Novel xanthine oxidase inhibitor studies. Part 3. Convenient and general syntheses of 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones as a new class of potential xanthine oxidase inhibitors

Abstract

Convenient and general syntheses of 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones (12), a new class of potent xanthine oxidase inhibitors, involving the oxidative cyclisation of 6-substituted 4-alkylidenehydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d ]pyrimidines (3 and 11) with 70% nitric acid as the key step, are reported. The hydrazones 3 and 11 were obtained by a versatile synthetic route via the key intermediates, 6-chloro-4-hydrazino-1H-pyrazolo[3,4-d ]pyrimidine 2 or oxypurinol 4, starting from 2,4,6-trichloropyrimidine-5-carbaldehyde 1. Their inhibitory activities against bovine milk xanthine oxidase in vitro are also described; i.e. the pyrazolotriazolopyrimidines 12 were several hundred times more potent than allopurinol.

---

Introduction

Allopurinol, a well known drug clinically used for treatment of gout and hyperuricemia resulting from uric acid,^2–4 has been reported as a potential inhibitor of xanthine oxidase (XO), which catalyzes the conversion of hypoxanthine and xanthine to uric acid.⁵ Allopurinol is relatively non-toxic and does not appear to interfere with anabolic processes within the cell, as judged by its lack of inhibition of the growth of bacteria or tumors.⁶ However, some allopurinol toxicities ⁷ and a life-threatening toxicity syndrome have been reported after its use.⁸ Although XO inhibitory activities have recently been discovered in some newly synthesized compounds and previously known compounds,^9–15 no clinically effective XO inhibitors for the treatment of hyperuricemia have been developed since allopurinol was introduced for clinical use in 1963.^2,6 We have recently discovered that 6-alkylidenehydrazino- or 6-arylmethylidenehydrazino-7H-purines (I) and the angular type purine analogues, 9H-1,2,4-triazolo[3,4-i]purines (II), have exhibited more potent bovine milk XO inhibitory activities than that of allopurinol.^1,16,17

In our recent communication,¹⁸ we reported the facile and general syntheses of 3- and/or 5-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidines (III) as a new class of potential xanthine oxidase inhibitors. Herein we report full details of the versatile and general syntheses of the 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones (III), involving the oxidative cyclisation of 6-substituted 4-alkylidenehydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d ]pyrimidines as the key step. Furthermore, we also report here their inhibitory activities against bovine milk xanthine oxidase in comparison with allopurinol in vitro.

Results and discussion

In the preceding paper,¹ we have clarified that 9H-1,2,4-triazolo[3,4-i ]purines (II), especially the 5-oxo or 5-thioxo derivatives, showed more potent bovine milk XO inhibitory activities than allopurinol. Therefore, in this paper we tried to prepare 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones (III), which are analogous to the triazolopurines (II), as another new class of potential XO inhibitors. Few methods for synthesis of the pyrazolotriazolopyrimidines (III) have been reported in the journal ^19,20 or patent ²¹ literature and several derivatives have been synthesised. However, none of the 5-substituted derivatives has been prepared up to now.

In the first place we tried to synthesise the key intermediate, 4-hydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-one 6 derived from barbituric acid. The requisite starting material, 2,4,6-trichloropyrimidine-5-carbaldehyde 1, was prepared according to a literature method.²² Treatment of 1 with anhydrous hydrazine (4 equiv.) in 2-methoxyethanol at 0 °C afforded 6-chloro-4-hydrazino-1H-pyrazolo[3,4-d ]pyrimidine 2 in 79% yield (Scheme 1). Subsequent reaction of compound 2 with appropriate aldehydes (1.2 equiv.) in dimethylfumamide (DMF) at room temperature gave the corresponding hydrazones 3a–g in 60–93% yields as shown in Tables 1 and 2. Further, heating compound 2 in concentrated hydrochloric acid (50 parts) under reflux for 1 hour gave oxypurinol 4 (58% yield), which was confirmed by direct comparison with an authentic sample.²³ The oxypurinol 4 was also obtained in a similar yield by heating the hydrazone 3b (R = Ph) in 10% hydrochloric acid (100 parts) for 3 hours. Thiation of oxypurinol 4 by phosphorous pentasulfide gave the 6-oxo-4-thioxo derivative 5 (76% yield) following a literature procedure ²³ and the reaction of 5 with excess 50% ethanolic hydrazine under reflux yielded the desired intermediate, 4-hydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-one 6, in 71% yield.

Table 1 Preparative, physical and analytical data for the compounds 3a–g, 8b–d,f

				(Found (%) (Required)
Compound (Formula)	Yield (%)	Mp/°C	Recrystn. solvent ^a (Rf, solvent system ^b)	C	H	N	m/z MH ^+
a All compounds 3 and 8 were obtained as colourless or pale yellow powdery crystals except for 3g (yellow). b Solvent systems: (A) AcOEt–n-hexane (4∶3 v/v), (B) AcOEt–EtOH (9∶1 v/v).
3a	60	250	AcOEt	52.9	6.5	28.5	295/297
C13H19ClN6			(0.78, A)	(53.0)	(6.5)	(28.5)
3b	93	>300	DMF	52.4	3.7	30.6	273/275
C12H9ClN6			(0.56, A)	(52.85)	(3.3)	(30.8)
3c	89	>300	EtOH–DMF	49.9	2.8	29.2	291/293
C12H8ClFN6			(0.60, A)	(49.6)	(2.8)	(28.9)
3d	76	>300	EtOH–DMF	46.7	2.4	27.0	307/309/311
C12H8Cl2N6			(0.64, A)	(46.9)	(2.6)	(27.4)
3e	78	>300	EtOH–DMF	54.0	4.1	29.1	287/289
C13H11ClN6			(0.61, A)	(54.5)	(3.9)	(29.3)
3f	79	>300	EtOH–DMF	51.7	3.8	27.7	303/305
C13H11ClN6O			(0.57, A)	(51.6)	(3.7)	(27.8)
3g	70	>300	EtOH–DMF	45.6	2.7	30.6	318/320
C12H8ClN7O2			(0.49, A)	(45.4)	(2.5)	(30.9)
8b	87	298–300	EtOH–DMF	60.7	4.8	29.9	357
C19H16N8·H2O			(0.60, B)	(60.95)	(4.85)	(29.9)
8c	93	>300	EtOH–DMF	54.1	4.1	26.4	393
C19H14F2N8·3/2 H2O			(0.61, B)	(54.4)	(4.1)	(26.7)
8d	75	>300	EtOH–DMF	50.2	3.7	24.6	425/427/429
C19H14Cl2N8·3/2 H2O			(0.68, B)	(50.45)	(3.8)	(24.8)
8f	77	276	EtOH–DMF	57.7	5.1	25.7	417
C21H20N8O2·H2O			(0.62, B)	(58.1)	(5.1)	(25.8)

