Synthesis, anti-varicella-zoster virus and anti-cytomegalovirus activity of quinazoline-2,4-diones containing isoxazolidine and phosphonate substructures

Dorota G Piotrowska; Graciela Andrei; Dominique Schols; Robert Snoeck; Magdalena Łysakowska

doi:10.1016/j.ejmech.2016.10.002

. 2016 Oct 4;126:84–100. doi: 10.1016/j.ejmech.2016.10.002

Synthesis, anti-varicella-zoster virus and anti-cytomegalovirus activity of quinazoline-2,4-diones containing isoxazolidine and phosphonate substructures

Dorota G Piotrowska ^a,^∗, Graciela Andrei ^b, Dominique Schols ^b, Robert Snoeck ^b, Magdalena Łysakowska ^a

PMCID: PMC7125519 PMID: 27750154

Abstract

Cycloadditions of N-substituted C-(diethoxyphosphoryl)nitrones to N-allylated quinazoline-2,4-diones functionalized at N3 with substituted benzoyl or benzyl groups proceeded with moderate to good diastereoselectivities (d.e. 28–68%). The synthesized isoxazolidine phosphonates were assessed for the antiviral activity against a broad range of DNA and RNA viruses. Compounds trans-13c, cis-13c/trans-13c (86:14), cis-15b/trans-15b (87:13) and trans-15d/cis-15d (95:5) exhibited the highest activity toward both TK⁺ and TK⁻ VZV strains (mean EC₅₀ values in the range of 3.0–8.7 μM). The EC₅₀'s for isoxazolidines trans-12a, cis-12a, cis-13a, trans-13d, cis-15a/trans-15a (50:50) ranged between 6.9 and 8.5 μM for VZV TK⁺ strain and between 10.7 and 13.2 μM for VZV TK^– strain. The isoxazolidine phosphonates cis-15/trans-15 having benzyl substituents both at N3 of the quinazoline-2,4-dione skeleton and at N2 of the isoxazolidine ring displayed some anti-cytomegalovirus potency but at the same time showed significant cytostatic activity for human embryonic lung fibroblasts (used to carry out the antiviral assays) as well as for other cell lines (i.e. CEM, L1210, HeLa and HMEC-1).

Keywords: Phosphonates; Isoxazolidines; Quinazoline-2,4-diones; Antiviral; Cytostatic

Graphical abstract

Highlights

•
1,3-Disubstituted quinazoline-2,4-diones containing isoxazolidine-3-yl-3-phosphonate moiety.
•
Anti-varicella-zoster virus active substituted quinazoline-2,4-diones.
•
Anti-cytomegalovirus active substituted quinazoline-2,4-diones.
•
Cytostatic potency of substituted quinazoline-2,4-diones.

1. Introduction

Herpesviruses are widespread among humans and may cause many diseases. The primary infection is usually followed by a life-long latency of the virus and its reactivation usually occurs during immunosuppression of the host. Infection with varicella-zoster virus (VZV) results in varicella (chickenpox) which usually takes a mild course in children but may be more severe in adults. Later on, after establishing latency in neural tissues, the virus can reactivate causing herpes zoster (shingles) which is often accompanied by neuralgic pain and can lead to post-herpetic neuralgia (PHN) as well as other complications such as loss of vision (zoster ophthalmicus) [1], [2].

In most cases immunocompetent patients infected by herpesviruses do not require antiviral therapy. However, reactivation of the virus is of significant concern in immunocompromised individuals, e.g. recipients of solid-organ and hematopoietic stem cell transplant, patients under aggressive chemotherapy or individuals with acquired immunodeficiency syndrome (AIDS). Under these circumstances, efficient antiviral drugs are of crucial importance. Effective treatments of herpesviridae species, including herpes simplex virus (HSV), VZV and human cytomegalovirus (HCMV) [3], [4] are available but they are hampered by emergence of drug resistance and significant drug toxicities for some anti-herpesvirus agents (such as ganciclovir, foscavir and cidofovir). Four compounds are currently licensed for the treatment of VZV infections, namely acyclovir, valaciclovir, famciclovir and brivudin [5], [6]. Regrettably, AIDS patients often do not respond well to acyclovir therapy or other antiviral drugs due to the emergence of thymidine kinase-deficient or thymidine kinase-altered mutations of VZV [7], [8]. Therefore, the extensive search for new anti-VZV agents with superior efficacy compared to currently approved drugs is of high importance.

Numerous structurally diversified compounds have already been synthesized and tested as new potential anti-VZV agents including bicyclic nucleoside analogues, non-nucleoside DNA polymerase inhibitors and N-(α-methylbenzyl)-N-arylthiourea analogues (Fig. 1 ) [9], [10], [11], [12], [13], [14], [15], [16], [17], [18].

Fig. 1 — Examples of anti-VZV active compounds.

On the other hand, the antiviral activity of several 1,3-disubstituted quinazoline-2,4-diones (Fig. 2 ) has been discovered in recent years [19]. A 3-benzylquinazolin-2,4-dione derivative 1 was reported to posses the anti-HIV-1 activity in MT-4 cells and inhibited the recombinant RT in vitro [20]. A quinazolinone-2,4-dione 2 was a potent inhibitor of RSV-induced cytopathic effect (EC₅₀ = 2.14 μM) [21]. Several other analogues, namely 3–5, proved to be very active toward Respiratory Syncytial Virus (RSV) [21]. Recently, the N3-benzoylquinazolinonedione moiety was successfully incorporated as a nucleobase mimetic into the 1,2,3-triazole analogues of nucleotides 6 [22] and 7 [23]. While the compound 6 showed a moderate activity against both herpes simplex viruses (HSV-1 and HSV-2) (EC₅₀ = 17 μM) as well as feline herpes virus (EC₅₀ = 24 μM), its dihydroxylated derivative (1R,2S)-7 proved to be even more potent (EC₅₀ = 2.9, 4 and 4 μM toward HSV-1, HSV-2 and feline herpes virus, respectively), while the enantiomer (1S,2S)-16 was inactive [23]. From several functionalized quinazoline-2,4-diones studied as allosteric inhibitors of the NS5B polymerase compounds 8–10 exhibited the highest affinity to the enzyme [24]. On the basis of these observations one may conclude that for the antiviral activity of quinazoline-2,4-diones substitution at N3 with aryl, benzyl or benzoyl groups is beneficial.

2. Results and discussion

2.1. Chemistry

Recently, we successfully accomplished the syntheses of homonucleoside analogues 11 which proved inactive against a broad spectrum of DNA and RNA viruses while some of them appeared slightly cytostatic toward several cancerous cell lines [25]. However, later on they were additionally screened for inhibition of VZV and HCMV replication and two compounds 11a (B = N-benzoyluracil) and 12a (R = benzoyl, R′ = methyl) showed noticeable activity toward VZV (Table 3). Based on this discovery we designed a new series of analogues (Scheme 1 ) installing at N3 of the quinazoline-2,4-dione skeleton either substituted benzoyl groups (compounds 2 and 3) or substituted benzyl residues (compounds 14 and 15).

Table 3.

Antiviral activity and cytotoxicity against varicella-zoster virus (VZV) in HEL cell cultures.

Compound	R′	R	Antiviral activity EC₅₀ (μM)^a		Cytotoxicity (μM)
Compound	R′	R	TK⁺ VZV strain	TK⁻ VZV strain	Cell morphology (MCC)^b	Cell growth (CC₅₀)^c
trans-11a	Me		83.6	>100	>100	n.d.
cis-11a	Me		65.7	88.4	>100	n.d.
trans-12a	Me	C₆H₅	7.5 ± 2.1^d	13.7 ± 4.7	≥100 ± 0	>100 ± 0
cis-12a	Me	C₆H₅	7.7 ± 2.4	10.9 ± 1.6	>100 ± 0	>100 ± 0
trans-13a	Bn	C₆H₅	8.5 ± 3.8	>20 ± 0	100 ± 0	28.9 ± 3.1
cis-13a	Bn	C₆H₅	8.5 ± 0.3	10.8 ± 1.7	100 ± 0	16.34 ± 0
trans-12b	Me	2-F-C₆H₄	36.57	34.2	>100	n.d.
cis-12b/trans-12b (87:13)	Me	2-F-C₆H₄	28.99	25.62	>100	n.d.
trans-13b/cis-13b (90:10)	Bn	2-F-C₆H₄	16.7 ± 4.7	20 ± 0	100 ± 0	21.4 ± 1.0
trans-12c	Me	3-F-C₆H₄	16.7 ± 4.7	15.0 ± 3.3	>100 ± 0	16.5 ± 2.5
cis-12c/trans-12c (94:6)	Me	3-F-C₆H₄	7.8 ± 3.6	21.4 ± 13.0	>100 ± 0	30.0 ± 11.2
trans-13c	Bn	3-F-C₆H₄	8.7 ± 4.3	8.5 ± 4.6	100 ± 0	9.8 ± 2.3
cis-13c/trans-13c (86:14)	Bn	3-F-C₆H₄	6.0 ± 6.9	8.5 ± 9.6	100 ± 0	19.3 ± 5.3
trans-12d	Me	4-F-C₆H₄	6.9 ± 5.3	10.7 ± 0.3	≥100 ± 0	14.7 ± 2.1
cis-12d	Me	4-F-C₆H₄	26.15	24.46	>100	n.d.
trans-13d	Bn	4-F-C₆H₄	7.5 ± 0.1	8.3 ± 0.2	100 ± 0	12.7 ± 2.7
cis-13d/trans-13d (85:15)	Bn	4-F-C₆H₄	7.4 ± 0.8	7.6 ± 1.1	100 ± 0	12.3 ± 2.0
trans-14a	Me	C₆H₅	>20	>20	100	n.d.
cis-14a/trans-14a (75:25)	Me	C₆H₅	>100	>100	>100	n.d.
trans-15a/cis-15a (90:10)	Bn	C₆H₅	>20	>20	100	n.d.
cis-15a/trans-15a (50:50)	Bn	C₆H₅	7.3 ± 1.0	13.2 ± 9.7	100 ± 0	9.0 ± 0.7
trans-14b	Me	2-F-C₆H₄	>20	>20	100	n.d.
cis-14b	Me	2-F-C₆H₄	58.48	>100	>100	n.d.
trans-15b	Bn	2-F-C₆H₄	4	>20	20	n.d.
cis-15b/trans-15b (87:13)	Bn	2-F-C₆H₄	4.7 ± 3.8	5.1 ± 1.6	100 ± 0	11.8 ± 4.6
trans-14c	Me	3-F-C₆H₄	55.7	>100	>100	n.d.
cis-14c/trans-14c (97:3)	Me	3-F-C₆H₄	7.0 ± 1.4	27.1 ± 10.0	≥100 ± 0	36.8 ± 3.1
trans-15c/cis-15c (90:10)	Bn	3-F-C₆H₄	>20	20	100	n.d.
cis-15c/trans-15c (80:20)	Bn	3-F-C₆H₄	>20	>20	100	n.d.
trans-14d	Me	4-F-C₆H₄	66.87	>20	10	n.d.
cis-14d/trans-14d (75:25)	Me	4-F-C₆H₄	35.54	25.17	100	n.d.
trans-15d/cis-15d (95:5)	Bn	4-F-C₆H₄	3.0 ± 2.3	3.6 ± 2.9	100 ± 0	6.6 ± 0
cis-15d/trans-15d (75:52)	Bn	4-F-C₆H₄	>4	>4	20	n.d.
Acyclovir			0.8 ± 0.1	50.3 ± 14.9	>440 ± 0	440 ± 0
Brivudin			0.005 ± 0.007	22.7 ± 3.1	>300 ± 0	300 ± 0

Open in a new tab

n.d. – not determined.

Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU).

Minimum cytotoxic concentration that causes a microscopically detectable alternation of cell morphology.

Cytotoxic concentration required to reduce cell growth by 50%.

Results are mean values ± STDEV of two independent experiments.

Scheme 1 — Retrosynthesis of quinazoline-2,4-diones 12–15.

As previously reported [25], N ¹-allyl-N ³-benzoylquinazoline-2,4-dione 18a was obtained in three steps in 20% overall yield starting from quinazoline-2,4-dione employing bis-N ¹,N ³-benzoylation with benzoyl chloride followed by the selective N ¹-debenzoylation and subsequent allylation. However, this procedure appeared tedious and the N ¹-debenzylation step was the least effective. For this reason another strategy for the syntheses of N ¹-allyl-N ³-benzoylquinazoline-2,4-diones 18a-18d was designed which relied on N-allylation of the commercially available isatoic anhydride 20 followed by a subsequent condensation of compound 21 with urea [26], [27] and concluded with N³-benzoylation of the resulted N-allylquinazoline-2,4-dione 22 with selected benzoyl chlorides (Scheme 2 ). Moreover, under these circumstances compounds 19a-d could be obtained in one step by benzylation of allylquinazoline-2,4-dione 22 (Scheme 2).

Scheme 2 — Synthesis of quinazoline-2,4-diones **18a-d** and **19a-d**.

1,3-Dipolar cycloadditions of nitrones 16 (R′ = Me) or 7 (R′ = Bn) with the respective N ¹-allyl-N ³-benzoylquinazoline-2,4-diones 18a-d were carried out at 60 °C in toluene or toluene-ethanol mixtures as solvents and afforded mixtures of diastereoisomeric isoxazolidines trans-12 and cis-12 or trans-13 and cis-13 (Scheme 3 , Table 1 ) with the trans-isomer predominating. The cis/trans ratios of diastereoisomeric products were determined on the basis of the ³¹P NMR spectral data. The reactions proceeded with moderate diastereoselectivities (d.e. 28–60%) and with good to excellent overall yields. The isolation of pure isomers was successfully accomplished chromatographically for major isomers trans-12a, trans-12b, trans-12c, trans-12d, trans-13c and trans-13d but also for minor isomers cis-12a, cis-12d and cis-13a.

Scheme 3 — Reaction and conditions: a) toluene or toluene-ethanol, 60 °C, 72 h.

Table 1.

Cycloadditions of the nitrone 16/17 and N¹-allyl-N³-benzoylquinazoline-2,4-diones 18a-d.

Nitrone 16/17 (R′)	Alkene 18 (R)	cis:trans ratio	Yield (%)
16 (Me)¹⁷	18a (Ph) [25]	20:80	cis-12a (11)^a, trans-12a (43)^a, cis-12a + trans-12a (25)^b
16 (Me)	18b (2-F-C₆H₄)	36:64	trans-12b (43)^a, cis-12b + trans-12b (49)^b
16 (Me)	18c (3-F-C₆H₄)	20:80	trans-12c (47)^a, cis-12c + trans-12c (46)^b
16 (Me)	18d (4-F-C₆H₄)	25:75	cis-12d (4.5)^a, trans-12d (25)^a, cis-12d + trans-12d (53)^b
17 (Bn)	18a (Ph)	27:73	cis-13a (7.1)^a, trans-13a (3.6)^a, cis-13a + trans-13a (74)^b
17 (Bn)	18b (2-F-C₆H₄)	32:68	cis-13b + trans-13b (92)^b
17 (Bn)	18c (3-F-C₆H₄)	28:72	trans-13c (13)^a, cis-13c + trans-13c (81)^b
17 (Bn)	18d (4-F-C₆H₄)	28:72	trans-13d (27)^a, cis-13d + trans-13d (61)^b

Open in a new tab

Yield of the pure isomer.

Yield of the pure mixture of cis- and trans-isomers.

To eliminate rigidity within the substituted quinazoline-2,4-dione moiety benzoyl substituents at N3 were replaced by the functionalized benzyl residues. 1,3-Dipolar cycloadditions of nitrones 16 (R′ = Me) or 7 (R′ = Bn) with the respective N ¹-allyl-N ³-benzylquinazoline-2,4-diones 19a-d were carried out under conditions already described for compounds 18. Diastereoisomeric cycloadducts trans-14 and cis-14 or trans-15 and cis-15 (Scheme 4, Table 2 ) were formed in good to excellent overall yields and with moderate diastereoselectivities (d.e. 28–60%) which were slightly higher for reactions of the nitrone 7 (R′ = Bn). Chromatographic isolation of pure isomers was achieved for trans-14a, cis-14b, trans-14b, trans-14c and trans-14d.

Scheme 4 — Reaction and conditions: a) toluene or toluene-ethanol, 60 °C, 72 h.

Table 2.

Cycloadditions of the nitrones 16 or 17 and N¹-allyl-N³-benzylquinazoline-2,4-diones 19a-d.

Nitrone 16/17 (R′)	Alkene 19 (R)	cis:trans ratio	Yield (%)
16 (Me)	19a (Ph)	22:78	trans-14a (37)^a, cis-14a + trans-14a (58)^b
16 (Me)	19b (2-F-C₆H₄)	22:78	cis-14b (6.5)^a, trans-14b (29)^a, cis-14b + trans-14b (56)^b
16 (Me)	19c (3-F-C₆H₄)	16:84	trans-14c (21)^a, cis-14c + trans-14c (75)^b
16 (Me)	19d (4-F-C₆H₄)	23:77	trans-14d (30)^a, cis-14d + trans-14d (56)^b
17 (Bn)	19a (Ph)	34:66	cis-15a + trans-15a (92)^b
17 (Bn)	19b (2-F-C₆H₄)	31:69	cis-15b (3.6)^a, cis-15b + trans-15b (86)^b
17 (Bn)	19c (3-F-C₆H₄)	35:65	cis-15c + trans-15c (96)^b
17 (Bn)	19d (4-F-C₆H₄)	33:67	cis-15d + trans-15d (95)^b

Open in a new tab

Yield of the pure isomer.

Yield of the pure mixture of cis- and trans-isomers.

2.2. Antiviral and cytostatic evaluation

The pure isomers of quinazoline-2,4-dione - conjugates [trans-11a, cis-11a, trans-12a, cis-12a, trans-13a, cis-13a, trans-12b, trans-12c, trans-13c, trans-12d, cis-12d, trans-13d, trans-14a, trans-14b, cis-14b, trans-15b, trans-14c, trans-14d] and the respective mixtures of cis/trans isomers [cis-12b/trans-12b (87:13), trans-13b/cis-13b (90:10), cis-2c/trans-2c (94:6), cis-13c/trans-13c (86:14), cis-13d/trans-13d (85:15), cis-14a/trans-14a (75:25), trans-15a/cis-15a (90:10), cis-15a/trans-15a (50:50), cis-15b/trans-15b (87:13), cis-14c/trans-14c (97:3), trans-15c/cis-15c (90:10), cis-15c/trans-15c (80:20), cis-14d/trans-14d (75:25), trans-15d/cis-15d (95:5), cis-15d/trans-15d (75:52)] were screened as inhibitors of a wide variety of DNA and RNA viruses using the following cell-based assays: (a) human embryonic lung (HEL) cells: herpes simplex virus-1 (KOS strain), herpes simplex virus-2 (G strain), thymidine kinase deficient (acyclovir resistant) herpes simplex virus-1 (TK^– KOS ACV^r strain), vaccinia virus, adenovirus-2, vesicular stomatitis virus, human coronavirus (229E), cytomegalovirus (AD-169 strain and Davis strain), varicella-zoster virus (TK⁺ VZV Oka strain and TK⁻ VZV 07-1 strain); (b) HeLa cell cultures: vesicular stomatitis virus, Coxsackie virus B4 and respiratory syncytial virus; (c) Vero cell cultures: parainfluenza virus 3, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, yellow fever virus; (e) Crandell-Rees feline kidney (CRFK) cell cultures: feline corona virus (FIPV) and feline herpes virus (FHV) and (d) Madin Darby canine kidney (MDCK) cell cultures: influenza A virus (H1N1 and H3N2 subtypes) and influenza B virus. Ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, zanamivir, alovudine, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight 10000, DS-10000), mycophenolic acid, Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA) were used as the reference compounds. The antiviral activity was expressed as the EC₅₀: the compound concentration required to reduce virus plaque formation (VZV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses).

Several synthesized quinazoline-2,4-diones inhibited the replication of both TK⁺ and TK⁻ VZV strains (Table 3 ). A 95:5 trans-15d/cis-15d mixture and a 87:13 cis-15b/trans-15b mixture emerged as the most active derivatives with EC₅₀'s of, respectively, 4.7 μM and 3 μM (VZV TK⁺ strain) and of 3.6 μM and 5.1 μM (VZV TK⁻ strain). These two quinazoline-2,4-diones were 4–6-fold less active against the TK⁺ virus but proved to be 10–14-fold more active against the TK^– strain when compared to the reference drug acyclovir. These data clearly indicate that these novel derivatives do not require activation by the viral TK. Although these quinazoline-2,4-diones did not significantly altered the morphology of cells in the antiviral assays, they showed considerable cytostatic activity (in the same range as the antiviral activity).

Compounds trans-13c, a 86:14 cis-13c/trans-13c mixture, trans-13d, and a 85:15 cis-13d/trans-13d mixture inhibited both VZV TK⁺ and TK^– viruses with EC₅₀'s in the range of 6.0–8.5 μM. Compounds trans-12a, cis-12a, cis-13a, trans-12d and a 50:50 cis-15a/trans-15a mixture also proved active against VZV TK⁺ strain (EC₅₀'s of 6.9–7.5 μM) and VZV TK⁻ strain (EC₅₀'s of 10–14 μM), slightly exceeding in potency against VZV TK⁻ strains the reference drugs acyclovir and brivudin (EC₅₀ = 50.3 μM and 22.7, respectively). However, majority of the studied compounds exhibited significant cytotoxicity and the 95:5 trans-15d/cis-15d mixture reduced cell growth (CC₅₀) at concentration as low as 6.6 μM which was almost two orders of magnitude lower than that for acyclovir (CC₅₀ = 440 μM).

