Targeting the subpocket in xanthine oxidase: Design, synthesis, and biological evaluation of 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.07.062

Xanthine oxidase is an important target for the treatment of hyperuricemia, gout and other related diseases. Analysis of the high-resolution structure of xanthine oxidase with febuxostat identified the existence of a subpocket formed by the residues Leu648, Asn768, Lys771, Leu1014 and Pro1076. In this study, we designed and synthesized a series of 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives (8a-8z) with a tetrazole group targeting this subpocket of the xanthine oxidase active site, and they were further evaluated for their inhibitory potency against xanthine oxidase in vitro. The results showed that all the tested compounds (8a-8z) exhibited an apparent xanthine oxidase inhibitory potency, with IC₅₀ values ranging from 0.0288 μM to 0.629 μM. Among them, compound 8u emerged as the most potent xanthine oxidase inhibitor, with an IC₅₀ value of 0.0288 μM, which was comparable to febuxostat (IC₅₀ = 0.0236 μM). The structure-activity relationship results revealed that the hydrophobic group at the 4′-position was indispensable for the inhibitory potency in vitro against xanthine oxidase. A Lineweaver-Burk plot revealed that the representative compound 8u acted as a mixed-type inhibitor for xanthine oxidase. Furthermore, molecular modeling studies were performed to gain insights into the binding mode of 8u with xanthine oxidase and suggested that the tetrazole group of the phenyl unit was accommodated in the subpocket, as expected. Moreover, a potassium oxonate-induced hyperuricemia model in rats was chosen to further confirm the hypouricemic effect of compound 8u, and the result demonstrated that compound 8u could effectively reduce serum uric acid levels at an oral dose of 5 mg/kg. In addition, acute oral toxicity study in mice indicated that compound 8u was nontoxic and tolerated at a dose up to 2000 mg/kg. Thus, compound 8u could be a potential and efficacious agent in treatment of hyperuricemia with low toxicity.

Full text of DOI:10.1016/j.ejmech.2019.07.062

Accepted Manuscript
Targeting the subpocket in xanthine oxidase: Design, synthesis, and biological
evaluation of 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-
carboxylic acid derivatives
Bing Zhang, Xiwen Dai, Ziyang Bao, Qing Mao, Yulin Duan, Yuwei Yang, Shaojie
Wang
PII:
S0223-5234(19)30683-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.062
EJMECH 11559
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 June 2019
Revised Date: 14 July 2019
Accepted Date: 21 July 2019
Please cite this article as: B. Zhang, X. Dai, Z. Bao, Q. Mao, Y. Duan, Y. Yang, S. Wang, Targeting
the subpocket in xanthine oxidase: Design, synthesis, and biological evaluation of 2-[4-alkoxy-3-(1H-
tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.062.
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. Graphical Abstract

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Targeting the subpocket in xanthine oxidase: design, synthesis, and biological
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evaluation
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2-[4-alkoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives
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Bing Zhang, Xiwen Dai, Ziyang Bao, Qing Mao, Yulin Duan, Yuwei Yang, Shaojie Wang*
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Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, 103 Culture Road, Shenhe District, Shenyang 110016, China
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*Corresponding authors.
Tel/Fax: +86-24-43520230, E-mail: sjwang_99@163.com (S. J. Wang).
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Abstract
Xanthine oxidase is an important target for the treatment of hyperuricemia, gout and other related diseases.
Analysis of the high-resolution structure of xanthine oxidase with febuxostat identified the existence of a
subpocket formed by the residues Leu648, Asn768, Lys771, Leu1014 and Pro1076. In this study, we designed
and synthesized a series of 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic
acid derivatives (8a-8z) with a tetrazole group targeting this subpocket of the xanthine oxidase active site, and
they were further evaluated for their inhibitory potency against xanthine oxidase in vitro. The results showed
that all the tested compounds (8a-8z) exhibited an apparent xanthine oxidase inhibitory potency, with IC values
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ranging from 0.0288 µM to 0.629 µM. Among them, compound 8u emerged as the most potent xanthine oxidase
inhibitor, with an IC50 value of 0.0288 µM, which was comparable to febuxostat (IC50 = 0.0236 µM). The
structure-activity relationship results revealed that the hydrophobic group at the 4′-position was indispensable
for the inhibitory potency in vitro against xanthine oxidase. A Lineweaver-Burk plot revealed that the
representative compound 8u acted as a mixed-type inhibitor for xanthine oxidase. Furthermore, molecular
modeling studies were performed to gain insights into the binding mode of 8u with xanthine oxidase and
suggested that the tetrazole group of the phenyl unit was accommodated in the subpocket, as expected.
Moreover, a potassium oxonate-induced hyperuricemia model in rats was chosen to further confirm the
hypouricemic effect of compound 8u, and the result demonstrated that compound 8u could effectively reduce
serum uric acid levels at an oral dose of 5 mg/kg. In addition, acute oral toxicity study in mice indicated that
compound 8u was nontoxic and tolerated at a dose up to 2000 mg/kg. Thus, compound 8u could be a potential
and efficacious agent in treatment of hyperuricemia with low toxicity.
Key words: 6-Oxo-1,6-dihydropyrimidine-5-carboxylic acid; Tetrazole; Xanthine oxidase inhibitor; Subpocket;
Synthesis
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1. Introduction
Xanthine oxidase (XO) is a molybdoenzyme catalyzing the oxidation of hypoxanthine to xanthine and
xanthine to urate in the last two steps of purine nucleotide metabolism [1-3]. Uric acid is the final product of
purine metabolism in humans, and the high level of uric acid in the plasma has been linked to a number of
pathologies, such as gout, cardiovascular diseases, hypertension and renal diseases [4-6]. Therefore, XO is
considered to be the most promising target for controlling the uric acid level and for the treatment of
hyperuricemia, gout and other related diseases [7].
Allopurinol (Fig. 1), a prototype XO inhibitor and a hypoxanthine isomer, is a widely used drug for
inhibiting XO in gout. However, in some cases, the interactions of purine analogs XO inhibitors on activities of
purine and pyrimidine metabolizing enzymes lead to the reported hypersensitivity (Stevens-Johnsons) syndrome
characterized by fever, skin rash, hepatitis, leukocytosis with eosinophilia and worsening renal function induced
in some of the patients [8-10].
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Therefore, finding the nonpurine compounds with potent XO-inhibition potency and fewer side effects than
the purine analogs is in great demand. Considering these side effects, some nonpurine based XO inhibitors, such
as febuxostat (approved in USA, 2009) [11], topiroxostat (approved in Japan, 2013) [12], Y-700 [13], isoxazole
derivatives [14], selenazole derivatives [15], imidazole derivatives [16], 2-(indol-2-yl)-thiazole derivatives [17],
1,2,3-triazole derivatives [18], pyrazole derivatives [19], isonicotinic acid derivatives [20], 2-mercaptopyridine
derivatives [21], isocytosine derivatives [22], pyrimidine derivatives [23], fused pyrano [3, 2-d] pyrimidine
derivatives [24], dihydropyridazine derivatives [25], naphthopyran derivatives [26] and naphthoflavone
derivatives [27] have been developed (Fig. 1). Additionally, other XO inhibitors with various chemotypes,
including 4-pyridyl-1H-imidazole derivatives [28], 5-(4-pyridyl)-1,2,4-triazole derivatives [29],
2-benzylamino-4-methyl-1,3-thiazole derivatives [30], 5-[4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl] benzonitrile
derivatives
[31],
N-(4-alkoxy-3-cyanophenyl)
isonicotinamide/nicotinamide
derivatives
[32],
4,6-diaryl/heteroaryl pyrimidone derivatives [33] and pyrimidine-2,4-diamine derivatives [34], have also been
published in the literature. However, febuxostat is no longer a precise and safe therapy for hyperuricemia
because the FDA added a black-box warning to this drug due to its heart-related complications [35]. Therefore,
there is still a need to explore new nonpurine XO inhibitors with fewer side effects for the treatment of
hyperuricemia and gout.
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Fig. 1. Chemical structures of allopurinol and nonpurine XO inhibitors.
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Analysis of the high-resolution structure (Fig. 2a) of XO with febuxostat could identify the existence of a
subpocket formed by Leu648, Asn768, Lys771, Leu1014 and Pro1076 residues (Fig. 2b), and it was observed
that the cyano group of febuxostat could interact with the Asn768 residue deep inside the subpocket (Fig. 2c). In
many biologically important enzyme targets, targeting the unique subpocket of an enzyme active site could
impart selectivity by modulators [36-40], which would help to further avoid some side effects [41-42]. As a
consequence, the subpocket of the XO active site could be utilized for the development of the new nonpurine
XO inhibitors with increased selectivity and fewer side effects. The tetrazole moiety is a low-toxic and
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practically nonmetabolized heterocyclic fragment acting as a hydrogen acceptor similar to the cyano group
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[43-45], and the tetrazole moiety has received major attention from medicinal chemists due to its wide
applications in the treatment of neonatal sepsis (latamoxef, approved in 1982) [46], hypertension (losartan,
approved in 1994) [47] and symptoms of intermittent claudication (cilostazol, approved in 1988) [48]. Thus, the
tetrazole moiety occupies an important position in the discovery of new drugs [49]. Therefore, we attempted to
introduce a tetrazole moiety targeting the subpocket at the XO active site, with the hope of the tetrazole moiety
filling the subpocket, which is useful for helping ligands anchored tightly to the binding site, enhancing the
binding affinity [32, 50-52] and potentially preventing some side effects.