---

Table 2 1R and ¹H NMR spectroscopic data for the compounds 3a–g, 8b–d,f

Compound	ν max(Nujol)/cm^−1	δ H[60 MHz; (CD3)2SO; Me4Si]
a This compound was measured at 200 MHz.
3a	3180, 3100 (NH)	0.86 (3 H, J 6.8, CHCH2[CH2]5CH3), 1.31 (10 H, br s, CHCH2[CH2]5CH3), 2.15–2.60 (2 H, m, CHCH2[CH2]5CH3), 7.61 (1 H, t, J 6.6, CH CH2[CH2]5CH3), 8.18 (1 H, s, 3-H), 12.00 (1 H, br, 4-NH), 12.90 (1 H, br, 1-NH)
3b	3190, 3080 (NH)	7.40–7.60 (3 H, m, Ph-m,pH), 7.70–7.95 (2 H, m, Ph-oH), 8.28 (1 H, s, 3-H), 8.40 (1 H, s, CH-Ar), 12.44 (1 H, s, 4-NH), 13.75 (1 H, br s, 1-NH)
3c	3200, 3100 (NH)	7.32 (2 H, dd, JH,H 8.8, JH,F 9.1, Ar-mH), 7.90 (2 H, dd, JH,H 8.8, JH,F 5.9, Ar-oH), 8.27 (1 H, s, 3-H), 8.40 (1 H, s, CH-Ar), 12.45 (1 H, s, 4-NH), 13.70 (1 H, br, 1-NH)
3d ^a	3180, 3080 (NH)	7.55 (2 H, d, J 8.6, Ar-mH), 7.85 (2 H, d, J 8.6, Ar-oH), 8.26 (1 H, s, 3-H), 8.40 (1 H, s, CH-Ar), 12.54 (1 H, br, 4-NH), 13.70 (1 H, br, 1-NH)
3e	3190, 3080 (NH)	2.37 (3 H, s, CH3), 7.30 (2 H, d, J 7.9, Ar-mH), 7.70 (2 H, d, J 7.9, Ar-oH), 8.29 (1 H, s, 3-H), 8.38 (1 H, s, CH-Ar), 12.40 (1 H, br, 4-NH), 13.50 (1 H, br, 1-NH)
3f	3210, 3100 (NH)	3.84 (3 H, s, OCH3), 7.06 (2 H, d, J 8.8, Ar-mH), 7.78 (2 H, d, J 8.8, Ar-oH), 8.24 (1 H, s, 3-H), 8.39 (1 H, s, CH-Ar), 12.35 (1 H, br s, 4-NH), 13.60 (1 H, br, 1-NH)
3g ^a	3190, 3090 (NH)	8.09 (2 H, d, J 8.8, Ar-oH), 8.32 (2 H, d, J 8.8, Ar-mH), 8.35 (1 H, s, 3-H), 8.46 (1 H, s, CH-Ar), 12.78 (1 H, br s, 4-NH), 13.90 (1 H, br, 1-NH)
8b	3240, 3180, 3140 (NH)	7.26–7.83 (10 H, m, Ph-H × 2), 8.19 (1 H, s, 6-CH-Ar), 8.24 (1 H, s, 3-H), 8.32 (1 H, s, 4-CH-Ar), 10.81 (1 H, br s, 6-NH), 11.78 (1 H, br, 4-NH), 13.07 (1 H, br, 1-NH)
8c	3250, 3170, 3130 (NH)	7.27 (2 H, dd, JH,H 8.8, JH,F 9.1, 6-Ar-mH), 7.32 (2 H, dd, JH,H 8.8, JH,F 9.1, 4-Ar-mH), 7.74 (2 H, dd, JH,H 8.8, JH,F 5.9, 6-Ar-oH), 7.87 (2 H, dd, JH,H 8.8, JH,F 5.8, 4-Ar-oH), 8.20 (1 H, s, 6-CH-Ar), 8.26 (1 H, s, 3-H), 8.33 (1 H, s, 4-CH-Ar), 10.93 (1 H, br s, 6-NH), 11.80 (1 H, br, 4-NH), 12.90 (1 H, br, 1-NH)
8d	3250, 3180, 3120 (NH)	7.45 (2 H, d, J 8.4, 6-Ar-mH), 7.51 (2 H, d, J 8.5, 4-Ar-mH), 7.74 (2 H, d, J 8.4, 6-Ar-oH), 7.80 (2 H, d, J 8.5, 4-Ar-oH), 8.17 (1 H, s, 6-CH-Ar), 8.28 (1 H, s, 3-H), 8.32 (1 H, s, 4-CH-Ar), 10.95 (1 H, br, 6-NH), 11.80 (1 H, br, 4-NH), 13.00 (1 H, br, 1-NH)
8f	3250, 3170, 3140 (NH)	3.75 (3 H, s, 6-OCH3), 3.81 (3 H, s, 4-OCH3), 6.95 (2 H, d, J 8.5, 6-Ar-mH), 7.06 (2 H, d, J 8.8, 4-Ar-mH), 7.60 (2 H, d, J 8.5, 6-Ar-oH), 7.77 (2 H, d, J 8.8, 4-Ar-oH), 8.12 (1 H, s, 6-CH-Ar), 8.20 (1 H, s, 3-H), 8.24 (1 H, s, 4-CH-Ar), 10.59 (1 H, br s, 6-NH), 11.66 (1 H, br, 4-NH), 12.83 (1 H, br, 1-NH)

---

Scheme 1 Reagents and conditions: i, anh. NH₂NH₂, 2-methoxyethanol, 0 °C, 0.5 h; ii, RCHO, DMF, r.t., 10 h; iii, conc. HCl reflux, 1 h; iv, 10% HCl, reflux, 3 h; v, P₂S₅, pyridine, reflux, 2 h; vi, 50% ethanolic NH₂NH₂, reflux, 10 min; vii, anh. NH₂NH₂, 2-methoxyethanol, 100 °C, 5 h; viii, 80% aq. NH₂NH₂, 80–90 °C, 4 h; ix, RCHO, DMF, r.t., 10 h; x, urea, 2-ethoxyethanol, reflux, 5 h; xi, urea, 2-ethoxyethanol, reflux, 10 h; xii, urea, 2-ethoxyethanol, 36 h.

On the other hand, heating compound 1 with excess anhydrous hydrazine at 100 °C or heating compound 2 with excess 80% hydrazine hydrate at 80–90 °C afforded the 4,6-dihydrazino derivative 7 in good yields. Subsequent reaction of compound 7 with appropriate aldehydes (3.0 equiv.) in DMF at room temperature gave the corresponding hydrazones 8b–d,f in excellent yields as shown in Tables 1 and 2. Next, in an attempt to convert the 6-chloro-4-hydrazino compound 2 to the 6-oxo-4-hydrazino derivative 6, compound 2 was reacted with urea (4.0 equiv.) in 2-ethoxyethanol under reflux for 5 hours. However, owing to the stability of the chloro group towards hydroxy substitution by urea or alkali, the intended compound 6 was not obtained, but 4-carbamoylhydrazino-6-chloro-1H-pyrazolo[3,4-d ]pyrimidine 9, which resulted from carbamoylation of the hydrazino group at the 4-position, was formed in 60% yield. Heating under reflux the product 9 with urea (3.0 equiv.) in 2-ethoxyethanol afforded the desired tricyclic compound, 3-amino-7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-one 10, in 18% yield. This compound 10 (29% yield) was also obtained by prolonged heating of 2 with urea under the same reaction conditions as mentioned above.

All new compounds 2, 3 and 6–10 exhibited satisfactory elemental combustion analyses except for 2 and 6 and FAB-MS, IR and ¹H NMR spectral data consistent with the structures. In particular, the structure of the product 10 was confirmed by the presence of a two-proton broad singlet signal at δ 7.94 and one-proton signals at δ 12.27 and 13.10 in the ¹H NMR spectrum attributable to the amino and imino groups and by the presence of peaks at 3370 (ν_as NH), 3260 (ν_s NH), 1720 (ν C[double bond, length half m-dash]O) and 1685 (δ NH) cm⁻¹ in the IR spectrum attributable to amino and carbonyl groups. It was clarified that the substitution reaction of the chloro group by hydroxy was difficult in the pyrazolopyrimidine ring 2, while in the pyrazolotriazolopyrimidine ring 10 it was easy.

The 4-hydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-one 6 as noted above was a versatile intermediate for the preparation of the 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidine ring system. Thus the hydrazinopyrazolopyrimidine 6 could be converted to the hydrazones 11b, e–r (63–95% yields) by reaction with an appropriate aldehyde (1.5 equiv.) in DMF at room temperature (Scheme 2 and Tables 3 and 4). The hydrazones 11b, e–p, r were subsequently cyclised to the corresponding 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones 12b, e–p, r by heating with 70% nitric acid (1.2 equiv.) at 100 °C in 60–91% yields (Method A) (Tables 5 and 6). In the case of compound 11q possessing a 4-hydroxybenzylidenehydrazino group as the substituent at the 4-position, the 3-(4-hydroxy-3-nitrophenyl) derivative 12s was obtained by oxidative cyclisation–nitration in 52% yield. Oxidative cyclisation was also accomplished by heating compounds 11e, g, h, k, m–o, q with diethyl azodicarboxylate (DEAD) (3–7 equiv.) under reflux in 50–67% yields (Method B). Moreover, the 3-alkyl derivatives 12a–d were synthesised by treatment of compound 6 with an appropriate trialkyl orthoester (5.0 equiv.) in trifluoroacetic acid at room temperature (Method C) or heating compound 6 with trialkyl orthoesters (3.0 equiv.) in DMF at 100 °C (Method D) in 54–83% yields. In the light of this multiple step synthesis, a one-pot oxidative cyclisation starting from the 6-chloro-4-hydrazones 3a–g would be attractive. Indeed, heating the hydrazones 3a–g with 70% nitric acid (5.0 equiv.) in DMF at 100 °C afforded the desired 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones 12e, g, h, k, m, n, r accompanied by hydrolytic dechlorination in 60–85% yields (Method E).