Among the investigated quinazoline-2,4-diones, the N3-benzoylated compounds (cis- and trans-12/13) were found inactive toward both human cytomegalovirus (HCMV) strains. On the other hand, isoxazolidine phosphonates having benzyl substituents both at N3 of the quinazoline-2,4-dione skeleton and at N2 of the isoxazolidine ring (cis- and trans-15) showed weak antiviral activity with EC₅₀ in the range of ≥3–≥14.5 μM (Table 4 ).

Table 4.

Antiviral activity and cytotoxicity against human cytomegalovirus in HEL cell cultures.

Compound	R′	R	Antiviral activity EC₅₀ (μM)^a		Cytotoxicity (μM)
Compound	R′	R	AD-169 strain	Davis strain	Cell morphology (MCC)^b	Cell growth (CC₅₀)^c
trans-11a	Me		>100	>100	100	n.d.
cis-11a	Me		>100	>100	100	n.d.
trans-12a	Me	C₆H₅	>100	>100	100	n.d.
cis-12a	Me	C₆H₅	>100	>100	100	n.d
trans-13a	Bn	C₆H₅	>20	>20	100	n.d.
cis-13a	Bn	C₆H₅	>20	>20	100	n.d.
trans-12b	Me	2-F-C₆H₄	>20	66.87	100	n.d.
cis-12b/trans-12b (87:13)	Me	2-F-C₆H₄	>100	63.14	100	n.d.
trans-13b/cis-13b (90:10)	Bn	2-F-C₆H₄	>20	>20	100	n.d.
trans-12c	Me	3-F-C₆H₄	>20	>20	100	n.d
cis-12c/trans-12c (94:6)	Me	3-F-C₆H₄	>100	>100	100	n.d.
trans-13c	Bn	3-F-C₆H₄	>20	>20	20	n.d.
cis-13c/trans-13c (86:14)	Bn	3-F-C₆H₄	>20	>20	100	n.d.
trans-12d	Me	4-F-C₆H₄	>20	>20	100	n.d.
cis-12d	Me	4-F-C₆H₄	>20	>20	100	n.d.
trans-13d	Bn	4-F-C₆H₄	>20	>20	20	n.d
cis-13d/trans-13d (85:15)	Bn	4-F-C₆H₄	>20	>20	20	n.d.
trans-14a	Me	C₆H₅	>100	>100	100	n.d
cis-14a/trans-14a (75:25)	Me	C₆H₅	>100	>100	100	n.d.
trans-15a/cis-15a (90:10)	Bn	C₆H₅	≥14.5 ± 7.8^d	13.1 ± 3.1	100 ± 0	41.7 ± 10.8
cis-15a/trans-15a (50:50)	Bn	C₆H₅	≥3.0 ± 1.4	6.5 ± 3.5	100 ± 0	9.0 ± 0.7
trans-14b	Me	2-F-C₆H₄	>100	100	20	n.d.
cis-14b	Me	2-F-C₆H₄	>100	100	100	n.d.
trans-15b	Bn	2-F-C₆H₄	>20	>4	20	n.d
cis-15b/trans-15b (87:13)	Bn	2-F-C₆H₄	8.94 ± 0	≥6.5 ± 3.5	100 ± 1	11.8 ± 4.6
trans-14c	Me	3-F-C₆H₄	76.47	63.14	>100	n.d.
cis-14c/trans-14c (97:3)	Me	3-F-C₆H₄	>20	44.72	100	n.d.
trans-15c/cis-15c (90:10)	Bn	3-F-C₆H₄	9.9 ± 1.4	8.94 ± 1	100 ± 0	20.8 ± 4.7
cis-15c/trans-15c (80:20)	Bn	3-F-C₆H₄	>20	>20	20	n.d.
trans-14d	Me	4-F-C₆H₄	>100	100	100	n.d.
cis-14d/trans-14d (75:25)	Me	4-F-C₆H₄	>20	>20	100	n.d.
trans-15d/cis-15d (95:5)	Bn	4-F-C₆H₄	≥6.5 ± 3.5	8.94 ± 0	100 ± 0	6.6 ± 0
cis-15d/trans-15d (75:52)	Bn	4-F-C₆H₄	>4	>4	20	n.d.
Ganciclovir			14.9 ± 8.1	6.5 ± 2.5	>350 ± 0	>350 ± 0
Cidofovir			1.44 ± 0.56	0.81 ± 0.07	>300 ± 0	>300 ± 0

Open in a new tab

n.d. – not determined.

Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU).

Minimum cytotoxic concentration that causes a microscopically detectable alternation of cell morphology.

Cytotoxic concentration required to reduce cell growth by 50%.

Results are mean values ± STDEV of two independent experiments.

Preliminary structure-activity relationship observations revealed a lack of significant differences in activity of cis vs. trans isoxazolidines and higher potency of isoxazolidines carrying N-benzyl substituents in comparison with their N-methyl counterparts especially well pronounced for the N ³-benzylquinazoline-2,4-diones 15. For the active compounds the introduction of a fluorine atom into the benzene ring either in benzyl or benzoyl residues did not improve their efficacy. While the quinazoline-2,4-diones substituted at N3 with benzoyl and benzyl moieties were found effective against VZV, only those carrying substituted benzyl components proved active toward HMCV.

All synthesized isoxazolidine phosphonates were also subjected to antiviral screening with other viruses, but only compound trans-12d appeared slightly active against other herpesviruses, adenovirus-2 and human Coronavirus (Table 5 ).

Table 5.

Antiviral activity and cytotoxicity in HEL cell cultures.

Compound	R′	R	Antiviral activity EC₅₀ (μM)^b					Minimum cytotoxic concentration (μM)^a
Compound	R′	R	Herpes simplex virus-1 (KOS)	Herpes simplex virus-2 (G)	Herpes simplex virus-1 TK^– KOS ACV^r	Adeno virus-2	Human Coronavirus (229E)	Minimum cytotoxic concentration (μM)^a
trans-12d	Me	4-F-C₆H₄	39.0 ± 15.6^c	12.0 ± 0	9.0 ± 1.4	17.5 ± 3.5	39.5 ± 7.8	≥100 ± 0
Brivudine			0.11	146	250	–	–	>250
Cidofovir			2	2	3.8	10	–	>250
Acyclovir			0.2	0.4	250	–	–	>250
Ganciclovir			0.032	0.055	4	–	–	>100
Zalcitabine			–	–	–	7.2	–	>250
Alovudine			–	–	–	10	–	>250
UDA			–	–	–	–	0.4	≥100
Ribavirin			–	–	–	–	112	≥250

Open in a new tab

Required to cause a microscopically detectable alteration of normal cell morphology.

Required to reduce virus-induced cytopathogenicity by 50%.

Results are mean values ± STDEV of two independent experiments.

2.3. Cytostatic activity

The 50% cytostatic inhibitory concentration (IC₅₀) causing a 50% decrease in cell proliferation was determined against murine leukemia L1210, human lymphocyte CEM, human cervix carcinoma HeLa and immortalized human dermal microvacsular endothelial cells (HMEC-1) (Table 6 ). Among all tested compounds only quinazoline-2,4-diones trans-15/cis-15 having benzyl substituents at N3 in the quinazolinone core and the benzyl group at N2 of the isoxazolidine unit showed significant cytostatic activity toward the tested cell lines. For the CEM cell line, these derivatives were as active as the reference drug 5-fluorouracil. It was noticed that the replacement of the benzyl component within the isoxazolidine moiety for the methyl group (trans-15/cis-15 vs. the respective trans-14/cis-14) resulted in decrease in potency by roughly an order of magnitude.

Table 6.

The inhibitory effect of the tested compounds against the proliferation of murine leukemia (L1210), human T-lymphocyte (CEM), human cervix carcinoma (HeLa) and immortalized human dermal microvascular endothelial cells (HMEC-1).

Compound	R′	R	IC₅₀^a (μM)
Compound	R′	R	L1210	CEM	HeLa	HMEC-1
trans-11a[25]	Me		>200	>200	>200	n.d^b
cis-11a[25]	Me		>200	>200	>200	n.d
trans-12a[25]	Me	C₆H₅	≥159	70 ± 22^c	96 ± 11	n.d.
cis-12a[25]	Me	C₆H₅	>200	74 ± 33	>200	n.d.
trans-13a	Bn	C₆H₅	154 ± 54	≥250	>250	>250
cis-13a	Bn	C₆H₅	155 ± 61	≥250	>250	>250
trans-12d	Me	4-F-C₆H₄	>250	>250	>250	>250
cis-12d	Me	4-F-C₆H₄	>250	>250	>250	>250
trans-13d	Bn	4-F-C₆H₄	123 ± 40	170 ± 22	>250	≥250
cis-13d/trans-13d (85:15)	Bn	4-F-C₆H₄	105 ± 46	132 ± 45	>250	≥250
trans-12b	Me	2-F-C₆H₄	>250	>250	>250	>250
cis-12b/trans-12b (87:13)	Me	2-F-C₆H₄	>250	>250	>250	>250
trans-13b/cis-13b (90:10)	Bn	2-F-C₆H₄	155 ± 79	158 ± 13	≥250	>250
trans-12c	Me	3-F-C₆H₄	228 ± 30	>250	>250	>250
cis-12c/trans-12c (94:6)	Me	3-F-C₆H₄	>250	>250	>250	>250
trans-13c	Bn	3-F-C₆H₄	170 ± 105	224 ± 37	>250	>250
cis-13c/trans-13c (86:14)	Bn	3-F-C₆H₄	185 ± 89	166 ± 118	>250	>250
trans-14a	Me	C₆H₅	141 ± 28	124 ± 9	119 ± 18	235 ± 22
cis-14a/trans-14a (75:25)	Me	C₆H₅	146 ± 1	104 ± 19	95 ± 41	222 ± 39
trans-15a/cis-15a (90:10)	Bn	C₆H₅	17 ± 7	15 ± 4	73 ± 9	28 ± 3
cis-15a/trans-15a (50:50)	Bn	C₆H₅	18 ± 1	10 ± 6	33 ± 21	28 ± 1
trans-14b	Me	2-F-C₆H₄	196 ± 74	99 ± 8	74 ± 28	189 ± 38
cis-14b	Me	2-F-C₆H₄	203 ± 8	181 ± 20	128 ± 40	≥250
trans-15b	Bn	2-F-C₆H₄	68 ± 4	98 ± 4	79 ± 4	145 ± 1
cis-15b/trans-15b (87:13)	Bn	2-F-C₆H₄	17 ± 1	20 ± 3	17 ± 0	23 ± 9
trans-14c	Me	3-F-C₆H₄	132 ± 5	100 ± 16	88 ± 9	152 ± 1
cis-14c/trans-14c (97:3)	Me	3-F-C₆H₄	118 ± 8	77 ± 20	86 ± 13	149 ± 1
trans-15c/cis-15c (90:10)	Bn	3-F-C₆H₄	17 ± 5	17 ± 2	63 ± 18	28 ± 4
cis-15c/trans-15c (80:20)	Bn	3-F-C₆H₄	89 ± 9	49 ± 12	73 ± 11	204 ± 66
trans-14d	Me	4-F-C₆H₄	126 ± 6	93 ± 10	82 ± 16	158 ± 8
cis-14d/trans-14d (75:25)	Me	4-F-C₆H₄	≥250	173 ± 51	158 ± 44	≥250
trans-15d/cis-15d (95:5)	Bn	4-F-C₆H₄	17 ± 0	13 ± 1	18 ± 1	26 ± 2
cis-15d/trans-15d (75:52)	Bn	4-F-C₆H₄	19 ± 0	17 ± 3	17 ± 1	27 ± 1
5-Fluorouracil			0.33 ± 0.17	18 ± 5	0.54 ± 0.12	n.d.

Open in a new tab

50% Inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%.

n.d. – not determined.

Results are mean values ± STDEV of two independent experiments.

3. Conclusions

Several series of {5-(2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl-2-methylisoxazolidin-3-yl})phosphonates (cis-12/trans-12 and cis-14/trans-14) and {5-(2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl-2-benzylisoxazolidin-3-yl})phosphonates (cis-13/trans-13 and cis-15/trans-15) modified at N3 in the quinazoline-2,4-dione moiety have been obtained by the 1,3-dipolar cycloaddition of N-substituted (C-diethoxyphosphoryl)nitrones 16 (R=Me) and 17 (R=Bn) and the respective N ¹-allylated quinazoline-2,4-diones substituted at N3 with benzoyl (compounds 18) or benzyl groups (compounds 19).

The synthesized isoxazolidine phosphonates were evaluated against a variety of DNA and RNA viruses and several derivatives appeared to be active against varicella-zoster virus and human cytomegalovirus. Among all tested compounds, a 95:5 trans-15d/cis-15d (95:5) (EC₅₀ = 3.0 μM) and a cis-15b/trans-15b (87:13) (EC₅₀ = 4.7 μM) showed the highest activity toward TK⁺ VZV strain. The potency of these derivatives was 4–6 fold lower than that of acyclovir, used as reference drug.

On the other hand, compounds trans-13c, mixture cis-13c/trans-13c (86:14), trans-13d, and a 85:15 mixture of cis-13d/trans-13d exhibited potency not only against TK⁺ VZV strain but also toward TK⁻ VZV strain and their anti-TK⁻ VZV activity was significantly higher than that of the reference drugs acyclovir and brivudin (EC₅₀ = 50.3 and 22.7 μM, respectively). The isoxazolidine phosphonates cis-15a-d/trans-15a-d having benzyl substituents both at N3 of the quinazoline-2,4-dione skeleton and at N2 of the isoxazolidine ring (cis- and trans-5) showed some activity toward human cytomegalovirus (EC₅₀ in the range of ≥3 to ≥14.5 μM).

The quinazoline-2,4-diones endowed with anti-VZV and anti-HCMV activity did not alter the morphology of cells used in the antiviral assays up to a concentration of 100 μM. However, these derivatives showed considerable cytostatic activity. Cytostatic activity of the obtained compounds was also evaluated on L1210, CEM, HeLa and HMEC-1 cells. Among all tested compounds, only quinazoline-2,4-diones (trans-15/cis-15) bearing benzyl substituents at N3 in the quinazolinone core and the benzyl group at N2 of the isoxazolidine unit showed significant (IC₅₀ = 10–98 μM) activity toward tested cell lines.

4. Experimental

4.1. General

¹H, ¹³C and ³¹P NMR spectra were taken in CDCl₃ on the Bruker Avance III spectrometers (600 MHz) with TMS as internal standard at 600, 151 and 243 MHz, respectively.

IR spectra were measured on an Infinity MI-60 FT-IR spectrometer. Melting points were determined on a Boetius apparatus and are uncorrected. Elemental analyses were performed by the Microanalytical Laboratory of this Faculty on Perkin-Elmer PE 2400 CHNS analyzer.

The following adsorbents were used: column chromatography, Merck silica gel 60 (70–230 mesh); analytical TLC, Merck TLC plastic sheets silica gel 60 F₂₅₄.

N-methyl- and N-benzyl-C-(diethoxyphosphoryl)nitrones 16 and 17 were obtained according to the literature procedures [28].

4.2. Synthesis of 1-allylquinazoline-2,4-dione (22)

To a solution of 1-allyl-1H-benzo[d] [1,3] oxazine-2,4-dione 21 (0.500 g, 2.46 mmol) in DMF (10 mL) urea (0.221 g, 3.69 mmol) was added. The reaction mixture was stirred for 5 h, the solvent was removed in vacuo and the residue was crystallized from ethanol to give pure 22 as a yellowish amorphous solid, m.p. = 218–219 °C.

IR (KBr, cm⁻¹) ν_max: 3165, 3031, 2926, 1673, 1605, 1502, 1398, 1295, 921, 763, 752, 503. ¹H NMR (600 MHz, CDCl₃): δ = 8.57 (bs, 1H, NH), 8.26–8.24 (m, 1H), 7.71–7.68 (m, 1H), 7.31–7.28 (m, 1H), 7.23–7.22 (m, 1H), 5.95 (ddt, ³ J = 17.3 Hz, ³ J = 10.1 Hz, ³ J = 5.0 Hz, 1H, CH₂ —CH CH₂), 5.31 (ddt, ³ J = 10.1 Hz, ⁴ J = 3.5 Hz, ² J = 0.7 Hz, 1H, CH₂ —CH CHH), 5.26 (d, ³ J = 17.3 Hz, ⁴ J = 3.5 Hz, ² J = 0.7 Hz, 1H, CH₂ —CH CHH), 4.79 (dt, ³ J = 5.0 Hz, ⁴ J = 3.5 Hz, 2H, CH ₂ —CH CH₂); ¹³C NMR (150 MHz, CDCl₃): δ = 161.60 (C O), 149.97 (C O), 141.06, 135.45, 131.05, 128.79, 123.21, 117.81, 116.03, 114.68, 45.15. Anal. calcd. For C₁₁H₁₀N₂O₃: C, 65.34; H, 4.98; N, 13.85. Found: C, 65.11; H, 4.74; N, 13.83.

4.3. The benzoylation of N-allylquinazoline-2,4-dione 22 – the general procedure

To a solution of N-allylquinazoline-2,4-dione 22 (1.00 mmol) in acetonitrile (10 mL) triethylamine was added (3.00 mmol) followed by the respective benzoyl chloride (2.20 mmol). The mixture was stirred at room temperature for 72 h. The solvent was removed in vacuo, the residue was dissolved in methylene chloride (10 mL) and washed with water (3 × 10 mL). The organic layer was dried (MgSO₄), concentrated and purified by column chromatography with methylene chloride-hexane mixture (7:3, v/v) and the appropriate fractions were crystallized from a chloroform-hexane mixture.

4.3.1. 1-Allyl-3-(2-fluoro)benzoyl-1H-quinazoline-2,4-dione (18b)

An amorphous solid, m.p. = 141–142 °C. IR (KBr, cm⁻¹) ν_max: 3253, 2923, 1739, 1693, 1658, 1608, 1478, 1454, 1172, 1013, 944, 755. ¹H NMR (600 MHz, CDCl₃): δ = 8.25 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H), 8.15 (dt, J = 7,9 Hz, J = 1.7 Hz, 1H), 7.73 (ddd, J = 8.6 Hz, J = 7.3 Hz, J = 1.6 Hz, 1H), 7.66–7.62 (m, 1H), 7.35–7.29 (m, 2H), 7.26 (d, J = 8.5 Hz, 1H), 7.12 (ddd, J = 11.7 Hz, J = 8.3 Hz, J = 1.0 Hz, 1H), 5.95 (ddt, ³ J = 17.2 Hz, ³ J = 10.2 Hz, ³ J = 4.9 Hz, 1H, CH₂ —CH CH₂), 5.32 (d, ³ J = 10.2 Hz, 1H, CH₂ —CH CHH), 5.28 (d, ³ J = 17.2 Hz, 1H, CH₂ —CH CHH), 4.82–4.79 (m, 2H); ¹³C NMR (150 MHz, CDCl₃): δ = 164.80 (C O), 162.09 (d, ¹ J _(CF) = 259.0 Hz), 160.79 (C O), 149.07 (C O), 140.41, 136.84 (d, ³ J _(CCCF) = 9.8 Hz), 135.85, 133.03, 130.73, 128.92, 125.03 (d, ⁴ J _(CCCCF) = 3.3 Hz), 123.43, 120.51 (d, ² J _(CCF) = 7.8 Hz), 117.88, 117.21 (d, ² J _(CCF) = 23.2 Hz), 114.84, 45.22. Anal. calcd. For C₁₈H₁₃FN₂O₃: C, 66.66; H, 4.04; N, 8.64. Found: C, 66.41; H, 3.73; N, 8.59.

4.3.2. 1-Allyl-3-(3-fluoro)benzoyl-1H-quinazoline-2,4-dione (18c)

An amorphous solid, m.p. = 125–127 °C. IR (KBr, cm⁻¹) ν_max: 3073, 2973, 1743, 1697, 1670, 1481, 1432, 1286, 1048, 779. ¹H NMR (600 MHz, CDCl₃): δ = 8.25 (d, J = 7.9 Hz), 7.81–7.75 (m, 2H), 7.67 (d, J = 8.8 Hz, 1H), 7.50 (dt, J = 8.0 Hz, J = 5.7 Hz, 1H), 7.38 (dt, J = 8.2 Hz, J = 2.1 Hz, 1H), 7.35–7.28 (m, 2H), 5.96 (ddt, ³ J = 17.7 Hz, ³ J = 10.9 Hz, ³ J = 5.0 Hz, 1H, CH₂ —CH CH₂), 5.35 (d, ³ J = 10.9 Hz, 1H, CH₂ —CH CHH), 5.30 (d, ³ J = 17.7 Hz, 1H, CH₂ —CH CHH), 4.80 (d, ³ J = 5.0 Hz, 2H, CH ₂ —CH CH₂); ¹³C NMR (150 MHz, CDCl₃): δ = 167.72 (d, ⁴ J _(C(O)CCCF) = 3.2 Hz, C O), 162.93 (d, ¹ J _(CF) = 248.9 Hz), 161.03 (C O), 149.13 (C O), 140.49, 136.03, 133.99 (d, ³ J _(CCCF) = 7.3 Hz), 130.89 (d, ³ J _(CCCF) = 7.7 Hz), 130.77, 129.09, 126.14 (d, ⁴ J _(CCCCF) = 2.8 Hz), 122.10 (d, ² J _(CCF) = 21.8 Hz), 118.39, 117.38 (d, ² J _(CCF) = 23.3 Hz), 115.59, 114.83, 45.44. Anal. calcd. For C₁₈H₁₃FN₂O₃: C, 66.66; H, 4.04; N, 8.64. Found: C, 66,63; H, 3.64; N, 8.82.

