Quinazolin-4-one derivatives: A novel class of non-competitive NR2C/D subunit-selective N-methyl-D-aspartate receptor antagonists

Abstract

We describe a new class of subunit-selective antagonists of N-methyl D-Aspartate (NMDA)-selective ionotropic glutamate receptors that contain the (E)-3-phenyl-2-styrylquinazolin-4(3H)-one backbone. The inhibition of recombinant NMDA receptor function induced by these quinazolin-4-one derivatives is non-competitive and voltage-independent, suggesting that this family of compounds does not exert action on the agonist binding site of the receptor or block the channel pore. The compounds described here resemble CP-465,022 ((S)-3-(2-chlorophenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one), a non-competitive antagonist of AMPA-selective glutamate receptors. However, modification of ring substituents resulted in analogues with greater than 100-fold selectivity for recombinant NMDA receptors over AMPA and kainate receptors. Furthermore, within this series of compounds, analogues were identified with 50-fold selectivity for recombinant NR2C/D-containing receptors over NR2A/B containing receptors. These compounds represent a new class of non-competitive subunit-selective NMDA receptor antagonists.

INTRODUCTION

N-methyl-D-aspartate (NMDA) receptors are ligand-gated cation-selective channels that mediate glutamatergic excitatory synaptic transmission in the central nervous system.¹ A wide range of roles have been proposed for NMDA receptors, including neuronal development^2-5 and synaptic plasticity underlying learning.^6-7 In addition, aberrant activation of NMDA receptors has been suggested to participate in neuropathological conditions such as stroke, epilepsy, schizophrenia, depression, Alzheimer's disease, Huntington's disease, and Parkinson's disease.^8-15

NMDA receptors are tetrameric assemblies of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four subtypes (NR2A, NR2B, NR2C, NR2D). The NR2 subunit appears to control pharmacological properties, response time course, and channel properties.^16-22 In particular, receptors containing NR2C or NR2D subunits are activated by glycine and glutamate with higher potency than receptors containing NR2A or NR2B. NMDA receptors containing NR2C or NR2D also show lower maximal open probability than receptors containing NR2A or NR2B. NR2D-containing NMDA receptors have been hypothesized to show exceptionally slow deactivation following removal of glutamate. NR2C and NR2D subunits also show localization patterns distinct from NR2A and NR2B, with prominent expression in cerebellum, discrete nuclei within the basal ganglia, and select populations of interneurons.^23-37 The distinct anatomical localization of the NR2 subunits raises the possibility that subunit-selective antagonists and allosteric modulators might provide well-tolerated therapeutic treatments for a wide range of indications.¹

Ifenprodil was the first subunit-selective NMDA receptor antagonist identified, showing over 200-fold selectivity at NR2B over NMDA receptors containing other NR2 subunits.^38-41 Despite intense interest in NMDA receptors and their hypothesized roles in numerous neurological disorders, no antagonists that are more than 10-fold selective for NR2A, NR2C, or NR2D have yet been identified.^42-46 The inability to identify subunit-selective competitive antagonists may reflect the highly conserved glutamate-binding pocket within the NR2 subunit.^47-48 Channel blockers are similarly poorly selective for NMDA receptors comprised of different NR2 subunits,⁴⁹ presumably due to structural conservation of the permeation pore. The lack of subunit-selective pharmacological tools for this receptor class has been an impediment to understanding the functional roles of the NR2A, NR2C, and NR2D subunits in neurons. Because NMDA receptor architecture appears to reflect the assembly of multiple extracellular semi-autonomous domains,^50-52 we hypothesized that the multiple protein-protein interfaces within the NMDA receptor may provide new targets for modulating NMDA receptor function. To search for modulators that might interact at these interfaces, we executed a multi-well fluorescence-based assay to identify novel non-competitive inhibitors of recombinant NMDA receptors.⁵³ Screening a diversity library of compounds with this assay allowed us to identify a new class of recombinant NMDA receptor antagonists that contains the quinazolin-4-one backbone, which is shared with the previously reported α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-selective noncompetitive antagonist CP-465,022 (1, Figure 1).^54-56 NMDA receptor inhibition was voltage-independent and non-competitive (Figure S1). Synthetic efforts towards optimizing backbone substituents led to the development of a structure-activity relationship (SAR), ultimately yielding novel compounds with between 50-100 fold selectivity for recombinant NR2C/D- over NR2A/B-containing receptors. The half-maximal inhibiting concentrations of members of this class are in the low micromolar range (IC₅₀ = 0.6-6 μM) at recombinant NMDA receptors, with no detectable activity at recombinant kainate receptors and variable activity at recombinant AMPA receptors.

Figure 1.

Structures for CP-465,022 (1), (E)-4-(2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (2), and (E)-3-(2-methoxyphenyl)-2-(2-nitrostyryl)quinazolin-4(3H)-one (3).

RESULTS

Chemistry

We evaluated both commercially available analogues and a number of synthetic styryl quinazolin-4-one analogues that were generated via a three step sequence which combines commercially available fragments: anthranilic acids, anilines, and aldehydes (Scheme 1). Briefly, a substituted anthranilic acid (4) was converted to the benzoxazin-4-one (5) by refluxing in acetic anhydride. The quinazolin-4-one core (6) was generated by a ring opening-ring closure reaction under acidic conditions in the presence of a substituted aniline. Finally, acid-catalyzed condensation reaction of 6 with a substituted aldehyde yielded the target (E)-3-phenyl-2-styrylquinazolin-4(3H)-one.

Scheme 1.

Synthesis of (E)-3-phenyl-2-styrylquinazolin-4(3H)-ones. (a) Ac₂O, reflux. (b) AcOH, reflux. (c) Ac₂O, AcOH, NaOAc, reflux.

An alternative methodology was utilized to synthesize analogues for which the starting material anthranilic acids were not commercially available (Scheme 2). Protection of the carboxylic acid of 78 with tert-butyldimethylsilyl chloride (TBDMSCl) and N-methylmorpholine gave 10.⁵⁷ Suzuki coupling between 10 and the appropriately substituted boronic acid afforded C ring R₈-substituted quinazolinones 55a and 56a. The TBDMS protecting group was removed with the addition of 1.0 N hydrochloric acid (HCl) during work-up. A condensation reaction of 55a or 56a with meta-nitrobenzaldehyde gave 55 and 56. Reduction of the styryl linker present in 80 with hydrogenolysis yielded the fully saturated linker compound 81 (Scheme 3).

Scheme 2.

Suzuki route for synthesis of (E)-3-phenyl-2-styrylquinazolin-4(3H)-ones. (a) TBDMSCl, NMM, THF. (b) RB(OH)₂, Pd(PPh₃)₄, aq. NaHCO₃, 1,2-DME, reflux. (c) Ac₂O, AcOH, NaOAc, reflux.

Scheme 3.

Synthesis of 81. (a) H₂, Pd/C, DMF.

Quniazolin-4-ones inhibit NMDA receptors

Completion of a fluorescence-based⁵⁸ screen of 83,880 compounds from ChemDiv and Asinex libraries of small molecules on NMDA receptor responses in both NR1/NR2D and NR1/NR2C cell lines yielded a 0.16% hit rate; all hits were confirmed by two-electrode voltage-clamp recording. A number of active compounds in this assay shared a quinazolin-4-one backbone, typified by (E)-4-(2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (2) and (E)-3-(2-methoxyphenyl)-2-(2-nitrostyryl)quinazolin-4(3H)-one (3) (see Figure 1). Quinazolin-4-one derivatives have previously been shown to be potent and relatively selective AMPA receptor antagonists.^54-56 Compounds 2 and 3 inhibited NR1/NR2C and NR1/NR2D responses completely with fitted IC₅₀ values in the micromolar range (IC₅₀ values 9 and 5 μM, respectively, at NR1/NR2D). Inhibition by 30 μM of compound 2 was non-competitive at NR1/NR2D receptors, in that it could not be surmounted by increasing the concentration of glutamate and glycine from 100/30 μM to 1000/300 μM (n=4; Figure S1 in Supporting Information). Moreover, inhibition of NR1/NR2D responses by 10 μM of compound 2 was voltage-independent, with no significant difference in inhibition at −40 mV compared to +40 mV (p>0.05; t-test; n=5; Figure S1 in Supporting Information). These data suggest that quinazolin-4-ones are noncompetitive antagonists of NR1/NR2D NMDA receptors that likely act at a site independent of the channel pore. Inhibition of NR1/NR2C receptors by compound 2 was similarly complete at saturating concentrations (n=8), non-competitive (n=6), and voltage-independent (n=6).

Positional Isomer Manipulation of Screening Hits 2 and 3

The screening hits, compounds 2 and 3, were inactive at kainate receptors, and 4-5-fold more potent (i.e. lower fitted IC₅₀) at NMDA receptors than AMPA receptors (Table 1). Analogues lacking substitutions on all aryl rings were inactive at NMDA and AMPA receptors (Figure S2 in Supporting Information), suggesting substitutions of aryl rings A and B are essential for activity. To evaluate whether we could improve the selectivity for NMDA receptors over AMPA receptors, positional isomer combinations of both 2 (A ring R₂ = NO₂, B ring R₄ = COOH) and 3 (A ring R₃ = NO₂, B ring R₆ = OMe) were evaluated (Table 1). All positional isomer modifications of A and B ring substitution led to significantly decreased potency (increased IC₅₀) compared to the screening hit 3, and thus no further work was conducted within this series. Two positional isomeric analogues of compound 2 appeared to inhibit NR1/NR2D responses with IC₅₀ values of 15 μM (7) and 6 μM (13). Notably, 2,7 and 13 all contained a para-carboxylic acid B ring substituent despite differential ortho-, meta-, or para-substitution of the A ring nitro group. This suggested optimal placement of the para-carboxylic acid B ring substituent would likely yield the most active analogues within this structural series. Moreover, a number of these compounds showed improved selectivity for NMDA receptors over the AMPA receptor GluR1, as well as improved selectivity for NR2D-containing NMDA receptors over receptors containing the NR2A subunit, although the substitution patterns controlling these effects were unclear. The IC₅₀ values for inhibition of NR2C- and NR2D-containing receptors were more similar than those for NR2A- and NR2B-containing receptors. These initial experiments suggested that it might be possible to identify derivatives within this class that selectively inhibit NR2C/D-containing recombinant NMDA receptors compared to AMPA, kainate, or NR2A/B-containing NMDA receptors.

Table 1.

Optimization of subunit selectivity through evaluation of Ring A and Ring B substituent positions.

IC50 values in μM were determined by fitting the Hill equation to average composite concentration-effect
Number	R1	R2	R3	R4	R5	R6	NR2A IC50	NR2B IC50	NR2C IC50	NR2D IC50	GluR1 IC50	GluR6 IC50	NR2Aa NR2D	GluR1b NR2D
7	NO2			COOH			64	93	15	15	>300	NE	4	>20
8	NO2				COOH		135	177	33	27	>300	NE	5	>10
9	NO2					COOH	>300	197	>300	278	>300	NE	1	1
2		NO2		COOH			21	30	12	9	32	NE	2	4
11		NO2			COOH		100	69	19	15	32	NE	7	2
12		NO2				COOH	>300	235	258	148	>300	NE	2	2
13			NO2	COOH			11	17	8	6	6	NE	2	1
14			NO2		COOH		17	20	8	7	5	NE	2	1
15 *	NO2			OCH3			>300	98	181	221	>300	NE	1	1
16 *	NO2				OCH3		>300	>300	>300	>300	>300	NE	1	1
17 *	NO2					OCH3	>300	>300	>300	290	>300	NE	1	1
18 *		NO2		OCH3			>300	>300	152	168	178	NE	2	1
19 *		NO2			OCH3		>300	262	205	186	>300	NE	2	2
20 *		NO2				OCH3	>300	>300	>300	>300	>300	NE	1	1
21 *			NO2	OCH3			>300	>300	143	125	223	NE	2	2
22 *			NO2		OCH3		>300	>300	>300	>300	>300	NE	1	1
3 *			NO2			OCH3	4	7	4	5	14	NE	1	3

curves from 3-17 oocytes injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1, or GluR6 cRNA. Oocytes were obtained from 1-3 frogs; NE indicates no effect. IC₅₀ values greater than 300 μM were determined as described in the Experimental Methods.