Table 3 Preparative, physical and analytical data for the compounds 11b, e–r

					(Found (%) (Required)
Compound (Formula)	Reaction temp/°C	Yield (%)	Mp/°C	Recrystn. solvent ^a (Rf, solvent system ^b)	C	H	N	m/z MH^+
a All compounds 11 were obtained as colourless powdery crystals except for 11m, r (pale yellow). b Solvent systems: (A) AcOEt–EtOH (4∶1 v/v), (B) AcOEt–EtOH (9∶1 v/v), (C) AcOEt–n-hexane–AcOH (8∶4∶1 v/v).
11b	r.t.	89	>300	EtOH–DMF	43.4	4.2	43.65	193
C7H8N6O				(0.47, A)	(43.75)	(4.2)	(43.7)
11e	r.t.	80	>300	EtOH–DMF	55.8	7.2	30.3	277
C13H20N6O·1/5 H2O				(0.65, A)	(55.8)	(7.35)	(30.0)
11f	r.t.	63	>300	EtOH–DMF	60.3	7.4	26.3	317
C16H24N6O				(0.47, B)	(60.7)	(7.65)	(26.6)
11g	r.t.	85	>300	water–DMF	55.8	4.1	32.8	255
C12H10N6O·1/5 H2O				(0.60, A)	(55.9)	(4.1)	(32.6)
11h	r.t.	74	>300	water–DMF	52.2	3.6	30.5	273
C12H9FN6O·1/4 H2O				(0.64, A)	(52.1)	(3.5)	(30.4)
11i	40	76	>300	EtOH–DMF	49.5	3.5	28.7	289/291
C12H9ClN6O				(0.64, A)	(49.9)	(3.1)	(29.1)
11j	40	95	>300	EtOH–DMF	49.3	3.4	28.9	289/291
C12H9ClN6O·1/5 H2O				(0.65, A)	(49.3)	(3.2)	(28.75)
11k	r.t.	95	>300	water–DMF	49.2	3.4	28.7	289/291
C12H9ClN6O·1/5 H2O				(0.63, A)	(49.3)	(3.2)	(28.75)
11l	r.t.	83	>300	EtOH–DMF	43.3	3.1	24.9	333/335
C12H9BrN6O				(0.65, A)	(43.3)	(2.7)	(25.2)
11m	r.t.	93	>300	DMF	57.4	4.5	31.1	269
C13H12N6O·1/5 H2O				(0.67, A)	(57.4)	(4.6)	(30.9)
11n	r.t.	87	>300	water–DMF	54.4	4.3	29.2	285
C13H12N6O2·1/5 H2O				(0.62, A)	(54.2)	(4.3)	(29.2)
11o	40	85	>300	water–DMF	52.1	3.6	27.7	299
C13H10N6O3				(0.67, A)	(52.35)	(3.4)	(28.2)
11p	40	80	>300	water–DMF	52.6	3.4	28.2	299
C13H10N6O3				(0.64, C)	(52.35)	(3.4)	(28.2)
11q	40	85	>300	EtOH–DMF	53.2	4.0	30.7	271
C12H10N6O2				(0.64, A)	(53.3)	(3.7)	(31.1)
11r	r.t.	88	>300	EtOH–DMF	45.45	3.5	30.8	300
C12H9N7O3·H2O				(0.60, A)	(45.4)	(3.5)	(30.9)

---

Table 4 IR and ¹H NMR spectroscopic data for the compounds 11b, e–r

Compound	ν max(Nujol)/cm^−1	δ H [200 MHz; (CD3)2SO; Me4Si]
a This compound was measured at 60 MHz.
11b	3175, 3130, 3070 (NH); 1700 (C[double bond, length half m-dash]O)	2.01 (3 H, d, J 5.4, CHCH3), 7.69 (1 H, q, J 5.4, CH CH3), 8.35 (1 H, s, 3-H), 10.40 (1 H, br, 4-NH), 10.80 (1 H, br s, 7-NH), 12.85 (1 H, br, 1-NH)
11e	3175, 3135, 3070 (NH); 1710 (C[double bond, length half m-dash]O)	0.86 (3 H, t, J 6.4, CHCH2CH2[CH2]4CH3), 1.29 (8 H, br s, CHCH2CH2[CH2]4CH3), 1.44–1.66 (2 H, m, CHCH2CH2[CH2]4CH3), 2.24–2.42 (2 H, m, CHCH2CH2[CH2]4CH3), 8.05 (1 H, t, J 5.4, CH CH2CH2[CH2]4CH3), 8.28 (1 H, s, 3-H), 10.22 (1 H, br, 4-NH), 10.82 (1 H, br s, 7-NH), 12.95 (1 H, br, 1-NH)
11f	3175, 3135, 3070 (NH); 1700 (C[double bond, length half m-dash]O)	1.28 (10 H, br s, CHCH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 1.44–1.64 (2 H, m, CHCH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 1.90–2.08 (2 H, m, CHCH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 2.22–2.42 (2 H, m, CHCH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 4.86–5.06 (2 H, m, CHCH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 5.64–5.90 (1 H, m, CHCH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 7.60–7.72 (1 H, m, CH CH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 8.28 (1H, s, 3-H), 10.28 (1 H, br, 4-NH), 10.82 (1 H, br s, 7-NH), 12.92 (1 H, br, 1-NH)
11g	3200, 3120, 3080 (NH); 1680 (C[double bond, length half m-dash]O)	7.40–7.53 (3 H, m, Ph-m,pH), 7.80–7.90 (2 H, m, Ph-oH), 8.40 (1 H, s, 3-H), 8.46 (1 H, s, CH-Ar), 10.40 (1 H, br, 4-NH), 11.01 (1 H, br, 7-NH), 13.10 (1 H, br, 1-NH)
11h	3180, 3140, 3070 (NH); 1680 (C[double bond, length half m-dash]O)	7.30 (2 H, dd, JH,H 8.8, JH,F 9.0, Ar-mH), 7.30 (2 H, dd, JH,H 8.8, JH,F 5.8, Ar-oH), 8.40 (1 H, s, 3-H), 8.45 (1H, s, CH-Ar), 10.40 (1 H, br, 4-NH), 11.00 (1 H, br, 7-NH), 13.09 (1 H, br, 1-NH)
11i ^a	3200, 3120, 3080 (NH); 1700 (C[double bond, length half m-dash]O)	7.33–7.60 (3 H, m, 3′-H, 4′-H and 5′-H), 8.12–8.28 (1 H, m, 6′-H), 8.48 (1H, s, 3-H), 8.72 (1 H, s, CH-Ar), 10.35 (1 H, br s, 4-NH), 11.10 (1 H, br, 7-NH), 13.40 (1 H, br, 1-NH)
11j	3170, 3140, 3060 (NH); 1720 (C[double bond, length half m-dash]O)	7.40–7.54 (2 H, m, 4′-H and 5′-H), 7.80–7.92 (2 H, m, 2′-H and 6′-H), 8.40 (1 H, s, 3-H), 8.43 (1 H, s, CH-Ar), 10.50 (1 H, br s, 4-NH), 11.01 (1 H, br s, 7-H), 13.10 (1 H, br s, 1-NH)
11k	3170, 3140, 3070 (NH); 1680 (C[double bond, length half m-dash]O)	7.48 (2 H, d, J 8.6, Ar-mH), 7.87 (2 H, d, J 8.6, Ar-oH), 8.37 (1 H, s, 3-H), 8.45 (1 H, s, CH-Ar), 10.50 (1 H, br, 4-NH), 11.00 (1 H, br, 7-NH), 13.14 (1 H, br, 1-NH)
11l	3170, 3140, 3060 (NH); 1685 (C[double bond, length half m-dash]O)	7.66 (2 H, d, J 8.6, Ar-mH), 7.80 (2 H, d, J 8.6, Ar-oH), 8.38 (1H, s, 3-H), 9.44 (1 H, s, CH-Ar), 10.50 (1 H, br, 4-NH), 11.00 (1 H, br s, 7-NH), 13.11 (1 H, br, 1-NH)
11m	3170, 3140, 3060 (NH); 1685 (C[double bond, length half m-dash]O)	2.36 (3 H, s, CH3), 7.28 (2 H, d, J 7.8, Ar-mH), 7.72 (2 H, d, J 7.8, Ar-oH), 8.35 (1 H, s, 3-H), 8.44 (1 H, s, CH-Ar), 10.45 (1 H, br, 4-NH), 11.00 (1 H, br, 7-NH), 13.10 (1 H, br, 1-NH)
11n ^a	3180, 3140, 3080 (NH); 1680 (C[double bond, length half m-dash]O)	3.80 (3 H, s, OCH3), 6.74 (2 H, d, J 8.5, Ar-mH), 7.58 (2 H, d, J 8.5, Ar-oH), 7.94 (1 H, s, 3-H), 7.99 (1 H, s, CH-Ar), 10.06 (1 H, br s, 4-NH), 10.92 (1 H, br s, 7-NH), 12.90 (1 H, br s, 1-NH)
11o	3180, 3140, 3070 (NH); 1655 (C[double bond, length half m-dash]O)	6.09 (2 H, s, OCH2O), 7.00 (1 H, d, J5′,6′ 8.2, 5′-H), 7.27 (1 H, d, J5′,6′ 8.2, J2′,6′ 1.6, 6′-H), 7.45 (1 H, d, J2′,6′ 1.6, 2′-H), 8.30 (1 H, s, 3-H), 8.45 (1 H, s, CH-Ar), 10.35 (1 H, br, 4-NH), 11.03 (1 H, br s, 7-NH), 13.04 (1 H, br, 1-NH)
11p ^a	3165, 3120, 3050 (NH); 1650, 1630 (C[double bond, length half m-dash]O)	8.00 (4 H, br, Ar-H), 8.50 (2 H, s, 3-H and CH-Ar), 11.25 (1 H, br, 4-NH), 11.95 (1 H, br, 7-NH), 12.35 (1 H, br, COOH ), 13.55 (1 H, br, 1-NH)
11q	3160, 3140, 3050 (NH); 1640 (C[double bond, length half m-dash]O)	6.84 (2 H, d, J 8.4, Ar-mH), 7.65 (2 H, d, J 8.4, Ar-oH), 8.26 (1 H, s, 3-H), 8.41 (1 H, s, CH-Ar), 9.67 (1 H, br s, OH), 10.86 (1 H, br s, 4-NH), 11.02 (1 H, br s, 7-NH), 12.90 (1 H, br, 1-NH)
11r	3165, 3120, 3080 (NH); 1690 (C[double bond, length half m-dash]O)	8.10 (1 H, d, J 8.8, Ar-oH), 8.30 (1 H, d, J 8.8, Ar-mH), 8.50 (1 H, s, 3-H), 8.52 (1 H, s, CH-Ar), 10.65 (1 H, br, 4-NH), 11.11 (1 H, br, 7-NH), 13.21 (1 H, br, 1-NH)