4.3.3. 1-Allyl-3-(4-fluoro)benzoyl-1H-quinazoline-2,4-dione (18d)

An amorphous solid, m.p. = 117.0–118.5 °C. IR (KBr, cm⁻¹) ν_max: 3084, 2987, 1744, 1700, 1661, 1495, 1411, 1242, 994, 757. ¹H NMR (600 MHz, CDCl₃): δ = 8.27 (dd, J = 7.9 Hz, J = 1.5 Hz, 1H), 8.04–8.02 (m, 2H), 7.76 (ddd, J = 8.7 Hz, J = 7.6 Hz, J = 1.6 Hz, 1H), 7.34 (dd, J = 7.7 Hz, J = 7.4 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.21–7.18 (m, 2H), 5.97 (ddt, ³ J = 17.0 Hz, ³ J = 10.7 Hz, ³ J = 5.2 Hz, 1H, CH₂ —CH CH₂), 5.35 (d, ³ J = 10.7 Hz, 1H, CH₂ —CH CHH), 5.30 (d, ³ J = 17.0 Hz, 1H, CH₂ —CH CHH), 4.81 (d, ³ J = 5.2 Hz, 2H, CH ₂ —CH CH₂); ¹³C NMR (150 MHz, CDCl₃): δ = 167.45 (C O), 166.92 (d, ¹ J _(CF) = 258.4 Hz), 161.06 (C O), 149.17 (C O), 140.49, 135.98, 133.31 (d, ³ J _(CCCF) = 9.9 Hz), 130.83, 128.34 (d, ⁴ J _(CCCCF) = 2.9 Hz), 123.58, 118.35, 116.54 (d, ² J _(CCF) = 22.4 Hz), 115.62, 114.81, 45.43. Anal. calcd. For C₁₈H₁₃FN₂O₃: C, 66.66; H, 4.04; N, 8.64. Found: C, 66,75; H, 3.95; N, 8.52.

4.4. The benzylation of N-allylquinazoline-2,4-dione 22 – the general procedure

To a solution of N-allylquinazoline-2,4-dione 22 (1.00 mmol) in acetonitrile (15 mL) potassium hydroxide (3.00 mmol) was added followed by the respective benzyl chloride (1.10 mmol). The reaction mixture was stirred at 105 °C for 4 h. The solvent was removed in vacuo, the residue was dissolved in methylene chloride (10 mL) and washed with water (3 × 10 mL). The organic layer was dried (MgSO₄), concentrated and the crude product was purified by column chromatography with methylene chloride-hexane mixture (7:3, v/v) and further crystallized from a chloroform-petroleum ether mixture.

4.4.1. 1-Allyl-3-benzyl-1H-quinazoline-2,4-dione (19a)

An amorphous solid, m.p. = 85–86 °C. IR (KBr, cm⁻¹) ν_max: 3085, 2853, 1699, 1657, 1609, 1484, 1456, 1435, 1269, 946, 759. ¹H NMR (600 MHz, CDCl₃): δ = 8.28–8.27 (m, 1H), 7.68–7.64 (m, 1H), 7.55–7.74 (m, 2H), 7.34–7.32 (m, 2H), 7.29–7.25 (m, 2H), 7.19–7.18 (m, 1H), 5.95 (ddt, ³ J = 17.0 Hz, ³ J = 10.3 Hz, ³ J = 5.0 Hz, 1H, CH₂ —CH CH₂), 5.32 (s, 2H, CH ₂Ph), 5.29 (d, ³ J = 10.3 Hz, 1H, CH₂ —CH CHH), 5.23 (d, ³ J = 17.3 Hz, 1H, CH₂ —CH CHH), 4.80 (d, ³ J = 4.9 Hz, 2H, CH ₂ —CH CH₂); ¹³C NMR (150 MHz, CDCl₃): δ = 161.76 (C O), 150.85 (C O), 139.90, 137.05, 134.99, 131.30, 129.16, 128.99, 128.42, 122.97, 117.64, 115.75, 114.14, 46.04, 45.05. Anal. calcd. For C₁₈H₁₆N₂O₂: C, 73.95; H, 5.52; N, 9.58. Found: C, 73.59; H, 5.52; N, 9.61.

4.4.2. 1-Allyl-3-(2-fluoro)benzyl-1H-quinazoline-2,4-dione (19b)

An amorphous solid, m.p. = 105–107 °C. IR (KBr, cm⁻¹) ν_max: 3090, 3067, 2975, 1707, 1661, 1608, 1480, 1416, 1290, 975, 917, 752. ¹H NMR (600 MHz, CDCl₃): δ = 8.28 (d, J = 7.9 Hz, 1H), 7.69–7.66 (m, 1H), 7.31–7.26 (m, 2H), 7.26–7.21 (m, 2H), 7.08–7.06 (m, 2H), 5.95 (ddt, ³ J = 17.3 Hz, ³ J = 10.2 Hz, ³ J = 5.0 Hz, 1H, CH₂ —CH CH₂), 5.42 (s, 2H, CH ₂Ph), 5.29 (d, ³ J = 10.2 Hz, 1H, CH₂ —CH CHH), 5.24 (d, ³ J = 17.3 Hz, 1H, CH₂ —CH CHH), 4.82 (d, ³ J = 5.0 Hz, 2H, CH ₂ —CH CH₂); ¹³C NMR (150 MHz, CDCl₃): δ = 161.68 (C O), 160.80 (d, ¹ J _(CF) = 247.6 Hz), 150.70 (C O), 139.95, 135.10, 131.24, 129.24, 129.23 (d, ⁴ J _(CCCCF) = 3.0 Hz), 128.95 (d, ³ J _(CCCF) = 7.9 Hz), 124.04 (d, ³ J _(CCCF) = 3.9 Hz), 123.92 (d, ² J _(CCF) = 14.3 Hz), 123.05, 117.66, 115.62, 115.48 (d, ² J _(CCF) = 21.8 Hz), 114.21, 46.03, 38.93 (d, ³ J _(CCCF) = 4.7 Hz). Anal. calcd. For C₁₈H₁₅FN₂O₂: C, 69.67; H, 4.87; N, 9.03. Found: C, 69.75; H, 4.53; N, 9.14.

4.4.3. 1-Allyl-3-(3-fluoro)benzyl-1H-quinazoline-2,4-dione (19c)

An amorphous solid, m.p. = 85–86 °C. IR (KBr, cm⁻¹) ν_max: 3083, 3017, 1701, 1656, 1483, 1401, 1346, 1209, 978, 943, 760. ¹H NMR (600 MHz, CDCl₃): δ = 8.28–8.27 (m, 1H), 7.68–7.66 (m, 1H), 7.32–7.27 (m, 3H), 7.24–7.20 (m, 2H), 6.98–6.96 (m, 1H), 5.95 (ddt, ³ J = 17.2 Hz, ³ J = 10.2 Hz, ³ J = 5.0 Hz, 1H, CH₂ —CH CH₂), 5.30–5.29 (m, 3H, CH ₂Ph, CH₂ —CH CHH), 5.24 (d, ³ J = 17.2 Hz, 1H, CH₂ —CH CHH), 4.81 (d, ³ J = 5.0 Hz, 2H, CH ₂ —CH CH₂); ¹³C NMR (150 MHz, CDCl₃): δ = 162.83 (d, ¹ J _(CF) = 245.8 Hz), 161.69 (C O), 150.77 (C O), 139.90, 139.40 (d, ³ J _(CCCF) = 7.6 Hz), 135.14, 131.20, 129.80 (d, ³ J _(CCCF) = 7.9 Hz), 129.18, 124.52 (d, ⁴ J _(CCCCF) = 3.1 Hz), 123.09, 117.72, 115.78 (d, ² J _(CCF) = 21.9 Hz), 115.62, 114.53 (d, ² J _(CCF) = 21.0 Hz), 114.21, 46.08, 45.05 (d, ⁴ J _(CCCCF) = 1.5 Hz). Anal. calcd. For C₁₈H₁₅FN₂O₂: C, 69.67; H, 4.87; N, 9.03. Found: C, 69.71; H, 4.48; N, 9.00.

4.4.4. 1-Allyl-3-(4-fluoro)benzyl-1H-quinazoline-2,4-dione (19d)

An amorphous solid, m.p. = 94.0–95.5 °C. IR (KBr, cm⁻¹) ν_max: 3092, 3021 2964, 1702, 1657, 1603, 1483, 1436, 1216, 1159, 961, 751. ¹H NMR (600 MHz, CDCl₃): δ = 8.28–8.26 (m, 1H), 7.67–7.64 (m, 1H), 7.57–7.54 (m, 2H), 7.29–7.26 (m, 1H), 7.20–7.18 (m, 1H), 7.02–6.99 (m, 2H), 5.98 (ddt, ³ J = 17.1 Hz, ³ J = 10.2 Hz, ³ J = 5.0 Hz, 1H, CH₂ —CH CH₂), 5.29 (d, ³ J = 10.2 Hz, 1H, CH₂ —CH CHH), 5.27 (s, 2H, CH ₂Ph), 5.23 (d, ³ J = 17.1 Hz, 1H, CH₂ —CH CHH), 4.80 (d, ³ J = 5.0 Hz, 2H, CH ₂ —CH CH₂); ¹³C NMR (150 MHz, CDCl₃): δ = 162.31 (d, ¹ J _(CF) = 246.8 Hz), 161.71 (C O), 150.78 (C O), 139.87, 135.07, 132.87 (d, ⁴ J _(CCCCF) = 3.3 Hz), 131.23, 131.05 (d, ³ J _(CCCF) = 7.6 Hz), 129.12, 123.04, 117.67, 115.67, 115.20 (d, ² J _(CCF) = 20.8 Hz), 114.18, 46.03, 44.31. Anal. calcd. For C₁₈H₁₅FN₂O₂: C, 69.67; H, 4.87; N, 9.03. Found: C, 69.31; H, 4.59; N, 9.18.

4.5. Cycloadditions of C-(diethoxyphosphoryl)nitrones 16 (R = Me) and 17 (R = Bn) and N¹-allylated quinazoline-2,4-diones 18 and 19 – the general procedure

Solutions of nitrones 16 or 17 (1.00 mmol) and the respective N ¹-allylated quinazoline-2,4-diones 18 or 19 (1.05 mmol) in toluene or a toluene-ethanol mixture were stirred at 60 °C until the starting nitrone disappeared. Solvents were removed in vacuo and the crude products (the respective mixtures of isoxazolidines cis-12/trans-12, cis-13/trans-13, cis-14/trans-14 or cis-15/trans-15) were purified on silica gel columns.

4.5.1. Diethyl trans-{5-[(3-(2-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-12b)

A colorless oil. IR (film, cm⁻¹) ν_max: 3451, 2981, 2924, 1748, 1702, 1666, 1609, 1481, 1390, 1234, 1052, 1023, 970, 758. ¹H NMR (600 MHz, CDCl₃): δ = 8.24 (d, J = 7.7 Hz, 1H), 8.14 (t, J = 7.5 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.76–7.73 (m, 1H), 7.66–7.63 (m, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.35–7.31 (m, 2H), 7.12 (dd, J = 11.4 Hz, J = 8.5 Hz, 1H), 4.51 (dd, ² J = 15.1 Hz, ³ J = 4.5 Hz, 1H, HCHN), 4.45–4.41 (m, 1H, HC5), 4.23–4.15 (m, 5H, 2 × CH ₂OP, HCHN), 3.02–3.00 (m, 1H, HC3), 2.86 (s, 3H, CH ₃N), 2.66 (dddd, ³ J _(H4α–P) = 19.6 Hz, ² J _{(H4α–H4β)} = 13.4 Hz, ³ J _(H4α–H3) = 7.1 Hz, ³ J _(H4β–H5) = 7.1 Hz, 1H, HαC4), 2.45 (dddd, ² J _{(H4β–H4α)} = 13.4 Hz, ³ J _(H4β–P) = 13.4 Hz, ³ J _(H4β–H5) = 9.6 Hz, ³ J _(H4β–H3) = 8.1 Hz, 1H, HβC4), 1.34 (t, ³ J = 7.1 Hz, 3H, 2 × CH ₃CH₂OP); ¹³ C NMR (151 MHz, CDCl₃): δ = 164.62 (C O), 162.07 (d, ¹ J _(CF) = 259.7 Hz), 160.65 (C O), 149.65 (C O), 140.71, 136.81 (d, ³ J _(CCCF) = 9.9 Hz), 135.68, 133.06, 128.89, 125.04 (d, ⁴ J _(CCCCF) = 3.4 Hz), 123.65, 120.52 (d, ² J _(CCF) = 7.8 Hz), 117.16 (d, ² J _(CCF) = 23.2 Hz), 115.76, 115.24, 75.22 (d, ³ J _(CCCP) = 7.2 Hz, C5), 63.92 (d, ¹ J _(CP) = 168.1 Hz, C3), 63.14 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.44 (d, ² J _(COP) = 7.2 Hz, CH₂OP), 46.27, 45.21, 36.85 (d, ² J _(CCP) = 1.3 Hz, C4), 16.49 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.43 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.67. Anal. calcd. for C₂₄H₂₇FN₃O₇P: C, 55.49; H, 5.24; N, 8.09. Found: C, 55.77; H, 5.04; N, 8.01.

4.5.2. Diethyl cis-{5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-12c)

A colorless oil. IR (film, cm⁻¹) ν_max: 3430, 2983, 1751, 1700, 1666, 1605, 1480, 1447, 1395, 1250, 1050, 1025, 970, 790, 757. (¹H NMR signals of cis- 12c were extracted from the spectrum of a 94:6 mixture of cis-12c and trans-12c) ¹H NMR (600 MHz, CDCl₃): δ = 8.21 (dd, J = 7.9 Hz, J = 1.3 Hz, 1H), 7.88–7.85 (m, 1H), 7.82–7.73 (m, 1H), 7.70–7.65 (m, 1H), 7.55–7.50 (m, 2H), 7.40–7.30 (m, 2H), 4.65–4.58 (m, 1H, HC5), 4.35 (dd, J = 14.8 Hz, J = 9.8 Hz, 1H, HCHN), 4.25–4.15 (m, 5H, 2 × CH ₂OP, HCHN), 2.99–2.94 (very broad m, 1H, HC3), 2.85 (s, 3H, CH ₃N), 2.85–2.77 (m, HαC4), 2.48–2.40 (broad m, 1H, HβC4), 1.41 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.40 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP); (¹³C NMR signals of cis- 12c were extracted from the spectrum of a 40:60 mixture of cis-12c and trans-12c) ¹³C NMR (151 MHz, CDCl₃): δ = ¹³C NMR (150 MHz, CDCl₃): δ = 167.83 (d, ⁴ J _(C(O)CCCF) = 3.0 Hz, C O), 162.94 (d, ¹ J _(CF) = 248.5 Hz), 161.23 (C O), 149.59 (C O), 141.75, 135.73, 133.89 (d, ³ J _(CCCF) = 9.6 Hz), 130.85 (d, ³ J _(CCCF) = 4.0 Hz), 128.46, 126.17 (d, ⁴ J _(CCCCF) = 2.1 Hz), 123.58, 122.08 (d, ² J _(CCF) = 21.7 Hz), 117.11 (d, ² J _(CCF) = 21.7 Hz), 116.24, 115.27, 75.04 (d, ³ J _(CCCP) = 6.9 Hz, C5), 63.62 (d, ¹ J _(CP) = 168.0 Hz, C3), 62.88 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 62.65 (d, ² J _(COP) = 7.2 Hz, CH₂OP), 46.59, 45.17, 35.53 (C4), 16.60 (d, ³ J _(CCOP) = 5.5 Hz, CH₃CH₂OP), 16.52 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.44. Anal. calcd. for C₂₄H₂₇FN₃O₇P: C, 55.49; H, 5.24; N, 8.09. Found: C, 55.28; H, 5.27; N, 7.93.

4.5.3. Diethyl trans-{5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-12c)

A colorless oil. IR (film, cm⁻¹) ν_max: 3424, 2980, 1752, 1703, 1665, 1608, 1481, 1442, 1389, 1262, 1052, 1024, 968, 794. ¹H NMR (600 MHz, CDCl₃): δ = 8.24 (dd, J = 7.9 Hz, J = 1.2 Hz, 1H), 7.80–7.75 (m, 2H), 7.70–7.65 (m, 1H), 7.55–7.50 (m, 2H), 7.40–7.32 (m, 2H), 4.54–4.44 (m, 2H, HCHN, HC5), 4.30–4.15 (m, 5H, 2 × CH ₂OP, HCHN), 3.10–3.00 (very broad m, 1H, HC3), 2.89 (s, 3H, CH ₃N), 2.72 (dddd, ³ J _(H4α–P) = 17.0 Hz, ² J _{(H4α–H4β)} = 13.4 Hz, ³ J _(H4α–H3) = 7.4 Hz, ³ J _(H4β–H5) = 7.4 Hz, 1H, HαC4), 2.48–2.40 (broad m, 1H, HβC4), 1.35 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.34 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = ¹³C NMR (150 MHz, CDCl₃): δ = 167.58 (d, ⁴ J _(C(O)CCCF) = 3.0 Hz, C O), 162.94 (d, ¹ J _(CF) = 249.1 Hz), 160.91 (C O), 149.67 (C O), 140.69, 135.93, 133.89 (d, ³ J _(CCCF) = 9.6 Hz), 130.89 (d, ³ J _(CCCF) = 7.7 Hz), 129.02, 126.19 (d, ⁴ J _(CCCCF) = 2.4 Hz), 123.86, 122.17 (d, ² J _(CCF) = 21.7 Hz), 117.11 (d, ² J _(CCF) = 23.2 Hz), 115.56, 115.26, 75.24 (broad s, C5), 63.62 (d, ¹ J _(CP) = 168.0 Hz, C3), 63.27 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.59 (d, ² J _(COP) = 6.9 Hz, CH₂OP), 45.98, 45.57, 35.93 (C4), 16.50 (d, ³ J _(CCOP) = 5.8 Hz, CH₃CH₂OP), 16.44 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.33. Anal. calcd. for C₂₄H₂₇FN₃O₇P: C, 55.49; H, 5.24; N, 8.09. Found: C, 55.31; H, 5.41; N, 7.94.

4.5.4. Diethyl cis-{5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-12d)

A colorless oil. IR (film, cm⁻¹) ν_max: 3460, 2923, 1750, 1700, 1699, 1663, 1600, 1485, 1297, 1245, 1025, 970, 760. ¹H NMR (600 MHz, CDCl₃): δ = 8.22–8.21 (m, 1H), 8.04–8.02 (m, 2H), 7.88–7.86 (m, 1H), 7.76–7.73 (m, 1H), 7.33–7.31 (m, 1H) 7.21–7.18 (m, 2H), 4.63–4.59 (m, 1H, HC5), 4.35 (dd, ² J = 14.8 Hz, ³ J = 9.8 Hz, 1H, HCHN), 4.30–4.22 (m, 4H, 2 × CH ₂OP), 4.20 (dd, ² J = 14.8 Hz, ³ J = 2.5 Hz, 1H, HCHN), 2.95 (ddd, ³ J _(H3–H4α) = 9.9 Hz, ³ J _(H3–H4β) = 7.7 Hz, ² J _(H3–P) = 2.3 Hz, 1H, HC3), 2.85 (s, 3H, CH ₃N), 2.84 (dddd, ³ J _(H4α–P) = 18.2 Hz, ² J _{(H4α–H4β)} = 12.7 Hz, ³ J _(H4α–H3) = 9.9 Hz, ³ J _(H4β–H5) = 8.8 Hz, 1H, HαC4), 2.39 (dddd, ² J _{(H4β–H4α)} = 12.7 Hz, ³ J _(H4β–P) = 11.5 Hz, ³ J _(H4β–H3) = 7.7 Hz, ³ J _(H4β–H5) = 3.6 Hz, 1H, HβC4), 1.42 (t, ³ J = 7.2 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 167.52 (C O), 166.92 (d, ¹ J _(CF) = 258.5 Hz), 161.25 (C O), 149.63 (C O), 141.78, 135.67, 133.34 (d, ³ J _(CCCF) = 9.9 Hz), 128.45, 128.38 (d, ⁴ J _(CCCCF) = 2.4 Hz), 123.52, 116.51 (d, ² J _(CCF) = 22.4 Hz), 116.21, 115.32, 75.00 (d, ³ J _(CCCP) = 7.0 Hz, C5), 63.13 (d, ¹ J _(CP) = 169.3 Hz, C3), 62.83 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 62.63 (d, ² J _(COP) = 7.0 Hz, CH₂OP), 46.58, 45.22 (d, ³ J = 4.2 Hz, 35.55 (C4), 16.59 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.52 (d, ³ J _(CCOP) = 5.9 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.46. Anal. calcd. for C₂₄H₂₇FN₃O₇P × H₂O: C, 53.63; H, 5.44; N, 7.82. Found: C, 53.68; H, 5.29; N, 7.98.