^a(IC₅₀ NR2A)/(IC₅₀ NR2D).

^b(IC₅₀ GluR1)/(IC₅₀ NR2D).

^*1-10 mM 2-hydroxypropyl-β-cyclodextrin was included for 30 and 100 μM concentrations of test compound. See Figure S2 (Supporting Information) for full general structure.

The effect of substituent position and identity on rings A,B,C

To further evaluate the effects of different aryl ring substituents, we measured the fitted IC₅₀ values for inhibition at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, and GluR1 receptor current responses under voltage clamp for each test compound. The position of a variety of A ring substituents was tested while holding the B ring substituent constant (R₄ = COOH) (Table 2). Given the lack of any detectable activity at recombinant kainate receptors (Table 1), responses at GluR6 were not studied further. A ring substitution at the meta position (R₂) was either equipotent or modestly improved potency compared to ortho (R₃) or para (R₁) A ring substitutions at NR2D-containing receptors among all analogues tested. For example, analogues containing meta methoxy (25) and trifluoromethyl (28) were most potent among positional isomers, whereas meta and ortho hydroxyl (31, 32) and nitro (2, 13) A ring substituents had nearly equivalent potencies.

Table 2.

Optimization of subunit selectivity through evaluation of Ring A substituents.

Number	R1	R2	R3	NR2A IC50	NR2B IC50	NR2C IC50	NR2D IC50	GluR1 IC50	NR2Aa NR2D	GluR1b NR2D
23				84	85	39	33	25	3	1
24	OCH3			100	98	42	31	250	3	8
25		OCH3		24	36	17	9	27	3	3
26			OCH3	88	51	32	16	38	6	2
27	CF3			61	55	23	15	233	4	16
28		CF3		13	26	8	12	22	1	2
29			CF3	>300	>300	88	28	>300	>10	>10
30	OH			38	35	50	36	110	1	3
31		OH		52	30	24	17	24	3	1
32			OH	20	18	20	18	36	1	2
7	NO2			64	93	15	15	>300	4	>20
2		NO2		21	30	12	9	32	2	4
13			NO2	11	17	8	6	6	2	1
33	CH3			41	138	28	17	>300	2	>17
34		CH3		21	34	28	18	>300	1	>16
35			CH3	86	93	80	81	62	1	1

IC₅₀ values in μM were determined by fitting the Hill equation to average composite concentration-effect curves from 4-17 oocytes injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, or GluR1 cRNA. Oocytes were obtained from 1-3 frogs. Compounds 2 and 7, previously shown in Table 1, are included here to facilitate comparison. IC₅₀ values greater than 300 μM were determined as described in the Experimental Methods.

^a(IC₅₀ NR2A)/(IC₅₀ NR2D)

^b(IC₅₀ GluR1)/(IC₅₀ NR2D). See Figure S2 (Supporting Information) for full general structure.

The positional preference for C ring substitutions was explored utilizing chlorinated derivatives while retaining optimal A ring (R₂ = NO₂) and B ring (R₄ = COOH) substitutions (Table 3; Fig. S2 in Supporting Information). Substitution at position R₈, specifically compound 37, gave both improved potency (IC₅₀ 1 μM) and selectivity (IC₅₀ NR2A/ IC₅₀ NR2D = 16) for mono-substituted compounds. Interestingly, the R₈,R₁₀-dichloro compound 40 showed enhanced NR2D selectivity (IC₅₀ NR2A/ IC₅₀ NR2D = 22) with decreased on-target potency (IC₅₀ 10 μM).

Table 3.

Optimization of subunit selectivity through evaluation of Ring C substituents.

Number	R1	R2	R7	R8	R9	R10	NR2A IC50	NR2B IC50	NR2C IC50	NR2D IC50	GluR1 IC50	NR2Aa NR2D	GluR1b NR2D
36		NO2	Cl				>300	110	22	14	>300	>20	>20
37		NO2		Cl			16	13	2	1	7	16	7
38		NO2			Cl		32	52	16	15	41	2	3
39 *		NO2				Cl	56	243	12	9	87	6	10
40		NO2		Cl		Cl	224	109	16	10	>300	22	30
41	NO2			Cl		Cl	>300	>300	9	6	>300	>50	>50
2		NO2		H			21	30	12	9	32	2	4
42		NO2		F			36	33	14	7	7	5	1
37		NO2		Cl			16	13	2	1	7	16	7
43		NO2		Br			17	13	2	1	8	17	8
44		NO2		I			18	6	1	0.6	31	18 d	52
45		NO2		CH3			12	15	5	2	27	6	14
46 *		NO2		OCH3			229	>300	6	3	>300	76	>100
47 *		NO2		NO2			NE	NE	204	90	>300	-	-
48		NO2		OH			237	>300	43	10	96	24	10
49		NO2		C6H5			11	3	2 c	2 c	5	6	3
50		NO2		R8-C4H4-R9			11	5	2 c	2 c	4	6	2
51		NO2		R8-OCH2CH2O-R9			>300	>300	281	213	>300	1	1
52		NO2		R8-OCH2O-R9			112	162	40	36	>300	3	>8
53 *		NO2		CH(CH3)2			101	98	6	7	200	14	29
54		NO2		CH2CH2CH3			28	18	3	4	50	7	13
55		NO2		2-thiophene			157	129	5	3	280	52	93
56		NO2		CHCHC6H5			19	30	4	4	131	5	33

IC₅₀ values in μM were determined by fitting the Hill equation to average composite concentration-effect curves from 7-26 oocytes injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 cRNA. Oocytes were obtained from 2-5 frogs. Compound 2, previously shown in Table 1, is included to facilitate comparison. IC₅₀ values greater than 300 μM were determined as described in Experimental Methods. NE indicates no effect.

^a(IC₅₀ NR2A)/(IC₅₀ NR2D)

^b(IC₅₀ GluR1)/(IC₅₀ NR2D)

^cdata were fitted with a variable minimum (see text)

^dselectivity was calculated from IC₅₀ values rounded to nearest value in μM.

^*1-10 mM 2-hydroxypropyl-β-cyclodextrin was included for 30 and/or 100 μM concentrations of test compound. See Figure S2 (Supporting Information) for full general structure.

Evaluation of a series of R₈ C ring substituents revealed a correlation between van der Waals radii of the series H, F, Cl, Br, I and both the IC₅₀ value at NR1/NR2D (r = −0.95; p<0.01; Figure S3 in Supporting Information) and the selectivity for NR2D over NR2A (r = 0.97; p<0.01). In particular, iodinated derivative 44 inhibited NR1/NR2D receptor responses with an IC₅₀ value of 600 nM and is 18-and 52-fold selective over recombinant NR2A and AMPA GluR1 receptors, respectively (Table 3). It is unclear whether the increased potency and selectivity of 44 can be attributed to an electropositive effect or a steric effect. Consistent with a steric effect, we observed that other analogues containing large R₈ substituents also improve NR2D apparent selectivity such as R₈ = Ph (49) and R₈,R₉-naphthyl (50) when it is assumed that inhibition is complete at saturating concentrations. However, the inhibition curves with these larger hydrophobic substituents revealed incomplete inhibition. Fitting the concentration-effect data for these compounds with a variable minimum (see Experimental) resulted in little difference in potency between the various NR2 subunits (see IC₅₀ values in Table 3). Nevertheless, the fitted curves reveal striking differences in the degree of inhibition. For example, a saturating concentration of compound 50 is predicted to reduce responses at receptors containing NR2A, NR2B, NR2C, or NR2D to 68, 50, 26, or 20% of control respectively. Similarly, a saturating concentration of compound 49 is predicted to reduce responses at saturating concentrations for receptors containing NR2A, NR2B, NR2C, or NR2D to 68, 51, 22, or 14% of control, respectively. These classes of compounds were not studied further.

Robust combined selectivity for NR2C and NR2D over NR2A, NR2B and GluR1 was achieved with methoxy at the R₈ position (46, IC₅₀ 3 μM), which was greater than 100-fold selective for NR1/NR2D over GluR1. This compound series has a maximum solubility of 70 μM (n=2), necessitating the use of 1 mM β-cyclodextrin for concentrations above 70 μM. IC₅₀ values for 46 at NR2C were similar to those at NR2D. Likewise, IC₅₀ values at NR2A were typically similar to NR2B, and were >50 fold higher than at NR2C/D containing receptors (see Table 3).

We subsequently tested the effect of altering the nitro and carboxylic acid substituents on the A and B rings, respectively, for compounds where R₈ was methoxy (C ring) (Table 4). Replacement of the p-carboxylic acid functionality on the B ring with hydrogen (57), cyano (58), amide (59), or methyl ester (60) moieties led to compounds that were either substantially less potent or inactive (Table 4). These results suggest an anionic component such as the carboxylate is crucial for binding. Substitutions of the A ring nitro group that maintained some activity included R₂ = OMe (61, NR2D IC₅₀ 9 μM) and R₂ = COOCH₃ (63, NR2D IC₅₀ 6 μM). Only modest activity was maintained with para substituents on the A ring (Table 4). These data suggest the binding pocket prefers the electron rich nitro and carboxylic acid groups on rings A and B, respectively.

Table 4.

Substitutions for Ring B carboxylic acid and Ring A nitro groups.

Number	R1	R2	R4	R8	NR2A IC50	NR2B IC50	NR2C IC50	NR2D IC50	GluR1 IC50	NR2Aa NR2D	GluR1b NR2D
57 *		NO2			>300	>300	>300	>300	>300	1	1
46 *		NO2	COOH	OCH3	229	>300	6	3	>300	76	>100
58		NO2	CN	OCH3	>300	>300	>300	>300	>300	1	1
59		NO2	CONH2	OCH3	>300	82	58	>300	>300	1	1
60		NO2	COOCH3	OCH3	218	46	39	38	138	6	4
61		OCH3	COOH	OCH3	73	109	18	9	220	8	24
62		COOH	COOH	OCH3	>300	>300	>300	131	>300	>2	>2
63		COOCH3	COOH	OCH3	80	39	15	6	41	13	7
64	COOCH3	COOCH3	COOH	OCH3	>300	>300	124	52	>300	>6	>6
7	NO2		COOH		64	93	15	15	>300	4	>20
65	SCH3		COOH		211	79	42	22	244	10	11
66	CN		COOH		94	106	28	34	>300	3	>9
67	N(CH3)2		COOH		>300	>300	63	53	>300	>5	>5
68	OCHF2		COOH		73	89	33	22	208	3	9
69	OCH2CHCH2		COOH		>300	144	111	30	>300	>10	>10
70	OCH2COOH		COOH		>300	>300	>300	>300	>300	1	1
71	COOH		COOH		>300	>300	>300	>300	>300	1	1

IC₅₀ values in μM were determined by fitting the Hill equation to average composite concentration-effect curves from 4-26 oocytes injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 cRNA. Oocytes were obtained from 1-4 frogs. Compounds 7 and 46, previously shown in Table 3, are included to facilitate comparison. IC₅₀ values greater than 300 μM were determined as described in the Experimental Methods.