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Fig. 2. Structure analysis of the XO-febuxostat complex. (a) Crystal structure of the XO-febuxostat complex as a
cartoon representation. (b) The subpocket is shown as a gray surface and febuxostat is represented as an orange
line. (c) Febuxostat is represented as an orange line, and key polar interactions are depicted with black dashed
lines.
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In our previous report, we explored several new classes of nonpurine XO inhibitors, including isoxazole
derivatives [14], imidazole derivatives [16] and 4-pyridyl-1H-imidazole derivatives [28]. Among them,
imidazole derivatives containing a 1-hydroxyimidazole moiety showed excellent inhibitory potency in vitro
against XO [16], and structure-activity relationship analysis and molecular modeling studies indicated that the
1-hydroxy moiety could form an extra H-bond with Thr1010 and contribute to the inhibitory potency [16,28].
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The results inspired us to further explore novel XO inhibitors based on an extra H-bond with Thr1010 in
addition to the interactions between the carboxyl group and Arg880 and Thr1010. Pyrimidine, a six membered
heterocyclic compound bearing two N-atoms in the ring, is usually used as an important pharmacophore for the
design of many drugs, such as anticancer, antiviral and antibacterial agents [53], and there are also many
successful examples of replacing five-membered heterocycles with the pyrimidine heterocycle in rational drug
design [54]. In addition, several nonpurine XO inhibitors containing a pyrimidine heterocycle were also reported
[21-24], so it was suggested that the pyrimidine group could be used as a potent pharmacophore to replace the
imidazole ring of imidazole derivatives [16]. Accordingly, we attempted to adopt the pyrimidine ring to replace
the imidazole ring by the bioisosteric replacement strategy and replace the cyano group with the tetrazole group
to design a series of 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid
derivatives (Fig. 3).
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In this paper, we described the synthesis, in vitro bioevaluation and structure-activity relationships of
2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives (8a-8z)
with a tetrazole group targeting the subpocket formed by Leu648, Asn768, Lys771, Leu1014 and Pro1076
residues in the XO active site. Moreover, we determined the inhibitory behavior of the representative compound
8u by using molecular modeling studies, steady-state kinetic analysis and a hyperuricemic rat model, as well as
its acute oral toxicity.
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Fig. 3. Design strategy.
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2. Results and discussion
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2.1. Chemistry
The synthesis of the 2-[4-alkoxy-3-(1H-tetrazol-1-yl)phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic
acids 8a-8y and the 2-[4-hydroxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1, 6-dihydropyrimidine-5-carboxylic acid
8z reported in this study are described in Scheme 1. The commercially available 4-hydroxybenzonitrile (1) was
nitrated with nitric acid in acetic acid to provide 4-hydroxy-3-nitrobenzonitrile (2) at a good yield, which was
then reduced under
a
hydrogen atmosphere catalyzed by Pd/C in methanol to obtain
3-amino-4-hydroxybenzonitrile (3) with high efficiency. Cyclization of the 3-amino-4-hydroxybenzonitrile (3)
with triethyl orthoformate and sodium azide in acetic acid generated the key intermediate (4) [55] with a
moderate yield, which was further alkylated with the appropriate alkyl halides in the presence of anhydrous
potassium carbonate and potassium iodide in DMF to give excellent yields of 4-alkoxy-3-(1H-tetrazol-1-yl)
benzonitriles 5a-5y. The resulting intermediates 5a-5y were treated with sodium methoxide and anhydrous
methanol to afford the imidates, which following aminolysis with ammonium chloride, gave
4-alkoxy-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride compounds 6a-6y [56] in moderate yields. The
condensation reaction of diethyl ethoxymethylenemalonate with 4-alkoxy-3-(1H-tetrazol-1-yl) benzimidamide
hydrochloride compounds 6a-6y in the presence of sodium ethoxide as an alkaline catalyst provided ethyl
2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylates 7a-7y [57] in good yields,
and this reaction was followed by hydrolysis reactions using an aqueous solution of lithium hydroxide to give
2-[4-alkoxy-3-(1H-tetrazol-1-yl)phenyl]-6-oxo-1,6-dihydropyrimidine-5- carboxylic acids 8a-8y.
The 2-[4-hydroxy-3-(1H-tetrazol-1-yl)phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid 8z was
obtained by removing the benzyl protecting group of the compound 8i under a hydrogen atmosphere at room
temperature catalyzed by Pd/C in DMF.
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The structures were elucidated by HRMS, IR, H NMR and C NMR spectra. All spectral data were in
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accordance with the assumed structures. HRMS analysis revealed the target compounds with [M-H] ion peaks.
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The IR spectra of the target compounds displayed hydroxyl stretching vibrations at 3402.8-3470.2 cm (8a-8y)
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and a phenolic hydroxyl stretching vibration at 3561.9 cm (8z). In H NMR spectra, the CH of the tetrazole
group was observed as a singlet at approximately 9.80 ppm in the series 8a-8z.
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Scheme 1. Reagents and conditions: i. HNO , HAc, 75°C; ii. Pd/C, H , MeOH, 25°C; iii. NaN , triethyl
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orthoformate, HAc, 60°C; iv. R Cl or R Br, K CO , KI, DMF, 50-65°C; v. MeONa, MeOH, 25°C, 36 h, then
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NH Cl, 50°C; vi. diethyl ethoxymethylenemalonate, NaH, EtOH, 25°C; and vii. LiOH, H O, THF, 50°C.
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2.2. Biological activity in vitro
The in vitro bovine XO inhibitory activity of compounds 8a-8z was measured spectrophotometrically by
determining uric acid production at 295 nm. Febuxostat and allopurinol were included as the positive controls.
The testing results are shown in Table 1. All compounds exhibited excellent inhibitory potency with IC values
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ranging from 0.0288 µM to 0.629 µM. In particular, compound 8u with a 3-chlorobenzyloxy group substituted
at the 4′-position emerged as the most potent XO inhibitor (IC50 = 0.0288 µM), which was comparable to
febuxostat (IC = 0.0236 µM) and had an IC value 264-fold higher than that of allopurinol (IC = 7.590 µM).
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Table 1
In vitro XO inhibitory potency of designed compounds
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Compound
R
IC50 (µM)
Compound
R
IC50 (µM)
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8
a
0.0920±0.00428
0.0737±0.00172
0.0644±0.00279
0.0541±0.00236
0.0437±0.00105
0.0569±0.000486
0.0692±0.00406
0.0500±0.00326
0.0461±0.00298
0.0585±0.0000781
0.0683±0.00339
8o
8p
8q
8r
8s
0.0507±0.00208
0.0531±0.00394
0.0691±0.00443
0.0552±0.00193
0.0516±0.00319
0.0477±0.00187
0.0288±0.00239
0.0450±0.00131
0.0917±0.00357
0.0639±0.00305
0.0838±0.00657
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0.0945±0.00295
8z
0.629±0.0374
0.0236±0.00230
7.590±0.215
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m
0.0894±0.00745
0.149±0.0160
Febuxostat
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n
Allopurinol
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a. All values are expressed as the mean ± standard error of the mean of triplicate determinations.
Among the tested compounds, increasing the size of the substituent at the 4′-position of the phenyl moiety
from a methoxy to an isopentyloxy group could steadily improve the XO inhibitory potency (8a < 8b < 8c < 8d,
IC50 = 0.0920 µM, 0.0737 µM, 0.0646 µM and 0.0541 µM, respectively). This finding indicated that increasing
the size of saturated alkoxy groups at the 4′-position of the phenyl moiety could favor the XO inhibitory potency,
which may be due to the improved hydrophobic interactions with nonpolar residues at the entrance of the pocket
[22], and similar inhibitory behavior could also be observed for selenazole derivatives [15]. The replacement of
a methoxy group with an allyloxy group at the 4′-position of the phenyl moiety led to approximately a 2-fold
increase in the inhibitory potency (8e vs 8a, IC50 = 0.0437 µM and 0.0920 µM, respectively), and when the
methyl substituent was introduced into the double bond of the allyloxy group (8e), the inhibitory potency
slightly decreased (8e > 8f > 8g, IC50 = 0.0437 µM, 0.0569 µM and 0.0692 µM, respectively). Meanwhile, a
decrease in the inhibitory potency was also observed by increasing the size of cycloalkoxy groups, such as
cyclopropylmethoxy, cyclopentyloxy and cyclohexylmethoxy groups (8i > 8j > 8k, IC50 = 0.0461 µM, 0.0585
µM and 0.0683 µM, respectively), implying that increasing the size of the olefinoxy groups or the cycloalkoxy
groups at the 4′-position of the phenyl moiety could damage the XO inhibitory potency, which may be due to the
steric hindrance of the substituents with the amino acids at the entrance of the active pocket. Moreover, the
introduction of the propargyloxy group at the 4′-position did not translate into improvement in the XO inhibitory
potency (8h vs 8e, IC = 0.0500 µM and 0.0437 µM, respectively).