4.5.5. Diethyl trans-{5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-12d)

A colorless oil. IR (film, cm⁻¹) ν_max: 3451, 2963, 1748, 1702, 1664, 1601, 1480, 1390, 1242, 1157, 1100, 1020, 971, 757. ¹H NMR (600 MHz, CDCl₃): δ = 8.26–8.24 (m, 1H), 8.04–8.02 (m, 2H), 7.78–7.75 (m, 1H), 7.51–7.50 (m, 1H), 7.36–7.34 (m, 1H), 7.21–7.18 (m, 2H), 4.35 (dd, ² J = 15.0 Hz, ³ J = 9.8 Hz, 1H, HCHN), 4.46–4.42 (m, 1H, HC5), 4.23–4.16 (m, 5H, 2 × CH ₂OP, HCHN), 3.05–2.99 (m, 1H, HC3), 2.88 (s, 3H, CH ₃N), 2.72 (dddd, ³ J _(H4α–P) = 19.4 Hz, ² J _{(H4α–H4β)} = 12.5 Hz, ³ J _(H4α–H3) = 7.0 Hz, ³ J _(H4β–H5) = 7.0 Hz, 1H, HαC4), 2.42 (dddd, ² J _{(H4β–H4α)} = 12.5 Hz, ³ J _(H4β–P) = 12.5 Hz, ³ J _(H4β–H5) = 10.1 Hz, ³ J _(H4β–H3) = 8.2 Hz, 1H, HβC4), 1.35 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.34 (t, ³ J = 7.2 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 167.28 (C O), 166.95 (d, ¹ J _(CF) = 258.5 Hz), 160.93 (C O), 149.69 (C O), 140.71, 135.81, 133.35 (d, ³ J _(CCCF) = 9.9 Hz), 128.98, 128.25 (d, ⁴ J _(CCCCF) = 2.7 Hz), 123.77, 116.53 (d, ² J _(CCF) = 22.7 Hz), 115.60, 115.20, 75.15 (d, ³ J _(CCCP) = 6.7 Hz, C5), 63.91 (d, ¹ J _(CP) = 167.9 Hz, C3), 63.14 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.50 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 46.25, 45.54, 36.04 (C4), 16.50 (d, ³ J _(CCOP) = 5.8 Hz, CH₃CH₂OP), 16.43 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.59. Anal. calcd. for C₂₄H₂₇FN₃O₇P × H₂O: C, 53.63; H, 5.44; N, 7.82. Found: C, 53.80; H, 5.32; N, 8.04.

4.5.6. Diethyl cis-{5-[(3-benzoyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-benzylisoxazolidin-3-yl}phosphonate (cis-13a)

A colorless oil. IR (film, cm⁻¹) ν_max: 3455, 2960, 1749, 1660, 1642, 1490, 1378, 1296, 1089, 1180, 1050, 1020, 970, 690. ¹H NMR (600 MHz, CDCl₃): δ = 8.13–8.11 (m, 1H), 7.98–7.97 (m, 2H), 7.67–7.65 (m, 1H), 7.52–7.49 (m, 2H), 7.41–7.39 (m, 1H), 7.35–7.34 (m, 2H), 7.30–7.29 (m, 3H), 7.15–7.12 (m, 2H), 4.64–4.62 (m, 1H, HC5), 4.44 (d, ² J = 13.7 Hz, 1H, HCHPh), 4.31–4.24 (m, 5H, 2 × CH ₂OP, HCHN), 4.21 (dd, ² J = 14.9 Hz, ³ J = 2.3 Hz, 1H, HCHN), 3.92 (d, ² J = 13.7 Hz, 1H, HCHPh), 3.24 (ddd, ³ J _(H3–H4α) = 10.2 Hz, ³ J _(H3–H4β) = 7.3 Hz, ² J _(H3–P) = 3.1 Hz, 1H, HC3), 2.81 (dddd, ³ J _(H4α–P) = 18.7 Hz, ² J _{(H4α–H4β)} = 12.9 Hz, ³ J _(H4α–H3) = 10.2 Hz, ³ J _(H4β–H5) = 10.2 Hz, 1H, HαC4), 2.31 (dddd, ² J _{(H4β–H4α)} = 12.9 Hz, ³ J _(H4β–P) = 12.9 Hz, ³ J _(H4β–H3) = 7.3 Hz, ³ J _(H4β–H5) = 4.1 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.1 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 168.78 (C O), 161.25 (C O), 149.75 (C O), 141.15, 136.61, 135.65, 134.91, 131.89, 130.47, 129.93, 129.13, 128.31, 128.18, 127.59, 123.15, 116.01, 115.08, 75.69 (d, ³ J _(CCCP) = 6.6 Hz, C5), 62.94 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.69 (d, ² J _(COP) = 7.1 Hz, CH₂OP), 62.36 (d,³ J _(CNCP) = 5.1 Hz,CH₂Ph), 60.63 (d, ¹ J _(CP) = 170.1 Hz, C3), 47.14 (CH₂N), 35.07 (s, C4), 16.61 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.55 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.64. Anal. calcd. for C₃₀H₃₂N₃O₇P: C, 62.39; H, 5.58; N, 7.28. Found: C, 62.58; H, 5.53; N, 7.18.

4.5.7. Diethyl trans-{5-[(3-benzoyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-benzylisoxazolidin-3-yl}phosphonate (trans-13a)

A colorless oil. IR (film, cm⁻¹) ν_max: 3455, 3062, 2982, 1750, 1701, 1665, 1608, 1480, 1390, 1238, 1052, 1023, 968, 757. ¹H NMR (600 MHz, CDCl₃): δ = 8.25–8.23 (m, 1H), 7.98–7.96 (m, 2H), 7.73–7.70 (m, 1H), 7.68–7.66 (m, 1H), 7.52–7.46 (m, 3H), 7.36–7.33 (m, 1H), 7.31–7.28 (m, 5H), 4.47 (d, ² J = 14.8 Hz, ³ J = 4.2 Hz, 1H, HCHN), 4.46–4.41 (m, 2H, HC5, HCHN), 4.26–4.17 (m, 5H, 2 × CH ₂OP, HCHPh), 3.91 (d, ² J = 13.9 Hz, 1H, HCHPh), 3.30 (ddd, ³ J _(H3–H4β) = 10.0 Hz, ³ J _(H3–H4α) = 6.5 Hz, ² J _(H3–P) = 2.7 Hz, 1H, HC3), 2.68 (dddd, ³ J _(H4α–P) = 19.0 Hz, ² J _{(H4α–H4β)} = 13.0 Hz, ³ J _(H4α–H3) = 6.5 Hz, ³ J _(H4β–H5) = 6.5 Hz, 1H, HαC4), 2.38 (dddd, ³ J _(H4β–P) = 14.9 Hz, ² J _{(H4β–H4α)} = 13.0 Hz, ³ J _(H4β–H3) = 10.0 Hz, ³ J _(H4β–H5) = 8.12 Hz, 1H, HβC4), 1.35 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP), 1.34 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 168.52 (C O), 160.99 (C O), 149.80 (C O), 140.81, 136.46, 135.66, 135.04, 131.68, 130.49, 129.64, 129.19, 128.87, 128.16, 127.52, 123.69, 115.59, 115.52, 75.48 (d, ³ J _(CCCP) = 6.4 Hz, C5), 63.31 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.71 (d,³ J _(CNCP) = 3.8 Hz, CH₂Ph), 62.51 (d, ² J _(COP) = 6.8 Hz, CH₂OP), 60.72 (d, ¹ J _(CP) = 170.2 Hz, C3), 45.22 (CH₂N), 35.19 (d, ² J _(CCP) = 1.8 Hz, C4), 16.57 (d, ³ J _(CCOP) = 5.7 Hz, CH₃CH₂OP), 16.49 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.67. Anal. calcd. for C₃₀H₃₂N₃O₇P: C, 62.39; H, 5.58; N, 7.28. Found: C, 62.18; H, 5.49; N, 7.07.

4.5.8. Diethyl cis-{2-benzyl-5-[(3-(2-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-13b)

A colorless oil. IR (film, cm⁻¹) ν_max: 3472, 2978, 1744, 1700, 1662, 1607, 1478, 1389, 1240, 1012, 970, 756. (¹H NMR signals of cis- 13b were extracted from the spectrum of a 90:10 mixture of cis-13b and trans-13b) ¹H NMR (600 MHz, CDCl₃): δ = 8.24–8.20 (m, 1H), 8.16–8.09 (m, 1H), 7.66–7.62 (m, 1H), 7.38–7.34 (m, 4H), 7.32–7.29 (m, 3H), 7.14–7.10 (m, 3H), 4.62 (dddd, ³ J _(H5–H4α) = 9.9 Hz, ³ J _(H5–CH) = 7.4 Hz, ³ J _(H5–H4β) = 4.2 Hz, ³ J _(H5–CH) = 4.2 Hz, 1H, HC5), 4.43 (d, ² J = 13.9 Hz, 1H, HCHPh), 4.31–4.18 (m, 5H, 2 × CH ₂OP, HCHN), 4.19 (dd, ² J = 14.8 Hz, ³ J _(HC–H5) = 7.3 Hz, 1H, HCHN), 3.92 (d, ² J = 13.9 Hz, 1H, HCHPh), 3.24 (ddd, ³ J _(H3–H4α) = 9.9 Hz, ³ J _(H3–H4β) = 7.6 Hz, ² J _(H3–P) = 3.1 Hz, 1H, HC3), 2.81 (dddd, ³ J _(H4α–P) = 18.7 Hz, ² J _{(H4α–H4β)} = 12.7 Hz, ³ J _(H4α–H3) = 9.9 Hz, ³ J _(H4β–H5) = 9.9 Hz, 1H, HαC4), 2.41 (dddd, ² J _{(H4β–H4α)} = 12.7 Hz, ³ J _(H4β–P) = 12.7 Hz, ³ J _(H4β–H3) = 7.6 Hz, ³ J _(H4β–H5) = 4.2 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.1 Hz, 3H, 2 × CH ₃CH₂OP); (¹³C NMR signals of cis- 13b were extracted from the spectrum of a 59:41 mixture of cis-13b and trans-13b) ¹³C NMR (151 MHz, CDCl₃): δ = 164.89 (C O), 162.08 (d, ¹ J _(CF) = 259.7 Hz), 160.94 (C O), 149.57 (C O), 141.06, 136.66 (d, ³ J _(CCCF) = 9.7 Hz), 136.60, 135.59, 132.96, 129.96, 128.79, 128.28, 127.56, 124.93 (d, ⁴ J _(CCCCF) = 3.8 Hz), 123.08, 120.55 (d, ² J _(CCF) = 8.0 Hz), 117.18 (d, ² J _(CCF) = 23.2 Hz), 115.96, 115.18, 75.67 (d, ³ J _(CCCP) = 7.2 Hz, C5), 62.93 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.65 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.69 (d, ³ J _(CNCP) = 4.0 Hz, CH₂Ph), 60.65 (d, ¹ J _(CP) = 170.1 Hz, C3), 47.07 (CH₂N), 35.12 (C4), 16.61 (d, ³ J _(CCOP) = 5.7 Hz, CH₃CH₂OP), 16.55 (d, ³ J _(CCOP) = 5.7 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.61. Anal. calcd. for C₃₀H₃₁FN₃O₇P: C, 60.50; H, 5.25; N, 7.06. Found: C, 60.41; H, 5.12; N, 6.82 (obtained on a 59:41 mixture of cis-13b and trans-13b).

4.5.9. Diethyl trans-{2-benzyl-5-[(3-(2-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-13b)

A colorless oil. IR (film, cm⁻¹) ν_max: 3063, 2930, 1749, 1702, 1666, 1609, 1480, 1454, 1391, 1159, 1051, 1022, 967, 775. (NMR signals of trans-13b were extracted from the spectra of a 10:90 mixture of cis-13b and trans-13b) ¹H NMR (600 MHz, CDCl₃): δ = 8.24–8.22 (m, 1H), 8.15–8.10 (m, 1H), 7.70–7.67 (m, 1H), 7.66–7.62 (m, 1H), 7.44–7.43 (m, 1H), 7.36–7.27 (m, 7H), 7.13–7.09 (m, 1H), 4.47–4.42 (m, 2H, HCHN, HC5), 4.43 (d, ² J = 13.8 Hz, 1H, HCHPh), 4.26–4.20 (m, 4H, 2 × CH ₂OP), 4.18 (dd, ² J = 14.8 Hz, ³ J _(HC–H5) = 7.1 Hz, 1H, HCHN), 3.87 (d, ² J = 13.8 Hz, 1H, HCHPh), 3.28 (ddd, ³ J _(H3–H4β) = 9.5 Hz, ³ J _(H3–H4α) = 6.5 Hz, ² J _(H3–P) = 2.8 Hz, 1H, HC3), 2.67 (dddd, ³ J _(H4α–P) = 19.2 Hz, ² J _{(H4α–H4β)} = 12.8 Hz, ³ J _(H4α–H3) = 6.5 Hz, ³ J _(H4β–H5) = 6.5 Hz, 1H, HαC4), 2.37 (dddd, ³ J _(H4β–P) = 14.8 Hz, ² J _{(H4β–H4α)} = 12.8 Hz, ³ J _(H4β–H3) = 9.5 Hz, ³ J _(H4β–H5) = 8.0 Hz, 1H, HβC4), 1.34 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.33 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 164.62 (C O), 162.05 (d, ¹ J _(CF) = 259.4 Hz), 160.66 (C O), 149.70 (C O), 140.77, 136.80 (d, ³ J _(CCCF) = 9.8 Hz), 136.60, 135.60, 133.06, 129.51, 128.78, 128.14, 127.45, 125.05 (d, ⁴ J _(CCCCF) = 3.6 Hz), 123.60, 120.51 (d, ² J _(CCF) = 8.1 Hz), 117.16 (d, ² J _(CCF) = 23.1 Hz), 115.70, 115.56, 75.50 (d, ³ J _(CCCP) = 6.5 Hz, C5), 63.25 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.76 (d, ³ J _(CNCP) = 4.9 Hz, CH₂Ph), 62.46 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 60.84 (d, ¹ J _(CP) = 169.9 Hz, C3), 44.91 (CH₂N), 34.96 (d, ² J _(CCP) = 1.6 Hz, C4), 16.54 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.47 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.72. Anal. calcd. for C₃₀H₃₁FN₃O₇P: C, 60.50; H, 5.25; N, 7.06. Found: C, 60.58.; H, 5.23; N, 6.97 (obtained on a 10:90 mixture of cis-13b and trans-13b).

4.5.10. Diethyl cis-{2-benzyl-5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-13c)

A colorless oil. IR (film, cm⁻¹) ν_max: 3063, 3031, 2982, 2930, 1751, 1702, 1665, 1608, 1480, 1389, 1284, 1159, 1051, 1022, 965, 793. (¹H NMR signals of cis- 13c were extracted from the spectrum of a 87:13 mixture of cis-13c and trans-13c) ¹H NMR (600 MHz, CDCl₃): δ = 8.13–8.11 (m, 1H), 7.76–7.73 (m, 1H), 7.68–7.66 (m, 1H), 7.51–7.46 (m, 4H), 7.33–7.29 (m, 4H), 7.17–7.12 (m, 1H), 4.63 (dddd, ³ J _(H5–H4α) = 10.1 Hz, ³ J _(H5–CH) = 8.5 Hz, ³ J _(H5–H4β) = 4.0 Hz, ³ J _(H5–CH) = 2.0 Hz, 1H, HC5), 4.44 (d, ² J = 13.4 Hz, 1H, HCHPh), 4.32–4.24 (m, 5H, 2 × CH ₂OP, HCHN), 4.20 (dd, ² J = 14.6 Hz, ³ J = 2.0 Hz, 1H, HCHN), 3.92 (d, ² J = 13.4 Hz, 1H, HCHPh), 3.24 (ddd, ³ J _(H3–H4α) = 10.1 Hz, ³ J _(H3–H4β) = 7.3 Hz, ² J _(H3–P) = 3.2 Hz, 1H, HC3), 2.82 (dddd, ³ J _(H4α–P) = 18.7 Hz, ² J _{(H4α–H4β)} = 13.0 Hz, ³ J _(H4α–H3) = 10.1 Hz, ³ J _(H4β–H5) = 10.1 Hz, 1H, HαC4), 2.42 (dddd, ² J _{(H4β–H4α)} = 13.0 Hz, ³ J _(H4β–P) = 11.5 Hz, ³ J _(H4β–H3) = 7.3 Hz, ³ J _(H4β–H5) = 4.0 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.0 Hz, 3H, 2 × CH ₃CH₂OP); (¹³C NMR signals of cis- 13c were extracted from the spectrum of a 59:41 mixture of cis-13c and trans-13c) ¹³C NMR (151 MHz, CDCl₃): δ = 167.89 (d, ⁴ J _(C(O)CCCF) = 3.0 Hz, C O), 162.93 (d, ¹ J _(CF) = 248.8 Hz), 161.21 (C O), 149.64 (C O), 141.13, 136.58, 135.79, 134.06 (d, ³ J _(CCCF) = 7.4 Hz), 130.85 (d, ³ J _(CCCF) = 7.1 Hz), 129.95, 128.31, 128.15, 127.59, 126.16 (d, ⁴ J _(CCCCF) = 2.8 Hz), 123.27, 122.05 (d, ² J _(CCF) = 21.7 Hz), 117.07 (d, ² J _(CCF) = 23.2 Hz), 116.11, 114.95, 75.63 (d, ³ J _(CCCP) = 6.5 Hz, C5), 62.93 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.69 (d, ² J _(COP) = 6.3 Hz, CH₂OP), 62.36 (d, ³ J _(CNCP) = 5.0 Hz, CH₂Ph), 60.61 (d, ¹ J _(CP) = 170.5 Hz, C3), 47.18 (CH₂N), 35.04 (C4), 16.62 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.55 (d, ³ J _(CCOP) = 5.2 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.61. Anal. calcd. for C₃₀H₃₁FN₃O₇P × H₂O: C, 58.82; H, 5.27; N, 6.86. Found: C, 58.64; H, 5.17; N, 6.82 (obtained on a 87:13 mixture of cis-13c and trans-13c).

4.5.11. Diethyl trans-{2-benzyl-5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-13c)

A colorless oil. IR (film, cm⁻¹) ν_max: 3064, 2983, 2931, 2907, 1752, 1703, 1665, 1608, 1480, 1390, 1285, 1147, 1052, 1023, 965, 756. ¹H NMR (600 MHz, CDCl₃): δ = 8.24–8.23 (m, 1H), 7.75–7.71 (m, 1H), 7.68–7.66 (m, 1H), 7.49–7.46 (m, 2H), 7.39–7.34 (m, 2H), 7.30–729 (m, 6H), 4.48–4.44 (m, 3H, H ₂CN, HC5), 4.24–4.17 (m, 5H, 2 × CH ₂OP, H'CHPh), 3.92 (d, ² J = 14.0 Hz, 1H, HCHPh), 3.32–3.29 (m, 1H, HC3), 2.69 (dddd, ³ J _(H4α–P) = 18.1 Hz, ² J _{(H4α–H4β)} = 12.5 Hz, ³ J _(H4α–H3) = 6.1 Hz, ³ J _(H4β–H5) = 6.1 Hz, 1H, HαC4), 2.41–2.34 (m, 1H, HβC4), 1.35 (t, ³ J = 6.1 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 167.60 (d, ⁴ J _(C(O)CCCF) = 2.9 Hz, C O), 162.94 (d, ¹ J _(CF) = 245.1 Hz), 160.92 (C O), 149.71 (C O), 140.79, 136.38, 135.79, 139.91 (d, ³ J _(CCCF) = 7.3 Hz), 130.89 (d, ³ J _(CCCF) = 7.8 Hz), 129.64, 128.90, 128.15, 127.53, 126.16 (d, ⁴ J _(CCCCF) = 2.4 Hz), 123.78, 122.13 (d, ² J _(CCF) = 21.7 Hz), 117.08 (d, ² J _(CCF) = 23.4 Hz), 115.59, 115.51, 75.48 (d, ³ J _(CCCP) = 6.3 Hz, C5), 63.31 (d, ² J _(COP) = 6.4 Hz, CH₂OP), 62.67 (br s, CH₂Ph), 62.52 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 60.75 (d, ¹ J _(CP) = 170.1 Hz, C3), 45.30 (CH₂N), 35.21 (C4), 16.55 (d, ³ J _(CCOP) = 5.5 Hz, CH₃CH₂OP), 16.47 (d, ³ J _(CCOP) = 5.5 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.58. Anal. calcd. for C₃₀H₃₁FN₃O₇P × H₂O: C, 58.82; H, 5.27; N, 6.86. Found: C, 58.74; H, 5.19; N, 6.92.

