mL) was added one spatula tip of 10% Pd/C. The mixture was
2
1
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
1
of compound 19b as a white solid (83% yield): mp 275 °C (dec); H NMR (DMSOꢀd ) δ 4.01 (t, J = 6.2
6
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,
1
H), 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
1
.5 min; flow rate 0.25 mL/min; column Luna Plus C18, 2.1 mm × 100 mm, 1.8 ꢁm): retention time
+
.32 min, m/z 224 [M + H] .
2-(2-(1H-Benzo[d]imidazol-1-yl)ethyl)-4-(benzyloxymethyl)-6-bromo-1,2,4-triazine-3,5(2H,4H)-
dione (17c). Compound 17c was prepared as described for the preparation of 17a except 2ꢀ(1Hꢀ
benzo[d]imidazolꢀ1ꢀyl)ethanol was used in place of 2ꢀ(pyridinꢀ2ꢀyl)ethanol and that the compound was
1
purified by trituration with EtOAc/hexanes: beige solid (72% yield); H NMR (CDCl ) δ 4.38 (t, J = 6.6
3
Hz, 2H), 4.56 (t, J = 6.4 Hz, 2H), 4.61 (s, 2H), 5.42 (s, 2H), 7.28ꢀ7.34 (m, 7H), 7.39 (m, 1H), 7.80 (m,
1H), 7.91 (s, 1H).
2
-(2-(1H-Benzo[d]imidazol-1-yl)ethyl)-6-(benzyloxy)-4-(benzyloxymethyl)-1,2,4-triazine-
3
,5(2H,4H)-dione (18c). Compound 18c was prepared from 17c as described for the preparation of 18a
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