^a(IC₅₀ NR2A)/(IC₅₀ NR2D)

^b(IC₅₀ GluR1)/(IC₅₀ NR2D)

^*1-10 mM 2-hydroxypropyl-β-cyclodextrin was included for 100 μM concentrations of test compound. See Figure S2 (Supporting Information) for full general structure.

Although compounds with R₂ = NO₂ (2) or R₃ = NO₂ (13) A ring substitution gave the best potency, improved selectivity for NMDA over AMPA receptors was noted for R₁ = NO₂ analogues (7 and 8 in Table 1), as well as modestly improved selectivity for NR2D over NR2A with R₅ = COOH B ring substitution (11 in Table 1). This led to a systematic comparison of ring substituents among these two positions (A ring: R₁, R₂ = NO₂; B ring: R₄, R₅ = COOH) for the most potent and selective ring C substitutions where R₈ was iodo or methoxy. Table 5 summarizes data describing these compounds, fitted as described in the Methods. The best potency was obtained for compounds with para-carboxylic acid substitution on the B ring (R₄ = COOH), in particular compounds 44, 46 (Fig. 2A). Among iodo-substituted compounds, 72, which contains a para-nitro A ring (R₁ = NO₂), inhibited NR2C/D containing receptors with the best apparent selectivity over NR2A, NR2B or AMPA receptors. We similarly saw somewhat improved selectivity with para-nitro in compound 41 (Table 3). Compound 72 had a maximum solubility of 10 μM, and thus required addition of 1 mM 2-hydroxypropyl-β-cyclodextrin at concentrations above 10 μM. We were unable to determine an IC₅₀ value for NR2A, NR2B, and GluR1 AMPA receptors, but this value would theoretically be greater than 300 μM given the level of inhibition observed at 100 μM of compound 72. Inhibition by 72 at NR1/NR2C and NR1/NR2D was incomplete, leading us to fit the data with a variable minimum (see Methods), which was on average 31% and 26% of control, respectively (Fig. 2B).

Table 5.

Optimization of Ring A, Ring B, Ring substituents.

Number	R1	R2	R4	R5	R8	NR2A IC50	NR2B IC50	NR2C IC50	NR2D IC50	GluR1 IC50	NR2Aa NR2D	GluR1b NR2D
72 *	NO2		COOH		I	>300	>300	2 c	1 c	>300	>150	>220
44		NO2	COOH		I	18	6	1	0.6	31	30	52
73	NO2			COOH	I	>300	279	9	8	>300	>37	>37
74		NO2		COOH	I	8	11	2	1	21	8	21
75	NO2		COOH		OCH3	197	206	13	7	>300	28	>42
46 *		NO2	COOH		OCH3	229	>300	6	3	>300	76	>100
76	NO2			COOH	OCH3	238	238	21	16	>300	15	>19
77		NO2		COOH	OCH3	208	>300	24	13	289	16	22

IC₅₀ values in μM were determined by fitting the Hill equation to average composite concentration-effect curves from 7-26 oocytes injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 cRNA. Oocytes were obtained from 2-5 frogs. Compounds 44 and 46, previously shown in Table 3, are included to facilitate comparison. IC₅₀ values greater than 300 μM were determined as described in the Experimental Methods.

^a(IC₅₀ NR2A)/(IC₅₀ NR2D)

^b(IC₅₀ GluR1)/(IC₅₀ NR2D)

^cindicates data fitted with variable minimum (see Fig 2B).

^*1 mM 2-hydroxypropyl-β-cyclodextrin was included for 30 and/or 100 μM concentrations of test compound. See Figure S2 (Supporting Information) for full general structure.

Figure 2.

Mean composite concentration-effect curves are shown for NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1, GluR6 for compounds (A) 46 and (B) 72; error bars are SEM. 2-hydroxypropyl-β-cyclodextrin (1 mM) was included in solutions of 30 and 100 μM 72 and 100 μM for 46. For compound 46, recordings of NMDA and AMPA receptor responses were made from 16-28 oocytes per receptor; oocytes were isolated from 3-5 different frogs. For compound 72, recordings of NMDA and AMPA receptor responses were made from 11-15 oocytes from 3 batches of oocytes from different frogs. No significant effect was observed for 30 μM 46 or 72 on GluR6 responses (6 oocytes, 1-2 batches of oocytes from different frogs for each compound).

Effect of changes to the backbone on subunit selectivity and potency at NR2C/D receptors

Modifications to the styryl A ring linker were also explored to determine the nature of the backbone structure in terms of both potency and subunit selectivity (Table 6). Complete removal of the ring, as in compound 78, which contains merely the quinazolin-4-one core structure, resulted in a total loss of inhibitory activity at concentrations up to 100 μM. We further investigated the saturation level of the styryl linker between the A ring and the quinazolin-4-one core structure. Reduction of the styryl linker of 80 (no A ring substitutions, B ring p-carboxylic acid, C ring R₈ methyl; IC₅₀ 26 μM) to the fully saturated linker analogue 81 (fitted IC₅₀ 282 μM; Table 6) resulted in 10-fold potency reduction. These data suggest the geometry of the trans-alkene linker is preferred for activity. Incorporation of larger ring systems pendant to the C ring led to compounds with modest activity, as shown by the various napthyl derivatives (82, 83, and 84) and phenyl-1,4-dioxane (86; Table 6), suggesting the presence of an extended space in the binding pocket for larger hydrophobic groups. Replacement of the A ring with a 3-pyridyl system also produced active compounds. The 3-pyridyl A ring analogues with the most potent C ring substitution (R₈ = iodine; Table 7) were subsequently evaluated. Compound 91 (R₆ = OMe) exhibited reasonable potency (IC₅₀ 2.5 μM) with modest selectivity (10-fold) over NR2A and good selectivity over GluR1 (66-fold). Introduction of a carboxylic acid at position R₄ (98) or R₅ (99) gave reasonable potency; however, selectivity over GluR1 was more modest.

Table 6.

Optimization of the linker and Ring A.

Number	R	R8	NR2A IC50	NR2B IC50	NR2C IC50	NR2D IC50	GluR1 IC50	NR2Aa NR2D	GluR1b NR2D
78	CH3	I	>300	>300	>300	>300	>300	1	1
79		I	53	66	13	4	23	13	6
80		CH3	39	25	22	26	21	2	1
81		CH3	>300	78	184	282	>300	1	1
82			21	31	14	6	>300	4	>50
83			63	36	20	11	103	6	9
84			25	68	22	6	82	4	14
85			>300	122	284	171	>300	1	1
86 *			>300	262	26	16	>300	>18	>18

IC₅₀ values in μM were determined by fitting the Hill equation to average composite concentration-effect curves from 3-12 oocytes injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 cRNA. Oocytes were obtained from 1-2 frogs. IC₅₀ values greater than 300 μM were determined as described in the Experimental Methods.

^a(IC₅₀ NR2A)/(IC₅₀ NR2D)

^b(IC₅₀ GluR1)/(IC₅₀ NR2D)

^*2-4 mM 2-hydroxypropyl-β-cyclodextrin was included for 1-100 μM concentrations of test compound. See Figure S2 (Supporting Information) for full general structure.

Table 7.

Optimization of Ring B substituents for pyridinyl analogues.

Number	R4	R5	R6	R8	NR2A IC50	NR2B IC50	NR2C IC50	NR2D IC50	GluR1 IC50	IC50NR2A IC50NR2D	IC50GluR1 IC50NR2D
87					40	51	140	35	74	1	2
88				I	132	>300	>300	108	>300	1	3
89 *	OCH3			I	>300	>300	>300	>300	>300	1	1
90 *		OCH3		I	>300	>300	>300	200	>300	1	1
91 *			OCH3	I	31	30	3	3	199	10	66
92 *	CH3			I	>300	>300	>300	>300	>300	1	1
93 *		CH3		I	>300	>300	241	>300	>300	1	1
94 *			CH3	I	>300	156	70	25	83	12	3
95 *	NO2			I	>300	>300	204	218	>300	1	1
96 *		NO2		I	>300	>300	>300	>300	>300	1	1
97 *			NO2	I	206	105	19	5	>300	41	60
98	COOH			I	26	31	6	4	61	7	15
99		COOH		I	45	49	7	4	64	11	16

IC₅₀ values in μM were determined by fitting the Hill equation to average composite concentration-effect curves from 3-20 oocytes injected with NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 cRNA. Oocytes were obtained from 1-3 frogs. IC₅₀ values greater than 300 μM were determined as described in the Experimental Methods. 1-10 mM 2-hydroxypropyl-β-cyclodextrin was included for 10, 30, and/or 100 μM concentrations of test compounds. See Figure S2 (Supporting Information) for full general structure.

DISCUSSION AND CONCLUSIONS

These data show that a previously unknown and novel binding site exists on recombinant NMDA receptors expressed in heterologous systems at which modulators can act with clear preference for NR2C/D subunits. The two most potent and selective compounds in terms of inhibition of NR2C/D-containing receptors to emerge from these experiments are compounds 46 and 72 (Fig. 2). Both 46 and 72 are highly selective for NMDA receptors over GluR6 kainate receptors. Compound 46 is over 50-fold selective for NR2D over NR2A/B, and at least 100-fold selective over GluR1. Compound 72 also appears to be selective over NR2A/B-containing receptors. Figure 2 compares the concentration-effect curves for each of these compounds at five different glutamate receptors. Overall, a small subset of compounds within this class is characterized by poor aqueous solubility at or above 100 μM. Thus, the compounds discussed here may be more selective than we have conservatively reported. These compounds represent the first set of non-competitive antagonists with selectivity for recombinant NMDA receptors containing NR2C/D subunits over receptors containing NR2A/B subunits. These data suggest that native NMDA receptors may have binding sites for this class of modulators, and that the concentration-effect relationship associated with this class of compounds may show significant differences between the NR2A/B and NR2C/D receptors. Thus, this class of molecule may serve as a starting point for the development of potent and selective subunit selective NMDA receptor inhibitors.

BIOLOGY EXPERIMENTAL

Two-electrode voltage-clamp electrophysiology

Two-electrode voltage-clamp recordings were performed on Xenopus oocytes expressing recombinant rat NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1, or GluR6 receptors. cDNAs for rat NR1-1a (GenBank accession numbers U11418 and U08261; hereafter NR1), NR2A (D13211), NR2B (U11419), NR2C (M91563), NR2D (L31611), GluR1 (X17184), GluR6 (Z11548) were provided by Drs. S. Heinemann (Salk Institute), S. Nakanishi (Kyoto University), and P. Seeburg (University of Heidelberg). Oocyte isolation and RNA injection were completed as described in detail elsewhere;⁵⁹ all protocols involving Xenopus laevis were approved by the Emory University Institutional Animal Care and Use Committee. During two-electrode voltage-clamp recordings, oocytes were placed into a perfusion chamber and continually washed with recording solution containing (in mM) 90 NaCl, 1.0 KCl, 0.5 BaCl₂, 0.005 EDTA, and 10 HEPES at pH 7.4 (23°C). Glass electrodes with a tip resistance of 0.5-2.5 MΩ were pulled from thin-walled glass capillary tubes and filled with 0.3-3.0 M KCl. An OC-725C amplifier (Warner Instrument Co) was used to hold the membrane potential of the oocytes at −40 mV during current recording. All compounds were made as 20 mM stock solutions in DMSO, and dissolved to reach the desired final concentration in recording solution containing 100 μM glutamate and 30 μM glycine for use on oocytes expressing NMDA receptors. Final DMSO content was 0.05-0.5% (vol/vol). Oocytes expressing GluR6 receptors were pre-treated with 10 μM concanavalin A for 10 minutes. Recombinant GluR1 and GluR6 receptors were activated by 100 μM glutamate. In order to prevent a gradual increase in current response over the course of the experiment, which appears to be a common feature of NR1/NR2A receptor responses in oocytes, some oocytes expressing NR1/NR2A were either pretreated with 50 μM BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester) for 10 minutes or injected with 50 nl of 2 mM K-BAPTA (potassium 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid).