4.5.12. Diethyl cis-{2-benzyl-5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-13d)

A colorless oil. IR (film, cm⁻¹) ν_max: 3460, 3063, 2990, 1750, 1700, 1669, 1610, 1490, 1391, 1252, 1022, 970, 757, 574. (¹H NMR signals of cis- 13d were extracted from the spectrum of a 92:8 mixture of cis-13d and trans-13d) ¹H NMR (600 MHz, CDCl₃): δ = 8.13–8.11 (m, 1H), 8.02–7.98 (m, 2H), 7.42–7.40 (m, 1H), 7.35–7.34 (m, 2H), 7.30–7.29 (m, 3H), 7.20–7.12 (m, 4H), 4.65–4.61 (m, 1H, HC5), 4.44 (d, ² J = 13.6 Hz, 1H, HCHPh), 4.32–4.23 (m, 5H, 2 × CH ₂OP, HCHN), 4.19 (dd, ² J = 14.9 Hz, ³ J _(HC–H5) = 2.5 Hz, 1H, HCHN), 3.92 (d, ² J = 13.6 Hz, 1H, HCHPh), 3.24 (ddd, ³ J _(H3 — _H4α) = 10.3 Hz, ³ J _(H3 — _H4β) = 7.3 Hz, ² J _(H3 — _P) = 3.2 Hz, 1H, HC3), 2.82 (dddd, ³ J _(H4α–P) = 18.7 Hz, ² J _{(H4α–H4β)} = 13.0 Hz, ³ J _(H4α–H3) = 10.3 Hz, ³ J _(H4β–H5) = 8.6 Hz, 1H, HαC4), 2.39 (dddd, ² J _{(H4β–H4α)} = 13.0 Hz, ³ J _(H4β–P) = 11.5 Hz, ³ J _(H4β–H3) = 7.3 Hz, ³ J _(H4β–H5) = 4.1 Hz, 1H, HβC4), 1.42 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.41 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP); (¹³C NMR signals of cis- 13d were extracted from the spectrum of a 85:15 mixture of cis-13d and trans-13d) ¹³C NMR (151 MHz, CDCl₃): δ = 167.58 (C O), 166.90 (d, ¹ J _(CF) = 258.7Hz), 161.22 (C O), 149.70 (C O), 141.14, 136.60, 135.74, 133.33 (d, ³ J _(CCCF) = 9.9Hz), 129.91, 128.39 (d, ⁴ J _(CCCCF) = 2.5 Hz), 128.32, 128.18, 127.59, 123.22, 116.50 (d, ² J _(CCF) = 22.2 Hz), 116.06, 115.01, 75.64 (d, ³ J _(CCCP) = 6.5 Hz, C5), 62.92 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 62.69 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 62.38 (d,³ J _(CNCP) = 5.0 Hz,CH₂Ph), 60.63 (d, ¹ J _(CP) = 170.2 Hz, C3), 47.15 (CH₂N), 35.04 (d, ² J _(CCP) = 1.3 Hz, C4), 16.62 (d, ³ J _(CCOP) = 5.5 Hz, CH₃CH₂OP), 16.51 (d, ³ J _(CCOP) = 6.5 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.64. Anal. calcd. for C₃₀H₃₁FN₃O₇P × H₂O: C, 58.82; H, 5.27; N, 6.86. Found: C, 58.93; H, 5.29; N, 6.81 (obtained on a 85:15 mixture of cis-13d and trans-13d).

4.5.13. Diethyl trans-{2-benzyl-5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-13d)

A colorless oil. IR (film, cm⁻¹) ν_max: 3458, 2982, 1749, 1701, 1665, 1600, 1479, 1390, 1242, 1022, 969, 756. ¹H NMR (600 MHz, CDCl₃): δ = 8.24–8.23 (m, 1H), 8.00–7.98 (m, 2H), 7.74–7.71 (m, 1H), 7.47–7.44 (m, 1H), 7.36–7.34 (m, 1H), 7.30–7.27 (m, 5H), 7.19–7.15 (m, 2H), 4.48–4.41 (m, 2H, HC5, HCHN), 4.45 (d, ² J = 13.9 Hz, 1H, HCHPh), 4.27–4.17 (m, 5H, 2 × CH ₂OP, HCHN), 3.91 (d, ² J = 13.9 Hz, 1H, HCHPh), 3.30 (ddd, ³ J _(H3–H4β) = 9.5 Hz, ³ J _(H3–H4α) = 6.6 Hz, ² J _(H3–P) = 2.8 Hz, 1H, HC3), 2.69 (dddd, ³ J _(H4α–P) = 19.1 Hz, ² J _{(H4α–H4β)} = 13.0 Hz, ³ J _(H4α–H3) = 6.6 Hz, ³ J _(H4β–H5) = 6.6 Hz, 1H, HαC4), 2.37 (dddd, ³ J _(H4β–P) = 14.9 Hz, ² J _{(H4β–H4α)} = 13.0 Hz, ³ J _(H4β–H3) = 9.5 Hz, ³ J _(H4β–H5) = 8.1 Hz, 1H, HβC4), 1.35 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.34 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 167.29 (C O), 166.93 (d, ¹ J _(CF) = 258.1 Hz), 160.93 (C O), 149.73 (C O), 140.79, 136.48, 135.72, 133.33 (d, ³ J _(CCCF) = 9.9 Hz), 129.61, 128.88, 128.23 (d, ⁴ J _(CCCCF) = 2.8 Hz), 128.1, 127.50, 123.73, 116.54 (d, ² J _(CCF) = 22.2 Hz), 115.55, 115.53, 75.43 (d, ³ J _(CCCP) = 6.3 Hz, C5), 63.27 (d, ² J _(COP) = 6.3 Hz, CH₂OP), 62.69 (d, ³ J _(CNCP) = 4.0 Hz, CH₂Ph), 62.50 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 60.77 (d, ¹ J _(CP) = 169.8 Hz, C3), 45.30 (CH₂N), 35.24 (d, ² J _(CCP) = 1.9 Hz, C4), 16.55 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.47 (d, ³ J _(CCOP) = 5.8 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.64. Anal. calcd. for C₃₀H₃₁FN₃O₇P × 1.5 H₂O: C, 57.88; H, 5.50; N, 6.75. Found: C, 58.05; H, 5.73; N, 6.81.

4.5.14. Diethyl cis-{5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14a)

A colorless oil. IR (film, cm⁻¹) ν_max: 3477, 2987, 1750, 1700, 1669, 1610, 1490, 1393, 1256, 1017. (NMR signals of cis- 14a were extracted from the spectrum of a 75:25 mixture of cis-14a and trans-14a) ¹H NMR (600 MHz, CDCl₃): δ = 8.24–8.22 (m, 1H), 7.76–7.74 (m, 1H), 7.67–7.63 (m, 1H), 7.53–7.52 (m, 2H), 7.33–7.30 (m, 2H), 7.26–7.23 (m, 2H), 5.31 (AB, J _AB = 13.9 Hz, 1H, HCHN), 5.26 (AB, J _AB = 13.9 Hz, 1H, HCHN), 4.60 (dddd, ³ J _(H5–H4α) = 9.6 Hz, ³ J _(H5–CH) = 7.3 Hz, ³ J _(H5–H4β) = 3.7 Hz, ³ J _(H5–CH) = 3.7 Hz, 1H, HC5), 4.29–4.22 (m, 5H, 2 × CH ₂OP, HCHN), 4.18 (dd, ² J = 14.9 Hz, ³ J = 3.7 Hz, 1H, HCHN), 2.93 (ddd, ³ J _(H3–H4α) = 10.0 Hz, ³ J _(H3–H4β) = 7.8 Hz, ² J _(H3–P) = 2.3 Hz, 1H, HC3), 2.82 (d, ⁴ J = 0.6 Hz, CH ₃N), 2.84–2.79 (m, 1H, HαC4), 2.41 (dddd, ² J _{(H4β–H4α)} = 12.8 Hz, ³ J _(H4β–P) = 12.8 Hz, ³ J _(H4β–H3) = 7.8 Hz, ³ J _(H4β–H5) = 3.7 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.1 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 161.94 (C O), 151.24 (C O), 141.15, 137.04, 134.73, 128.97, 128.56, 128.40, 127.54, 128.79, 122.93, 115.55, 115.43, 75.08 (d, ³ J _(CCCP) = 7.3 Hz, C5), 63.22 (d, ¹ J _(CP) = 168.7 Hz, C3), 62.83 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 62.64 (d, ² J _(COP) = 6.8 Hz, CH₂OP), 47.19 (s, CH₂N), 45.22 (d, ³ J _(CNCP) = 3.6 Hz, CH₃N), 44.92 (CH₂Ph), 35.67 (d, ² J _(CCP) = 1.7 Hz, C4), 16.59 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.51 (d, ³ J _(CCOP) = 5.5 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.51. Anal. calcd. for C₂₄H₃₀N₃O₆P: C, 53.19; H, 6.20; N, 8.62. Found: C, 53.35; H, 5.99; N, 8.36 (obtained on a 75:25 mixture of cis-14a and trans-14a).

4.5.15. Diethyl trans-{5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14a)

A colorless oil. IR (film, cm⁻¹) ν_max: 3474, 2980, 1703, 1660, 1662, 1609, 1483, 1349, 1238, 1023, 966, 759. ¹H NMR (600 MHz, CDCl₃): δ = 8.26–8.24 (m, 1H), 7.66–7.63 (m, 1H), 7.52–7.51 (m, 2H), 7.40–7.38 (m, 1H), 7.32–7.28 (m, 2H), 7.28–7.24 (m, 2H), 5.32 (AB, J _AB = 13.9 Hz, 1H, HCHN), 5.27 (AB, J _AB = 13.9 Hz, 1H, HCHN), 4.51 (dd, ² J = 14.9 Hz, ³ J _(HC–H5) = 4.1 Hz, 1H, HCHN), 4.42–4.37 (m, 1H, HC5), 4.21–4.14 (m, 5H, 2 × CH ₂OP, HCHN), 3.04–2.98 (m, 1H, HC3), 2.86 (s, CH ₃N), 2.68 (dddd, ³ J _(H4α–P) = 19.3 Hz, ² J _{(H4α–H4β)} = 12.6 Hz, ³ J _(H4α–H3) = 7.1 Hz, ³ J _(H4β–H5) = 7.1 Hz, 1H, HαC4), 2.41 (dddd, ² J _{(H4β–H4α)} = 12.6 Hz, ³ J _(H4β–P) = 12.6 Hz, ³ J _(H4β–H3) = 9.7 Hz, ³ J _(H4β–H5) = 4.4 Hz, 1H, HβC4), 1.35 (t, ³ J = 7.3 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 161.63 (C O), 151.29 (C O), 140.14, 136.93, 134.87, 128.04, 128.9, 128.40, 127.58, 128.79, 123.16, 115.67, 114.57, 75.35 (d, ³ J _(CCCP) = 7.3 Hz, C5), 63.95 (d, ¹ J _(CP) = 173.5 Hz, C3), 63.14 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.40 (d, ² J _(COP) = 7.1 Hz, CH₂OP), 46.24 (CH₃N), 45.98 (CH₂N), 45.04 (CH₂Ph), 35.99 (C4), 16.50 (d, ³ J _(CCOP) = 5.9 Hz, CH₃CH₂OP), 16.45 (d, ³ J _(CCOP) = 5.9 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.74. Anal. calcd. for C₂₄H₃₀N₃O₆P × H₂O: C, 57.02; H, 6.38; N, 8.31. Found: C, 57.26; H, 6.09; N, 8.33.

4.5.16. Diethyl cis-{5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14b)

A colorless oil. IR (film, cm⁻¹) ν_max: 2999, 1780, 1720, 1666, 1617, 1450, 1386, 1249, 1032. ¹H NMR (600 MHz, CDCl₃): δ = 8.25–8.24 (m, 1H), 7.79–7.77 (m, 1H), 7.68–7.66 (m, 1H), 7.32–7.26 (m, 2H), 7.25–7.22 (m, 1H), 7.08–7.05 (m, 2H), 5.41 (AB, J _AB = 14.6 Hz, 1H, HCHN), 5.37 (AB, J _AB = 14.6 Hz, 1H, HCHN), 4.62–4.59 (m, 1H, HC5), 4.31–4.20 (m, 5H, 2 × CH ₂OP, HCHN), 4.20 (dd, ² J = 15.2 Hz, ³ J _(HC–H5) = 7.4 Hz, 1H, HCHN), 2.95–2.92 (m, 1H, HC3), 2.83 (dddd, ³ J _(H4α–P) = 18.3 Hz, ² J _{(H4α–H4β)} = 11.5 Hz, ³ J _(H4α–H3) = 9.4 Hz, ³ J _(H4β–H5) = 9.4 Hz, 1H, HαC4), 2.82 (s, CH ₃N), 2.38 (dddd, ² J _{(H4β–H4α)} = 11.5 Hz, ³ J _(H4β–P) = 11.5 Hz, ³ J _(H4β–H3) = 7.9 Hz, ³ J _(H4β–H5) = 3.4 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.0 Hz, 6H, 2 × CH ₃CH₂OP); (¹³C NMR signals of cis- 14b were extracted from the spectrum of a 80:20 mixture of cis-14b and trans-14b) ¹³C NMR (151 MHz, CDCl₃): δ = 161.91 (s, C O), 160.77 (d, ¹ J _(CF) = 247.7 Hz), 150.05 (C O), 141.22, 134.84, 131.24, 129.34 (d, ³ J _(CCCF) = 3.5 Hz), 128.93 (d, ³ J _(CCCF) = 7.8 Hz), 124.06 (d, ⁴ J _(CCCCF) = 3.2 Hz), 123.95 (d, ² J _(CCF) = 14.3 Hz), 123.00, 115.63, 115.43 (d, ² J _(CCF) = 21.8 Hz), 114.66, 75.10 (d, ³ J _(CCCP) = 7.1 Hz, C5), 63.21 (d, ¹ J _(CP) = 169.2 Hz, C3), 62.82 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.55 (d, ² J _(COP) = 6.8 Hz, CH₂OP), 47.18 (CH₂N), 45.19 (d, ³ J _(CNCP) = 3.8 Hz, CH₃N), 38.65 (d, ³ J _(CCCF) = 4.7 Hz, CH₂Ph), 35.65 (d, ² J _(CCP) = 1.6 Hz, C4), 16.58 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.50 (d, ³ J _(CCOP) = 5.8 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.50. Anal. calcd. for C₂₄H₂₉FN₃O₆P × 1.5 H₂O: C, 54.13; H, 6.06; N, 7.89. Found: C, 54.16; H, 5.96; N, 8.37.

4.5.17. Diethyl trans-{5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14b)

A colorless oil. IR (film, cm⁻¹) ν_max: 3063, 2981, 1707, 1665, 1610, 1483, 1455, 1410, 1347, 1232, 1052, 1023. ¹H NMR (600 MHz, CDCl₃): δ = 8.27–8.26 (m, 1H), 7.69–7.67 (m, 1H), 7.43–7.42 (m, 1H), 7.30–7.27 (m, 2H), 7.25–7.22 (m, 1H), 7.08–7.05 (m, 2H), 5.40 (AB, J _AB = 14.8 Hz, 1H, HCHN), 5.38 (AB, J _AB = 14.8 Hz, 1H, HCHN), 4.53 (dd, ² J = 15.0 Hz, ³ J _(HC–H5) = 4.1 Hz, 1H, HCHN), 4.41 (dddd, ³ J _(H5–H4β) = 11.9 Hz, ³ J _(H5–CH) = 7.0 Hz, ³ J _(H5–H4α) = 7.0 Hz, ³ J _(H5–CH) = 4.1 Hz, 1H, HC5), 4.21–4.15 (m, 5H, 2 × CH ₂OP, HCHN), 3.04–2.98 (m, 1H, HC3), 2.86 (s, CH ₃N), 2.68 (dddd, ³ J _(H4α–P) = 19.4 Hz, ² J _{(H4α–H4β)} = 13.2 Hz, ³ J _(H4α–H3) = 7.0 Hz, ³ J _(H4β–H5) = 7.0 Hz, 1H, HαC4), 2.38–2.38 (m, 1H, HβC4), 1.35 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.34 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 161.58 (C O), 160.76 (d, ¹ J _(CF) = 247.1 Hz), 150.13 (C O), 140.19, 134.98, 129.26 (d, ⁴ J _(CCCCF) = 3.8 Hz), 129.10, 128.96 (d, ³ J _(CCCF) = 8.0 Hz), 124.03 (d, ³ J _(CCCF) = 3.3 Hz), 123.81 (d, ² J _(CCF) = 14.6 Hz), 123.23, 115.55, 115.44 (d, ² J _(CCF) = 21.8 Hz), 114.66, 75.32 (d, ³ J _(CCCP) = 7.0 Hz, C5), 63.94 (d, ¹ J _(CP) = 169.2 Hz, C3), 63.12 (d, ² J _(COP) = 6.4 Hz, CH₂OP), 62.40 (d, ² J _(COP) = 6.8 Hz, CH₂OP), 46.22 (CH₃N), 46.00 (CH₂N), 33.91 (d, ³ J _(CCCF) = 4.5 Hz, CH₂Ph), 35.96 (C4), 16.48 (d, ³ J _(CCOP) = 6.7 Hz, CH₃CH₂OP), 16.43 (d, ³ J _(CCOP) = 6.9 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.72. Anal. calcd. for C₂₄H₂₉FN₃O₆P × 1.5 H₂O: C, 54.13; H, 6.06; N, 7.89. Found: C, 54.22; H, 6.20; N, 5.87.

4.5.18. Diethyl cis-{5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14c)

A colorless oil. IR (film, cm⁻¹) ν_max: 3065, 2981, 2908, 1704, 1660, 1608, 1481, 1345, 1138, 1050, 1022, 968, 794, 758. (¹H NMR signals of cis-14c were extracted from the spectrum of a 97:3 mixture of cis-14c and trans-14c) ¹H NMR (600 MHz, CDCl₃): δ = 8.24–8.22 (m, 1H), 7.77–7.76 (m, 1H), 7.67–7.65 (m, 1H), 7.30–7.26 (m, 3H), 7.25–7.22 (m, 1H), 6.98–6.94 (m, 1H), 5.29 (AB, J _AB = 14.0 Hz, 1H, HCHN), 5.25 (AB, J _AB = 14.0 Hz, 1H, HCHN), 4.60 (dddd, ³ J _(H5–H4α) = 11.9 Hz, ³ J _(H5–CH) = 6.0 Hz, ³ J _(H5–H4β) = 3.0 Hz, ³ J _(H5–CH) = 3.0 Hz, 1H, HC5), 4.31–4.29 (m, 5H, 2 × CH ₂OP, HCHN), 4.21 (dd, ² J = 14.7 Hz, ³ J _(HC–H5) = 3.0 Hz, 1H, HCHN), 2.94 (ddd, ³ J _(H3–H4α) = 9.9 Hz, ³ J _(H3–H4β) = 7.7 Hz, ² J _(H3–P) = 2.2 Hz, 1H, HC3), 2.86–2.80 (m, 1H, HαC4), 2.82 (s, CH ₃N), 2.40 (dddd, ² J _{(H4β–H4α)} = 12.5 Hz, ³ J _(H4β–P) = 11.6 Hz, ³ J _(H4β–H3) = 7.7 Hz, ³ J _(H4β–H5) = 3.0 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.1 Hz, 6H, 2 × CH ₃CH₂OP); (¹³C NMR signals of cis-14c were extracted from the spectrum of a 69:31 mixture of cis-14c and trans-14c) ¹³C NMR (150 MHz, CDCl₃): δ = 162.79 (d, ¹ J _(CF) = 246.2 Hz), 161.87 (C O), 151.15 (C O), 141.14, 139.36 (d, ³ J _(CCCF) = 7.5 Hz), 134.88, 129.86 (d, ³ J _(CCCF) = 8.5 Hz), 128.56, 124.52 (d, ⁴ J _(CCCCF) = 1.7 Hz), 123.05, 115.81 (d, ² J _(CCF) = 21.9 Hz), 115.62, 114.64, 114.50 (d, ² J _(CCF) = 21.0 Hz), 75.03 (d, ³ J _(CCCP) = 7.4 Hz, C5), 63.17 (d, ¹ J _(CP) = 169.3 Hz, C3), 62.60 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.57 (d, ² J _(COP) = 7.1 Hz, CH₂OP), 47.20 (CH₂N), 45.17 (d, ³ J _(CNCP) = 4.1 Hz, CH₃N), 44.41 (CH₂Ph), 35.63 (d, ² J _(CCP) = 1.4 Hz, C4), 16.59 (d, ³ J _(CCOP) = 5.9 Hz, CH₃CH₂OP), 16.51 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.48. Anal. calcd. for C₂₄H₂₉FN₃O₆P × H₂O: C, 55.06; H, 5.97; N, 8.03. Found: C, 54.98; H, 5.88; N, 7.91 (obtained on a 69:31 mixture of cis-14c and trans-14c).