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Among the benzyloxy containing compounds, the insertion of the electron donating or electron withdrawing
substituents, such as fluoro, chloro, bromo and methoxy substituents, at para position of the benzyloxy group
led to a 1.3-1.8-fold increase in the inhibitory potency compared with the unsubstituted benzyloxy derivative (8o,
8p, 8q, 8r vs 8l, IC50 = 0.0507 µM, 0.0531 µM, 0.0691 µM, 0.0552 µM and 0.0945 µM, respectively). This
finding demonstrated that modulation of the electron density of the benzyloxy aromatic ring could benefit the
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inhibitory potency through introducing fluoro, chloro, bromo and methoxy substituents into the para position of
the benzyloxy group, except compounds with a methyl or tert-butyl group at the para position of the benzyloxy
group (8m and 8n, IC50 =0.0894 µM and 0.149 µM, respectively). Then, the compounds with fluoro, chloro,
bromo and methoxy groups at the meta position of the benzyloxy group were prepared (8t, 8u and 8t, IC50
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0.0477 µM, 0.0288 µM and 0.0450 µM, respectively). Interestingly, the chloro substitution at the meta position,
as in the case of compound 8u, showed a remarkable inhibitory potency (IC50 = 0.0288 µM), 2.4-fold higher
than that of compound 8p with chloro substitution at the para position (IC50 = 0.0691 µM), which indicated that
a chlorine atom substituted at the meta position of the benzyloxy group was beneficial for increasing the
inhibitory potency. To further examine the influence of the positions and number of chlorine atoms on the
benzyloxy group, the corresponding ortho-monochloro, ortho and para-dichloro and ortho-dichloro substituted
derivatives were synthesized (8w, 8x and 8y, IC50 = 0.0917 µM, 0.0639 µM and 0.0838 µM, respectively).
Among them, the ortho substituted derivative 8w showed a 3.2-fold decrease in inhibitory potency in
comparison to compound 8u (8w < 8u, IC50 = 0.0917 µM and 0.0288 µM, respectively), and the dichloro
substituted derivatives also had a slight decrease in the inhibitory potency (8x, 8y < 8u, IC = 0.0639 µM,
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0.0838 µM and 0.0288 µM, respectively). Presumably, the chloro monosubstitution at the meta position kept the
chlorobenzyloxy moiety in a more favorable position so that it could form better hydrophobic interactions with
nonpolar residues at the entrance of the XO active pocket.
Compound 8z has a polar hydroxy group substituted at the 4′-position, and it showed an IC value of 629
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µM, which was 4.2-21.8-fold weaker than those of other compounds (8a-8y) with hydrophobic alkoxy groups
substituted at the 4′-position. In addition, the hydrophobic group at the 4′-position was indispensable for the
inhibitory potency in vitro against XO, and this finding was consistent with the conclusion of the isocytosine
derivatives [22].
2.3. Molecular modeling
To explore a probable interaction model of inhibitors and the XO active site, molecular docking of the
compound 8u in the substrate binding pocket of XO was performed using the Glide XP docking protocol (2016,
Schrodinger Suite). Since molybdenum-pterion sites of both XO and bovine xanthine dehydrogenase (XDH) are
structurally equivalent [58], the X-ray crystal structure of the XDH/febuxostat complex (PDB code 1N5X) used
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in the docking studies was obtained from the RCSB Protein Data Bank (PDB) [16]. The protein was prepared by
removing all water molecules and adding all hydrogen atoms using Protein Preparation Wizard (2016,
Schrodinger Suite). The carboxyl groups of compound 8u and febuxostat were calculated in dissociated forms
using the LIGPREP module (2016, Schrodinger Suite).
The binding model of the representative compound 8u was illustrated by Discovery Studio Visualizer 2017
and Pymol (Fig. 4). According to our docking studies, the binding site residues and overall binding mode of 8u
were similar to that observed with febuxostat (Fig. 4a, Fig. 4b), and the results showed that the tetrazole moiety
of the phenyl unit was able to be accommodated by the subpocket formed by Leu648, Asn768, Lys771, Leu1014
and Pro1076, which further interacted with Asn768 and Lys771 through two hydrogen bonds, as expected (Fig.
4c). In the deepest part of the channel, the carbonyl group of the pyrimidine engaged in two hydrogen bonds
with the side chain hydroxy group of Thr1010 and the backbone amino group of Thr1010 and two additional
hydrogen bonds with the guanidine group of Arg880. Moreover, the carboxyl group of the pyrimidine formed an
electrostatic interaction and a hydrogen bond with Arg880 and two additional hydrogen bonds with Mo-OH. The
pyrimidine ring as a whole was sandwiched between Phe914 and Phe1009 via “face-to-face” and “face-to-edge”
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π
-
π
stacking interactions, respectively. In addition, the N-3 atom of the pyrimidine acted as a H-bond acceptor
and linked to the amino acid residue Glu802 via a hydrogen bond. Furthermore, several hydrophobic
interactions, including a strong interaction between Phe649 and the 3-chlorobenzyl group of the phenyl unit,
π-π
were also observed at the mouth of the pocket with Leu648, Phe649, Leu873, Val1011 and Leu1014 (Fig. 4c).
As designed, the carbonyl group, which exercised a role similar to the carboxyl group of febuxostat, was
able to interact simultaneously with Thr1010 and Arg880. Meanwhile, the carboxyl group was closer to the deep
part of the active pocket, still retaining the key interactions with Arg880 and surprisingly increasing two extra
hydrogen bonds with Mos3004.
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Fig. 4. Binding modes of 8u and febuxostat within the XO binding pocket. (a) Protein (PDB code 1N5X,
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rainbow) is shown as a cartoon, and small molecules are shown as a line. The subpocket of the binding pocket is
shown as a surface colored in gray. 8u (cyan) and febuxostat (orange) occupy the same binding site in XO. (b)
XO residues interacting with febuxostat are depicted by gray lines. Hydrogen bonds of febuxostat are shown as
black dashed lines. (c) XO residues interacting with 8u (cyan) are depicted by gray lines. Hydrogen bonds,
electrostatic interactions and π-π stacking interactions of 8u are shown as green, orange and purple dashed lines,
respectively.
2.4. Steady-state kinetic analysis
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To determine the action mode of compounds 8a-8z with XO, enzyme kinetics studies of the representative
compound 8u were performed (Fig. 5). As show in Fig.5, the analysis on the data of compound 8u indicated that
Vmax decreased with changing slope (K /Vmax) in the presence of increasing concentrations of inhibitor, wherein,
m
the K and V values in the presence of 0.0133 to 0.106 µM compound 8u were 11.48, 24.63, 44.17, 74.54 µM
m
max
and 0.151, 0.141, 0.124, 0.0961 µM /min, while, for the positive control, the K and V values were 5.33 µM
m
max
and 0.155 µM /min, respectively. This behavior showed that 8u acted as a mixed-type mode inhibitor with
respect to xanthine for binding to XO, which was similar to febuxostat. Moreover, it was found that the
intersecting lines on the graph converge to the second quadrant, which indicated that the value of α (a constant
that defines the degree to which inhibitor binding affects the affinity of the enzyme for the substrate) was greater
than 1 [6,8]. This confirmed that the inhibitor preferentially bound to the free enzyme and not the enzyme–
substrate complex. In addition, dose-dependent inhibition of XO by 8u was exhibited (Fig. 6).
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Fig. 5. Lineweaver-Burk plots of XO inhibition by compound 8u.
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Fig. 6. The inhibition of XO by compound 8u. Values are means ± SDs, n = 3.
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2.5. Acute oral toxicity study
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To explore the preliminary toxicity profile of the most potent compound 8u, the acute oral toxicity study
was performed at dose up to 2000 mg/kg, which was about 400 times over the effective dose of 5mg/kg,
according to OECD guidelines 2001 [59]. No signs and symptoms of toxicity and mortality were observed
within 24 h after the administration of test compound.
2.6. Hypouricemic effect in vivo
Since the compound 8u showed more potent inhibitory activity than all other compounds tested in vitro, its
in vivo hypouricemic effect in the acute hyperuricemia rat model was investigated and compared with those of
febuxostat and allopurinol (Fig. 7). As expected, an intraperitoneal injection of 300 mg/kg potassium oxonate
markedly increased serum uric acid levels 3 h after drug administration in the model group compared with the
blank group (P < 0.001 for Blank vs Model), confirming that the model was successfully established. In
addition, administration of a single oral dose of 5 mg/kg 8u was able to significantly reduce the serum levels of
uric acid at 3 h (P < 0.05 for 8u vs Model), although the hypouricemic action was slightly lower than that of
febuxostat and allopurinol at an oral dose of 5 mg/kg and 10 mg/kg, respectively. Furthermore, the compound 8u
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①
was able to reduce the serum uric acid levels comparable to the blank group from 3 h to 8 h ( P > 0.05 for 8u vs
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Blank). The results of in vivo hypouricemic activity evaluation suggested that compound 8u was a potential and
1
efficacious agent in the treatment of hyperuricemia.