For every test compound, we recorded 5-7 concentrations in at least 4 oocytes per concentration on each of five different receptors. We subsequently determined the IC₅₀ (half-maximally effective concentration of inhibitor) by fitting the equation

$$\mathit{Response}=(100-\mathit{minimum})∕(1+{(\left[\mathrm{I}\right]∕{\mathrm{IC}}_{50})}^{\mathrm{N}})+\mathit{minimum}$$ (1)

to the mean composite concentration-response data normalized to the response in the absence of inhibitor (100%). N is the Hill slope, [ I ] is the inhibitor concentration, and minimum is the residual inhibition at saturating concentrations of ligand. Because inhibition was complete for most compounds tested, the minimum was fixed to 0 for all fitted curves, unless otherwise indicated. For a few compounds with bulky hydrophobic C ring substituents (49, 50, 72), minimum was allowed to vary. Compounds that showed a response greater than 75% of control in the presence of 100 μM test compound are reported as having an IC₅₀ value >300 μM, which is theoretically predicted by the Hill equation for a slope of 1.

The maximum solubility was determined in a subset of 24 compounds representing various classes of structure using a BMG Labtech Nephelostar nephelometer (Offenburg, Germany), according to manufacturer's instructions. Maximum solubility of each test compound was determined in oocyte recording solution (components given below) and 1% DMSO. Only responses for concentrations below the experimentally determined limit of solubility were measured; whenever necessary we repeated experiments with 1-10 mM 2-hydroxypropyl-β-cyclodextrin added to the recording solution to ensure that the compounds remained in solution up to 100 μM. 2-hydroxypropyl-β-cyclodextrin had no detectable effect on NMDA response amplitude (data not shown).

CHEMISTRY EXPERIMENTAL

Compounds not described below were purchased from commercial vendors. Provided samples were of greater than 90% purity, as determined by the suppliers, via HPLC or NMR.

All reagents were obtained from commercial suppliers and used without further purification. Reaction progress was monitored by thin layer chromatography (TLC) on pre-coated glass plates (silica gel 60 F254, 0.25 mm). Proton and carbon NMR spectra were recorded on an INOVA-400 (400 MHz), VNMRS 400 (400 MHz), INOVA-600 (600 MHz), or Mercury 300 Vx (300 MHz). The spectra obtained were referenced to the residual solvent peak. Mass spectra were performed by the Emory University Mass Spectroscopy Center on either a VG 70-S Nier Johnson or JEOL instrument. Elemental analyses were performed by Atlantic Microlab Inc. C, H, N agreed with proposed structures within ±0.4% of theoretical values unless indicated. Flash chromatography was performed on a Teledyne ISCO Combiflash Companion with prepackaged Teledyne RediSep disposable normal phase silica columns. Melting temperatures were determined on a Mel-Temp apparatus and are uncorrected.

General procedure for synthesis of (E)-3-phenyl-2-styrylquinazolin-4(3H)-one products (Procedure C)

The quinazolinone (6, 1.0 equiv), benzaldehyde (1.33 equiv), and sodium acetate (1.63 equiv) were suspended in a mixture of glacial acetic acid (58 equiv) and acetic anhydride (7.0 equiv). The mixture was refluxed until TLC indicated the reaction was finished (generally 12-18 hours). After cooling to room temperature, the mixture was filtered and washed with methanol. Further purification was performed (chromatography or recrystallization) as necessary.

(E)-4-(2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (2)

Compound 2 was prepared via procedure C with compound 2b (0.100 g, 0.36 mmol) and meta-nitrobenzaldehyde (0.072 g, 0.48 mmol, 1.3 equiv). The crude material was purified via silica gel chromatography (ISCO, RediSep 12 g column, silica cake, 5-10% MeOH/DCM gradient) to give an off-white solid (0.030 g, 20%). ¹H NMR (600 MHz, DMSO-d₆) δ 8.26 (s, 1H), 8.17 (d, J=7.6 Hz, 4H), 8.01 (d, J=15.2 Hz, 1H), 7.92 (t, J=7.1 Hz, 1H), 7.86 (d, J=7.6 Hz, 1H), 7.81 (d, J=8.1 Hz, 1H), 7.65-7.62 (mult, 3H), 7.58 (t, J=7.6 Hz, 1H), 6.51 (d, J=15.7 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.7, 161.1, 150.6, 148.3, 147.2, 140.7, 136.7, 136.6, 135.0, 133.3, 131.6, 130.6, 130.5, 129.5, 127.4, 127.1, 126.5, 124.0, 122.8, 122.4, 120.7, 103.3. HRMS calcd for C₂₃H₁₅N₃O₅, 414.10911 [M+H]⁺; found, 414.10791 [M+H]⁺. Anal. (C₂₃H₁₅N₃O₅·0.25AcOH) C, H, N. MP > 260 °C.

(E)-2-(2-(4-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (9)

Compound 9 was prepared via procedure C with compound 9b (1.00 g, 3.57 mmol) and para-nitrobenzaldehyde (0.717 g, 4.75 mmol, 1.33 equiv) to give the title compound as a yellow solid (1.18 g, 80%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.20-8.14 (mult, 4H), 7.96 (d, J= 15.9 Hz, 1H), 7.92-7.81 (mult, 3H), 7.74 (t, J= 7.6 Hz, 1H), 7.68 (d, J= 7.9 Hz, 2H), 7.61-7.56 (mult, 2H), 6.50 (d, J=15.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.7, 161.3, 151.0, 147.5, 147.3, 141.2, 136.6, 136.5, 134.9, 133.8, 131.8, 130.7, 130.0, 128.6, 127.4, 127.0, 126.5, 124.1, 124.0, 120.7. HRMS calcd for C₂₃H₁₅N₃O₅, 414.10911 [M+H]⁺; found, 414.10903 [M+H]⁺. Anal.(C₂₃H₁₅N₃O₅) C, H, N. MP > 260° C.

tert-Butyldimethylsilyl 4-(6-iodo-2-methyl-4-oxoquinazolin-3(4H)-yl)benzoate (10)

Carboxylic acid 78 (0.300 g, 0.74 mmol) was dissolved in THF (10.0 mL). To this solution was added N-methylmorpholine (0.081 mL, 0.74 mmol, 1.0 equiv) and TBDMSCl (0.111 g, 0.74 mmol, 1.0 equiv). The mixture was stirred at room temperature for 1 hour. The yellow suspension was concentrated in vacuo, and the resulting residue was partitioned between ethyl acetate and water. The mixture was extracted 3x with ethyl acetate. The combined organics were washed with brine, dried over MgSO₄, and concentrated in vacuo to give a white solid. The crude material was purified using silica gel chromatography (ISCO, RediSep 12 g column, 100% EtOAc) to give a white foam (0.280 g, 73%). ¹H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 8.8 Hz, 2H), 8.02 (dd, J₁ = 8.7 Hz, J₂ = 2.1 Hz, 1H), 7.41 (d, J = 8.6 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H), 2.22 (s, 3H), 1.05 (s, 9H), 0.41 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 165.5, 160.8, 154.3, 146.9, 143.7, 141.5, 135.9, 132.9, 132.1, 129.0, 128.4, 122.5, 91.3, 25.8, 24.6, 18.0, −4.6. HRMS calcd for C₂₂H₂₅IN₂O₃Si, 521.07585 [M+H]⁺; found, 521.07527 [M+H]⁺.

(E)-2-(2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (12)

Compound 12 was prepared via procedure C with compound 9b (0.250 g, 0.89 mmol) and meta-nitrobenzaldehyde (0.179 g, 1.19 mmol, 1.33 equiv) to give the title compound as a yellow solid (0.202 g, 55%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.22-8.13 (mult, 4H), 7.99 (d, J=15.2 Hz, 1H), 7.93-7.80 (mult, 4H), 7.75 (t, J=7.3 Hz, 1H), 7.65-7.60 (mult, 2H), 7.57 (t, J=7.0 Hz, 1H), 6.47 (d, J=15.6 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 165.7, 161.3, 151.1, 148.3, 147.4, 136.6, 136.6, 136.6, 134.9, 133.8, 133.3, 131.8, 130.7, 130.5, 129.9, 129.4, 127.3, 126.8, 126.5, 124.0, 122.6, 122.1, 120.7. HRMS calcd for C₂₃H₁₅N₃O₅, 414.10911 [M+H]⁺; found, 414.10822 [M+H]⁺. Anal. (C₂₃H₁₅N₃O₅) C, H, N. MP > 260° C.

(E)-4-(5-chloro-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (36)

Compound 36 was prepared via procedure C using compound 36b (0.300 g, 0.95 mmol) and meta-nitrobenzaldehyde (0.192 g, 1.27 mmol, 1.33 equiv) to give the title compound as a bright yellow solid (0.251 g, 59%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (bs, 1H), 8.22 (s, 2H), 8.19-8.15 (mult, 2H), 7.99 (d, J=15.7 Hz, 1H), 7.83-7.78 (mult, 2H), 7.65 (d, J=8.6 Hz, 2H), 7.61 (t, J=7.8 Hz, 2H), 7.55 (d, J=7.4 Hz, 1H), 6.42 (d, J=15.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 159.1, 151.2, 149.7, 148.2, 140.6, 137.3, 136.3, 134.7, 133.3, 132.9, 131.6, 130.7, 130.5, 129.5, 129.3, 126.9, 124.1, 122.4, 122.3, 117.6. HRMS calcd for C₂₃H₁₄ClN₃O₅, 448.07014 [M+H]⁺; found, 448.06970 [M+H]⁺. Anal.(C₂₃H₁₄ClN₃O₅) C, H, N. MP > 260 °C.

(E)-4-(6-chloro-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (37)

Compound 37 was prepared via procedure C using 37b (0.300 g, 0.950 mmol) and meta-nitrobenzaldehyde (0.192 g, 1.30 mmol, 1.33 equiv). The crude material was purified using recrystallization with methanol and dioxane. Further trituration with water yielded the title compound as a yellow solid (0.060 g, 14%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.19-1.66 (mult, 3H), 8.05 (d, J=2.2 Hz, 1H), 8.01 (d, J=15.6 Hz, 1H), 7.94 (dd, J₁=8.7 Hz, J₂=2.2 Hz, 1H), 7.85-7.81 (mult, 2H), 7.66-7.61 (mult, 3H), 6.48 (d, J=15.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.4, 160.9, 151.8, 148.9, 146.6, 141.0, 137.9, 137.1, 135.7, 134.0, 133.0, 132.4, 131.9, 131.4, 131.8, 130.2, 130.0, 126.1, 124.8, 123.1, 122.6. HRMS calcd for C₂₃H₁₄ClN₃O₅, 446.05426 [M-H]⁺; found, 446.05511 [M-H]⁺. Anal.(C₂₃H₁₄ClN₃O₅·0.75 H₂O) C, H, N. MP > 260 °C.