---

Table 5 Preparative, physical and analytical data for the compounds 12a–s

	Reaction conditions ^a						(Found (%) (Required)
Compound (Formula)	Method	Temp/°C	Time/h	Yield ^a (%)	Mp/°C	Recrystn. solvent ^b (Rf, solvent system ^c)	C	H	N	m/z MH^+
a The reaction conditions and yields depend on the particular method. b All compounds 12 were obtained as colourless powdery crystals except for 12b, h, k, l, (colourless needles) and 12r, s (yellow powder). c Solvent systems: (A) AcOEt–EtOH (4∶1 v/v), (B) AcOEt–EtOH (9∶1 v/v), (C) AcOEt–n-hexane–AcOH (8∶4∶1 v/v).
12a	(C )	r.t.	1	66	>300	DMF	39.9	2.7	46.5	177
C6H4N6O·1/4 H2O	(D)	100	1	65		(0.32, A)	(39.9)	(2.5)	(46.5)
12b	(A)	100	2.5	64	>300	EtOH–DMF	43.5	3.5	43.7	191
C7H6N6O·1/5 H2O	(C )	r.t	1	83		(0.38, A)	(43.4)	(3.3)	(43.4)
	(D)	100	1	55
12c	(D)	100	1	54	>300	EtOH–DMF	46.8	4.2	41.4	205
C8H8N6O						(0.45, A)	(47.1)	(3.95)	(41.2)
12d	(D)	100	1	55	>300	EtOH–DMF	51.1	5.5	36.0	233
C10H12N6O·1/5 H2O						(0.55, A)	(50.9)	(5.3)	(35.6)
12e	(A)	100	3	77	>300	EtOH–DMF	55.7	6.7	30.2	275
C13H18N6O·1/4 H2O	(B)	reflux	7	60		(0.64, A)	(56.0)	(6.7)	(30.1)
	(E )	100	1	75
12f	(A)	100	2.5	67	>300	EtOH–DMF	60.3	7.35	26.3	315
C16H22N6O·1/4 H2O						(0.34, B)	(60.3)	(7.1)	(26.35)
12g	(A)	100	1	91	>300	water–DMF	55.9	3.45	32.75	253
C12H8N6O/1/4 H2O	(B)	reflux	5	60		(0.60, A)	(56.1)	(3.3)	(32.7)
	(E )	100	1	85
12h	(A)	100	1	91	>300	EtOH–DMF	52.6	2.9	30.65	271
C12H7FN6O·1/4 H2O	(B)	reflux	8	65		(0.62, A)	(52.5)	(2.75)	(30.6)
	(E )	100	3	72
12i	(A)	100	2	70	>300	water–DMF	50.0	2.8	28.9	287/289
C12H7ClN6O						(0.63, A)	(50.3)	(2.5)	(29.3)
12j	(A)	100	2	76	>300	water–DMF	49.9	2.8	29.2	287/289
C12H7ClN6O·1/5 H2O						(0.64, A)	(49.65)	(2.6)	(28.95)
12k	(A)	100	2	90	>300	water–DMF	49.7	2.8	29.2	287/289
C12H7ClN6O·1/5 H2O	(B)	reflux	8	67		(0.63, A)	(49.65)	(2.6)	(28.95)
	(E )	100	1	71
12l	(A)	100	5	74	>300	EtOH–DMF	43.2	2.5	24.9	331/333
C12H7BrN6O·1/5 H2O						(0.64, A)	(43.1)	(2.2)	(25.1)
12m	(A)	100	9	60	>300	water–DMF	58.0	4.0	31.3	267
C13H10N6O·1/5 H2O	(B)	reflux	9	54		(0.67, A)	(57.9)	(3.9)	(31.1)
	(E )	100	3	67
12n	(A)	100	3	88	>300	water–DMF	54.5	3.9	29.3	283
C13H10N6O2·1/4 H2O	(B)	reflux	9	57		(0.60, A)	(54.45)	(3.7)	(29.3)
	(E )	100	3	61
12o	(A)	100	1	81	>300	EtOH–DMF	52.2	3.1	28.0	297
C13H8N6O3·1/4 H2O	(B)	reflux	9	55		(0.62, A)	(51.9)	(2.85)	(27.9)
12p	(A)	100	1.5	69	>300	EtOH–DMF	51.8	3.2	27.6	297
C13H8N6O3·1/3 H2O						(0.64, C)	(51.7)	(2.9)	(27.8)
12q	(B)	reflux	9	50	>300	EtOH–DMF	52.8	2.9	31.0	269
C12H8N6O2·1/4 H2O						(0.51, A)	(52.85)	(3.1)	(30.8)
12r	(A)	100	5	60	>300	EtOH–DMF	48.1	2.8	32.2	298
C12H7N7O3·1/4 H2O	(E )	100	5	60		(0.60, A)	(47.8)	(2.5)	(32.5)
12s	(A)	100	1	52	>300	DMF	45.4	2.6	30.6	314
C12H7N7O4·1/4 H2O						(0.46, A)	(45.4)	(2.4)	(30.9)