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Fig. 7. Effect of compound 8u, febuxostat and allopurinol on the serum uric acid levels in the potassium
oxonate-induced hyperuricemic rat model. (a) Time course changes in the serum uric acid levels after oral
administration of Febuxostat, Allopurinol and 8u in a potassium oxonate induced hyperuricemic rat. (b) The
serum uric acid levels 3 h after oral administration of Febuxostat, Allopurinol and 8u in a potassium oxonate
induced hyperuricemic rat. (c) The AUC (uric acid, 3-8 h) after oral administration of 8u, febuxostat and allopurinol in
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a potassium oxonate induced hyperuricemic rat. Data are expressed as the mean ± S.D. *P < 0.05, *P < 0.001
①
and ****P < 0.0001 vs hyperuricemic rat (model), P>0.05 vs Blank.
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3. Conclusion
We
designed, synthesized
and identified
a
series of 2-[4-alkoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives as novel XO inhibitors with a tetrazole
group targeting the subpocket formed by residues Leu648, Asn768, Lys771, Leu1014 and Pro1076 at the XO
active site. Specifically, compound 8u, which was comparable to febuxostat, emerged as the most potent XO
inhibitor. The Lineweaver-Burk plot showed that compound 8u acted as a mixed-type XO inhibitor. The
structure-activity relationship analysis demonstrated that the hydrophobic group at the 4′-position was
indispensable for the inhibitory potency in vitro against XO. Furthermore, molecular docking studies provided
the molecular basis for rationalizing the activity of the designed compounds and suggested that the subpocket
centered around Asn768 was able to accommodate the tetrazole group, which further provided a potential
strategy for the design of nonpurine XO inhibitors. The results of in vivo hypouricemic activity evaluation
suggested that compound 8u could effectively reduce serum uric acid levels at an oral dose of 5 mg/kg. In
addition, acute oral toxicity study in mice indicated that compound 8u was nontoxic and tolerated at dose up to
2000 mg/kg. Thus, compound 8u could be a potential and efficacious agent in treatment of hyperuricemia with
low toxicity.
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4. Experimental protocols
4.1 Chemistry
Unless otherwise indicated, reagents and solvents were purchased from commercial sources and used
without further purification. All reactions were monitored by TLC using silica gel aluminum cards (0.2 mm
thickness) with 254 nm and 365 nm fluorescent indicator. Melting points were recorded on a YRT-3 melting
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apparatus and were uncorrected. H NMR spectra were recorded on a Bruker 400 MHz spectrometer or a Bruker
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600 MHz spectrometer, and C NMR spectra were recorded on a Bruker 400 MHz spectrometer or a Bruker
600 MHz spectrometer. Chemical shifts were expressed in parts per million using tetramethylsilane as an
internal reference and DMSO-d as the solvent. IR spectra were determined as KBr pellets on a Bruker IFS-55
6
spectrometer and expressed in reciprocal centimeters. ESI-MS data were gathered using an Agilent 1100
instrument. ESI-HRMS data were recorded in the Agilent 6540 Series Q-TOF-MS system.
4.1.1. Synthesis of 4-hydroxy-3-nitrobenzonitrile (2)
A solution of nitric acid (24.4 g, 0.252 mol) in acetic acid (80 mL) was added dropwise at 75 °C to a stirred
solution of 4-hydroxybenzonitrile (30.0 g, 0.252 mol) in acetic acid (200 mL). Upon completion of the addition,
the mixture was heated under reflux for anther 1 h, then it was poured into ice water, and the precipitate was
filtered, washed with water to yield the compound 2 (39.0 g, 94.2%) as a yellow solid, mp 144.2°C-145.6°C.
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MS (ESI) m/z: 163.0 [M - H] ; H NMR (600 MHz, DMSO-d ) δ 12.34 (s, 1H), 8.42 (d, J = 2.1 Hz, 1H), 7.94
6
(dd, J = 8.7, 2.1 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H).
4.1.2. Synthesis of 3-amino-4-hydroxybenzonitrile (3)
A mixture of the compound 2 (39.0 g, 0.238 mol) and 10% Pd/C (3.9 g) in methanol was stirred at room
temperature for 12 h under hydrogen atmosphere. After the completion of the reaction, the Pd/C was filtered out
and the filtrate was evaporated to give a brown solid (29.5 g, 92.3%), which was used directly in the next step.
+
1
Mp 135.6 °C-136.6 °C. MS (ESI) m/z: 480.8 [2M + Na] ; H NMR (600 MHz, DMSO-d ) δ 9.79 (s, 1H), 8.26
6
(d, J = 2.1 Hz, 1H), 8.07 (dd, J = 8.8, 2.1 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 4.92 (hept, J = 6.0 Hz, 1H), 1.27 (d,
J = 6.0 Hz, 6H).
4.1.3. Synthesis of 4-hydroxy-3-(1H-tetrazol-1-yl) benzonitrile (4)
A mixture of 4-hydroxy-3-nitro benzonitrile (29.5 g, 0.220 mol), triethyl orthoformate (39.1 g, 0.264 mol)
and sodium azide (14.4 g, 0.222 mol) was added to acetic acid (88.5 mL). The mixture was stirred at 60 °C for
12 h under nitrogen atmosphere. After completion of the reaction, the precipitate was filtered and recrystallized
from ethanol to give the compound 4 (21.53 g, 52.3%) as a gray white solid, mp 194.8°C-195.0°C. MS (ESI)
-
1
m/z: 185.9 [M - H] ; H NMR (600 MHz, DMSO-d ) δ 12.15 (s, 1H), 9.83 (s, 1H), 8.20 (d, J = 2.1 Hz, 1H), 7.89
6
(dd, J = 8.6, 2.1 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H).
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4.1.4. Synthesis of 4-methoxy-3-(1H-tetrazol-1-yl) benzonitrile (5a)
A mixture of the compound 4 (6 g, 32.06 mmol), methyl iodide (5.46 g, 38.47 mmol), anhydrous potassium
carbonate (8.85 g, 42.7 mmol) and DMF (32 mL) was reacted at ambient temperature for 6 h under nitrogen
atmosphere. After the reaction was completed, the mixture was poured into water (200 mL). The precipitate was
filtered, washed with water, and recrystallized (petroleum ether : ethyl acetate = 1:2) to yield the compound 5a
+
+ 1
(3.65 g, 39.1%) as a white solid, mp 172.3 °C-174.1 °C . MS (ESI) m/z: 202.4 [M + H] ; 224.4 [M + Na] ; H
NMR (600 MHz, DMSO-d ) δ 9.85 (s, 1H), 8.28 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.56 (d, J =
6
8.8 Hz, 1H), 3.96 (s, 3H).
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4.1.5. General procedure for synthesis of 4-alkoxy-3-(1H-tetrazol-1-yl) benzonitriles (5b-5y)
A mixture of compound 4 (6 g, 32.06 mmol), alkyl halides or benzyl halides (38.47 mmol), anhydrous
potassium carbonate (8.85 g, 42.7 mmol), potassium iodide (710.48 mg, 4.28 mmol) and DMF (32 mL) was
reacted at 50°C for 8 h under nitrogen atmosphere. After the reaction was completed, the mixture was poured
into water (200 mL). The precipitate was filtered, washed with water, and recrystallized (petroleum ether: ethyl
acetate = 1:2) to yield 4-alkoxy-3-(1H-tetrazol-1-yl) benzonitriles (5b-5y).
4.1.5.1. Synthesis of 4-isopropoxy-3-(1H-tetrazol-1-yl) benzonitrile (5b)
+
1
A white solid, yield: 66.5%. Mp 135.6 °C-136.6 °C. MS (ESI) m/z: 480.8 [2M + Na] ; H NMR (600 MHz,
DMSO-d ) δ 9.79 (s, 1H), 8.26 (d, J = 2.1 Hz, 1H), 8.07 (dd, J = 8.8, 2.1 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 4.92
6
(hept, J = 6.0 Hz, 1H), 1.27 (d, J = 6.0 Hz, 6H).
4.1.5.2. Synthesis of 4-isobutoxy-3-(1H-tetrazol-1-yl) benzonitrile (5c)
+
+
A white solid, yield: 69.3%. Mp 139.2 °C-140.6 °C. MS (ESI) m/z: 244.4 [M + H] ; 266.3 [M + Na] ; 508.9
+
1
[2M + Na] ; H NMR (600 MHz, DMSO-d ) δ 9.79 (s, 1H), 8.28 (d, J = 2.1 Hz, 1H), 8.10 (dd, J = 8.8, 2.1 Hz,
6
1H), 7.54 (d, J = 8.8 Hz, 1H), 3.98 (d, J = 6.3 Hz, 2H), 1.95 (hept, J = 6.6 Hz, 1H), 0.84 (d, J = 6.7 Hz, 6H).