(E)-4-(7-chloro-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (38)

Compound 38 was prepared via procedure C using compound 38b (0.200 g, 0.640 mmol) and meta-nitrobenzaldehyde (0.128 g, 0.850 mmol, 1.33 equiv) to give the title compound as a yellow solid (0.135 g, 47%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.18-8.13 (mult, 4H), 8.00 (d, J=15.6 Hz, 1H), 7.85 (d, J=8.3 Hz, 1H), 7.83 (d, J=1.9 Hz, 1H), 7.65 (d, J=8.6 Hz, 3H), 7.60 (dd, J₁=8.3 Hz, J₂=1.9 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 160.6, 152.0, 148.3, 148.2, 140.4, 139.5, 137.6, 136.3, 133.4, 131.7, 130.7, 130.6, 130.5, 129.4, 128.6, 127.2, 126.3, 124.2, 122.4, 119.5 HRMS calcd for C₂₃H₁₄ClN₃O₅, 448.07014 [M+H]⁺; found, 448.06890 [M+H]⁺. Anal.( C₂₃H₁₄ClN₃O₅) C, H, N. MP > 260 °C.

(E)-4-(8-chloro-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (39)

Compound 39 was prepared via procedure C using compound 39b (0.400 g, 1.27 mmol) and meta-nitrobenzaldehyde (0.255 g, 1.69 mmol, 1.33 equiv) to yield the title compound as a yellow solid (0.071 g, 12%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.24 (bs, 1H), 8.26 (s, 1H), 8.17 (d, J=8.2 Hz, 3H), 8.10-8.02 (mult, 3H), 7.86 (d, J=7.8 Hz, 1H), 7.67-7.62 (mult, 3H), 7.53 (t, J=7.8 Hz, 1H), 6.51 (d, J=15.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ1 67.3, 161.4, 151.9, 148.9, 144.3, 141.0, 138.4, 137.0, 135.5, 134.0, 132.3, 131.5, 131.4, 131.1, 130.0, 127.9, 126.2, 124.8, 123.2, 123.0. HRMS calcd for C₂₃H₁₄ClN₃O₅, 448.07014 [M+H]⁺; found 448.07015 [M+H]⁺. Anal.( C₂₃H₁₄ClN₃O₅) C, H, N. MP > 260 °C.

(E)-4-(6,8-dichloro-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (40)

Compound 40 was prepared via procedure C using 41b (0.250 g, 0.716 mmol) and meta-nitrobenzaldehyde (0.144 g, 0.952 mmol, 1.3 equiv) to give the title compound as a yellow solid, which was obtained by filtration (0.139 g, 40%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.34 (bs, 1H), 8.29 (s, 1H), 8.24 (d, J=2.4 Hz, 1H), 8.16-8.20 (m, 3H), 8.08 (s, 1H), 8.04-8.05 (m, 1H) 7.88 (d, J=7.8 Hz, 1H), 7.62-7.78 (m, 3H), 6.51 (d, J=15.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 159.9, 151.8, 148.3, 142.8, 140.2, 138.2, 136.3, 134.5, 133.4, 131.8, 130.8, 130.7, 130.6, 129.3, 124.7, 124.3, 123.2, 122.8, 122.4. HRMS calcd for C₂₃H₁₃Cl₂N₃O₅, 482.03114 [M+H]⁺; found 482.03068 [M+H]⁺. Anal. (C₂₃H₁₃Cl₂N₃O₅) C, H, N. MP > 260°C.

(E)-4-(6,8-dichloro-2-(4-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (41)

Compound 41 was prepared via procedure C using compound 41b (0.250 g, 0.716 mmol) and para-nitrobenzaldehyde (0.144 g, 0.952 mmol, 1.3 equiv) to yield the title compound as a yellow solid (0.050 g, 14%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.34 (bs, 1H), 8.24 (d, J=2.7, 1H) 8.16-8.20 (mult, 4H), 8.05 (d, J=2.4, 1H), 8.02 (d, J=15.7, 1H), 7.71 (d, J=9.0, 2H), 7.65 (d, J=8.6, 2H), 6.53 (d, J=15.3, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 159.9, 151.6, 147.7, 142.7, 140.8, 140.1, 138.0, 134.5, 132.4, 131.8, 130.9, 130.8, 129.3, 129.0, 124.7, 124.2, 123.7, 123.2. HRMS calcd for C₂₃H₁₃Cl₂N₃O₅, 482.03114 [M+H]⁺; found, 482.0974 [M+H]⁺. Anal. (C₂₃H₁₃Cl₂N₃O₅) C, H, N. MP > 260 °C.

(E)-4-(6-fluoro-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (42)

Compound 42 was prepared via procedure C using 42b (0.400 g, 1.34 mmol) and meta-nitrobenzaldehyde (0.270 g, 1.78 mmol, 1.33 equiv). The crude material was purified by recrystallization with methanol and dioxane and further purification by trituration with methanol and ethyl acetate to remove residual dioxane to yield the title compound as a pale yellow solid (0.268 h, 43%). ¹H NMR (600 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.18-8.16 (mult, 3H), 7.99 (d, J=15.5 Hz, 1H), 7.89-7.80 (mult, 4H), 7.66-7.62 (mult, 3H), 6.49 (d, J=15.7, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 160.5, 159.8 (d, J=245 Hz), 150.2, 148.3, 144.1, 140.5,136.8, 136.5, 133.3, 131.6, 130.7, 130.5, 130.3, 130.0, 129.4, 124.0, 123.3 (d, J=24 Hz), 122.4 (d, J=15 Hz) 121.9 (d, J=8.4 Hz), 111.2 (d, J=24 Hz). HRMS calcd for C₂₃H₁₄FN₃O₅, 432.09969 [M+H]⁺; found, 432.09850 [M+H]⁺. Anal.(C₂₃H₁₄FN₃O₅) C, H, N. MP > 260 °C.

(E)-4-(6-bromo-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (43)

Compound 43 was prepared via procedure C using compound 43b (0.300 g, 0.840 mmol) and meta-nitrobenzaldehyde (0.168 g, 1.10 mmol, 1.33 equiv). The crude material was recrystallized using methanol and dioxane and further purified by hot trituration with methanol and ethyl acetate to remove residual dioxane to yield the title compound as a yellow solid (0.120 g, 30%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.22 (s, 1H), 8.19-8.14 (mult, 4H), 8.04 (dd, J₁= 8.5 Hz, J₂=2.3 Hz, 1H), 8.00 (d, J=2.3 Hz, 1H), 7.82 (d, J=7.6 Hz, 1H), 7.73 (d, J=8.5 Hz, 1H), 7.64-7.59 (mult, 3H), 6.46 (d, J=15.5 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.8, 160.0, 151.1, 148.2, 146.1, 140.0, 137.7, 137.2, 136.4, 133.2, 132.5, 130.7, 130.5, 129.6, 129.2, 128.5, 124.1, 122.4, 122.2, 119.3. HRMS calcd for C₂₃H₁₄BrN₃O₅, 492.01962 [M+H]⁺; found, 492.00228 [M+H]⁺. Anal. (C₂₃H₁₄BrN₃O₅) C, H, N. MP > 260 °C.

(E)-4-(6-methyl-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (45)

Compound 45 was prepared via procedure C using compound 45b (0.507 g, 1.72 mmol) and meta-nitrobenzaldehyde (0.346 g, 2.29 mmol, 1.33 equiv) to yield the title compound as a yellow solid (0.498 g, 68%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.34 (bs, 1H), 8.22 (s, 1H), 8.18-8.14 (mult, 3H), 7.96 (d, J=15.6 Hz, 1H), 7.93 (s, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.73-7.77 (mult, 2H), 7.64-7.60 (mult, 3H), 6.47 (d, J=15.6 Hz, 1H), 2.48 (s, 3H). ¹³C (100 MHz, DMSO-d₆) δ 166.7, 161.0, 149.8, 148.2, 145.2, 140.8, 136.9, 136.6, 136.3, 133.3, 131.6, 130.7, 130.5, 129.5, 127.2, 125.8, 123.9, 122.8, 122.3, 120.4, 20.9. HRMS calcd for C₂₄H₁₇N₃O₅, 428.12476 [M+H]⁺; found 428.12422 [M+H]⁺. Anal.(C₂₄H₁₇N₃O₅) C, H, N. MP > 260 °C.

(E)-4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (46)

Compound 46 was prepared via procedure C using compound 46b (0.120 g, 0.390 mmol) and meta-nitrobenzaldehyde (0.078 g, 0.51 mmol, 1.3 equiv) to yield the title compound as a yellow solid (0.094 g, 55%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.16 (d, J=8.3 Hz, 3H), 7.94 (d, J=15.6 Hz, 1H), 7.84 (d, J=7.9 Hz, 2H), 7.62 (d, J=9.8 Hz, 1H), 7.65-7.61 (mult, 3H), 7.54-7.53 (mult, 2H), 6.49 (d, J=15.6 Hz, 1H), 3.83 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)_.δ167.7, 161.5, 158.8, 149.2, 148.9, 142.5, 140.9, 137.4, 136.4, 133.8, 133.7, 131.2, 129.9, 129.8, 129.7, 125.2, 124.5, 123.4, 122.9, 122.2, 56.4. HRMS calcd for C₂₄H₁₇N₃O₆, 444.11968 [M+H]⁺; found, 444.11849 [M+H]⁺. Anal.(C₂₄H₁₇N₃O₆ ·0.50 H₂O) C, H, N. MP > 260 °C.

(E)-4-(6-nitro-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (47)

Compound 47 was prepared via procedure C using compound 47b (0.250 g, 0.769 mmol) and 3-nitrobenzaldehyde (0.154 g, 1.33 equiv, 1.02 mmol). The crude material was purified by recrystallization with ethyl acetate and hexanes to give a yellow solid (0.321 g, 91%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.35 (bs, 1H), 8.84 (d, J=3.0 Hz, 1H), 8.65 (dd, J₁=9.0 Hz, J₂=2.6 Hz, 1H), 8.32 (s, 1H), 8.15-8.21 (mult, 4H), 7.98 (d, J=9.0 Hz, 1H), 7.91 (d, J=8.1 Hz, 1H), 7.63-7.77 (mult, 3H), 6.54 (d, J=15.4 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 160.6, 153.9, 151.5, 148.3, 144.8, 140.0, 139.1, 136.2, 133.6, 131.9, 130.8, 130.6, 129.3, 129.0, 128.9, 124.5, 122.9, 122.6, 122.2, 120.8. HRMS calcd for C₂₃H₁₄N₄O₇, 459.09414 [M+H]⁺; found 459.09363 [M+H]⁺. Anal.(C₂₃H₁₄N₄O₇) C, H, N. MP > 260 °C.

(E)-4-(6-hydroxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (48)

Compound 48 was prepared via procedure C using compound 48b (0.262 g, 0.770 mmol) and meta-nitrobenzaldehyde (0.156 g, 1.0 mmol, 1.33 equiv). The crude material was purified by recrystallization with methanol and dioxane, and further purified by hot gravity filtration after trituration with ethyl acetate and methanol (0.095 g, 29%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.23 (s, 1H), 8.15 (d, J=8.6 Hz, 3H), 7.90 (d, J=15.6 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.69 (d, J=9.0 Hz, 1H), 7.62-7.59 (mult, 3H), 7.45 (d, J=3.1 Hz, 1H), 7.36 (dd, J₁=8.6 Hz, J₂=2.7 Hz, 1H), 6.47 (d, H=15.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 160.8, 156.7, 148.3, 147.6, 140.9, 140.4, 136.8, 135.3, 133.2, 131.4, 130.6, 130.5, 129.5, 129.2, 124.5, 123.7, 122.8, 122.2, 121.7, 109.4. HRMS calcd for C₂₃H₁₅N₃O₆, 430.10404 [M+H]⁺; found, 430.10455 [M+H]⁺ Anal. (C₂₃H₁₅N₃O₆·0.25 H₂O) C, H, N. MP > 260 °C.