---

Table 6 IR and ¹H NMR spectroscopic data for the compounds 12a–s

Compound	ν max(Nujol)/cm^−1	δ H [200 MHz; (CD3)2SO; Me4Si]
a This compound was measured at 60 MHz.
12a	3120, 3030 (NH); 1740 (C[double bond, length half m-dash]O)	8.34 (1 H, s, 9-H), 8.62 (1 H, s, 3-H), 12.58 (1 H, br s, 6-NH), 13.60 (1 H, br s, 7-NH)
12b	3110, 3050 (NH); 1700 (C[double bond, length half m-dash]O)	2.40 (3 H, s, CH3), 8.54 (1 H, s, 9-H), 12.46 (1 H, br s, 6-NH), 13.57 (1 H, br s, 7-NH)
12c	3100, 3060 (NH); 1700 (C[double bond, length half m-dash]O)	1.29 (3 H, t, J 7.6, CH2CH3), 2.77 (2 H, q, J 7.6, CH2CH3), 8.57 (1 H, s, 9-H), 12.43 (1 H, br s, 6-NH), 13.55 (1 H, br s, 7-NH)
12d	3090, 3060 (NH); 1700 (C[double bond, length half m-dash]O)	0.92 (3 H, t, J 7.6, CH2CH2CH2CH3), 1.36 (2 H, sextet, J 7.6, CH2CH2CH2CH3), 1.72 (2 H, quintet, J 7.6, CH2CH2CH2CH3), 2.74 (2 H, t, J 7.6, CH2CH2CH2CH3), 8.57 (1 H, s, 9-H), 12.44 (1 H, br s, 6-NH), 13.55 (1 H, br s, 7-NH)
12e ^a	3110, 3070 (NH); 1700 (C[double bond, length half m-dash]O)	0.86 (3 H, t, J 6.5, CH2CH2[CH2]4CH3), 1.30 (8 H, br s, CH2CH2[CH2]4CH3), 1.50–1.80 (2 H, m, CH2CH2[CH2]4CH3), 2.50–2.95 (2 H, m, CH2CH2[CH2]4CH3), 8.53 (1 H, s, 9-H), 12.41 (1 H, br s, 6-NH), 13.50 (1 H, br, 7-NH)
12f	3150, 3070 (NH); 1710 (C[double bond, length half m-dash]O)	1.28 (10 H, br s, CH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 1.62–1.80 (2 H, m, CH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 1.92–2.06 (2 H, m, CH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 2.72 (2 H, t, J 7.3, CH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 4.87–5.04 (2 H, m, CH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 5.66–5.89 (1 H, m, CH2CH2[CH2]5CH2CH[double bond, length half m-dash]CH2), 8.57 (1 H, s, 9-H), 12.43 (1 H, br s, 6-NH), 13.55 (1 H, br s, 7-NH)
12g ^a	3150, 3050 (NH); 1720 (C[double bond, length half m-dash]O)	7.40–7.70 (3 H, m, Ph-m,pH), 7.90–8.35 (2 H, m, Ph-oH), 8.68 (1 H, s, 9-H), 12.60 (1 H, br s, 6-NH), 13.60 (1 H, br, 7-NH)
12h ^a	3110, 3090 (NH); 1710 (C[double bond, length half m-dash]O)	7.37 (2 H, dd, JH,H 8.8, JH,F 9.1, Ar-mH), 8.22 (2 H, dd, JH,H 8.8, JH,F 5.9, Ar-oH), 8.66 (1 H, s, 9-H), 12.59 (1 H, br s, 6-NH), 13.65 (1 H, br, 7-NH)
12i	3150, 3070 (NH); 1710 (C[double bond, length half m-dash]O)	7.44–7.78 (3 H, m, 3′-H, 4′-H and 5′-H), 7.95–8.09 (1 H, m, 6′-H), 8.69 (1 H, s, 9-H), 12.67 (1 H, br s, 6-NH), 13.58 (1 H, br, 7-NH)
12j ^a	3150, 3100 (NH); 1720 (C[double bond, length half m-dash]O)	7.54–7.63 (2 H, m, 4′-H and 5′-H), 7.95–8.25 (2 H, m, 2′-H and 6′-H), 8.67 (1 H, s, 9-H), 12.62 (1 H, br s, 6-NH), 13.60 (1 H, br s, 7-NH)
12k	3160, 3100 (NH); 1700 (C[double bond, length half m-dash]O)	7.62 (2 H, d, J 8.6, Ar-mH), 8.17 (2 H, d, J 8.6, Ar-oH), 8.70 (1 H, s, 9-H), 12.62 (1 H, br s, 6-NH), 13.66 (1 H br s, 7-NH)
12l	3150, 3060 (NH); 1720 (C[double bond, length half m-dash]O)	7.76 (2 H, d, J 8.6, Ar-mH), 8.10 (2 H, d, J 8.6, Ar-oH), 8.69 (1 H, s, 9-H), 12.62 (1 H, br s, 6-NH), 13.65 (1 H, br s, 7-NH)
12m	3180, 3100 (NH); 1720 (C[double bond, length half m-dash]O)	2.39 (3 H, s, CH3), 7.35 (2 H, d, J 8.0, Ar-mH), 8.05 (2 H, d, J 8.0, Ar-oH), 8.66 (1 H, s, 9-H), 12.54 (1 H, br s, 6-NH), 13.61 (1 H, br s, 7-NH)
12n ^a	3160, 3100 (NH); 1730 (C[double bond, length half m-dash]O)	3.85 (3 H, s, OCH3), 7.10 (2 H, d, J 8.8, Ar-mH), 8.12 (2 H, d, J 8.8, Ar-oH), 8.64 (1 H, s, 9-H), 12.60 (1 H, br s, 6-NH), 13.50 (1 H, br, 7-NH)
12o	3160, 3050 (NH); 1720 (C[double bond, length half m-dash]O)	6.13 (2 H, s, OCH2O), 7.08 (1 H, d, J5′,6′ 8.1, 5′-H), 7.58 (1 H, d, J2′,6′ 1.6, 2′-H), 7.72 (1 H, d, J5′,6′ 8.1, J2′,6′ 1.6, 6′-H), 8.68 (1 H, s, 9-H), 12.55 (1 H, br s, 6-NH), 13.62 (1 H, br s, 7-NH)
12p ^a	3160, 3090 (NH); 1700, 1660 (C[double bond, length half m-dash]O)	8.10 (2 H, d, J 8.8, Ar-mH), 8.32 (2 H, d, J 8.8, Ar-oH), 8.69 (1 H, s, 9-H), 12.70 (1 H, br s, 6-NH), 13.50 (2 H, br, 7-NH and COOH)
12q	3160, 3050 (NH); 1718 (C[double bond, length half m-dash]O)	6.90 (2 H, d, J 8.8, Ar-mH), 7.99 (2 H, d, J 8.8, Ar-oH), 8.66 (1 H, s, 9-H), 9.94 (1 H, s, OH), 12.51 (1 H, br s, 6-NH), 13.60 (1 H, br, 7-NH)
12r ^a	3170, 3100 (NH); 1720 (C[double bond, length half m-dash]O)	8.39 (4 H, br s, Ar-H), 8.69 (1 H, s, 9-H), 12.70 (1 H, br s, 6-NH), 13.60 (1 H, br 7-NH)
12s	3160, 3080 (NH); 1720 (C[double bond, length half m-dash]O)	7.31 (1 H, d, J5′,6′ 8.8, 5′-H), 8.28 (1 H, dd, J5′,6′ 8.8. J2′,6′ 2.2, 6′-H), 8.60 (1 H, d, J2′,6′ 2.2, 2′-H), 8.70 (1 H, s, 9-H), 11.59 (1 H, s, OH), 12.62 (1 H, br s, 6-NH), 13.65 (1 H, br s, 7-NH)

---

Scheme 2 Reagents and conditions: i, RCHO, DMF, r.t. or 40 °C, 10 h; ii, 70% HNO₃, DMF, 100 °C, 1–9 h; iii, DEAD, DMF, reflux, 5–9 h; iv, RC(OEt)₃, TFA, r.t., 1 h; v, RC(OEt)₃ or RC(OMe)₃, DMF, 100 °C, 1 h.

All new compounds 11 and 12 exhibited satisfactory elemental combustion analyses and FAB MS, IR and ¹H NMR spectral data consistent with the structures as indicated in Tables 3–6.

Xanthine oxidase inhibitory results

The novel pyrazolopyrimidines 2, 3, 6, 8 and 11 and pyrazolotriazolopyrimidines 12 prepared in this study were tested as inhibitors of bovine milk xanthine oxidase in a similar assay method ¹⁴ as previously reported. The inhibition (%) and IC₅₀ (μM) values of the compounds tested against bovine milk xanthine oxidase are shown in Table 7. Thus the introduction of both an aryl aldehyde hydrazone at the 4-position and an oxo group at the 6-position of the 1H-pyrazolo[3,4-d ]pyrimidine ring led to markedly better activities in xanthine oxidase inhibition, these compounds being two orders of magnitude more active than allopurinol: IC₅₀ values for 11g–k and 11m–q were ca. 0.08–0.4 μM, whereas that for allopurinol was 24.3 μM. Most of the pyrazolotriazolopyrimidines 12a–s showed potent inhibitory activities, being two or three orders of magnitude more active than allopurinol. Of these compounds, 12k (R = 4-ClC₆H₄) was the most active; it showed a 760-fold (IC₅₀ = 0.032 μM) more potent bovine milk XO inhibitory activity than that of allopurinol.