4.1.5.3. Synthesis of 4-(isopentyloxy)-3-(1H-tetrazol-1-yl) benzonitrile (5d)
+
+
A white solid, yield: 81.9%. Mp 108.7 °C-190 °C. MS (ESI) m/z: 258.3 [M + H] ; 280.2 [M + Na] ; 536.8
+
1
[2M + Na] ; H NMR (600 MHz, DMSO-d ) δ 9.79 (s, 1H), 8.27 (d, J = 2.1 Hz, 1H), 8.10 (dd, J = 8.8, 2.1 Hz,
6
1H), 7.57 (d, J = 8.8 Hz, 1H), 4.22 (t, J = 6.3 Hz, 2H), 1.57 (dq, J = 12.2, 6.0 Hz, 3H), 0.83 (d, J = 6.3 Hz, 6H).
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4.1.5.4. Synthesis of 4-(allyloxy)-3-(1H-tetrazol-1-yl) benzonitrile (5e)
+
+
A white solid, yield: 94.5%. Mp 112.4°C-113.5°C. MS (ESI) m/z: 228.3 [M + H] ; 250.2 [M + Na] ; 476.9
+
1
[2M + Na] ; H NMR (600 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.11 (dd, J = 8.8, 2.0 Hz,
1H), 7.54 (d, J = 8.8 Hz, 1H), 5.98 (ddt, J = 15.8, 10.4, 5.1 Hz, 1H), 5.31 – 5.23 (m, 2H), 4.81 (d, J = 5.1 Hz,
2H).
4.1.5.5. Synthesis of 4-[(2-methylallyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5f)
+
+
A white solid, yield: 85.4%. Mp 115.9°C -117.5°C. MS (ESI) m/z : 242.3 [M + H] ; 264.2 [M + Na] ; 504.8
+
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[2M + Na] ; H NMR (600 MHz, DMSO-d ) δ 9.81 (s, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.11 (dd, J = 8.8, 2.0 Hz,
6
1H), 7.53 (d, J = 8.8 Hz, 1H), 4.92 (d, J = 19.0 Hz, 2H), 4.71 (s, 2H), 1.65 (s, 3H).
4.1.5.6. Synthesis of 4-[(3-methylbut-2-en-1-yl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5g)
+
+
A white solid, yield: 70.2%. Mp 126.3°C-127.2°C. MS (ESI) m/z: 256.2 [M + H] ; 278.2 [M + Na] ; 532.8
+
1
[2M + Na] ; H NMR (600 MHz, DMSO-d ) δ 9.79 (s, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.09 (dd, J = 8.8, 2.0 Hz,
6
1H), 7.55 (d, J = 8.8 Hz, 1H), 5.37 (t, J = 6.6 Hz, 1H), 4.78 (d, J = 6.7 Hz, 2H), 1.69 (d, J = 23.2 Hz, 6H).
4.1.5.7. Synthesis of 4-(prop-2-yn-1-yloxy)-3-(1H-tetrazol-1-yl) benzonitrile (5h)
1
A brown solid, yield: 82.4%. Mp 138.4°C-138.9°C. H NMR (600 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.37 –
8.27 (m, 1H), 8.21 – 8.12 (m, 1H), 7.61 (d, J = 8.8 Hz, 1H), 5.15 – 5.03 (m, 2H), 3.74 (d, J = 2.6 Hz, 1H).
4.1.5.8. Synthesis of 4-(cyclopropylmethoxy)-3-(1H-tetrazol-1-yl) benzonitrile (5i)
1
A brown solid, yield: 91.3%. Mp 139.1°C-140.2°C. H NMR (600 MHz, DMSO-d ) δ 9.81 (s, 1H), 8.28 (d,
6
J = 2.1 Hz, 1H), 8.07 (dd, J = 8.7, 2.1 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 4.09 (d, J = 7.1 Hz, 2H), 1.20 (dddd, J
= 15.1, 10.3, 5.3, 2.4 Hz, 1H), 0.56 – 0.49 (m, 2H), 0.34 – 0.27 (m, 2H).
4.1.5.9. Synthesis of 4-(cyclopentyloxy)-3-(1H-tetrazol-1-yl) benzonitrile (5J)
+
+
1
A white solid, yield: 64.3%. Mp 148°C-149.1°C. MS (ESI) m/z: 256.3 [M + H] ; 278.2 [M + Na] ; H
NMR (600 MHz, DMSO-d ) δ 9.75 (s, 1H), 8.26 (d, J = 2.1 Hz, 1H), 8.07 (dd, J = 8.8, 2.1 Hz, 1H), 7.54 (d, J =
6
8.8 Hz, 1H), 5.11 (tt, J = 5.6, 2.5 Hz, 1H), 1.95 – 1.82 (m, 2H), 1.71 – 1.62 (m, 2H), 1.59 – 1.47 (m, 4H).
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4.1.5.10. Synthesis of 4-(cyclohexylmethoxy)-3-(1H-tetrazol-1-yl) benzonitrile (5k)
1
A white solid, yield: 63.2%. Mp 120.6°C-122.2°C. H NMR (600 MHz, DMSO-d ) δ 9.78 (s, 1H), 8.27 (d, J
6
= 2.1 Hz, 1H), 8.09 (dd, J = 8.8, 2.1 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 4.01 (d, J = 6.2 Hz, 2H), 1.71 – 1.56 (m,
6H), 1.17 (qt, J = 12.3, 3.2 Hz, 2H), 1.07 (qt, J = 12.6, 3.2 Hz, 1H), 0.91 (qd, J = 12.4, 3.4 Hz, 2H).
4.1.5.11. Synthesis of 4-(benzyloxy)-3-(1H-tetrazol-1-yl) benzonitrile (5l)
+
1
A white solid, yield: 66.6%. Mp 148.3°C-149.0°C. MS (ESI) m/z: 576.8 [2M + Na] ; H NMR (600 MHz,
DMSO-d ) δ 9.83 (s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.41
6
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– 7.31 (m, 6H), 5.36 (s, 2H).
4.1.5.12. Synthesis of 4-[(4-methylbenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5m)
+
1
An off-white solid, yield: 96.5%. Mp 161.2°C-162.5°C. MS (ESI) m/z : 604.6 [2M + Na] ; H NMR (600
MHz, DMSO-d ) δ 9.80 (s, 1H), 8.29 (d, J = 2.1 Hz, 1H), 8.11 (dd, J = 8.8, 2.1 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H),
6
7.26 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 7.8 Hz, 2H), 5.29 (s, 2H), 2.28 (s, 3H).
4.1.5.13. Synthesis of 4-{[4-(tert-butyl) benzyl] oxy}-3-(1H-tetrazol-1-yl) benzonitrile (5n)
+
1
An off-white solid, yield: 80.2%. Mp 125.6 °C-128.4 °C. MS (ESI) m/z: 356.4 [M + Na] ; H NMR (600
MHz, DMSO-d ) δ 9.84 (s, 1H), 8.30 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H),
6
7.42 – 7.37 (m, 2H), 7.33 – 7.26 (m, 2H), 5.32 (s, 2H), 1.26 (s, 9H).
4.1.5.14. Synthesis of 4-[(4-methoxybenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5o)
+
1
A white solid, yield: 68.4%. Mp 148.9°C-150.2°C. MS (ESI) m/z: 330.2 [M + Na] ; H NMR (600 MHz,
DMSO-d ) δ 9.79 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.33
6
(d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 5.26 (s, 2H), 3.74 (s, 3H).
4.1.5.15. Synthesis of 4-[(4-fluorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5p)
+
+
1
A white solid, yield: 52.7%. Mp 160.7°C-161.6°C. MS (ESI) m/z: 296.4 [M + H] ; 318.2 [M + Na] ; H
NMR (600 MHz, DMSO-d ) δ 9.83 (s, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.12 (dd, J = 8.8, 2.0 Hz, 1H), 7.63 (d, J =
6
8.8 Hz, 1H), 7.45 (dd, J = 8.4, 5.6 Hz, 2H), 7.21 (t, J = 8.8 Hz, 2H), 5.34 (s, 2H).
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4.1.5.16. Synthesis of 4-[(4-chlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5q)
1
A white solid, yield: 61.1%. Mp 195.9°C-197°C. H NMR (600 MHz, DMSO-d ) δ 9.84 (s, 1H), 8.31 (d, J =
6
2.0 Hz, 1H), 8.12 (dd, J = 8.8, 2.0 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz,
2H), 5.35 (s, 2H).
4.1.5.17. Synthesis of 4-[(4-bromobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5r)
+
+ 1
A white solid, yield: 59.4%. Mp 181.3°C-183.6°C. MS (ESI) m/z: 373.1 [M + H] ; 395.2 [M + Na] ; H
NMR (600 MHz, DMSO-d ) δ 9.84 (s, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.12 (dd, J = 8.8, 1.8 Hz, 1H), 7.61 (d, J =
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8.8 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 5.34 (s, 2H).