4.5.19. Diethyl trans-{5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14c)

A colorless oil. IR (film, cm⁻¹) ν_max: 3062, 2981, 1703, 1657, 1610, 1483, 1400, 1346, 1251, 1051, 967, 787. δ = ¹H NMR (600 MHz, CDCl₃): δ = 8.24 (d, J = 7.7 Hz, 1H), 7.67–7.65 (m, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.30–7.24 (m, 3H), 7.25–7.20 (m, 1H), 6.97–6.93 (m, 1H), 5.28 (AB, J _AB = 14.0 Hz, 1H, HCHN), 5.25 (AB, J _AB = 14.0 Hz, 1H, HCHN), 4.51 (dd, ² J = 14.9 Hz, ³ J _(HC–H5) = 4.1 Hz, 1H, HCHN), 4.45–4.37 (m, 1H, HC5), 4.24–4.15 (m, 5H, 2 × CH ₂OP, HCHN), 3.05–3.00 (m, 1H, HC3), 2.85 (s, CH ₃N), 2.76–2.64 (m, 1H, HαC4), 2.43–36 (m, 1H, HβC4), 1.35 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.34 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (150 MHz, CDCl₃): δ = 162.80 (d, ¹ J _(CF) = 245.8 Hz), 161.58 (C O), 151.22 (C O), 140.12, 139.26 (d, ³ J _(CCCF) = 7.5 Hz), 135.04, 129.88 (d, ³ J _(CCCF) = 8.5 Hz), 129.08, 124.52 (d, ⁴ J _(CCCCF) = 2.4 Hz), 123.29, 115.76 (d, ² J _(CCF) = 21.8 Hz), 115.56, 114.64 114.56 (d, ² J _(CCF) = 22.8 Hz), 75.31 (d, ³ J _(CCCP) = 7.1 Hz, C5), 63.93 (d, ¹ J _(CP) = 169.9 Hz, C3), 63.18 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.43 (d, ² J _(COP) = 7.2 Hz, CH₂OP), 46.24 (d, ³ J _(CNCP) = 3.8 Hz, CH₃N), 46.02 (CH₂N), 44.54 (CH₂Ph), 35.06 (C4), 16.50 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.44 (d, ³ J _(CCOP) = 6.0 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.73. Anal. calcd. for C₂₄H₂₉FN₃O₆P × H₂O: C, 55.06; H, 5.97; N, 8.03. Found: C, 55.24; H, 5.55; N, 7.95.

4.5.20. Diethyl cis-{5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14d)

A colorless oil. IR (film, cm⁻¹) ν_max: 2989, 1711, 1673, 1617, 1483, 1393, 1250, 1020, 970, 770. (NMR signals of cis-14d were extracted from the spectrum of a 75:25 mixture of cis-14d and trans-14d) ¹H NMR (600 MHz, CDCl₃): δ = 8.21–8.20 (m, 1H), 7.75–7.74 (m, 1H), 7.66–7.62 (m, 1H), 7.54–7.51 (m, 2H), 7.29–7.22 (m, 1H), 7.00–6.96 (m, 2H), 5.4125 (AB, J _AB = 13.8 Hz, 1H, HCHN), 5.21 (AB, J _AB = 13.8 Hz, 1H, HCHN), 4.58 (dddd, ³ J _(H5–H4β) = 11.8 Hz, ³ J _(H5–CH) = 7.0 Hz, ³ J _(H5–H4α) = 3.6 Hz, ³ J _(H5–CH) = 3.6 Hz, 1H, HC5), 4.29–4.21 (m, 4H, 2 × CH ₂OP), 4.19 (dd, ² J = 14.2 Hz, ³ J _(HC–H5) = 3.6 Hz, 1H, HCHN), 4.17 (dd, ² J = 14.2 Hz, ³ J _(HC–H5) = 7.0 Hz, 1H, HCHN), 2.93 (ddd, ³ J _(H3–H4α) = 9.8 Hz, ³ J _(H3–H4β) = 7.8 Hz, ² J _(H3–P) = 2.0 Hz, 1H, HC3), 2.85–2.78 (m, 1H, HαC4), 2.81 (s, CH ₃N), 2.39 (dddd, ² J _{(H4β–H4α)} = 11.8 Hz, ³ J _(H4β–P) = 11.8 Hz, ³ J _(H4β–H3) = 7.8 Hz, ³ J _(H4β–H5) = 3.6 Hz, 1H, HβC4), 1.40 (t, ³ J = 7.0 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 162.27 (d, ¹ J _(CF) = 246.3 Hz), 161.88 (C O), 151.16 (C O), 141.12, 134.79, 132.85 (d, ⁴ J _(CCCCF) = 3.2 Hz), 131.00 (d, ³ J _(CCCF) = 7.8 Hz), 128.50, 122.98, 115.59, 115.35, 115.17 (d, ² J _(CCF) = 21.4 Hz), 75.03 (d, ³ J _(CCCP) = 7.3 Hz, C5), 63.20 (d, ¹ J _(CP) = 169.0 Hz, C3), 62.82 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.55 (d, ² J _(COP) = 6.7 Hz, CH₂OP), 47.18 (CH₂N), 45.16 (d, ³ J _(CNCP) = 3.8 Hz, CH₃N), 44.16 (CH₂Ph), 35.65 (C4), 16.57 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.50 (d, ³ J _(CCOP) = 5.9 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.48. Anal. calcd. for C₂₄H₂₉FN₃O₆P × H₂O: C, 55.06; H, 5.97; N, 8.03. Found: C, 54.89; H, 5.92; N, 8.04 (obtained on a 75:25 mixture of cis-14d and trans-14d).

4.5.21. Diethyl trans-{5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14d)

A colorless oil. IR (film, cm⁻¹) ν_max: 3060, 2981, 1704, 1651, 1609, 1607, 1484, 1400, 1223, 1096, 1024, 966, 771, 756. ¹H NMR (600 MHz, CDCl₃): δ = 8.25–8.23 (m, 1H), 7.66–7.64 (m, 1H), 7.54–7.51 (m, 2H), 7.39–7.38 (m, 1H), 7.29–7.25 (m, 1H), 7.00–6.97 (m, 2H), 5.25 (AB, J _AB = 13.8 Hz, 1H, HCHN), 5.23 (AB, J _AB = 13.8 Hz, 1H, HCHN), 4.50 (dd, ² J = 15.0 Hz, ³ J _(HC–H5) = 4.3 Hz, 1H, HCHN), 4.39 (dddd, ³ J _(H5–H4β) = 9.8 Hz, ³ J _(H5–CH) = 7.0 Hz, ³ J _(H5–H4α) = 7.0 Hz, ³ J _{(H5–HδCHN)} = 4.3 Hz, 1H, HC5), 4.21–4.15 (m, 5H, 2 × CH ₂OP, HCHN), 3.02–2.98 (m, 1H, HC3), 2.85 (s, CH ₃N), 2.69 (dddd, ³ J _(H4α–P) = 19.4 Hz, ² J _{(H4α–H4β)} = 12.5 Hz, ³ J _(H4α–H3) = 7.0 Hz, ³ J _(H4β–H5) = 7.0 Hz, 1H, HαC4), 2.41 (dddd, ² J _{(H4β–H4α)} = 12.5 Hz, ³ J _(H4β–P) = 12.5 Hz, ³ J _(H4β–H5) = 9.8 Hz, ³ J _(H4β–H3) = 8.0 Hz, 1H, HβC4), 1.35 (t, ³ J = 7.2 Hz, 3H, CH ₃CH₂OP), 1.34 (t, ³ J = 7.5 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 162.30 (d, ¹ J _(CF) = 246.3 Hz), 161.60 (C O), 151.22 (C O), 140.11, 134.94, 132.76 (d, ⁴ J _(CCCCF) = 3.2 Hz), 130.85 (d, ³ J _(CCCF) = 8.0 Hz), 129.01, 123.22, 115.62, 115.19 (d, ² J _(CCF) = 21.4 Hz), 114.61, 75.33 (d, ³ J _(CCCP) = 7.4 Hz, C5), 63.94 (d, ¹ J _(CP) = 168.6 Hz, C3), 63.8213 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.42 (d, ² J _(COP) = 7.1 Hz, CH₂OP), 46.20 (d, ³ J _(CNCP) = 1.9 Hz, CH₃N), 45.99 (CH₂N), 44.29 (CH₂Ph), 36.00 (C4), 16.48 (d, ³ J _(CCOP) = 7.2 Hz, CH₃CH₂OP), 16.44 (d, ³ J _(CCOP) = 5.9 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.73. Anal. calcd. for C₂₄H₂₉FN₃O₆P × H₂O: C, 55.06; H, 5.97; N, 8.03. Found: C, 54.82; H, 5.82; N, 7.93.

4.5.22. Diethyl cis-{2-benzyl-5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15a)

A colorless oil. IR (film, cm⁻¹) ν_max: 2999, 17123, 1685, 1614, 1599, 1459, 1411, 1260, 1023. (NMR signals of cis- 15a were extracted from the spectrum of a 50:50 mixture of cis-15a and trans-15a) ¹H NMR (600 MHz, CDCl₃): δ = 8.14–8.13 (m, 1H), 7.64–7.61 (m, 1H), 7.53–7.50 (m, 2H), 7.37–7.28 (m, 9H), 7.10–7.04 (m, 1H), 5.31 (AB, J _AB = 13.9 Hz, 1H, HCHN), 5.23 (AB, J _AB = 13.9 Hz, 1H, HCHN), 4.61 (dddd, ³ J _(H5–H4α) = 9.5 Hz, ³ J _(H5–CH) = 7.7 Hz, ³ J _(H5–H4β) = 4.7 Hz, ³ J _(H5–CH) = 3.7 Hz, 1H, HC5), 4.43 (dd, ² J = 13.7 Hz, ³ J _(HC–H5) = 4.7 Hz, 1H, HCHN), 4.31–4.15 (m, 6H, 2 × CH ₂OP, HCHN, HCHPh), 3.89 (d, ² J = 13.6 Hz, 1H, HCHPh), 3.23 (ddd, ³ J _(H3–H4α) = 9.5 Hz, ³ J _(H3–H4β) = 7.8 Hz, ² J _(H3–P) = 2.8 Hz, 1H, HC3), 2.84 (dddd, ³ J _(H4α–P) = 18.5 Hz, ² J _{(H4α–H4β)} = 12.8 Hz, ³ J _(H4α–H3) = 9.5 Hz, ³ J _(H4β–H5) = 9.5 Hz, 1H, HαC4), 2.42 (dddd, ² J _{(H4β–H4α)} = 12.8 Hz, ³ J _(H4β–P) = 12.8 Hz, ³ J _(H4β–H3) = 7.8 Hz, ³ J _(H4β–H5) = 4.7 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP), 1.40 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 161.95 (C O), 151.30 (C O), 140.51, 137.06, 136.57, 134.82, 129.95, 129.02, 128.96, 128.41, 128.28, 127.55, 127.46, 122.66, 115.38, 115.11, 75.82 (d, ³ J _(CCCP) = 6.8 Hz, C5), 62.96 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.61 (d, ² J _(COP) = 6.9 Hz, CH₂OP), 62.29 (d, ³ J _(CNCP) = 5.2 Hz, CH₂Ph), 60.62 (d, ¹ J _(CP) = 170.1 Hz, C3), 47.77 (CH₂N), 44.91 (CH₂Ph), 35.15 (C4), 16.62 (d, ³ J _(CCOP) = 6.0 Hz, CH₃CH₂OP), 16.50 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.66. Anal. calcd. for C₃₀H₃₄N₃O₆P × 1.5 H₂O: C, 61.01; H, 6.31; N, 7.11. Found: C, 60.85; H, 6.53; N, 7.18 (obtained on a 50:50 mixture of cis-15a and trans-15a).

4.5.23. Diethyl trans-{2-benzyl-5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15a)

A colorless oil. IR (film, cm⁻¹) ν_max: 3087, 2981, 1703, 1658, 1608, 1607, 1453, 1400, 1236, 1022, 963, 757, 701. (NMR signals of trans- 15a were extracted from the spectrum of a 24:76 mixture of cis-15a and trans-15) ¹H NMR (600 MHz, CDCl₃): δ = 8.25–8.23 (m, 1H), 7.63–7.60 (m, 1H), 7.53–7.50 (m, 2H), 7.35–7.25 (m, 10H), 5.30 (AB, J _AB = 13.9 Hz, 1H, HCHN), 5.26 (AB, J _AB = 13.9 Hz, 1H, HCHN), 4.48 (dd, ² J = 14.9 Hz, ³ J _(HC–H5) = 3.4 Hz, 1H, HCHN), 4.42 (d, ² J = 13.7 Hz, 1H, HCHPh), 4.45–4.40 (m, 1H, HC5), 4.29–4.14 (m, 5H, 2 × CH ₂OP, HCHN), 3.89 (d, ² J = 13.7 Hz, 1H, HCHPh), 3.29–3.22 (m, 1H, HC3), 2.67 (dddd, ³ J _(H4α–P) = 18.7 Hz, ² J _{(H4α–H4β)} = 12.8 Hz, ³ J _(H4α–H3) = 6.4 Hz, ³ J _(H4β–H5) = 6.4 Hz, 1H, HαC4), 2.46–2.32 (m, 1H, HβC4), 1.36 (t, ³ J = 6.6 Hz, 3H, CH ₃CH₂OP), 1.35 (t, ³ J = 6.6 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 161.65 (s, C(O)), 151.32 (s, C(O)), 140.18, 136.94, 136.51, 134.79, 129.64, 128.92, 128.43, 128.19, 128.12, 127.59, 127.46, 123.13, 115.61, 114.94, 75.64 (d, ³ J _(CCCP) = 6.4 Hz, C5), 62.30 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.70 (d, ³ J _(CNCP) = 5.2 Hz, CH₂Ph), 62.44 (d, ² J _(COP) = 6.8 Hz, CH₂OP), 60.78 (d, ¹ J _(CP) = 169.7 Hz, C3), 45.74 (CH₂N), 45.05 (CH₂Ph), 35.15 (C4), 16.57 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.51 (d, ³ J _(CCOP) = 5.7 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.83. Anal. calcd. for C₃₀H₃₄N₃O₆P × 1.5H₂O: C, 61.01; H, 6.31; N, 7.11. Found: C, 60.81; H, 6.19; N, 7.07 (obtained on a 10:90 mixture of cis-15a and trans-15a).

4.5.24. Diethyl cis-{2-benzyl-5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15b)

A colorless oil. IR (film, cm⁻¹) ν_max: 2978, 1744, 1700, 1662, 1607, 1478, 1389, 1240, 1012, 756. (NMR signals of cis- 15b were extracted from the spectrum of a 85:15 mixture of cis-15b and trans-15b) ¹H NMR (600 MHz, CDCl₃): ¹H NMR (600 MHz, CDCl₃): δ = 8.17–8.13 (m, 1H), 7.34–7.31 (m, 3H), 7.29–7.26 (m, 5H), 7.11–7.04 (m, 4H), 5.39 (AB, ² J _AB = 14.8 Hz, 1H, N—CH _2a), 5.34 (AB, ² J _AB = 14.8 Hz, 1H, N—CH _2b), 4.61 (dddd, ³ J _(H5–H4α) = 10.2 Hz, ³ J _(H5–CH) = 7.9 Hz, ³ J _(H5–H4β) = 4.3 Hz, ³ J _(H5–CH) = 4.3 Hz, 1H, HC5), 4.43 (d, ² J = 13.7 Hz, 1H, HCHPh), 4.32–4.17 (m, 6H, 2 × CH ₂OP, HCHN), 3.90 (d, ² J = 13.7 Hz, 1H, HCHPh), 3.23 (ddd, ³ J _(H3–H4α) = 10.2 Hz, ³ J _(H3–H4β) = 7.4 Hz, ² J _(H3–P) = 3.1 Hz, 1H, HC3), 2.82 (dddd, ³ J _(H4α–P) = 18.3 Hz, ² J _{(H4α–H4β)} = 12.8 Hz, ³ J _(H4α–H3) = 10.2 Hz, ³ J _(H4β–H5) = 10.2 Hz, 1H, HαC4), 2.41 (dddd, ² J _{(H4β–H4α)} = 12.8 Hz,³ J _(H4β–P) = 11.9 Hz, ³ J _(H4β–H3) = 7.4 Hz, ³ J _(H4β–H5) = 4.3 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.40 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 161.91 (s, C O), 160.76 (d, ¹ J _(CF) = 247.5 Hz), 151.10 (s, C O), 140.58, 136.58, 134.95, 129.92, 129.34 (d, ³ J _(CCCF) = 3.8 Hz), 128.93 (d, ³ J _(CCCF) = 7.9 Hz), 128.36, 128.28, 127.55, 124.05 (d, ⁴ J _(CCCCF) = 3.3 Hz), 123.97 (d, ² J _(CCF) = 14.3 Hz), 122.74, 115.53, 115.44 (d, ² J _(CCF) = 21.7 Hz), 115.00, 75.84 (d, ³ J _(CCCP) = 6.6 Hz, C5), 62.95 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.62 (d, ² J _(COP) = 6.9 Hz, CH₂OP), 62.29 (d, ³ J _(CNCP) = 5.2 Hz, CH₂Ph), 60.64 (d, ¹ J _(CP) = 169.9 Hz, C3), 47.77 (CH₂N), 38.64 (d, ³ J _(CCCF) = 4.5 Hz, CH₂Ph), 35.14 (C4), 16.61 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.55 (d, ³ J _(CCOP) = 5.8 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.62. Anal. calcd. for C₃₀H₃₃FN₃O₆P: C, 61.69; H, 5.72; N, 7.23. Found: C, 61.65; H, 5.60; N, 7.35.

4.5.25. Diethyl trans-{2-benzyl-5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15b)

A colorless oil. IR (film, cm⁻¹) ν_max: 2981, 1706, 1664, 1609, 1482, 1454, 1400, 1286, 1231, 1097, 1052, 1022, 756. ¹H NMR (600 MHz, CDCl₃): δ = 8.27–8.26 (m, 1H), 7.67–7.64 (m, 1H), 7.40–7.39 (m, 1H), 7.31–7.26 (m, 7H), 7.25–7.22 (m, 1H), 7.08–7.04 (m, 2H), 5.40 (AB, J _AB = 14.8 Hz, 1H, HCHN), 5.37 (AB, J _AB = 14.8 Hz, 1H, HCHN), 4.50 (dd, ² J = 15.1 Hz, ³ J _(HC–H5) = 4.3 Hz, 1H, HCHN), 4.43 (d, ² J = 13.7 Hz, 1H, HCHPh), 4.40–4.39 (m, 1H, HC5), 4.25–4.17 (m, 5H, 2 × CH ₂OP, HCHN), 3.89 (d, ² J = 13.7 Hz, 1H, HCHPh), 3.28 (ddd, ³ J _(H3–H4β) = 9.4 Hz, ³ J _(H3–H4α) = 6.2 Hz, ² J _(H3–P) = 2.6 Hz, 1H, HC3), 2.67 (dddd, ³ J _(H4α–P) = 19.3 Hz, ² J _{(H4α–H4β)} = 13.8 Hz, ³ J _(H4α–H3) = 6.2 Hz, ³ J _(H4β–H5) = 6.2 Hz, 1H, HαC4), 2.35 (dddd, ³ J _(H4β–P) = 14.8 Hz, ² J _{(H4β–H4α)} = 13.8 Hz, ³ J _(H4β–H3) = 9.4 Hz, ³ J _(H4β–H5) = 8.3 Hz, 1H, HβC4), 1.36 (t, ³ J = 7.4 Hz, 3H, CH ₃CH₂OP), 1.35 (t, ³ J = 6.8 Hz, 3H, CH ₃CH₂OP); (¹³C NMR signals of trans-15b were extracted from the spectrum of a 7:93 mixture of cis-15b and trans-15b) ¹³C NMR (151 MHz, CDCl₃): δ = 161.61 (C O), 160.77 (d, ¹ J _(CF) = 247.1 Hz), 151.17 (C O), 140.26, 136.50, 134.90, 129.63, 129.26 (d, ³ J _(CCCF) = 3.5 Hz), 129.02, 128.98 (d, ³ J _(CCCF) = 7.9 Hz), 128.10, 127.45, 124.05 (d, ⁴ J _(CCCCF) = 3.4 Hz), 123.81 (d, ² J _(CCF) = 14.4 Hz), 123.21, 115.51, 115.46 (d, ² J _(CCF) = 21.7 Hz), 115.01, 75.63 (d, ³ J _(CCCP) = 6.5 Hz, C5), 63.27 (d, ² J _(COP) = 6.4 Hz, CH₂OP), 62.68 (d, ³ J _(CNCP) = 5.1 Hz, CH₂Ph), 62.43 (d, ² J _(COP) = 7.0 Hz, CH₂OP), 60.77 (d, ¹ J _(CP) = 170.3 Hz, C3), 45.75 (CH₂N), 38.95 (d, ³ J _(CCCF) = 4.9 Hz, CH₂Ph), 35.12 (d, ² J _(CCP) = 2.3 Hz, C4), 16.54 (d, ³ J _(CCOP) = 5.4 Hz, CH₃CH₂OP), 16.48 (d, ³ J _(CCOP) = 5.4 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.77. Anal. calcd. for C₃₀H₃₃FN₃O₆P: C, 61.69; H, 5.72; N, 7.23. Found: C, 61.75; H, 5.83; N, 7.43.