Table 7 Inhibitory activities of the compounds 2, 3, 4, 6, 8, 11 and 12 against bovine milk xanthine oxidase in comparison with allopurinol

	Inhibition (%)
Compound No.	10	3	1	0.3	0.1	0.03	IC50/μM
a 30 μM: 53.9%, 100 μM: 63.9%. b This value is inaccurate because of insolubility in DMSO. c 0.01 μM: 12.6%. d 0.01 μM: 9.6%. e 0.01 μM: 30.4%, 0.003 μM, 16.4%. f 0.01 μM: 37.3%. g 0.01 μM: 28.2%, 0.003 μM: 13.6%. h 0.01 μM: 29.0%. i Allo: allopurinol.
2	21.4	11.8	8.3				>10
3b	7.6						>10
3c	21.0	16.0	11.5				>10
3d	12.0	9.5	9.4				>10
3e	14.2	13.8	10.8				>10
3f	41.0						>10
4 ^a	39.8						22.1
6	24.1						>10
8b	7.2						>10
8c	16.3						>10
8d	12.4						>10
8f	17.2						>10
11b	58.6	45.0	34.8	15.8	6.9	6.2	4.670
11e	71.7	69.2	60.6	44.6	25.8	13.3	0.450
11f	53.3	36.5	22.4	13.0	9.7	4.6	7.894
11g	75.6	73.0	66.3	49.8	29.9		0.305
11h	68.5	69.9	63.6	47.0	29.7		0.373
11i	68.9	69.6	68.5	64.4	53.8	36.3	0.077
11j	67.6	66.4	63.4	54.2	38.7	20.7	0.223
11k	66.1	65.6	62.9	53.8	39.5	22.7	0.224
11l ^b	45.1	44.2	42.1	49.6	40.1	23.9	>10
11m	68.9	66.5	63.8	52.8	37.0	18.6	0.247
11n	74.5	73.3	70.0	59.2	41.0		0.172
11o	68.4	65.9	61.6	47.0	29.0	14.1	0.385
11p	68.1	65.2	60.3	46.8	29.3	14.8	0.399
11q	62.8	66.2	60.3	48.2	40.3	16.7	0.359
11r	57.3	52.1	46.9	39.5	28.7	14.0	1.925
12a	69.2	67.5	65.2	56.6	41.8	23.9	0.184
12b	71.5	68.8	65.1	52.5	37.3	20.7	0.250
12c ^c	57.0	55.2	52.0	42.2	28.8	17.8	0.782
12d ^d	57.6	55.1	53.0	46.1	34.9	18.9	0.529
12e	69.8	69.1	67.9	62.8	54.4	40.2	0.069
12f	66.8	65.0	63.4	59.6	48.4	32.0	0.117
12g	67.8	65.3	64.2	59.8	49.7	32.4	0.103
12h	69.5	68.8	67.7	64.6	56.7	39.6	0.062
12i	68.5	68.6	67.1	63.5	54.8	38.9	0.070
12j	68.9	69.6	68.8	66.7	61.3	47.4	0.038
12k ^e	72.3	70.6	70.3	68.3	62.9	49.3	0.032
12l ^f	70.1	66.4	67.4	63.9	60.4	48.9	0.034
12m ^g	72.2	70.7	70.0	67.0	60.9	46.0	0.041
12n	71.6	71.0	70.4	66.9	58.6	42.3	0.053
12o	70.2	69.6	68.6	66.6	61.0	46.3	0.041
12p	69.0	67.1	65.7	60.6	50.2	34.3	0.098
12q ^h	67.6	66.0	65.4	62.9	57.0	42.9	0.055
12r	69.8	68.2	68.7	64.4	57.5	39.6	0.060
12s	69.1	69.7	69.0	67.7	64.0	46.9	0.037
Allo ^i	38.2	19.9	9.9	4.6	3.2		24.3

---

Conclusion

Thus, this simple and general methodology provided a facile and convenient route to the preparation of 3-substituted 7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones (12), which were obtained by oxidative cyclisation of the corresponding 4-aldehyde hydrazones of 1H-pyrazolo[3,4-d ]pyrimidines (3 and 11) with 70% nitric acid as the key step, as a new class of potential xanthine oxidase inhibitors. Their inhibitory activities against bovine milk xanthine oxidase in vitro were investigated, and some 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-ones (11) exhibited from several times to several hundred times more potent activities than allopurinol. In addition, the tricyclic compounds, 3-aryl-7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-ones (12) showed potent inhibitory activities, being ca. three orders of magnitude more active than allopurinol. They did not show any appreciable inhibition against the proliferation of T-cell acute lymphoblastic leukemia (CCRF-HSB-2) however.† Biological testing of the compounds in vivo is now ongoing and the results will be reported later.

Experimental

General

Mps were obtained on a Yanagimoto micro melting point apparatus and were uncorrected. Microanalyses were measured by a Yanaco CHN Corder MT-5 apparatus. Mass spectra were recorded at 70 eV ionizing voltage with FAB ionization using a VG-70SE spectrometer and 3-nitrobenzyl alcohol or glycerol as a matrix. IR spectra were recorded using a JASCO FT/IR-200 spectrophotometer as Nujol mulls. ¹H NMR spectra were obtained using Hitachi FT-NMR R-1500 (60 MHz) and Varian VXR 200 MHz spectrometers. In all cases, chemical shifts are in ppm relative to SiMe₄ as internal standard and J values are given in Hz. All reagents were of commercial quality from freshly opened containers and were used without further purification. Organic solvents were dried by standard methods and distilled before use. Reaction progress was monitored by analytical thin layer chromatography (TLC) on pre-coated glass plates (silica gel 70 FM Plate-Wako) using the following solvent systems: (A) AcOEt–EtOH (4∶1 v/v), (B) EtOH, (C) MeOH and others cited in the Tables. The products were visualized by UV light. Column chromatography was run on Daisogel IR-60 (63/210 μM, Daiso Co.). The reaction temperatures are indicated as the temperature of oil bath.

6-Chloro-4-hydrazino-1H-pyrazolo[3,4-d ]pyrimidine 2

To a stirring solution of 2,4,6-trichloropyrimidine-5-carbaldehyde 1 ²² (3.0 g, 14.2 mmol) in 2-methoxyethanol (20 cm³) at 0 °C was added a solution of anhydrous hydrazine (1.82 g, 56.8 mmol) diluted with 2-methoxyethanol (18 cm³) in limited amounts for 30 min. After the reaction was complete, the precipitated crystals were collected by filtration and washed with water and EtOH to afford the pyrazolopyrimidine2 (2.06 g, 79%) as pale yellow powdery crystals, mp > 300 °C; R_f (A) 0.64; ν_max/cm⁻¹ 3350 and 3260 (NH₂), 3160 and 3100 (NH) and δ_max/cm⁻¹ 1660 (NH₂); δ_H [60 MHz; (CD₃)₂SO] 4.20 (2 H, br, NH₂), 8.50 (1 H, s, 3-H), 9.45 (1 H, br, 4-NH) and 13.40 (1 H, br, 1-NH); m/z (FAB, 3-nitrobenzyl alcohol matrix) 185 (MH⁺) and 187 (MH⁺ + 2). The product 2 was obtained as a single compound and was used for the following reactions without further purification because it was difficult to purify since it was insoluble in usual solvents.

4-Alkylidenehydrazino- and 4-arylmethylidenehydrazino-6-chloro-1H-pyrazolo[3,4-d ]pyrimidines 3a–g; General procedure

A mixture of the hydrazinopyrazolopyrimidine 2 (1.0 g, 5.42 mmol) and an appropriate alkyl aldehyde or aryl aldehyde (6.50 mmol) in DMF (50 cm³) was stirred at room temperature for 10 hours. After the reaction was complete, the solution was evaporated under reduced pressure and the residue was triturated with EtOH or AcOEt to give crystals, which were collected by filtration and recrystallized from an appropriate solvent to afford the corresponding hydrazones3a–g as shown in Tables 1 and 2.