(E)-4-(2-(3-nitrostyryl)-4-oxo-6-phenylquinazolin-3(4H)-yl)benzoic acid (49)

Compound 49 was prepared via procedure C using 49b (0.200 g, 0.560 mmol) and meta-nitrobenzaldehyde (0.113 g, 0.750 g, 1.33 equiv) to yield the title compound as a yellow solid (0.210 g, 76%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.35 (d, J=2.0 Hz, 1H), 8.26-8.25 (mult, 1H), 8.23 (d, J=2.3 Hz, 1H), 8.19-8.15 (mult, 3H), 8.03 (d, J=15.7 Hz, 1H), 7.89 (d, J=8.2 Hz, 2H), 7.86 (d, J=7.8 Hz, 2H), 7.67-7.65 (mult, 3H), 7.53 (t, J=7.4 Hz, 2H), 7.43 (t, J=7.0 Hz, 1H), 6.51 (d, J=15.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.8, 161.1, 150.6, 148.2, 146.5, 140.4, 138.7, 138.6, 136.8, 136.5, 133.4, 133.3, 132.2, 130.7, 130.5, 129.4, 129.2, 128.1, 126.8, 124.0, 123.7, 122.7, 122.4, 121.0. HRMS calcd for C₂₉H₁₉N₃O₅, 490.14044 [M+H]⁺; found, 490.13965 [M+H]⁺. Anal.(C₂₉H₁₉N₃O₅) C, H, N. MP > 260 °C.

(E)-4-(2-(3-nitrostyryl)-4-oxobenzo[g]quinazolin-3(4H)-yl)benzoic acid (50)

Compound 50 was prepared via procedure C using compound 50b (0.250 g, 0.760 mmol) and meta-nitrobenzaldehyde (0.152 g, 1.01 mmol, 1.33 equiv). The crude material was purified by hot gravity filtration with methanol (0.194 g, 55%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (bs, 1H), 8.87 (s, 1H), 8.28 (s, 1H), 8.25 (mult, 2H), 8.20-8.16 (mult, 4H), 8.02 (d, J=15.7 Hz, 1H), 7.86 (d, J=7.8 Hz, 1H), 7.33-7.59 (mult, 5H), 6.52 (d, J=15.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.8, 161.6, 149.6, 148.3, 142.6, 140.9, 136.7, 136.5, 133.3, 131.5, 131.2, 130.7, 130.5, 129.7, 129.4, 128.8, 128.0, 126.6, 124.9, 124.0, 123.0, 122.3, 119.8. HRMS calcd for C₂₇H₁₇N₃O₅, 464.12474 [M+H]⁺ ; found, 464.125661 [M+H]⁺. Anal. (C₂₇H₁₇N₃O₅) C, H, N. MP > 260 °C.

(E)-4-(2-(3-nitrostyryl)-4-oxo-7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-3(4H)-yl)benzoic acid (51)

Compound 51 was prepared via procedure C using compound 51b (0.150 g, 0.44 mmol) and meta-nitrobenzaldehyde (0.089 g, 0.59 mmol, 1.33 equiv). The crude material was purified by hot gravity filtration with methanol to give a yellow solid (0.056 g, 27%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (s, 1H), 8.14 (d, J = 8.2 Hz, 1H), 8.03 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 15.7 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.51 (s, 1H), 7.26 (d, J = 8.2 Hz, 2H), 7.21 (s, 1H), 6.49 (d, J = 15.7 Hz, 1H), 4.44-4.38 (mult, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.1, 160.3, 149.8, 149.7, 148.3, 143.7, 142.7, 142.0, 136.8, 136.7, 135.5, 132.9, 130.6, 130.1, 127.6, 123.8, 123.0, 122.1, 115.0, 114.2, 113.4, 112.6, 64.4, 64.3. HRMS calcd for C₂₅H₁₇N₃O₇, 472.11459 [M+H]⁺; found, 472.11429 [M+H]⁺. Anal.(C₂₅H₁₇N₃O₇) C, H, N. MP > 260 C.

(E)-4-(6-(3-nitrostyryl)-8-oxo-[1,3]dioxolo[4,5-g]quinazolin-7(8H)-yl)benzoic acid (52)

Compound 52 was prepared via procedure C using compound 52b (0.100 g, 0.31 mmol) and meta-nitrobenzaldehyde (0.062 g, 0.41 mmol, 1.33 equiv). The crude material was purified by hot gravity filtration with methanol to give a yellow solid (0.048 g, 34%). ¹H NMR (400 MHz, d₆-DMSO) δ 8.21 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.91 (d, J = 15.6 Hz, 1H), 7.76 (d, J = 7.0 Hz, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.46 (s, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.24 (s, 1H), 6.49 (d, J = 15.6 Hz, 1H), 6.27 (s, 2H). HRMS calcd for C₂₄H₁₅N₃O₇, 458.09894 [M+H]⁺; found, 458.09772 [M+H]⁺. Anal.(C₂₄H₁₅N₃O₇) C, H, N. MP > 260°C.

(E)-4-(6-isopropyl-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (53)

Compound 53 was prepared via procedure C using 53b (0.150 g, 0.47 mmol) and meta-nitrobenzaldehyde (0.094 g, 0.62 mmol, 1.3 equiv) to yield the title compound as a yellow solid (0.137 g, 65%). ¹H NMR (400 MHz, DMSOd₆) δ 13.34 (bs, 1H), 8.26 (d, 1H), 8.16 (d, J=8.6 Hz, 2H), 8.00 (d, J=15.7 Hz, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.87-7.83 (mult, 3H), 7.75 (d, J=8.2 Hz, 1H), 7.66-7.61 (mult, 3H), 6.49 (d, J=15.7 Hz, 1H), 3.11 (sept, J=7.0 Hz, 1H), 1.29 (d, J=7.0 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.8, 161.1, 149.9, 148.3, 147.6, 145.6, 140.7, 136.6, 136.3, 133.8, 133.3, 131.8, 130.7, 130.5, 129.4, 127.5, 123.9, 123.1, 122.7, 122.3, 120.4, 33.3, 23.7. HRMS calcd for C₂₆H₂₁N₃O₅, 456.15604 [M+H]⁺; found, 456.15557 [M+H]⁺. Anal. (C₂₆H₂₁N₃O₅) C, H, N. MP > 260 °C.

(E)-4-(2-(3-nitrostyryl)-4-oxo-6-propylquinazolin-3(4H)-yl)benzoic acid (54)

Compound 54 was prepared via procedure C using compound 54b (0.578 g, 1.79 mmol) and meta-nitrobenzaldehyde (0.360 g, 2.38 mmol). The crude material was purified by silica gel chromatography (ISCO, RediSep 24 g column, silica cake, 0-10% MeOH/DCM gradient) to give a yellow solid (0.086 g, 11%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (s, 1H), 8.16 (d, J=8.6 Hz, 3H), 7.96 (d, J=15.6, 2H), 7.85 (d, J=7.6 Hz, 1H), 7.79-7.73 (mult, 3H), 7.64 (t, J=7.9 Hz, 3H), 6.50 (d, J=15.6 Hz, 1H), 2.75 (t, J=7.3 Hz, 2H), 1.67 (sext, J=7.3 Hz, 2H), 0.93 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.8, 161.1, 149.9, 148.3, 145.5, 141.5, 140.8, 136.6, 136.3, 135.7, 133.3, 131.6, 130.7, 130.5, 129.5, 127.3, 125.4, 123.9, 122.8, 122.3, 120.4, 36.8, 24.1, 13.5. HRMS calcd for C₂₆H₂₁N₃o₅, 456.15604 [M+H]⁺; found, 456.15568 [M+H]⁺. Anal.(C₂₆H₂₁N₃O₅) C, H, N. MP > 260 °C.

(E)-4-(2-(3-nitrostyryl)-4-oxo-6-(thiophen-2-yl)quinazolin-3(4H)-yl)benzoic acid (55)

Compound 55a (0.077 g, 0.212 mmol) was dissolved in a mixture of acetic acid (0.71 mL, 12.3 mmol, 58 equiv) and acetic anhydride (0.14 mL, 1.4 mmol, 7.0 equiv). To this solution was added meta-nitrobenzaldehyde (0.043 g, 0.283 mmol, 1.33 equiv) and sodium acetate (0.028 g, 0.35 mmol, 1.63 equiv). The mixture was refluxed for 6 hours. After cooling to room temperature, the resultant yellow solid was collected by filtration and washed with methanol. The crude material was purified using silica gel chromatography (silica cake, ISCO, RediSep 24 g column, 0-10% MeOH/DCM gradient) to give a yellow solid (0.049 g, 48%). ¹H NMR (400 MHz, d₆-DMSO) δ 13.35 (bs, 1H), 8.31-8.25 (mult, 3H), 8.17 (d, J = 8.2 Hz, 3H), 8.03 (d, J = 15.7 Hz, 1H), 7.87 (t, J = 7.4 Hz, 2H), 7.74 (dd, J₁ = 3.5 Hz, J₂=1.2 Hz, 1H), 7.68-7.63 (mult, 4H), 7.21 (dd, J₁ = 5.1 Hz, J₂ = 3.51 Hz, 1H), 6.51 (d, J = 15.7 Hz, 1H). ¹³C NMR (150 MHz, d₆-DMSO) δ 166.7, 160.9, 150.5, 148.3, 146.4, 141.9, 140.6, 136.8, 136.5, 131.3, 132.3, 131.9, 131.7, 130.7, 130.5, 129.4, 129.0, 128.3,126.8, 124.9, 124.0, 122.6, 122.4, 121.9, 121.2. HRMS calcd for C₂₇H₁₇N₃O₅S, 496.09683 [M+H]⁺; found, 496.10655 [M+H]⁺. Anal. (C₂₇H₁₇N₃O₅S) C, H, N. MP > 260°C.