4.1.5.18. Synthesis of 4-[(3-methoxybenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5s)
+
+ 1
A white solid, yield: 85.6%. Mp 134.4°C-135.6°C .MS (ESI) m/z: 308.3 [M + H] ; 330.2 [M + Na] ; H
NMR (400 MHz, DMSO-d ) δ 9.85 (s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.12 (dd, J = 8.8, 2.1 Hz, 1H), 7.61 (d, J =
6
8.8 Hz, 1H), 7.28 (t, 1H), 6.97 – 6.85 (m, 3H), 5.33 (s, 2H), 3.73 (s, 3H).
4.1.5.19. Synthesis of 4-[(3-fluorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5t)
+
+
An off-white solid, yield: 87.0%. Mp 142.6°C -144.7°C .MS (ESI) m/z: 296.3 [M + H] ; 318.2 [M + Na] ;
1
H NMR (400 MHz, DMSO-d ) δ 9.87 (s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 8.13 (dd, J = 8.8, 2.2 Hz, 1H), 7.61 (d,
6
J = 8.8 Hz, 1H), 7.49 – 7.38 (m, 1H), 7.24 – 7.19 (m, 2H), 7.19 – 7.13 (m, 1H), 5.38 (s, 2H).
4.1.5.20. Synthesis of 4-[(3-chlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5u)
1
An off-white solid, yield: 54.3%. Mp 157.2°C-158.5°C. H NMR (600 MHz, DMSO-d ) δ 9.88 (s, 1H), 8.32
6
(d, J = 2.1 Hz, 1H), 8.14 (dd, J = 8.8, 2.1 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.44 – 7.38
(m, 2H), 7.34 (dd, J = 5.0, 3.4 Hz, 1H), 5.37 (s, 2H).
4.1.5.21. Synthesis of 4-[(3-bromobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5v)
+
1
An off-white solid, yield: 66.5%. Mp 161.9 °C -162.2 °C. MS (ESI) m/z: 378.2 [M + Na] ; H NMR (600
MHz, DMSO-d ) δ 9.87 (s, 1H), 8.32 (d, J = 2.1 Hz, 1H), 8.14 (dd, J = 8.8, 2.1 Hz, 1H), 7.64 – 7.57 (m, 2H),
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7.54 (dt, J = 7.5, 1.8 Hz, 1H), 7.40 – 7.31 (m, 2H), 5.36 (s, 2H).
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4.1.5.22. Synthesis of 4-[(2-chlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5w)
+
1
A white solid, yield: 63.5%. Mp 149.5°C-151.7°C. MS (ESI) m/z: 644.8 [2M + Na] ; H NMR (400 MHz,
DMSO-d ) δ 9.75 (s, 1H), 8.32 (d, J = 2.1 Hz, 1H), 8.15 (dd, J = 8.7, 2.1 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.50
6
(dd, J = 7.7, 1.6 Hz, 1H), 7.45 (dd, J = 7.1, 2.2 Hz, 1H), 7.43 – 7.32 (m, 2H), 5.41 (s, 2H).
4.1.5.23. Synthesis of 4-[(2, 6-dichlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5x)
+
1
A white solid, yield: 83.8%. Mp 178.0°C-178.9°C. MS (ESI) m/z: 368.4 [M + Na] ; H NMR (400 MHz,
DMSO-d ) δ 9.57 (s, 1H), 8.32 (d, J = 2.1 Hz, 1H), 8.20 (dd, J = 8.7, 2.1 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.56
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– 7.50 (m, 2H), 7.48 – 7.42 (m, 1H), 5.48 (s, 2H).
4.1.5.24. Synthesis of 4-[(2, 4-dichlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzonitrile (5y)
1
A white solid, yield: 92.2%. Mp 176.7°C-177.3°C. H NMR (400 MHz, DMSO-d ) δ 9.75 (s, 1H), 8.32 (d, J
6
= 2.1 Hz, 1H), 8.14 (dd, J = 8.8, 2.1 Hz, 1H), 7.76 – 7.63 (m, 2H), 7.54 – 7.41 (m, 2H), 5.40 (s, 2H).
4.1.6. General procedure for synthesis of 4-alkoxy-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6a-6y)
A 500 mL flask was charged with 250 mL of anhydrous methanol, 17.40 mmol of the compounds 5a-5y, and
5.22 mmol of sodium methoxide. The complex was protected from moisture and stirred for 36 h. Then, 34.8
mmol NH Cl was added and stirring was continued at 50°C for 6 h. Unreacted NH Cl was filtered, and the
4
4
reaction mixture was concentrated under reduced pressure. The crude residue was refluxed with ethyl acetate
and the precipitate was collected by filtration to give the compounds 6a-6y, which were used for the next
reaction without further purification.
4.1.6.1. Synthesis of 4-methoxy-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6a)
+
1
A white solid, yield: 31.8%. MS (ESI) m/z: 218.2 [M + H] ; H NMR (600 MHz, DMSO-d ) δ 9.96 (s, 1H),
6
9.22 (s, 3H), 8.35 (d, J = 2.4 Hz, 1H), 8.24 (dd, J = 8.9, 2.5 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 4.01 (s, 3H).
4.1.6.2. Synthesis of 4-isopropoxy-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6b)
+
+ 1
A white solid, yield: 24.0%. MS (ESI) m/z: 247.2 [2M + H] ; 269.1 [2M + Na] H NMR (600 MHz,
DMSO-d ) δ 9.85 (s, 1H), 9.26 (s, 4H), 8.25 (d, J = 2.4 Hz, 1H), 8.08 (dd, J = 8.9, 2.5 Hz, 1H), 7.65 (d, J = 9.0
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Hz, 1H), 4.96 (hept, J = 6.1 Hz, 1H), 1.30 (d, J = 6.0 Hz, 6H).
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4.1.6.3. Synthesis of 4-isobutoxy-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6c)
+
1
A white solid, yield: 61.6%. MS (ESI) m/z: 261.3 [M + H] ; H NMR (600 MHz, DMSO-d ) δ 9.87 (s, 1H),
6
8.89 (s, 3H), 8.30 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.8, 2.5 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 4.01 (d, J = 6.4
Hz, 2H), 1.97 (dp, J = 13.2, 6.6 Hz, 1H), 0.86 (d, J = 6.7 Hz, 6H).
4.1.6.4. Synthesis of 4-(isopentyloxy)-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6d)
+
1
A white solid, yield: 34.5%. MS (ESI) m/z: 275.2 [M + H] ; H NMR (600 MHz, DMSO-d ) δ 9.86 (s, 1H),
6
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1
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9.14 (s, 4H), 8.28 (d, J = 2.4 Hz, 1H), 8.16 (dd, J = 8.9, 2.4 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 4.25 (t, J = 6.3 Hz,
2H), 1.59 (dq, J = 12.1, 6.1, 5.6 Hz, 3H), 0.85 (d, J = 6.3 Hz, 6H).
4.1.6.5. Synthesis of 4-(allyloxy)-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6e)
+
+
- 1
A white solid, yield: 36.3%. MS (ESI) m/z: 245.1 [M + H] ; 488.9 [2M + H] ; 243.3 [M - H] ; H NMR
(600 MHz, DMSO-d ) δ 9.94 (s, 1H), 8.63 (s, 6H), 8.33 (s, 1H), 8.20 (d, J = 8.9 Hz, 1H), 7.60 (d, J = 8.8 Hz,
6
1H), 6.00 (ddt, J = 15.6, 9.9, 4.6 Hz, 1H), 5.28 (dd, J = 28.0, 13.9 Hz, 2H), 4.84 (d, J = 3.9 Hz, 2H).
4.1.6.6. Synthesis of 4-[(2-methylallyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6f)
+
+
- 1
A white solid, yield: 33.6%. MS (ESI) m/z: 259.1 [M + H] ; 517.0 [2M + H] ; 257.3 [M - H] ; H NMR
(600 MHz, DMSO-d ) δ 9.91 (s, 1H), 8.32 (d, J = 2.3 Hz, 1H), 8.20 (dd, J = 8.9, 2.4 Hz, 1H), 8.13 – 7.96 (m,
6
10H), 7.59 (d, J = 8.9 Hz, 1H), 4.93 (d, J = 4.0 Hz, 2H), 4.74 (s, 2H), 1.66 (s, 3H).
4.1.6.7. Synthesis of 4-[(3-methylbut-2-en-1-yl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6g)
+
+
1
A white solid, yield: 47.3%. MS (ESI) m/z: 273.2 [M + H] ; 544.9 [2M + H] ; H NMR (600 MHz,
DMSO-d ) δ 9.87 (s, 1H), 9.22 (s, 0H), 8.30 (d, 2H), 8.18 (dd, 2H), 7.61 (d, J = 8.9 Hz, 1H), 5.39 (s, 2H), 4.81
6
(d, J = 6.5 Hz, 3H), 1.70 (d, J = 17.8 Hz, 7H).