1H-Pyrazolo[3,4-d ]pyrimidine-4,6(5H,7H )-dione 4 (oxypurinol)

(1) A mixture of the hydrazino derivative 2 (0.20 g, 1.08 mmol) with concentrated hydrochloric acid (10 cm³) was heated under reflux for 1 hour. After the reaction was complete, the solution was treated with activated charcoal and evaporated under reduced pressure; the residue was recrystallized from water to afford oxypurinol [95 mg, 58%; mp > 300 °C; R_f (A) 0.48; ν_max/cm⁻¹ 3180, 3150 and 3120 (NH) and 1720 (C[double bond, length half m-dash]O)], which was identical with an authentic sample.²³

(2) The hydrazone 3b (0.50 g, 1.83 mmol) in 10% aqueous HCl (50 cm³) was heated under reflux for 3 hours. After the reaction was complete, the solution was treated with activated charcoal and cooled to afford a deposit, which was collected by filtration. The filtrate was evaporated under reduced pressure and the residue was recrystallized from water to get the second crop. The product was identical with oxypurinol (150 mg, 54%).

4-Hydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-one 6

To a mixture of hydrazine monohydrate (5.0 g, 99.9 mmol) and ethanol (5 cm³) was added 4,5-dihydro-4-thioxo-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-one 5 ²³ (1.0 g, 5.95 mmol) and the mixture was heated under reflux for 10 min. After the reaction was complete, the precipitated crystals were collected by filtration and washed with water and EtOH to afford the hydrazino derivative6 (0.70 g, 71%) as colourless powdery crystals, mp > 300 °C; R_f (B) 0.28; ν_max/cm⁻¹ 3360 and 3310 (NH₂), 3200, 3150 and 3100 (NH), 1710 (C[double bond, length half m-dash]O) and δ_max/cm⁻¹ 1670 (NH₂); δ_H [60 MHz; CF₃CO₂D] 8.72 (1 H, s, 3-H); m/z (FAB, 3-nitrobenzyl alcohol matrix) 167 (MH⁺). The product 6 was obtained as a single compound and was used for the following reactions without further purification because it was difficult to purify since it was insoluble in usual solvents.

4,6-Dihydrazino-1H-pyrazolo[3,4-d ]pyrimidine 7

(1) To a stirring solution of 2,4,6-trichloropyrimidine-5-carbaldehyde 1 ²² (3.0 g, 14.2 mmol) in 2-methoxyethanol (20 cm³) at 0 °C was added anhydrous hydrazine (9.1 g, 283.9 mmol) dropwise. Then, the stirred mixture was heated at 100 °C for 5 hours. After the reaction was complete, the precipitated crystals were collected by filtration, washed with water and EtOH and recrystallized from water to afford the dihydrazino derivative7 (1.94 g, 76%) as colourless powdery crystals, mp > 300 °C (Found: C, 33.1; H, 4.55; N, 61.5. C₅H₈N₈·1/5 H₂O requires C, 32.7; H, 4.6; N, 61.0%); R_f (C) 0.20; ν_max/cm⁻¹ 3335, 3270 and 3230 (NH₂), 3180, 3120 and 3110 (NH) and δ_max/cm⁻¹ 1650 and 1640 (NH₂); δ_H [60 MHz; CF₃CO₂D] 8.81 (1H, s, 3-H); m/z (FAB, 3-nitrobenzyl alcohol matrix) 181 (MH⁺).

(2) To a stirring solution of 80% aqueous hydrazine hydrate (20 cm³, 320 mmol) was added 6-chloro-4-hydrazino-1H-pyrazolo[3,4-d ]pyrimidine 2 (2.0 g, 10.8 mmol) and the mixture was heated at 80–90 °C for 4 hours. After the same work-up as noted above, recrystallization of the crude crystals from water gave the dihydrazino derivative7 (1.40 g, 72%).

4,6-Bis(arylmethylidenehydrazino)-1H-pyrazolo[3,4-d ]pyrimidines 8b–d, f. General procedure

A mixture of the dihydrazino derivative 7 (0.60 g, 3.33 mmol) and an appropriate aldehyde (9.99 mmol) in DMF (20 cm³) was stirred at room temperature for 10 hours. After the reaction was complete, the solution was evaporated under reduced pressure and the residue was triturated with EtOH to give crystals, which were collected by filtration and recrystallized from a mixture of EtOH and DMF to afford the corresponding bishydrazones8b–d, f as shown in Tables 1 and 2.

4-Carbamoylhydrazino-6-chloro-1H-pyrazolo[3,4-d ]pyrimidine 9

A mixture of the hydrazinopyrazolopyrimidine 2 (0.5 g, 2.71 mmol) and urea (0.65 g, 10.8 mmol) in 2-ethoxyethanol (25 cm³) was heated under reflux for 5 hours. After the reaction was complete, the solution was evaporated under reduced pressure to afford a solid. The solid was collected by filtration, washed with water and recrystallized from water to afford the carbamoylhydrazino derivative9 (0.37 g, 60%) as colourless powdery crystals, mp > 300 °C (Found: C, 31.1; H, 2.9; N, 43.0. C₆H₆ClN₇O·1/7 H₂O requires C, 31.3; H, 2.75; N, 42.6%); R_f (B) 0.70; ν_max/cm⁻¹ 3410 and 3360 (NH₂), 3200, 3100 and 3040 (NH), 1675 (C[double bond, length half m-dash]O) and δ_max/cm⁻¹ 1675 (NH₂); δ_H [200 MHz; (CD₃)₂SO] 6.25 (2 H, br, NH₂), 7.94 (1 H, s, 3-H), 8.66 and 10.00 (each 1 H, each br s, 2 × NH) and 13.67 (1 H, br s, 1-NH); m/z (FAB, glycerol matrix) 228 (MH⁺) and 230 (MH⁺ + 2).

3-Amino-7H-pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-one 10

(1) The reaction mixture in the same reaction and under the same conditions as in the above preparation for 9 was heated under reflux for 36 hours. After the same work-up as noted above, recrystallization of the crude crystals from water gave the pyrazolotriazolopyrimidine 10 (0.15 g, 29%) as colourless powdery crystals, mp > 300 °C (Found: C, 37.1; H, 3.1; N, 49.9. C₆H₅N₇O·1/4 H₂O requires C, 36.8; H, 2.8; N, 50.1%); R_f (A) 0.57; ν_max/cm⁻¹ 3370 and 3260 (NH₂), 3180 and 3100 (NH), 1720 (C[double bond, length half m-dash]O) and δ_max/cm⁻¹ 1685 (NH₂); δ_H [200 MHz; (CD₃)₂SO] 7.83 (1 H, s, 9-H), 7.94 (2 H, br s, NH₂), 12.27 (1 H, br s, 6-NH) and 13.10 (1 H, br s, 7-NH); m/z (FAB, glycerol matrix) 192 (MH⁺).

(2) A mixture of the pyrazolopyrimidine 9 (0.2 g, 0.88 mmol) and urea (0.16 g, 2.66 mmol) in 2-ethoxyethanol (10 cm³) was heated under reflux for 10 hours. After the same work-up as noted above, recrystallization of the crude crystals from water gave the pyrazolotriazolopyrimidine10 (30 mg, 18%).

4-Alkylidenehydrazino- and 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-ones 11b, e–r. General procedure

A mixture of the hydrazinopyrazolopyrimidine 6 (1.0 g, 6.02 mmol) and an appropriate alkyl aldehyde or aryl aldehyde (9.03 mmol) in DMF (50 cm³) was stirred at room temperature or 40 °C for 10 hours. After the reaction was complete, the solution was evaporated under reduced pressure and the residue was triturated with EtOH or AcOEt to give crystals, which were collected by filtration and recrystallized from an appropriate solvent to afford the corresponding hydrazones11b, e–r as shown in Tables 3 and 4.

7H-Pyrazolo[4,3-e]-1,2,4-triazolo[4,3-c]pyrimidin-5(6H )-one 12a and its 3-substituted derivatives 12b–s. General procedure

(1) Method A: A mixture of an appropriate 4-alkylidenehydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-one 11b, e–r (2.0 mmol) with 70% nitric acid (0.22 cm³, 2.4 mmol) in DMF (30–50 cm³) was heated at 100 °C for 1–9 hours. After the reaction was complete, the precipitated crystals were collected by filtration and combined with further material obtained by concentration of the filtrate under reduced pressure. The crystals were recrystallized from an appropriate solvent to afford the corresponding pyrazolotriazolopyrimidines12b, e–p, r, s as shown in Tables 5 and 6.