4-(2-(3-Nitrostyryl)-4-oxo-6-styrylquinazolin-3(4H)-yl)benzoic acid (56)

Compound 56a (0.219 g, 0.57 mmol) was dissolved in a mixture of acetic acid (1.9 mL, 33 mmol, 58 equiv) and acetic anhydride (0.38 mL, 4.0 mmol, 7.0 equiv). To this solution was added meta-nitrobenzaldehyde (0.115 g, 0.76 mmol, 1.33 equiv) and sodium acetate (0.077 g, 0.93 mmol, 1.63 equiv). The mixture was refluxed for 6 hours. The mixture was cooled to room temperature and the resulting yellow solid was collected by filtration and washed with methanol. The crude material was purified using silica gel chromatography (silica cake, ISCO, RediSep 24 g column, 0-10% MeOH/DCM gradient) to give a yellow solid (0.146 g, 50%). ¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 8.17 (d, J=7.8 Hz, 1H), 8.11 (d, J=8.2 Hz, 2H), 8.03 (d, J=15.3 Hz, 1H), 7.97 (s, 1H), 7.85-7.82 (mult, 2H), 7.65 (t, J=8.2 Hz, 2H), 7.52 (d, J=8.6 Hz, 2H), 7.44-7.24 (mult, 3H), 7.37-7.35 (mult, 2H), 6.52 (d, J=15.6 Hz, 1H). HRMS calcd for C₃₁H₂₁N₃O₅, 516.15606 [M+H]⁺; found, 516.14182 [M+H]⁺. Anal. (C₃₁H₂₁N₃O₅) C, H, N. MP > 260 °C

(E)-4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzonitrile (58)

Compound 58 was prepared via procedure C using compound 58a (0.150 g, 0.520 mmol) and meta-nitrobenzaldehyde (0.103 g, 0.690 mmol, 1.33 equiv). The crude material was purified by recrystallization with hexanes and chloroform to yield the title compound as a bright yellow solid (0.112 g, 51%). ¹H NMR (400 MHz, CDCl₃) δ 8.19-8.17 (mult, 2H), 8.00 (d, J=15.7 Hz, 1H), 7.93 (d, J=8.6 Hz, 2H), 7.76 (d, J=9.0 Hz, 1H), 7.66 (d, J=2.7 Hz, 1H), 7.63-7.61 (mult, 1H), 7.54 (d, J=8.6 Hz, 1H), 7.51 (d, J=8.6 Hz, 2H), 7.45 (dd, J₁=9.0 Hz, 1H), 3.95 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 161.8, 159.3, 148.9, 147.2, 142.1, 141.2, 137.3, 137.0, 134.1, 132.9, 130.3, 130.1, 129.6, 125.7, 124.3, 122.8, 122.1, 121.8, 118.0, 114.0, 106.9, 56.1. HRMS calcd for C₂₄H₁₆N₄O₄, 425.12514 [M+H]⁺; found, 425.12473 [M+H]⁺. Anal.(C₂₄H₁₆N₄O₄) C, H, N. MP > 260 °C

(E)-4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzamide (59)

Compound 59 was prepared via procedure C using compound 59a (0.200 g, 0.650 mmol) and meta-nitrobenzaldehyde (0.130 g, 0.860 mmol, 1.33 equiv). The crude material was purified by hot gravity filtration after boiling with methanol and collection of the title compound as the resulting yellow solid (0.162 g, 57%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.24 (s, 1H), 8.16 (d, J=8.6 Hz, 2H), 8.09 (d, J=8.6 Hz, 2H), 7.94 (d, J=15.7 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.77 (d, J=9.8 Hz, 1H), 7.64 (t, J=7.8 Hz, 1H), 7.58 (d, J=8.6 Hz, 2H), 7.54-7.51 (mult, 3H), 6.49 (d, J=15.7 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (100 MHz, , DMSO-d₆) δ 167.1, 160.9, 158.1, 148.6, 148.3, 141.7, 139.4, 136.7, 135.7, 134.9, 133.1, 130.5, 129.2, 129.0, 128.9, 124.5, 123.8, 122.8, 122.2, 121.6, 55.8. HRMS calcd for C₂₄H₁₈N₄O₅, 443.13564; found, 443.13529 [M+H]⁺. Anal.( C₂₄H₁₈N₄O₅) C, H, N. MP > 260 °C.

(E)-methyl 4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoate (60)

Compound 60 was prepared via procedure C using compound 60a (0.200 g, 0.650 mmol) and meta-nitrobenzaldehyde (0.124 g, 0.860 mmol, 1.33 equiv) to yield the title compound as a yellow solid (0.143 g, 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.29 (d, J=8.2 Hz, 2H), 8.15 (d, J=6.7 Hz, 2H), 7.96 (d, J=15.7 Hz, 1H), 7.76 (d, J=9.0 Hz, 1H), 7.67 (d, J=2.7 Hz, 1H), 7.59 (d, J=7.8 Hz, 1H), 7.50 (t, J=7.8 Hz, 1H), 7.45-7.43 (mult, 3H), 6.42 (d, J=15.7 Hz, 1H), 4.01 (s, 3H), 3.95 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.3, 161.9, 159.1, 148.8, 148.3, 142.2, 141.1, 137.2, 136.8, 132.9, 131.6, 131.5, 130.0, 129.5, 129.2, 125.5, 124.1, 122.7, 122.6, 121.9, 106.8, 56.4, 53.1. HRMS calcd for C₂₅H₁₉N₃O₆, 458.13534 [M+H]⁺; found, 458.13473 [M+H]⁺. Anal. (C₂₅H₁₉N₃O₆) C, H, N. MP > 260 °C.

(E)-4-(6-methoxy-2-(3-methoxystyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (61)

Compound 61 was prepared via procedure C using compound 46b (0.200 g, 0.650 mmol) and meta-anisaldehyde (0.104 mL, 0.860 mmol, 1.33 equiv) to yield the title compound as a yellow solid (0.150 g, 54%). ¹H NMR (300 MHz, DMSO-d₆) δ 13.32 (bs, 1H), 8.16 (d, J=8.5 Hz, 2H), 7.78 (d, J=15.2 Hz, 1H), 7.75 (d, J=9.4 Hz, 1H), 7.52-7.49 (mult, 2H), 7.26 (t, J=7.9 Hz, 1H), 6.92 (d, J=8.5 Hz, 3H), 6.28 (d, J=15.5 Hz, 1H), 3.90 (s, 3H), 3.72 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 160.9, 159.5 157.7, 148.8, 141.9, 141.0, 138.0, 136.3, 131.6, 130.6, 130.1, 129.5, 129.0, 124.5, 121.3, 120.2, 119.1, 115.1, 113.5, 106.5, 55.7, 55.1. HRMS calcd for C₂₅H₂₀N₂O₅, 429.14514 [M+H]⁺; found, 429.14404 [M+H]⁺. Anal. (C₂₅H₂₀N₂O₅) C, H, N. MP > 260 °C.

(E)-3-(2-(3-(4-carboxyphenyl)-6-methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzoic acid (62)

Compound 62 was prepared via procedure C using compound 46b (0.200 g, 0.650 mmol) and meta-formylbenzoic acid (0.129 g, 0.860 mmol, 1.33 equiv) to yield the title compound as a yellow solid (0.259 g, 45%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (d, J=8.6 Hz, 2H), 7.89 (mult, 3H), 7.76 (d, J=9.5 Hz, 1H), 7.63 (d, J=8.6 Hz, 2H), 7.53-7.50 (mult, 2H), 7.58 (t, J=7.9 Hz, 1H), 6.37 (d, J=15.6 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.8, 166.7, 160.9, 158.1, 148.7, 141.8, 141.0, 137.2, 135.3, 131.52, 131.49, 130.7, 130.2, 129.5, 129.1, 128.1, 124.5, 121.4, 120.9, 106.5, 55.8. HRMS calcd for C₂₅H₁₈N₂O₆, 443.12444 [M+H]⁺; found, 443.12393 [M+H]⁺. Anal.(C₂₅H₁₈N₂O₆) C, H, N. MP > 260 °C.

(E)-4-(6,7-dimethoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (63)

Compound 63 was prepared via procedure C using compound 63b (0.200 g, 0.59 mmol) and meta-nitrobenzaldehyde (0.118 g, 0.780 mmol, 1.33 equiv). The crude material was purified by hot gravity filtration after boiling with methanol to yield the title compound as a yellow solid (0.165 g, 59%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (bs, 1H), 8.18-8.14 (mult, 4H), 7.94 (d, J=15.2 Hz, 1H), 7.81 (d, J=7.8 Hz, 1H), 7.64-7.60 (mult, 3H), 7.42 (s, 1H), 7.26 (s, 1H), 6.45 (d, J=15.2 Hz, 1H), 3.97 (s, 3H), 3.88 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.1, 167.3, 160.3, 155.0, 149.4, 149.0, 148.3, 143.3, 136.7, 135.6, 133.0, 130.5, 130.3, 123.8, 122.9, 122.1, 114.2, 107.8, 105.8, 56.1, 55.8. HRMS calcd for C₂₅H₁₉N₃O₇, 474.13024 [M+H]⁺; found, 474.13077 [M+H]⁺. Anal.(C₂₅H₁₉N₃O₇·0.25 H₂O) C, H, N. MP > 260 °C.

(E)-4-(2-(3,4-diacetoxystyryl)-6-methoxy-4-oxoquinazolin-3(4H)-yl)benzoic acid (64)

Compound 64 was prepared via procedure C using 46b (0.400 g, 1.29 mmol) and 3,4-dihydroxybenzaldehyde (0.237 g, 1.71 g, 1.33 equiv). The crude material was purified by silica gel chromatography (ISCO, RediSep 40 g column, silica cake, 0-10% MeOH/DCM gradient) to yield the title compound as a yellow solid (0.221 g, 33%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.15 (d, J=8.3 Hz, 2H), 7.91 (d, J=15.6 Hz, 1H), 7.76 (d, J=7.6 Hz, 1H), 7.60 (d, J=8.3 Hz, 2H), 7.52 (d, J=7.3 Hz, 2H), 7.25 (d, J=8.3 Hz, 1H), 6.32 (d, J=15.6 Hz, 1H), 3.90 (s, 3H), 2.27 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.81, 168.76, 161.5, 158.6, 149.4, 143.3, 143.0, 142.5, 141.5, 137.2, 134.4, 132.3, 131.3, 130.0, 129.8, 126.3, 125.1, 124.9, 123.4, 123.3, 122.0, 121.6, 107.2, 56.4, 21.02, 20.98. HRMS calcd for C₂₈H₂₂N₂O₈, 515.1510 [M]⁺; found, 515.14526 [M]⁺. Anal.(C₂₈H₂₂N₂O₈) C, H, N. MP 247-252°C.

(E)-4-(6-iodo-2-(4-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (72)

Compound 72 was prepared via procedure C using compound 72b (0.200 g, 0.490 mmol) and para-nitrobenzaldehyde (0.099 g, 0.65 mmol, 1.3 equiv) to yield the title compound as a yellow solid (0.174 g, 66%). ¹H NMR (400 MHz, DMSOd₆) δ 8.32 (s, 1H), 8.16-8.12 (mult, 4H), 8.05 (s, 1H), 7.98 (d, J=15.6 Hz, 1H), 7.77-7.68 (mult, 3H), 7.61-7.54 (mult, 2H), 6.41 (d, J=15.6 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.6, 159.9, 151.4, 148.2, 146.4, 143.1, 137.0, 136.5, 136.4, 134.6, 133.3, 130.5, 130.1, 129.8, 129.3, 124.0, 122.5, 122.4, 122.1, 91.9.HRMS calcd for C₂₃H₁₄IN₃O₅, 540.00574 [M+H]⁺; found, 540.00559 [M+H]⁺. Anal. (C₂₃H₁₄IN₃O₅) C, H, N. MP > 260 °C.

(E)-3-(6-iodo-2-(4-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (73)

Compound 73 was prepared via procedure C using compound 73b (0.800 g, 1.97 mmol) and para-nitrobenzaldehyde (0.396 g, 2.62 mmol, 1.33 equiv) to yield the title compound as a yellow solid (0.572 g, 54%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.29 (bs, 1H), 8.32 (mult, 1H), 8.16-8.09 (mult, 5H), 7.94 (d, J=15.3 Hz, 1H), 7.77 (s, 2H), 7.65-7.54 (mult, 3H), 6.47 (d, J=15.3 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.8, 158.7, 151.6, 148.7, 147.2, 142.8, 136.7, 136.6, 135.9, 134.3, 133.2, 130.8, 130.6, 129.4, 128.9, 128.8, 123.9, 123.5, 123.4, 122.1, 92.2 HRMS calcd for C₂₃H₁₄IN₃O₅, 540.00574 [M+H]⁺.; found, 540.00548 [M+H]⁺. Anal. (C₂₃H₁₄IN₃O₅) C, H, N. MP > 260 °C.