4.5.26. Diethyl cis-{2-benzyl-5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15c)

A colorless oil. IR (film, cm⁻¹) ν_max: 2986, 1704, 1660, 1609, 1484, 1400, 1250, 1023, 966, 760. (NMR signals of cis- 15c were extracted from the spectrum of a 65:35 mixture of cis-15c and trans-15c) ¹H NMR (600 MHz, CDCl₃): δ = 8.14–8.13 (m, 1H), 7.33–7.28 (m, 8H), 7.22–7.21 (m, 1H), 7.11–7.06 (m, 2H), 6.97–6.95 (m, 1H), 5.27 (AB, J _AB = 14.1 Hz, 1H, HCHN), 5.23 (AB, J _AB = 14.1 Hz, 1H, HCHN), 4.64–4.58 (m, 1H, HC5), 4.43 (dd, ² J = 13.7 Hz, ³ J _(HC–H5) = 4.2 Hz, 1H, HCHN), 4.30–4.16 (m, 6H, 2 × CH ₂OP, HCHN, HCHPh), 3.89 (d, ² J = 13.7 Hz, 1H, HCHPh), 3.23 (ddd, ³ J _(H3–H4α) = 9.6 Hz, ³ J _(H3–H4β) = 7.5 Hz, ² J _(H3–P) = 2.5 Hz, 1H, HC3), 2.84 (dddd, ³ J _(H4α–P) = 20.3 Hz, ² J _{(H4α–H4β)} = 11.9 Hz, ³ J _(H4α–H3) = 9.6 Hz, ³ J _(H4β–H5) = 9.6 Hz, 1H, HαC4), 2.43 (dddd, ² J _{(H4β–H4α)} = 11.9 Hz, ³ J _(H4β–P) = 11.9 Hz, ³ J _(H4β–H3) = 7.5 Hz, ³ J _(H4β–H5) = 4.4 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.0 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 162.81 (d, ¹ J _(CF) = 246.1 Hz), 161.85 (C O), 151.22 (C O), 140.53, 139.42 (d, ³ J _(CCCF) = 7.5 Hz), 136.58, 134.95, 129.92, 129.84 (d, ³ J _(CCCF) = 8.4 Hz), 128.26, 128.10, 127.53, 124.54 (d, ⁴ J _(CCCCF) = 2.5 Hz), 122.75, 115.82 (d, ² J _(CCF) = 21.6 Hz), 115.47, 115.01, 114.49 (d, ² J _(CCF) = 21.4 Hz), 75.47 (d, ³ J _(CCCP) = 6.7 Hz, C5), 62.94 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.60 (d, ² J _(COP) = 7.0 Hz, CH₂OP), 62.30 (d, ³ J _(CNCP) = 4.8 Hz, CH₂Ph), 60.66 (d, ¹ J _(CP) = 170.2 Hz, C3), 47.81 (CH₂N), 44.41 (CH₂Ph), 35.17 (C4), 16.60 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.54 (d, ³ J _(CCOP) = 5.8 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.60. Anal. calcd. for C₃₀H₃₃FN₃O₆P: C, 61.69; H, 5.72; N, 7.23. Found: C, 61.80; H, 5.95; N, 7.25 (obtained on a 65:35 mixture of cis-15c and trans-15c).

4.5.27. Diethyl trans-{2-benzyl-5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15c)

A colorless oil. IR (film, cm⁻¹) ν_max: 3457, 3063, 2982, 1705, 1700, 1661, 1610, 1483, 1346, 1250, 1235, 1023, 970, 763. (NMR signals of trans-15c were extracted from the spectrum of a 13:87 mixture of cis-15c and trans-15c) ¹H NMR (600 MHz, CDCl₃): δ = 8.25–8.24 (m, 1H), 7.65–7.62 (m, 1H), 7.38–7.36 (m, 1H), 7.33–7.25 (m, 8H), 7.22–7.20 (m, 1H), 6.97–6.94 (m, 1H), 5.28 (AB, J _AB = 14.0 Hz, 1H, HCHN), 5.25 (AB, J _AB = 14.0 Hz, 1H, HCHN), 4.49 (dd, ² J = 15.1 Hz, ³ J _(HC–H5) = 4.3 Hz, 1H, HCHN), 4.43 (d, ² J = 13.8 Hz, 1H, HCHPh), 4.40 (dddd, ³ J _(H5–H4β) = 8.3 Hz, ³ J _(H5–CH) = 6.9 Hz, ³ J _(H5–H4α) = 6.6 Hz, ³ J _(H5–CH) = 4.3 Hz, 1H, HC5), 4.25–4.15 (m, 5H, 2 × CH ₂OP, HCHN), 3.89 (d, ² J = 13.8 Hz, 1H, HCHPh), 3.28 (ddd, ³ J _(H3–H4β) = 9.1 Hz, ³ J _(H3–H4α) = 6.6 Hz, ² J _(H3–P) = 2.6 Hz, 1H, HC3), 2.68 (dddd, ³ J _(H4α–P) = 19.0 Hz, ² J _{(H4α–H4β)} = 12.9 Hz, ³ J _(H4α–H3) = 6.6 Hz, ³ J _(H4β–H5) = 6.6 Hz, 1H, HαC4), 2.35 (dddd, ³ J _(H4β–P) = 14.8 Hz, ² J _{(H4β–H4α)} = 12.9 Hz, ³ J _(H4β–H3) = 9.1 Hz, ³ J _(H4β–H5) = 8.3 Hz, 1H, HβC4), 1.36 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP), 1.35 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 162.81 (d, ¹ J _(CF) = 246.5 Hz), 161.58 (C O), 151.26 (C O), 140.19, 139.28 (d, ³ J _(CCCF) = 7.6 Hz), 136.51, 134.95, 129.90 (d, ³ J _(CCCF) = 9.6 Hz), 129.62, 128.96, 128.10, 127.45, 124.45 (d, ⁴ J _(CCCCF) = 3.2 Hz), 123.23, 115.77 (d, ² J _(CCF) = 21.7 Hz), 115.51, 115.00, 114.51 (d, ² J _(CCF) = 20.9 Hz), 75.61 (d, ³ J _(CCCP) = 6.5 Hz, C5), 63.28 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.70 (d, ³ J _(CNCP) = 4.8 Hz, CH₂Ph), 62.44 (d, ² J _(COP) = 7.0 Hz, CH₂OP), 60.80 (d, ¹ J _(CP) = 170.0 Hz, C3), 45.81 (CH₂N), 44.56 (CH₂Ph), 35.17 (C4), 16.54 (d, ³ J _(CCOP) = 5.5 Hz, CH₃CH₂OP), 16.47 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.76. Anal. calcd. for C₃₀H₃₃FN₃O₆P × 1.5H₂O: C, 59.21; H, 5.96; N, 6.90. Found: C, 59.38; H, 5.98; N, 6.82 (obtained on a 13:87 mixture of cis-15c and trans-15c).

4.5.28. Diethyl cis-{2-benzyl-5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15d)

A colorless oil. IR (film, cm⁻¹) ν_max: 2989, 1706, 1660, 1510, 1489, 1398, 1348, 1225, 1052, 1024, 965, 754. (NMR signals of cis-15d were extracted from the spectrum of a 82:18 mixture of cis-15d and trans-15d) ¹H NMR (600 MHz, CDCl₃): δ = 8.14–8.12 (m, 1H), 7.54–7.51 (m, 2H), 7.37–7.29 (m, 2H), 7.28–7.26 (m, 5H), 7.10–7.05 (m, 1H), 7.00–6.97 (m, 2H), 5.25 (AB, J _AB = 13.9 Hz, 1H, HCHN), 5.20 (AB, J _AB = 13.9 Hz, 1H, HCHN), 4.61 (dddd, ³ J _(H5–H4β) = 9.9 Hz, ³ J _(H5–CH) = 7.8 Hz, ³ J _(H5–H4α) = 3.6 Hz, ³ J _(H5–CH) = 3.6 Hz, 1H, HC5), 4.43 (d, ² J = 13.7 Hz, 1H, HCHPh), 4.32–4.23 (m, 4H, 2 × CH ₂OP), 4.21 (dd, ² J = 12.2 Hz, ³ J _(HC–H5) = 7.8 Hz, 1H, HCHN), 4.19 (dd, ² J = 12.2 Hz, ³ J _(HC–H5) = 3.6 Hz, 1H, HCHN), 3.89 (d, ² J = 13.7 Hz, 1H, HCHPh), 3.23 (ddd, ³ J _(H3–H4α) = 9.9 Hz, ³ J _(H3–H4β) = 7.5 Hz, ² J _(H3–P) = 3.1 Hz, 1H, HC3), 2.84 (dddd, ³ J _(H4α–P) = 18.2 Hz, ³ J _{(H4α–H4β)} = 12.8 Hz, ³ J _(H4α–H3) = 9.9 Hz, ³ J _(H4β–H5) = 9.9 Hz, 1H, HαC4), 2.42 (dddd, ² J _{(H4β–H4α)} = 12.8 Hz, ³ J _(H4β–P) = 11.8 Hz, ³ J _(H4β–H3) = 7.5 Hz, ³ J _(H4β–H5) = 3.6 Hz, 1H, HβC4), 1.41 (t, ³ J = 7.1 Hz, 3H, CH ₃CH₂OP), 1.40 (t, ³ J = 7.0 Hz, 3H, CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 162.29 (d, ¹ J _(CF) = 246.4 Hz), 161.90 (C O), 151.25 (C O), 140.50, 136.57, 134.89, 132.87 (d, ⁴ J _(CCCCF) = 3.2 Hz), 131.03 (d, ³ J _(CCCF) = 8.5 Hz), 129.91, 128.27, 128.25, 127.53, 122.72, 115.58, 115.18 (d, ² J _(CCF) = 21.0 Hz, C3′, C5′), 114.96, 75.78 (d, ³ J _(CCCP) = 6.6 Hz, C5), 62.94 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.61 (d, ² J _(COP) = 6.6 Hz, CH₂OP), 62.30 (d, ³ J _(CNCP) = 4.8 Hz, CH₂Ph), 60.64 (d, ¹ J _(CP) = 170.0 Hz, C3), 47.77 (CH₂N), 44.17 (CH₂Ph), 35.15 (d, ² J _(CCP) = 1.5 Hz, C4), 16.61 (d, ³ J _(CCOP) = 5.7 Hz, CH₃CH₂OP), 16.55 (d, ³ J _(CCOP) = 5.7 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 22.62. Anal. calcd. for C₃₀H₃₃FN₃O₆P × 1.5H₂O: C, 59.21; H, 5.96; N, 6.90. Found: C, 59.46; H, 5.99; N, 6.92 (obtained on a 82:18 mixture of cis-15d and trans-15d).

4.5.29. Diethyl trans-{2-benzyl-5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15d)

A colorless oil. IR (film, cm⁻¹) ν_max: 2983, 1703, 1700, 1658, 1608, 1483, 1400, 1223, 1050, 1024, 966, 754. (NMR signals of trans-15d were extracted from the spectrum of a 12:88 mixture of cis-15d and trans-15d) ¹H NMR (600 MHz, CDCl₃): δ = 8.25–8.23 (m, 1H), 7.64–7.62 (m, 1H), 7.53–7.51 (m, 2H), 7.30–7.25 (m, 7H), 7.00–6.97 (m, 2H), 7.00–6.97 (m, 2H), 5.25 (AB, J _AB = 13.8 Hz, 1H, HCHN), 5.22 (AB, J _AB = 13.8 Hz, 1H, HCHN), 4.48 (d, ² J = 13.8 Hz, 1H, HCHPh), 4.43–4.39 (m, 2H, HC5, HCHN), 4.29–4.17 (m, 4H, 2 × CH ₂OP), 4.16 (dd, ² J = 15.7 Hz, ³ J _(HC–H5) = 5.7 Hz, 1H, HCHN), 3.89 (d, ² J = 13.8 Hz, 1H, HCHPh), 3.29–3.26 (m, 1H, HC3), 2.67 (dddd, ³ J _(H4α–P) = 18.5 Hz, ² J _{(H4α–H4β)} = 13.0 Hz, ³ J _(H4α–H3) = 6.5 Hz, ³ J _(H4β–H5) = 6.5 Hz, 1H, HαC4), 2.38–2.31 (m, 1H, HβC4), 1.37 (t, ³ J = 6.0 Hz, 6H, 2 × CH ₃CH₂OP); ¹³C NMR (151 MHz, CDCl₃): δ = 162.30 (d, ¹ J _(CF) = 246.2 Hz), 161.67 (C O), 151.27 (C O), 140.17, 136.50, 134.85, 132.47 (d, ⁴ J _(CCCCF) = 3.1 Hz), 130.97 (d, ³ J _(CCCF) = 8.0 Hz), 129.61, 128.92, 128.10, 127.46, 123.19, 115.57, 115.21 (d, ² J _(CCF) = 21.6 Hz), 114.96, 75.61 (d, ³ J _(CCCP) = 6.2 Hz, C5), 63.27 (d, ² J _(COP) = 6.5 Hz, CH₂OP), 62.69 (d, ³ J _(CNCP) = 4.5 Hz, CH₂Ph), 62.44 (d, ² J _(COP) = 7.0 Hz, CH₂OP), 60.80 (d, ¹ J _(CP) = 169.8 Hz, C3), 47.78 (CH₂N), 44.31 (CH₂Ph), 35.17 (d, ² J _(CCP) = 2.0 Hz, C4), 16.54 (d, ³ J _(CCOP) = 5.6 Hz, CH₃CH₂OP), 16.48 (d, ³ J _(CCOP) = 5.7 Hz, CH₃CH₂OP); ³¹P NMR (243 MHz, CDCl₃): δ = 21.78. Anal. calcd. for C₃₀H₃₃FN₃O₆P: C, 61.69; H, 5.72; N, 7.23. Found: C, 61.89; H, 5.97; N, 7.28 (obtained on a 12:88 mixture of cis-15d and trans-15d).

4.6. Antiviral activity assays

The compounds were evaluated against different herpesviruses, including herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK^–) HSV-1 KOS strain resistant to ACV (ACV^r), herpes simplex virus type 2 (HSV-2) strain G, varicella-zoster virus (VZV) strain Oka, TK^– VZV strain 07-1, human cytomegalovirus (HCMV) strains AD-169 and Davis as well as feline herpes virus (FHV), the poxvirus vaccinia virus (Lederle strain), para-influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, respiratory syncytial virus (RSV), feline coronovirus (FIPV) and influenza A virus subtypes H1N1 (A/PR/8), H3N2 (A/HK/7/87) and influenza B virus (B/HK/5/72) and human immune deficiency virus (5HVV-1 and HIV-2). The antiviral assays, other than HIV, were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey kidney cells (Vero), human epithelial cervix carcinoma cells (HeLa), Crandell-Rees feline kidney cells (CRFK), or Madin Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) and the cell cultures were incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation (VZV) was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC₅₀ or compound concentration required to reduce virus-induced cytopathicity or viral plaque formation by 50%. Cytotoxicity of the test compounds was expressed as the minimum cytotoxic concentration (MCC) or the compound concentration that caused a microscopically detectable alteration of cell morphology.

4.7. Cytostatic activity against immortalized cell lines

Murine leukemia (L1210), human T-lymphocyte (CEM), human cervix carcinoma (HeLa) and immortalized human dermal microvascular endothelial cells (HMEC-1) were suspended at 300,000–500,000 cells/mL of culture medium, and 100 μL of a cell suspension was added to 100 μL of an appropriate dilution of the test compounds in 200 μL-wells of 96-well microtiter plates. After incubation at 37 °C for two (L1210), three (CEM) or four (HeLa) days, the cell number was determined using a Coulter counter. The IC50 was defined as the compound concentration required to inhibit cell proliferation by 50%.

Acknowledgments

The authors wish to express their gratitude to Mrs. Edyta Grzelewska, Mrs. Leentje Persoons, Mrs. Frieda De Meyer, Mrs. Ellen De Waegenaere and Mrs. Lizette van Berckelaer for excellent technical assistance. The synthetic part of this work was supported by the Medical University of Lodz internal funds (503/3-014-01/503-31-001 and 502-03/3-014-01/502-34-066). The biological part of this work was supported by the KU Leuven (GOA 15/19 TBA).

Footnotes

^{Appendix A}

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2016.10.002.

Appendix A. Supplementary data

The following is the supplementary data related to this article:

Figures S1–S30

mmc1.pdf^{(2.7MB, pdf)}

References

1.Weller T.H. Varicella and herpes zoster – changing concepts of the natural history, control, and importance of a Not-So-Benign Virus (First and second of two parts) N. Engl. J. Med. 1983;309:1362–1368. doi: 10.1056/NEJM198312013092205. 1434–1440. [DOI] [PubMed] [Google Scholar]
2.Straus S.E., Ostrove J.M., Inchauspe G., Felser J.M., Freifeld A., Croen K.D., Sawyer M.H. Varicella-zoster virus infections: biology, natural history, treatment and prevention. Ann. Intern. Med. 1988;108:221–237. doi: 10.7326/0003-4819-108-2-221. [DOI] [PubMed] [Google Scholar]
3.Reiter C., Frohlich T., Zeino M., Marschall M., Bahsi H., Leidenberger M., Friedrich O., Kappes B., Hampel F., Efferth T., Tsogoeva S.B. New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids. Eur. J. Med. Chem. 2015;97:164–172. doi: 10.1016/j.ejmech.2015.04.053. [DOI] [PubMed] [Google Scholar]
4.Stipkovic Babic M., Makuc D., Plavec J., Martinovic T., Kraljevic Pavelic S., Pavelic K., Snoeck R., Andrei G., Schols D., Wittine K., Mintas M. Novel halogenated 3-deazapurine, 7-deazapurine and alkylated 9-deazapurine derivatives of L-ascorbic or imino-L-ascorbic acid: synthesis, antitumour and antiviral activity evaluations. Eur. J. Med. Chem. 2015;102:288–302. doi: 10.1016/j.ejmech.2015.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Jucker E., editor. vol. 56. Springer Basel AG; Basel: 2001. Progress in drug research; pp. 77–120. (Chapter 2: Current and Potential Therapies for the Treatment of Herpesvirus Infection). by Villarreal E. C. [DOI] [PubMed] [Google Scholar]
6.Andrei G., Snoeck R. Advances in the treatment of varicella-zoster virus infections. Adv. Pharmacol. 2013;67:107–168. doi: 10.1016/B978-0-12-405880-4.00004-4. [DOI] [PubMed] [Google Scholar]
7.Linnemann C.C., Jr., Biron K.K., Hoppenjans W.G., Solinger A.M. Emergence of acyclovir-resistant varicella zoster virus in an AIDS patient on prolonged acyclovir therapy. AIDS. 1990;4:577–579. doi: 10.1097/00002030-199006000-00014. [DOI] [PubMed] [Google Scholar]
8.Jacobson M.A., Berger T.G., Fikrig S., Becherer P., Moohr J.W., Stanat S.C., Biron K.K. Acyclovir-resistant varicella zoster virus infection after chronic oral acyclovir therapy in patients with the acquired immunodeficiency syndrome (AIDS) Ann. Intern. Med. 1990;112:187–191. doi: 10.7326/0003-4819-112-3-187. [DOI] [PubMed] [Google Scholar]
9.Migliore M. FV-100: the most potent and selective anti-varicella zoster virus agent reported to data. Antivir. Chem. Chemother. 2009;20:107–115. doi: 10.3851/IMP1472. 20. [DOI] [PubMed] [Google Scholar]
10.Brideau R.J., Knechtel M.L., Huang A., Vaillancourt V.A., Vera E.E., Oien N.L., Hopkins T.A., Wieber J.L., Wilkinson K.F., Rush B.D., Schwende F.J., Wathen M.W. Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses. Antivir. Res. 2002;54:19–28. doi: 10.1016/s0166-3542(01)00208-x. [DOI] [PubMed] [Google Scholar]
11.Véron J.-B., Enguehard-Gueiffier C., Snoeck R., Andrei G., De Lcercq E., Gueiffier A. Influence of 6 or 8-substitution on the antiviral activity of 3-phenethylthiomethylimidazo[1,2-a]pyridine against human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) Bioorg. Med. Chem. 2007;15:7209–7219. doi: 10.1016/j.bmc.2007.03.061. [DOI] [PubMed] [Google Scholar]
12.Schnute M.E., Anderson D.J., Brideau R.J., Ciske F.L., Collier S.E., Cudahy M.M., Eggen M.J., Genin M.J., Hopkins T.A., Judge T.M., Kim E.J., Knechtel M.L., Nair S.K., Nieman J.A., Oien N.L., Scott A., Tanis S.P., Vaillancourt V.A., Wathen M.W., Wieber J.L. 2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines as broad-spectrum inhibitors of human herpesvirus polymerases. Bioorg. Med. Chem. Lett. 2007;17:3349–3353. doi: 10.1016/j.bmcl.2007.03.102. [DOI] [PubMed] [Google Scholar]
13.Nieman J.A., Nair S.K., Heasley S.E., Schultz B.L., Zerth H.M., Nugent R.A., Chen K., Stephanski K.J., Hopkins T.A., Knechtel M.L., Oien N.L., Wieber J.L., Wathen M.W. Modifications of C-2 on the pyrroloquinoline template aimed at the development of potent herpesvirus antivirals with improved aqueous solubility. Bioorg. Med. Chem. Lett. 2010;20:3039–3042. doi: 10.1016/j.bmcl.2010.03.115. [DOI] [PubMed] [Google Scholar]
14.Robins M.J., Nowak I., Rajwanshi V.K., Miranda K., Cannon J.F., Peterson M.A., Andrei G., Snoeck R., De Clercq E., Balzarini J. Synthesis and antiviral evaluation of 6-(Alkyl-heteroaryl)furo[2,3-d]pyrimidin-2(3H)-one nucleosides and analogues with ethynyl, ethenyl, and ethyl spacers at C6 of the furopyrimidine core. J. Med. Chem. 2007;50:3897–3905. doi: 10.1021/jm070210n. [DOI] [PubMed] [Google Scholar]
15.Robins M.J., Miranda K., Rajwanshi V.K., Peterson M.A., Andrei G., Snoeck R., De Clercq E., Balzarini J. Synthesis and biological evaluation of 6-(Alkyn-1-yl)furo[2,3-d]pyrimidin-2(3H)-one base and nucleoside derivatives. J. Med. Chem. 2006;49:391–398. doi: 10.1021/jm050867d. [DOI] [PubMed] [Google Scholar]
16.Brideau R.J., Knechtel M.L., Huang A., Vaillancourt V.A., Vera E.E., Oien N.L., Hopkins T.A., Wieber J.L., Wilkinson K.F., Rush B.D., Schwende F.J., Wathen M.W. Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses. Antivir. Res. 2002;54:19–28. doi: 10.1016/s0166-3542(01)00208-x. [DOI] [PubMed] [Google Scholar]
17.Chono K., Katsumata K., Kontani T., Kobayashi M., Sudo K., Yokota T., Konno K., Shimizu Y., Suzuki H. ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicellazoster virus and herpes simplex virus types 1 and 2. J. Antimicrob. Chemother. 2010;65:1733–1741. doi: 10.1093/jac/dkq198. [DOI] [PubMed] [Google Scholar]
18.Sergerie Y., Rivest S., Boivin G. Tumor necrosis factor-α and interleukin-1β play a critical role in the resistance against lethal herpes simplex virus encephalitis. J. Infect. Dis. 2007;196:853–860. doi: 10.1086/520094. [DOI] [PubMed] [Google Scholar]
19.Khan I., Ibrar A., Ahmed W., Saeed A. Synthetic approaches, functionalization and therapeutic potential of quinazoline and quinazolinone skeletons: the advances continue. Eur. J. Med. Chem. 2015;90:124–169. doi: 10.1016/j.ejmech.2014.10.084. [DOI] [PubMed] [Google Scholar]
20.Novikov M.S., Valuev-Elliston V.T., Babkov D.A., Paramonova M.P., Ivanov A.V., Gavryushov S.A., Khandazhinskaya A.L., Kochetkov S.N., Pannecouque C., Andrei G., Snoeck R., Balzarini J., Seley-Radtke K.L. N1,N3-disubstituted uracils as nonnucleoside inhibitors of HIV-1 reverse transcriptase. Bioorg. Med. Chem. 2013;21:1150–1158. doi: 10.1016/j.bmc.2012.12.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Matharu D.S., Flaherty D.P., Simpson D.S., Schroeder C.E., Chung D., Yan D., Noah J.W., Jonsson C.B., White E.L., Aubé J., Plemper R.K., Severson W.E., Golden J.E. Optimization of potent and selective quinazolinediones: inhibitors of respiratory syncytial virus that block RNA-dependent RNAPolymerase complex activity. J. Med. Chem. 2014;57:10314–10328. doi: 10.1021/jm500902x. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Głowacka I.E., Balzarini J., Wróblewski A.E. The synthesis, antiviral, cytostatic and cytotoxic evaluation of a new series of acyclonucleotide analogues with a 1,2,3-triazole linker. Eur. J. Med. Chem. 2013;70:703–722. doi: 10.1016/j.ejmech.2013.10.057. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Głowacka I.E., Balzarini J., Andrei G., Snoeck R., Schols D., Piotrowska D.G. Design, synthesis, antiviral and cytostatic activity of ɷ-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues. Bioorg. Med. Chem. 2014;22:3629–3641. doi: 10.1016/j.bmc.2014.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Malancona S., Donghi M., Ferrara M., Hernando J.I.M., Pompei M., Pesci S., Ontoria J.M., Koch U., Rowley M., Summa V. Allosteric inhibitors of hepatitis C virus NS5B polymerase thumb domain site II: structure-based design and synthesis of new templates. Bioorg. Med. Chem. 2010;18:2836–2848. doi: 10.1016/j.bmc.2010.03.024. [DOI] [PubMed] [Google Scholar]
25.Łysakowska M., Balzarini J., Piotrowska D.G. Design, synthesis, antiviral and cytostatic evaluation of novel isoxazolidine analogues of homonucleotides. Arch. Pharm. Chem. Life Sci. 2014;347:341–353. doi: 10.1002/ardp.201300382. [DOI] [PubMed] [Google Scholar]
26.Süsse M., Johne S. Chinazolincarbonsäuren, 10. Mitt. Synthese von 1-Methyl-2,4-dioxo-chinazolin-3-yl-essigsäure, 2,4-Dioxo-chinazolin-1-yl-essigsäuren, 2,4-Dioxo-1,3-chinazolindiessigsäuren und deren Estern. Monatsh. Chem. 1987;118:71–79. [Google Scholar]
27.Abbas S.Y., El-Bayouki K.A.M., Basyouni W.M. Utilization of isatoic anhydride in the syntheses of various types of quinazoline and quinazolinone derivatives. Synth. Commun. 2016;46:993–1035. [Google Scholar]
28.Piotrowska D.G. N-Substituted C-diethoxyphosphorylated nitrones as useful synthons for the synthesis of α-aminophosphonates. Tetrahedron Lett. 2006;47:5363–5366. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figures S1–S30

mmc1.pdf^{(2.7MB, pdf)}

[bib1] 1.Weller T.H. Varicella and herpes zoster – changing concepts of the natural history, control, and importance of a Not-So-Benign Virus (First and second of two parts) N. Engl. J. Med. 1983;309:1362–1368. doi: 10.1056/NEJM198312013092205. 1434–1440. [DOI] [PubMed] [Google Scholar]

[bib2] 2.Straus S.E., Ostrove J.M., Inchauspe G., Felser J.M., Freifeld A., Croen K.D., Sawyer M.H. Varicella-zoster virus infections: biology, natural history, treatment and prevention. Ann. Intern. Med. 1988;108:221–237. doi: 10.7326/0003-4819-108-2-221. [DOI] [PubMed] [Google Scholar]

[bib3] 3.Reiter C., Frohlich T., Zeino M., Marschall M., Bahsi H., Leidenberger M., Friedrich O., Kappes B., Hampel F., Efferth T., Tsogoeva S.B. New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids. Eur. J. Med. Chem. 2015;97:164–172. doi: 10.1016/j.ejmech.2015.04.053. [DOI] [PubMed] [Google Scholar]

[bib4] 4.Stipkovic Babic M., Makuc D., Plavec J., Martinovic T., Kraljevic Pavelic S., Pavelic K., Snoeck R., Andrei G., Schols D., Wittine K., Mintas M. Novel halogenated 3-deazapurine, 7-deazapurine and alkylated 9-deazapurine derivatives of L-ascorbic or imino-L-ascorbic acid: synthesis, antitumour and antiviral activity evaluations. Eur. J. Med. Chem. 2015;102:288–302. doi: 10.1016/j.ejmech.2015.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Jucker E., editor. vol. 56. Springer Basel AG; Basel: 2001. Progress in drug research; pp. 77–120. (Chapter 2: Current and Potential Therapies for the Treatment of Herpesvirus Infection). by Villarreal E. C. [DOI] [PubMed] [Google Scholar]

[bib6] 6.Andrei G., Snoeck R. Advances in the treatment of varicella-zoster virus infections. Adv. Pharmacol. 2013;67:107–168. doi: 10.1016/B978-0-12-405880-4.00004-4. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Linnemann C.C., Jr., Biron K.K., Hoppenjans W.G., Solinger A.M. Emergence of acyclovir-resistant varicella zoster virus in an AIDS patient on prolonged acyclovir therapy. AIDS. 1990;4:577–579. doi: 10.1097/00002030-199006000-00014. [DOI] [PubMed] [Google Scholar]

[bib8] 8.Jacobson M.A., Berger T.G., Fikrig S., Becherer P., Moohr J.W., Stanat S.C., Biron K.K. Acyclovir-resistant varicella zoster virus infection after chronic oral acyclovir therapy in patients with the acquired immunodeficiency syndrome (AIDS) Ann. Intern. Med. 1990;112:187–191. doi: 10.7326/0003-4819-112-3-187. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Migliore M. FV-100: the most potent and selective anti-varicella zoster virus agent reported to data. Antivir. Chem. Chemother. 2009;20:107–115. doi: 10.3851/IMP1472. 20. [DOI] [PubMed] [Google Scholar]

[bib10] 10.Brideau R.J., Knechtel M.L., Huang A., Vaillancourt V.A., Vera E.E., Oien N.L., Hopkins T.A., Wieber J.L., Wilkinson K.F., Rush B.D., Schwende F.J., Wathen M.W. Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses. Antivir. Res. 2002;54:19–28. doi: 10.1016/s0166-3542(01)00208-x. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Véron J.-B., Enguehard-Gueiffier C., Snoeck R., Andrei G., De Lcercq E., Gueiffier A. Influence of 6 or 8-substitution on the antiviral activity of 3-phenethylthiomethylimidazo[1,2-a]pyridine against human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) Bioorg. Med. Chem. 2007;15:7209–7219. doi: 10.1016/j.bmc.2007.03.061. [DOI] [PubMed] [Google Scholar]

[bib12] 12.Schnute M.E., Anderson D.J., Brideau R.J., Ciske F.L., Collier S.E., Cudahy M.M., Eggen M.J., Genin M.J., Hopkins T.A., Judge T.M., Kim E.J., Knechtel M.L., Nair S.K., Nieman J.A., Oien N.L., Scott A., Tanis S.P., Vaillancourt V.A., Wathen M.W., Wieber J.L. 2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines as broad-spectrum inhibitors of human herpesvirus polymerases. Bioorg. Med. Chem. Lett. 2007;17:3349–3353. doi: 10.1016/j.bmcl.2007.03.102. [DOI] [PubMed] [Google Scholar]

[bib13] 13.Nieman J.A., Nair S.K., Heasley S.E., Schultz B.L., Zerth H.M., Nugent R.A., Chen K., Stephanski K.J., Hopkins T.A., Knechtel M.L., Oien N.L., Wieber J.L., Wathen M.W. Modifications of C-2 on the pyrroloquinoline template aimed at the development of potent herpesvirus antivirals with improved aqueous solubility. Bioorg. Med. Chem. Lett. 2010;20:3039–3042. doi: 10.1016/j.bmcl.2010.03.115. [DOI] [PubMed] [Google Scholar]

[bib14] 14.Robins M.J., Nowak I., Rajwanshi V.K., Miranda K., Cannon J.F., Peterson M.A., Andrei G., Snoeck R., De Clercq E., Balzarini J. Synthesis and antiviral evaluation of 6-(Alkyl-heteroaryl)furo[2,3-d]pyrimidin-2(3H)-one nucleosides and analogues with ethynyl, ethenyl, and ethyl spacers at C6 of the furopyrimidine core. J. Med. Chem. 2007;50:3897–3905. doi: 10.1021/jm070210n. [DOI] [PubMed] [Google Scholar]

[bib15] 15.Robins M.J., Miranda K., Rajwanshi V.K., Peterson M.A., Andrei G., Snoeck R., De Clercq E., Balzarini J. Synthesis and biological evaluation of 6-(Alkyn-1-yl)furo[2,3-d]pyrimidin-2(3H)-one base and nucleoside derivatives. J. Med. Chem. 2006;49:391–398. doi: 10.1021/jm050867d. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Brideau R.J., Knechtel M.L., Huang A., Vaillancourt V.A., Vera E.E., Oien N.L., Hopkins T.A., Wieber J.L., Wilkinson K.F., Rush B.D., Schwende F.J., Wathen M.W. Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses. Antivir. Res. 2002;54:19–28. doi: 10.1016/s0166-3542(01)00208-x. [DOI] [PubMed] [Google Scholar]

[bib17] 17.Chono K., Katsumata K., Kontani T., Kobayashi M., Sudo K., Yokota T., Konno K., Shimizu Y., Suzuki H. ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicellazoster virus and herpes simplex virus types 1 and 2. J. Antimicrob. Chemother. 2010;65:1733–1741. doi: 10.1093/jac/dkq198. [DOI] [PubMed] [Google Scholar]

[bib18] 18.Sergerie Y., Rivest S., Boivin G. Tumor necrosis factor-α and interleukin-1β play a critical role in the resistance against lethal herpes simplex virus encephalitis. J. Infect. Dis. 2007;196:853–860. doi: 10.1086/520094. [DOI] [PubMed] [Google Scholar]

[bib19] 19.Khan I., Ibrar A., Ahmed W., Saeed A. Synthetic approaches, functionalization and therapeutic potential of quinazoline and quinazolinone skeletons: the advances continue. Eur. J. Med. Chem. 2015;90:124–169. doi: 10.1016/j.ejmech.2014.10.084. [DOI] [PubMed] [Google Scholar]

[bib20] 20.Novikov M.S., Valuev-Elliston V.T., Babkov D.A., Paramonova M.P., Ivanov A.V., Gavryushov S.A., Khandazhinskaya A.L., Kochetkov S.N., Pannecouque C., Andrei G., Snoeck R., Balzarini J., Seley-Radtke K.L. N1,N3-disubstituted uracils as nonnucleoside inhibitors of HIV-1 reverse transcriptase. Bioorg. Med. Chem. 2013;21:1150–1158. doi: 10.1016/j.bmc.2012.12.027. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Matharu D.S., Flaherty D.P., Simpson D.S., Schroeder C.E., Chung D., Yan D., Noah J.W., Jonsson C.B., White E.L., Aubé J., Plemper R.K., Severson W.E., Golden J.E. Optimization of potent and selective quinazolinediones: inhibitors of respiratory syncytial virus that block RNA-dependent RNAPolymerase complex activity. J. Med. Chem. 2014;57:10314–10328. doi: 10.1021/jm500902x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 22.Głowacka I.E., Balzarini J., Wróblewski A.E. The synthesis, antiviral, cytostatic and cytotoxic evaluation of a new series of acyclonucleotide analogues with a 1,2,3-triazole linker. Eur. J. Med. Chem. 2013;70:703–722. doi: 10.1016/j.ejmech.2013.10.057. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Głowacka I.E., Balzarini J., Andrei G., Snoeck R., Schols D., Piotrowska D.G. Design, synthesis, antiviral and cytostatic activity of ɷ-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues. Bioorg. Med. Chem. 2014;22:3629–3641. doi: 10.1016/j.bmc.2014.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib24] 24.Malancona S., Donghi M., Ferrara M., Hernando J.I.M., Pompei M., Pesci S., Ontoria J.M., Koch U., Rowley M., Summa V. Allosteric inhibitors of hepatitis C virus NS5B polymerase thumb domain site II: structure-based design and synthesis of new templates. Bioorg. Med. Chem. 2010;18:2836–2848. doi: 10.1016/j.bmc.2010.03.024. [DOI] [PubMed] [Google Scholar]

[bib25] 25.Łysakowska M., Balzarini J., Piotrowska D.G. Design, synthesis, antiviral and cytostatic evaluation of novel isoxazolidine analogues of homonucleotides. Arch. Pharm. Chem. Life Sci. 2014;347:341–353. doi: 10.1002/ardp.201300382. [DOI] [PubMed] [Google Scholar]

[bib26] 26.Süsse M., Johne S. Chinazolincarbonsäuren, 10. Mitt. Synthese von 1-Methyl-2,4-dioxo-chinazolin-3-yl-essigsäure, 2,4-Dioxo-chinazolin-1-yl-essigsäuren, 2,4-Dioxo-1,3-chinazolindiessigsäuren und deren Estern. Monatsh. Chem. 1987;118:71–79. [Google Scholar]

[bib27] 27.Abbas S.Y., El-Bayouki K.A.M., Basyouni W.M. Utilization of isatoic anhydride in the syntheses of various types of quinazoline and quinazolinone derivatives. Synth. Commun. 2016;46:993–1035. [Google Scholar]

[bib28] 28.Piotrowska D.G. N-Substituted C-diethoxyphosphorylated nitrones as useful synthons for the synthesis of α-aminophosphonates. Tetrahedron Lett. 2006;47:5363–5366. [Google Scholar]

PERMALINK

Synthesis, anti-varicella-zoster virus and anti-cytomegalovirus activity of quinazoline-2,4-diones containing isoxazolidine and phosphonate substructures

Dorota G Piotrowska

Graciela Andrei

Dominique Schols

Robert Snoeck

Magdalena Łysakowska

Abstract

Graphical abstract

Highlights

1. Introduction

Fig. 1.

Fig. 2.

2. Results and discussion

2.1. Chemistry

Table 3.

Scheme 1.

Scheme 2.

Scheme 3.

Table 1.

Scheme 4.

Table 2.

2.2. Antiviral and cytostatic evaluation

Table 4.

Table 5.

2.3. Cytostatic activity

Table 6.

3. Conclusions

4. Experimental

4.1. General

4.2. Synthesis of 1-allylquinazoline-2,4-dione (22)

4.3. The benzoylation of N-allylquinazoline-2,4-dione 22 – the general procedure

4.3.1. 1-Allyl-3-(2-fluoro)benzoyl-1H-quinazoline-2,4-dione (18b)

4.3.2. 1-Allyl-3-(3-fluoro)benzoyl-1H-quinazoline-2,4-dione (18c)

4.3.3. 1-Allyl-3-(4-fluoro)benzoyl-1H-quinazoline-2,4-dione (18d)

4.4. The benzylation of N-allylquinazoline-2,4-dione 22 – the general procedure

4.4.1. 1-Allyl-3-benzyl-1H-quinazoline-2,4-dione (19a)

4.4.2. 1-Allyl-3-(2-fluoro)benzyl-1H-quinazoline-2,4-dione (19b)

4.4.3. 1-Allyl-3-(3-fluoro)benzyl-1H-quinazoline-2,4-dione (19c)

4.4.4. 1-Allyl-3-(4-fluoro)benzyl-1H-quinazoline-2,4-dione (19d)

4.5. Cycloadditions of C-(diethoxyphosphoryl)nitrones 16 (R = Me) and 17 (R = Bn) and N1-allylated quinazoline-2,4-diones 18 and 19 – the general procedure

4.5.1. Diethyl trans-{5-[(3-(2-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-12b)

4.5.2. Diethyl cis-{5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-12c)

4.5.3. Diethyl trans-{5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-12c)

4.5.4. Diethyl cis-{5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-12d)

4.5.5. Diethyl trans-{5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-12d)

4.5.6. Diethyl cis-{5-[(3-benzoyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-benzylisoxazolidin-3-yl}phosphonate (cis-13a)

4.5.7. Diethyl trans-{5-[(3-benzoyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-benzylisoxazolidin-3-yl}phosphonate (trans-13a)

4.5.8. Diethyl cis-{2-benzyl-5-[(3-(2-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-13b)

4.5.9. Diethyl trans-{2-benzyl-5-[(3-(2-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-13b)

4.5.10. Diethyl cis-{2-benzyl-5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-13c)

4.5.11. Diethyl trans-{2-benzyl-5-[(3-(3-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-13c)

4.5.12. Diethyl cis-{2-benzyl-5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-13d)

4.5.13. Diethyl trans-{2-benzyl-5-[(3-(4-fluorobenzoyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-13d)

4.5.14. Diethyl cis-{5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14a)

4.5.15. Diethyl trans-{5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14a)

4.5.16. Diethyl cis-{5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14b)

4.5.17. Diethyl trans-{5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14b)

4.5.18. Diethyl cis-{5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14c)

4.5.19. Diethyl trans-{5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14c)

4.5.20. Diethyl cis-{5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (cis-14d)

4.5.21. Diethyl trans-{5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]-2-methylisoxazolidin-3-yl}phosphonate (trans-14d)

4.5.22. Diethyl cis-{2-benzyl-5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15a)

4.5.23. Diethyl trans-{2-benzyl-5-[(3-benzyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15a)

4.5.24. Diethyl cis-{2-benzyl-5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15b)

4.5.25. Diethyl trans-{2-benzyl-5-[(3-(2-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15b)

4.5.26. Diethyl cis-{2-benzyl-5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15c)

4.5.27. Diethyl trans-{2-benzyl-5-[(3-(3-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15c)

4.5.28. Diethyl cis-{2-benzyl-5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (cis-15d)

4.5.29. Diethyl trans-{2-benzyl-5-[(3-(4-fluorobenzyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl]isoxazolidin-3-yl}phosphonate (trans-15d)

4.6. Antiviral activity assays

4.7. Cytostatic activity against immortalized cell lines

Acknowledgments

Footnotes

Appendix A. Supplementary data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

4.5. Cycloadditions of C-(diethoxyphosphoryl)nitrones 16 (R = Me) and 17 (R = Bn) and N¹-allylated quinazoline-2,4-diones 18 and 19 – the general procedure