4.1.6.8. Synthesis of 4-(prop-2-yn-1-yloxy)-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6h)
+
+ 1
A white solid, yield: 36.7%. MS (ESI) m/z: 243.4 [M + H] ; 265.3 [M + Na] ; H NMR (600 MHz,
DMSO-d ) δ 9.92 (s, 1H), 9.01 (s, 5H), 8.34 (s, 1H), 8.23 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 5.11 (s,
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2H), 3.77 (s, 1H).
4.1.6.9. Synthesis of 4-(cyclopropylmethoxy)-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6i)
+
1
A white solid, yield: 53.7%. MS (ESI) m/z: 259.3 [M + H] ; H NMR (600 MHz, DMSO-d ) δ 9.90 (s, 1H),
6
8.32 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.9, 2.5 Hz, 1H), 7.72 (s, 29H), 7.58 (d, J = 8.9 Hz, 1H), 4.11 (d, J = 7.1
Hz, 2H), 1.27 – 1.16 (m, 1H), 0.57 – 0.48 (m, 2H), 0.35 – 0.28 (m, 2H).
4.1.6.10. Synthesis of 4-(cyclopentyloxy)-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6J)
+
+
1
A white solid, yield: 26.9%. MS (ESI) m/z: 273.2 [M + H] ; 295.2 [M + Na] ; H NMR (600 MHz,
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DMSO-d ) δ 9.83 (s, 1H), 8.40 (s, 1H), 8.29 (d, J = 2.4 Hz, 1H), 8.16 (dd, J = 8.9, 2.4 Hz, 4H), 7.60 (d, J = 8.9
6
Hz, 1H), 5.17 – 5.13 (m, 1H), 1.97 – 1.88 (m, 2H), 1.74 – 1.65 (m, 2H), 1.60 – 1.51 (m, 4H).
4.1.6.11. Synthesis of 4-(cyclohexylmethoxy)-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6k)
+
1
A white solid, yield: 42.5%. MS (ESI) m/z: 301.3 [M + H] ; H NMR (600 MHz, DMSO-d ) δ 9.85 (s, 1H),
6
8.26 (d, J = 2.5 Hz, 1H), 8.14 (dd, J = 9.0, 2.5 Hz, 2H), 7.96 (s, 10H), 7.61 (d, J = 8.9 Hz, 1H), 4.04 (d, J = 6.2
Hz, 2H), 1.73 – 1.57 (m, 6H), 1.25 – 1.13 (m, 2H), 1.12 – 1.03 (m, 1H), 0.99 – 0.89 (m, 2H).
4.1.6.12. Synthesis of 4-(benzyloxy)-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6l)
+
1
A white solid, yield: 46.4%. MS (ESI) m/z: 295.2 [M + H] ; H NMR (600 MHz, DMSO-d ) δ 9.91 (s, 1H),
6
8.86 (s, 4H), 8.29 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 8.9, 2.5 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H), 7.42 – 7.36 (m,
4H), 7.36 – 7.32 (m, 1H), 5.39 (s, 2H).
4.1.6.13. Synthesis of 4-[(4-methylbenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6m)
+
- 1
A white solid, yield: 42.5%. MS (ESI) m/z: 309.4 [M + H] ; 307.3 [M - H] ; H NMR (600 MHz, DMSO-d )
6
δ 9.89 (s, 1H), 8.92 (s, 4H), 8.28 (d, J = 2.4 Hz, 1H), 8.13 (dd, J = 8.9, 2.5 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H),
7.29 (d, J = 7.8 Hz, 2H), 7.19 (d, J = 7.7 Hz, 2H), 5.34 (s, 2H), 2.29 (s, 3H).
4.1.6.14. Synthesis of 4-{[4-(tert-butyl) benzyl] oxy}-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6n)
+
- 1
A white solid, yield: 46.1%. MS (ESI) m/z: 351.3 [M + H] ; 349.4 [M - H] ; H NMR (600 MHz, DMSO-d )
6
δ 9.92 (s, 1H), 8.30 (d, J = 2.4 Hz, 1H), 8.16 (dd, J = 8.9, 2.4 Hz, 1H), 7.70 (d, J = 9.0 Hz, 1H), 7.43 – 7.37 (m,
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4.1.6.15. Synthesis of 4-[(4-methoxybenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6o)
+
- 1
A white solid, yield: 74.6%. MS (ESI) m/z: 325.2 [M + H] ; 323.0 [M - H] ; H NMR (600 MHz, DMSO-d )
6
δ 9.93 (s, 1H), 8.36 (d, J = 2.4 Hz, 1H), 8.24 (dd, J = 8.9, 2.4 Hz, 1H), 8.10 (s, 15H), 7.73 (d, J = 9.0 Hz, 1H),
7.37 (dd, 2H), 6.94 (dd, 2H), 5.32 (s, 2H), 3.75 (s, 3H).
4.1.6.16. Synthesis of 4-[(4-fluorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6p)
+
- 1
A white solid, yield: 38.6%. MS (ESI) m/z: 313.2 [M + H] ; 311.3 [M - H] ; H NMR (600 MHz, DMSO-d )
6
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δ 9.93 (s, 1H), 8.97 (s, 6H), 8.32 (s, 1H), 8.19 (dd, J = 9.0, 2.4 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.48 (dd, J =
8.4, 5.4 Hz, 2H), 7.29 – 7.13 (m, 2H), 5.38 (s, 2H).
4.1.6.17. Synthesis of 4-[(4-chlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6q)
+
- 1
A white solid, yield: 39.0%. MS (ESI) m/z: 329.1 [M + H] ; 327.1 [M - H] ; H NMR (600 MHz, DMSO-d )
6
δ 9.93 (s, 1H), 9.37 (s, 5H), 8.31 (d, J = 2.2 Hz, 1H), 8.16 (dd, J = 9.0, 2.4 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H),
7.45 (dd, J = 2.3 Hz, 4H), 5.39 (s, 2H).
4.1.6.18. Synthesis of 4-[(4-bromobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6r)
+
+
1
A white solid, yield: 34.6%. MS (ESI) m/z: 373.1 [M + H] ; 395.2 [M + Na] ; H NMR (600 MHz,
DMSO-d ) δ 9.94 (s, 1H), 9.47 (s, 4H), 8.32 (s, 1H), 8.17 (d, J = 8.9 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.59 (d, J
6
= 7.9 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 5.38 (s, 2H).
4.1.6.19. Synthesis of 4-[(3-methoxybenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6s)
+
+
- 1
A white solid, yield: 34.6%. MS (ESI) m/z: 325.2 [M + H] ; 347.2 [M + Na] ; 323.0 [M - H] ; H NMR (400
MHz, DMSO-d ) δ 9.97 (s, 1H), 8.36 (d, J = 2.3 Hz, 1H), 8.23 (dd, 1H), 8.12 (s, 8H), 7.67 (d, J = 8.9 Hz, 1H),
6
7.28 (t, J = 7.8 Hz, 1H), 7.01 – 6.92 (m, 2H), 6.88 (dd, J = 8.3, 2.5 Hz, 1H), 5.36 (s, 2H).
4.1.6.20. Synthesis of 4-[(3-fluorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6t)
+
+
-
1
A white solid, yield: 66.3%. MS (ESI) m/z: 313.2 [M + H] ; 335.2 [M + Na] ; 311.0 [M - H] ; H NMR (400
MHz, DMSO-d ) δ 9.98 (s, 1H), 8.36 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 8.9, 2.4 Hz, 1H), 7.95 (s, 16H), 7.67 (d, J
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= 8.9 Hz, 1H), 7.48 – 7.39 (m, 1H), 7.30 – 7.21 (m, 2H), 7.20 – 7.12 (m, 1H), 5.41 (s, 2H).
4.1.6.21. Synthesis of 4-[(3-chlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6u)
+
- 1
A white solid, yield: 26.7%. MS (ESI) m/z: 329.2 [M + H] ; 327.0 [M - H] ; H NMR (600 MHz, DMSO-d )
6
δ 10.00 (s, 1H), 8.37 (s, 1H), 8.24 (d, J = 8.8 Hz, 1H), 7.67 (s, 59H), 7.48 (s, 1H), 7.43 – 7.33 (m, 3H), 5.40 (s,
2H).
4.1.6.22. Synthesis of 4-[(3-bromobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6v)
+
- 1
A white solid, yield: 64.1%. MS (ESI) m/z: 373.3 [M + H] ; 370.9 [M - H] ; H NMR (600 MHz, DMSO-d )
6
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δ 9.98 (s, 1H), 8.36 (d, J = 2.4 Hz, 1H), 8.22 (dd, J = 8.9, 2.5 Hz, 1H), 8.09 (s, 8H), 7.66 (d, J = 9.0 Hz, 1H),
7.62 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H), 5.40 (s, 2H).