(E)-3-(6-iodo-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (74)

Compound 74 was prepared via procedure C using compound 73b (0.800 g, 1.97 mmol) and meta-nitrobenzaldehyde (0.396 g, 2.62 mmol, 1.33 equiv) to yield the title compound as a yellow solid (0.626 g, 59%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.16-8.12 (mult, 4H), 8.05 (s, 1H), 7.98 (d, J=15.6 Hz, 1H), 7.77-7.68 (mult, 3H), 7.61-7.54 (mult, 2H), 6.41 (d, J=15.6 Hz, 1H).¹³C NMR (150 MHz, DMSO-d₆) δ 166.6, 159.9, 151.4, 148.2, 146.4, 143.1, 137.0, 136.5, 136.4, 134.6, 133.3, 130.5, 130.1, 129.8, 129.3, 124.0, 122.5, 122.4, 122.1, 91.9.HRMS calcd for C₂₃H₁₄IN₃O₅, 540.00574 [M+H]⁺; found, 540.00546 [M+H]⁺. Anal. (C₂₃H₁₄IN₃O₅), C, H, N. MP > 260 °C.

(E)-4-(6-methoxy-2-(4-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (75)

Compound 75 was prepared via procedure C using compound 46b (0.730 g, 2.35 mmol) and para-nitrobenzaldehyde (0.473 g, 3.13 mmol, 1.3 equiv) to yield the title compound as a yellow solid (0.559 g, 54%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.32 (bs, 1H), 8.17 (t, J=8.8 Hz, 4H), 7.92 (d, J=15.3, 1H), 7.78 (dd, J₁=10 Hz, J₂=4 Hz, 1H), 7.68 (d, J=8.9 Hz, 1H), 7.63 (d, J=8.2 Hz, 1H) 7.58-7.52 (mult, 2H), 6.51 (d, J=15.7 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 158.4, 148.3, 147.4, 141.7, 141.3, 140.7, 135.7, 130.7, 129.4, 128.6, 124.5, 124.2, 106.6, 55.8. HRMS calc for C₂₄H₁₇N₂O₆, 444.11964 [M+H]⁺; found, 444.11978 [M+H]⁺. Anal.(C₂₄H₁₇N₂O₆) C, H, N. MP > 260 °C.

(E)-3-(6-methoxy-2-(4-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (76)

Compound 76 was prepared via procedure C using compound 76a (0.730 g, 2.35 mmol) and para-nitrobenzaldehyde (0.473 g, 3.13 mmol, 1.3 equiv) to yield the title compound as a yellow solid (0.728g, 70%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.34 (bs, 1H), 8.13-8.18 (mult, 3H), 8.04 (s, 1H), 7.91 (d, J=15.7 Hz, 1H), 7.74-7.79 (mult, 3H), 7.65 (d, J=9.0 Hz, 2H), 7.51-7.54 (mult, 2H), 6.50 (d, J=15.7 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.5, 161, 158.2, 148.6, 147.4, 141.7, 141.4, 137.1, 135.5, 133.4, 132.5, 130.1, 130, 129.2, 128.5, 124.4, 124.2, 124.2, 121.7, 106.6, 55.8. HRMS calcd for C₂₄H₁₇N₂O₆, 444.11964 [M+H]⁺; found, 444.11978 [M+H]⁺. Anal.(C₂₄H₁₇N₂O₆) C, H, N. MP > 260 °C.

(E)-3-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)benzoic acid (77)

Compound 77 was prepared via procedure C using compound 76a (0.194 g, 0.626 mmol) and meta-nitrobenzaldehyde (0.126 g, 0.833 mmol, 1.3 equiv.) to yield the title compound as a yellow solid (0.087g, 33%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.32 (bs, 1H), 8.21 (s, 1H), 8.13-8.15 (mult, 2H), 8.03 (s, 1H), 7.92 (d, J=15.7 Hz, 1H), 7.82 (d, J=7.8 Hz, 1H), 7.73-7.77 (mult, 3H), 7.61 (t, J=8.0 Hz, 1H), 7.51-7.53 (mult, 2H), 6.47 (d, J=15.7 Hz, 1H), 3.89 (s, 3H). (100 MHz, DMSO-d₆) δ 166.6, 161.0, 158.1, 148.7, 148.3, 141.7, 137.2, 136.8, 135.7, 133.5, 133.2, 132.4, 130.5, 130.1, 130, 129.2, 124.4, 123.8, 122.9, 122.1, 121.6, 106.6, 55.8. HRMS calcd for C₂₄H₁₇N₂O₆, 444.11964 [M+H]⁺; found, 444.11957 [M+H]⁺. Anal.(C₂₄H₁₇N₂O₆) C, H, N. MP > 260 °C.

4-(6-Iodo-2-methyl-4-oxoquinazolin-3(4H)-yl)benzoic acid (78)

Compound 78 was prepared via procedure B using compound 78a (2.00 g, 7.00 mmol) and para-aminobenzoic acid (1.15 g, 8.40 mmol, 1.20 equiv). The crude material was recrystallized with ethyl acetate and methanol to give an off-white solid (1.27 g, 45%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.27 (bs, 1H), 8.36 (d, J=2.0 Hz, 1H), 8.15-8.11 (mult, 3H), 7.61 (d, J=8.5 Hz, 2H), 7.47 (d, J=8.5 Hz, 1H), 2.11 (s, 3H).¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 160.0, 1547, 146.6, 143.0, 141.5, 134.5, 131.5, 130.7, 128.9, 122.3, 91.3, 24.2. HRMS calcd for C₁₆H₁₁IN₂O₃, 406.98934 [M+H]⁺; found, 406.98893 [M+H]⁺.

(E)-4-(6-Iodo-4-oxo-2-styrylquinazolin-3(4H)-yl)benzoic acid (79)

Compound 79 was prepared via procedure C using compound 45b (0.300 g, 0.740 mol) and benzaldehyde (0.100 mL, 0.980 mmol, 1.33 equiv). The crude material was purified by hot gravity filtration of the title compound as a yellow solid after boiling in methanol (0.070 g, 19%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (d, J=1.9 Hz, 1H), 8.19-8.15 (mult, 3H), 7.92 (d, J=15.5 Hz, 1H), 7.62-7.57 (mult, 3H), 7.39-7.35 (mult, 5H), 6.30 (d, J=15.5 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.8, 160.0, 151.6, 146.7, 143.2, 140.4, 139.7, 134.6, 132.3, 130.7, 130.0,129.3, 129.2, 129.0, 127.7, 122.4, 119.5, 114.0, 91.5. HRMS calcd for C₂₃H₁₅IN₂O₃, 495.02064; found, 495.01987 [M+H]⁺. Anal.(C₂₃H₁₅IN₂O₃) C, H, N. MP > 260 °C.

4-(6-Methyl-4-oxo-2-phenethylquinazolin-3(4H)-yl)benzoic acid (81)

Compound 80 (0.100 g, 0.26 mmol) was dissolved in DMF (5.0 mL). Palladium on carbon (10%, 0.10 g, 10 wt %) was added. A balloon filled with hydrogen was added and the mixture was stirred at room temperature for 1 hour. The mixture was filtered over a pad of celite, and the resulting solution was concentrated in vacuo to give a white solid. The solid was triturated with DCM and obtained by filtration (0.016 g, 16%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.27 (bs, 1H), 8.09 (d, J=8.2 Hz, 2H), 7.93 (s, 1H), 7.71 (d, J=8.6 Hz, 1H), 7.65 (d, J=8.2 Hz, 1H), 7.54 (d, J=8.1 Hz, 2H), 7.22 (t, J=7.4 Hz, 2H), 7.14 (t, J=7.0 Hz, 1H), 7.06 (d, J=7.0 Hz, 2H), 2.98 (t, J=7.4 Hz, 2H), 2.58 (t, J=7.4 Hz, 2H), 2.47 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 161.2, 154.6, 145.1, 141.3, 140.8, 136.4, 136.1, 131.5, 130.5, 129.2, 128.4, 128.2, 126.9, 126.1, 125.7, 120.2, 37.1, 32.0, 20.8. HRMS calcd for C₂₄H₂₀N₂O₃, 385.15534 [M+H]⁺; found, 385.15459 [M+H]⁺. Anal.(C₂₄H₂₀N₂O₃) C: 72.02, H: 5.03, N: 7.19. MP > 260 °C

(E)-4-(6-iodo-4-oxo-2-(2-(pyridin-3-yl)vinyl)quinazolin-3(4H)-yl)benzoic acid (98)

Compound 98 was prepared via procedure C using compound 72b (0.240 g, 0.591 mmol) and nicotinaldehyde (0.084 g, 0.074 mL, 0.786 mmol, 1.33 equiv). The reaction mixture was filtered and washed with MeOH to give the title compound as a yellow solid (0.170 g, 58%). %). ¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (bs, 1H), 8.67 (d, J=2.4 Hz, 1H), 8.52 (dd, J₁=4.7 Hz, J₂=1.6 Hz, 1H), 8.41 (d, J=2.0 Hz, 1H), 8.19 (dd, J₁=8.4 Hz, J₂=8.4 Hz, 1H), 8.15 (d, J=8.6 Hz, 2H), 7.93 (d, J=15.7 Hz, 1H), 7.79 (d, J=8.2 Hz, 1H), 7.58-7.63 (mult, 3H), 7.37 (dd, J₁=8.0 Hz, J₂=4.9 Hz, 1H), 6.43 (d, J=15.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 159.9, 151.3, 149.5, 146.6, 143.3, 140.5, 136.3, 133.9, 131.7, 130.7, 130.5, 129.3, 124.1, 122.5, 121.6, 91.9. HRMS calcd for C₂₂H₁₄IN₃O₃,494.00006 [M-H]⁺; found, 494.00150 [M-H]⁺. Anal. (C₂₂H₁₄IN₃O₃) C, H, N. MP > 260 °C.

(E)-3-(6-iodo-4-oxo-2-(2-(pyridin-3-yl)vinyl)quinazolin-3(4H)-yl)benzoic acid (99)

Compound 99 was prepared via procedure C using compound 73b (0.300 g, 0.739 mmol) and nicotinaldehyde (0.105 g, 0.092 mL, 0.982 mmol, 1.3 equiv.) The reaction mixture was recrystallized from ethyl acetate and hexanes to give the title compound as a yellow solid (0.111 g, 30%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (bs, 1H), 8.65 (s, 1H), 8.51 (d, J=3.9 Hz, 1H), 8.39 (s, 1H), 8.16 (t, J=9.4 Hz, 2H), 8.05 (s, 1H), 7.92 (d, J=15.7 Hz, 1H), 7.74-7.78 (mult, 3H), 7.58 (d, J=8.6 Hz, 1H), 7.34-7.37 (mult, 1H), 6.42 (d, J=15.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.5, 160.1, 151.6, 150.5, 149.5, 146.6, 143.2, 136.9, 136.1, 134.7, 133.9, 133.4, 132.4, 130.5, 130.2, 130.1, 129.9, 129.4, 124.1, 122.6, 121.7, 91.8. HRMS calcd for C₂₂H₁₄IN₃O₃ 494.00006 [M-H]⁺; found, 494.00165 [M-H]⁺. Anal. (C₂₂H₁₄IN₃O₃) C: 45.18, H: 2.23, N: 7.16. MP > 260 °C.

ABBREVIATIONS

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

BAPTA-AM

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester

cDNA

complementary DNA

DMSO

dimethyl sulfoxide

HPLC

high performance liquid chromatography

IC₅₀

concentration of a test compound that produces half maximal inhibition

K-BAPTA

potassium 1,2-bis(oaminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

NMDA

N-methyl-D-aspartate

NMR

nuclear magnetic resonance