(2) Method B: A mixture of an appropriate 4-alkylidenehydrazino- or 4-arylmethylidenehydrazino-1H-pyrazolo[3,4-d ]pyrimidin-6(7H )-one 11e, g, h, k, m–o, q (2.0 mmol) with DEAD (0.35 g, 2.0 mmol) in DMF (50 cm³) was heated under reflux. After heating for several hours, further DEAD (2.0 mmol amounts; total 3–7 equiv.) was added to the heated solution at hourly intervals until the hydrazone 11 disappeared. After the reaction was complete, the solution was evaporated under reduced pressure to leave a solid, which was purified by column chromatography on silica gel using AcOEt as eluent and recrystallized from an appropriate solvent to give the corresponding pyrazolotriazolopyrimidines12e, g, h, k, m–o, q as shown in Tables 5 and 6.

(3) Method C: A mixture of the hydrazinopyrazolopyrimidine 6 (0.60 g, 3.6 mmol) with an appropriate triethyl orthoester (18.0 mmol) in trifluoroacetic acid (9 cm³) was stirred at room temperature for 1 hour. After the reaction was complete, the precipitated crystals were collected by filtration and recrystallized from an appropriate solvent to afford the corresponding pyrazolotriazolopyrimidines12a, b as shown in Tables 5 and 6.

(4) Method D: A mixture of the hydrazinopyrazolopyrimidine 6 (0.60 g, 3.6 mmol) with an appropriate triethyl or trimethyl orthoester (10.8 mmol) in DMF (30–40 cm³) was heated at 100 °C for 1 hour. After the reaction was complete, the solution was evaporated under reduced pressure and the residue was triturated with EtOH or AcOEt to give crystals, which were collected by filtration and recrystallized from an appropriate solvent to afford the corresponding pyrazolotriazolopyrimidines12a–d as shown in Tables 5 and 6.

(5) Method E: A mixture of an appropriate 4-alkylidenehydrazino- or 4-arylmethylidenehydrazino-6-chloro-1H-pyrazolo[3,4-d ]pyrimidine 3a–g (2.0 mmol) with 70% nitric acid (0.9 cm³, 10.0 mmol) in DMF (30–50 cm³) was heated at 100 °C for 1–5 hours. After the reaction was complete, the precipitated crystals were collected by filtration and further crystals were obtained by concentration of the filtrate under reduced pressure. The combined crystals were recrystallized from an appropriate solvent to afford the corresponding pyrazolotriazolopyrimidines12e, g, h, k, m, n, r as shown in Tables 5 and 6.

Xanthine oxidase assay

All test compounds and allopurinol were dissolved in dimethyl sulfoxide (DMSO) and diluted with 50 mM sodium phosphate buffer (pH 7.4) for in vitro experiments. The final concentration of DMSO in the reaction solution was 0.1%.

Bovine milk xanthine oxidase (XO) (10 mU ml⁻¹) was incubated with 100 μM xanthine in the presence and absence of the test compound (0.003–10 μM) at 25 °C for 15 min. Uric acid formation was determined by absorbance at 292 nm using a Hitachi 228-A spectrophotometer, and the inhibition rate (%) for the formation of uric acid and IC₅₀ values of the test compounds were determined. The inhibition rate (I) of the test compound at each concentration was calculated by eqn. (1), where T is the optical density of a solution of xanthine and XO, D is the optical density of a solution of test compound, xanthine and XO and D_B is the optical density of a solution of test compound and XO.

I (%) = 100 − [(D − D_B)/T] × 100 (1)

The inhibitory activity of allopurinol against bovine milk xanthine oxidase was also examined as a positive control. Each experiment was repeated at least twice at different concentrations (0.003–10 μM). The values of IC₅₀, i.e. the μM concentration of inhibitor necessary for 50% inhibition, were determined from the dose–response curve from the relation of the logarithmic concentration (μM) and the inhibition (%).

Acknowledgements

We are grateful to the SC-NMR Laboratory of Okayama University for 200 MHz proton NMR experiments. This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 09680570) from the Japan Society for the Promotion of Science.

References

1. Part 2 T. Nagamatsu, H. Yamasaki, T. Fujita, K. Endo and H. Machida, J. Chem. Soc., Perkin Trans. 1, 1999, 3117.
2. R. W. Rundles, J. B. Wyngaarden, G. H. Hitchings, G. B. Elion and H. R. Silberman, Trans. Assoc. Am. Physicians, 1963, 76, 126.
3. T. F. Yü and A. B. Gutman, Am. J. Med., 1964, 37, 885.
4. J. R. Klinenberg, S. E. Goldfinger and J. E. Seegmiller, Ann. Intern. Med., 1965, 62, 639.
5. G. B. Elion, Ann. Rheumat. Dis., 1966, 25, 608.
6. G. B. Elion, S. Callahan, H. Nathan, S. Bieber, R. W. Rundles and G. H. Hitchings, Biochem. Pharmacol., 1963, 12, 85.
7. J. L. Young, R. B. Boswell and A. S. Nies, Arch. Intern Med., 1974, 134, 553.
8. K. R. Hande, R. M. Noone and W. J. Stone, Am. J. Med., 1984, 76, 47.
9. D. E. Duggan, R. M. Noll, J. E. Baer, F. C. Novello and J. J. Baldwin, J. Med. Chem., 1975, 18, 900.
10. R. L. Wortmann, A. S. Ridolfo, R. W. Lightfoot Jr. and I. H. Fox, J. Rheumatol., 1985, 12, 540.
11. A. Bindoli, M. Valente and L. Cavallini, Pharmacol. Res. Commun., 1985, 17, 831.
12. T. Spector, W. W. Hall, D. J. Porter, C. U. Lambe, D. J. Nelson and T. A. Krenitsky, Biochem. Pharmacol., 1989, 38, 4315.
13. S. Sato , K. Tatsumi and T. Takahashi , Purine and Pyrimidine Metabolism in Man VII, Part A: Chemotherapy, ATP Depletion and Gout, eds. R. A. Harkness, G. B. Elion and N. Zöllner, Plenum Press, New York, 1991, p. 135..
14. Y. Osada, M. Tsuchimoto, H. Fukushima, K. Takahashi, S. Kondo, M. Hasegawa and K. Komoriya, Eur. J. Pharmacol., 1993, 241, 183.
15. G. Biagi, I. Giorgi, O. Livi, V. Scartoni, I. Tonetti and L. Costantino, Farmaco, 1995, 50, 257.
16. T. Nagamatsu , Y. Watanabe , K. Endo and M. Imaizumi , PCT Int. Appl. WO 96 26,208/1996(Chem. Abstr., 1996, 125, 247848j)..
17. T. Nagamatsu , T. Abiru , Y. Watanabe and K. Endo , Jpn. Kokai Tokkyo Koho JP 07,242,694/1995(Chem. Abstr., 1996, 124, 117896s)..
18. T. Nagamatsu and T. Fujita, Chem. Commun., 1999, 1461.
19. G. A. Bhat and L. B. Townsend, J. Chem. Soc., Perkin Trans. 1, 1981, 2387.
20. F. Gatta, M. Luciani and G. Palazzo, J. Heterocycl. Chem., 1989, 26, 613.
21. U. D. Treuner and H. Breuer , USP 4 053 474, 1977(Chem. Abstr., 1977, 88, 37826s); USP 4 124 764, 1978 (Chem. Abstr., 1978, 90, 87508b); Ger. Offen. 2 838 029, 1979 (Chem. Abstr., 1979, 91, 39492r)..
22. F. Yoneda, Y. Sakuma, S. Mizumoto and R. Ito, J. Chem. Soc., Perkin Trans. 1, 1976, 1805.
23. R. K. Robins, J. Am. Chem. Soc., 1956, 78, 784.

---

Footnote

† We have found that some derivatives exhibited poor inhibitory activities against the proliferation of T-cell acute lymphoblastic leukemia (CCRF-HSB-2): the IC₅₀ for 11j, 11 μM; for 11o, 45 μM; for 12i, 25 μM; for 12j, 14 μM; for 12k, 35 μM; for 12l, 23 μM; for 12n, 36 μM; for 12q, 34 μM; for 12r, 36 μM; for 12s, 35 μM and for arabinosylcytosine, 0.061 μM.

---