4.1.6.23. Synthesis of 4-[(2-chlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6w)
+
+
- 1
A white solid, yield: 43.1%. MS (ESI) m/z: 329.3 [M + H] ; 351.3 [M + Na] ; 327.1 [M - H] ; H NMR (400
MHz, DMSO-d ) δ 9.86 (s, 1H), 8.43 (s, 1H), 8.35 (d, J = 2.3 Hz, 1H), 8.26 (s, 10H), 7.75 (d, J = 8.9 Hz, 1H),
6
7.53 – 7.46 (m, 2H), 7.43 – 7.32 (m, 2H), 5.44 (s, 2H).
4.1.6.24. Synthesis of 4-[(2, 6-dichlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6x)
+
A white solid, yield: 39.8%. MS (ESI) m/z: 363.1 [M + H] .
4.1.6.25. Synthesis of 4-[(2, 4-dichlorobenzyl) oxy]-3-(1H-tetrazol-1-yl) benzimidamide hydrochloride (6y)
-
A white solid, yield: 36.4%. MS (ESI) m/z: 363.0 [M - H] ; 1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H),
8.43 (s, 1H), 8.39 (d, J = 2.3 Hz, 1H), 8.28 (d, J = 6.5 Hz, 1H), 7.86 (s, 12H), 7.69 (s, 1H), 7.57 (d, J = 8.3 Hz,
1H), 7.48 (d, J = 7.9 Hz, 1H), 5.45 (s, 2H).
4.1.7. General procedure for synthesis of ethyl 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,
6-dihydropyrimidine-5-carboxylates (7a-7y)
To a solution of sodium hydride (3 g, 0.125 mol) in ethanol (10 mL) were added compounds 7a-7y (1.97
mmol) and diethyl ethoxymethylenemalonate (460 mg, 2.16 mmol). The reaction mixture was stirred at room
temperature until the material spot disapperaed by TLC. After the reaction completed, the mixture was added 5
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mL 6 M hydrochloric acid and stirred for 0.5 h, then the precipitate was collected by filtration. The resulting
residue was refluxed (ethanol or ethanol: dechloromethane=1:1) to yield ethyl 2-[4-alkoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1, 6-dihydropyrimidine-5-carboxylates (7a-7y).
4.1.7.1.
Synthesis
of
ethyl
2-[4-methoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,
6-dihydropyrimidine-5-carboxylate (7a)
-
1
A white solid, yield: 48.6%. Mp: degraded at 216.7°C. MS (ESI) m/z: 341.1 [M - H] ; H NMR (600 MHz,
DMSO-d ) δ 13.22 (s, 1H), 9.87 (s, 1H), 8.64 (s, 1H), 8.57 (s, 1H), 8.45 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 8.9 Hz,
6
1H), 4.25 (q, J = 7.0 Hz, 2H), 3.98 (s, 3H), 1.28 (t, J = 7.0 Hz, 3H).
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4.1.7.2.
Synthesis
of
ethyl
2-[4-isopropoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,
6-dihydropyrimidine-5-carboxylate (7b)
-
1
A white solid, yield: 48.4%. Mp 183.1°C-184.5°C. MS (ESI) m/z: 369.2 [M - H] ; H NMR (600 MHz,
DMSO-d ) δ 13.23 (s, 1H), 9.81 (s, 1H), 8.65 (s, 1H), 8.58 (s, 1H), 8.43 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 9.0 Hz,
6
1H), 4.94 (hept, J = 6.0 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.30 (d, J = 6.0 Hz, 6H), 1.28 (t, J = 7.1 Hz, 2H).
4.1.7.3.
Synthesis
of
ethyl
2-[4-isobutoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,
6-dihydropyrimidine-5-carboxylate (7c)
+
+
A white solid, yield: 70.1%. Mp 204.1°C-204.7°C. MS (ESI) m/z: 385.4 [M + H] ; 407.3 [M + Na] ; 383.2
-
1
[M - H] ; H NMR (600 MHz, DMSO-d ) δ 13.20 (s, 1H), 9.81 (s, 1H), 8.64 (s, 1H), 8.55 (d, J = 2.0 Hz, 1H),
6
8.44 (dd, J = 8.9, 2.4 Hz, 1H), 7.54 (d, J = 9.0 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 4.00 (d, J = 6.4 Hz, 2H), 1.99
(hept, J = 6.6 Hz, 1H), 1.28 (t, J = 7.1 Hz, 3H), 0.87 (d, J = 6.7 Hz, 6H).
4.1.7.4.
Synthesis
of
ethyl
2-[4-(isopentyloxy)-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,
6-dihydropyrimidine-5-carboxylate (7d)
+
+
A white solid, yield: 40.2%. Mp 204.3°C-204.8°C. MS (ESI) m/z: 399.3 [M + H] ; 421.2 [M + Na] ; 437.2
+
- 1
[M + K] ; 397.0 [M - H] ; H NMR (600 MHz, DMSO-d ) δ 13.21 (s, 1H), 9.80 (s, 1H), 8.63 (s, 1H), 8.55 (s,
6
1H), 8.43 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 4.29 – 4.20 (m, 4H), 1.65 – 1.57 (m, 3H), 1.28 (t, J = 7.1
Hz, 3H), 0.86 (d, J = 6.1 Hz, 6H).
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4.1.7.5.
Synthesis
of
ethyl
2-[4-(allyloxy)-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,
6-dihydropyrimidine-5-carboxylate (7e)
+
+
A white solid, yield: 69.8%. Mp: degraded at 231.2°C. MS (ESI) m/z: 369.3 [M + H] ; 391.2 [M + Na] ;
+
- 1
407.2 [M + K] ; 367.0 [M - H] ; H NMR (600 MHz, DMSO-d ) δ 9.85 (s, 1H), 8.56 (s, 1H), 8.52 (s, 1H), 8.48
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(d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 6.00 (ddt, J = 16.3, 10.5, 5.1 Hz, 1H), 5.26 (dd, J = 25.2, 13.9 Hz,
2H), 4.74 (d, J = 5.1 Hz, 2H), 4.16 (q, J = 7.1 Hz, 2H), 1.25 (t, J = 6.7 Hz, 3H).
4.1.7.6.
Synthesis
of
ethyl
2-{4-[(2-methylallyl)
oxy]-3-(1H-tetrazol-1-yl)
phenyl}-6-oxo-1,
6-dihydropyrimidine-5-carboxylate (7f)
+
- 1
A white solid, yield: 61.4%. Mp: degraded at 219.8°C. MS (ESI) m/z: 405.4 [M + Na] ; 381.1 [M - H] ; H
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NMR (600 MHz, DMSO-d ) δ 13.22 (s, 1H), 9.84 (s, 1H), 8.64 (s, 1H), 8.55 (s, 1H), 8.44 (d, J = 8.9 Hz, 1H),
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7.55 (d, J = 9.0 Hz, 1H), 4.95 (d, J = 8.4 Hz, 2H), 4.73 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.68 (s, 3H), 1.28 (t, J =
7.1 Hz, 3H).
4.1.7.7. Synthesis of ethyl 2-{4-[(3-methylbut-2-en-1-yl) oxy]-3-(1H-tetrazol-1-yl) phenyl}-6-oxo-1,
6-dihydropyrimidine-5-carboxylate (7g)
+
+
A white solid, yield: 63.4%. Mp 204.2°C-204.5°C. MS (ESI) m/z: 397.4 [M + H] ; 419.3 [M + Na] ; 395.1
-
1
[M - H] ; H NMR (600 MHz, DMSO-d ) δ 13.21 (s, 1H), 9.81 (s, 1H), 8.65 (s, 1H), 8.56 (d, J = 1.8 Hz, 1H),
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8.44 (dd, J = 8.9, 2.4 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 5.41 (t, J = 6.5 Hz, 1H), 4.80 (d, J = 6.6 Hz, 2H), 4.26 (q,
J = 7.1 Hz, 2H), 1.73 (s, 3H), 1.69 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H).
4.1.7.8.
Synthesis
of
ethyl
6-oxo-2-[4-(prop-2-yn-1-yloxy)-3-(1H-tetrazol-1-yl)
phenyl]-1,
6-dihydropyrimidine-5-carboxylate (7h)
-
1
A white solid, yield: 55.6%. Mp 211.3°C-212.0°C. MS (ESI) m/z: 365.0 [M - H] ; H NMR (400 MHz,
DMSO-d ) δ 13.22 (s, 1H), 9.84 (s, 1H), 8.66 (s, 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.47 (dd, J = 9.0, 2.3 Hz, 1H),
6
7.61 (d, J = 9.0 Hz, 1H), 5.09 (d, J = 2.4 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 3.73 (t, J = 2.4 Hz, 1H), 1.29 (t, J =
7.1 Hz, 3H).
4.1.7.9.
6-dihydropyrimidine-5-carboxylate (7i)
A white solid, yield: 58.4%. Mp: degraded at 210.1°C. MS (ESI) m/z: 405.4 [M + Na] ; 381.3 [M - H] ; H
Synthesis
of
ethyl
2-[4-(cyclopropylmethoxy)-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,
+
- 1
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