Acid-Catalyzed Synthesis of Isatoic Anhydride-8-Secondary Amides Enables IASA Transformations for Medicinal Chemistry

Abstract

Quinazolin-dione-N-3-alklyl derivatives are the core scaffolds for several categories of bioactive small molecules, but current synthetic methods are costly, involve environmental hazards, and are not uniformly scalable. Here, we report an inexpensive, flexible, and scalable method for the one-pot synthesis of substituted quinazolin-dione-N-3-alkyls (isomers of isatoic-8-secondary amides (IASAs)) from isatin that take advantage of in situ capture of imidic acid under acidic conditions. We further show that this method can be used for the synthesis of a wide variety of derivatives with medicinal uses.

Graphical Abstract

INTRODUCTION

Quinazolinedione-N3-alkyl and 2-substituted quinazolines are heterocyclic derivatives that occur in a wide range of natural products and are incorporated into pharmaceutical agents for the treatment of infection, epilepsy, immune disorders, diabetes, and cancer. Specific targets include sirtuins,¹ β-secretase,² hepatitis C virus (NS5B polymerase),³ B-Raf,⁴ phosphoinositide3-kinase (PI3K),⁵ and poly(ADP-ribose) glycohydrolase (PARG)⁶ (Figure 1). Synthetic methods to achieve quinazoline structures include starting from isatoic anhydride⁷ and cyclization of anthranilic acid using chloroformate,^8–10 phosgene,^11,12 CDI,¹³ phosphoradiate,¹⁴ or isocyanate,¹⁵ or urea at high temperatures.¹⁶ It can also be achieved by N3 alkylation of quinazolinone using halo-anilines,¹⁷ triflate,¹⁸ thionitrile,¹⁹ or boronic acid.²⁰ However, these methods do not allow functionalization of early intermediates, resulting in the need to functionalize in later stages of synthesis through protection and deprotection strategies.^21–27

Figure 1.

Structures of quinazolines containing pharmaceutical agents.

Here, we describe the flexible chemistry for generation of novel isatoic anhydride-8-sec amides (Scheme 1), which can be used to achieve diverse products, such as quinazoline-N3 alkyl-8-acid or amides, 2-substituted quinazoline-8-amides, and 2,4-disubstituted quinazoline-8-amides. This work builds upon our prior studies showing that 8-substituted quinazoline could be synthesized by utilizing isatoic anhydride-8-amide (IASA).²⁸ However, this work is different because we present the direct preparation of novel isatoic anhydride-8-secondary amides in the presence of secondary alcohol. Subsequently, this method gives sufficiently pure products of IASA to enable several new quinazoline derivatives. These reactions require fewer precautions than prior methods, are redox-stable, scalable, and inexpensive.

Scheme 1.

Synthesis of Isatoic Anhydride-8-Secondary Amide (IASA)

RESULT AND DISCUSSION

We embarked on this line of work with the goal of obtaining isatoic anhydride-8-secondary amides (IASA). First, we attempted using excess amine,²⁹ but this gave multiple products (Figure S1). In another approach, we treated IAA with nitrous acid³⁰ to convert it to isatoic anhydride-8-acid.^31,32 However, again multiple products formed (Figure S1). We considered our prior experience showing that isatin-7-acid could be cleaved by sodium azide in an acidic medium and hypothesized that doing this reaction in the presence of alcohols could yield the desired secondary amide (Scheme 1).

To test this hypothesis, we prepared IAA in an acidic medium and added primary alcohols to generate the carbonium ion in situ. This yielded the corresponding 2-amino isophthalamic acid esters³³ in a good yield. The time course of the reaction, tracked by NMR, shows that IAA is first formed, followed by hydrolysis to an 2-amino isophthalamic acid 2, followed by esterification to yield a 2-amino isophthalamic acid ester as the final product 2a (80%) and 2b (83%) (Figure S2A–C). When a secondary alcohol, such as isopropyl alcohol, was used, we obtained isatoic anhydride-8-isopropyl amide 3a (Scheme 2), as expected. The reaction was clean and easily worked up using filtration to obtain spectrally pure products (Figure S3). We speculated that the reaction is driven by the stability of the carbonium ion, with primary carbonium ions being less stable than secondary ones.

Scheme 2.

Ring Expansion of Isatin: Cyclization toward C-7 Position Generation of IASA

To further test this hypothesis, we carried out reactions using several diversified secondary alcohols and obtained products in 70–88% yield (3b–3h). For branched-chain alcohols, such as 2-butanol, 3-pentanol, 2-hexanol, and 4-heptanol, the products 3b–3e were isolated. Selectivity was observed for cyclic alkanols, such as cyclopentanol and cyclohexanol. In those cases, the addition of imidic acid to cyclic-sec-carbonium ion occurred from one side only, yielding single isomers, 3f–3h (Scheme 1). Further, the reaction of 5-bromoisatin with cyclopentyl alcohol gave 3h with 70% yield, demonstrating that substitutions at the 5th position of isatin did not affect the formation of IASA. Since phenyl diazonium salt can give an aryl secondary carbocation intermediate, we attempted to synthesize IAA-phenyl amide but isolated IAA (3i) exclusively.

We next turned to an investigation of adding tertiary butyl alcohols. We obtained multiple products, which we suspected arose from the trace of water in the tert-butyl starting materials. We repeated the reaction with another tertiary alcohol, dihydromyrcenol, but results were the same. The results confirm prior literature reports,³⁴ that tertiary alcohols in the presence of HN₃ and sulfuric acid lead to the formation of enamines, which can be hydrolyzed to ketones in an acidic medium. We further explored the effect of substituting other positions, focusing on those that would be predicted to be involved in the mechanism. Use of isatin-7-methyl ester instead of secondary amide led to the formation of the primary amide (2a), presumably because of the spontaneous conversion of nitrile to amide. To understand the function of the seventh position, we attempted reactions with N-alkyl substituted isatin-7-acid or 7 esters (2d and 2e). Nitrile was isolated,³⁵ suggesting that the seventh position acid or N-alkyl prevents nitrile hydrolysis. With N-propanoic acid isatin³⁶ (2f) or 7-fluoro isatin³⁷ (2g), the corresponding primary amides were obtained (Figure S4).

We previously demonstrated that placing an acid at the 4th position of isatin can yield phthalimides.²⁸ We anticipated that doing the same experiments in the presence of alcohols would lead to the formation of N-alkyl phthalimide, but obtained phthalimide³⁸ exclusively (Scheme 3A, 4a). This suggests that in situ-generated imidic acid prefers an intramolecular nucleophilic substitution over an intermolecular reaction with the carbonium ion. The presence of carboxylic acid at the 5th position (Scheme 3B, 4b) gave only the primary amide (4-amino isophthalamic acid), suggesting that in this scenario, the nitrile is hydrolyzed before it can react with the carbonium ion.

Scheme 3.

Reaction of C-4 and C-5 Substituted Isatin: (A) Formation of Amino Phthalimide; and (B) Formation of Isophthalamic Acid

Mechanism of IASA and Phthalimide Formation.

The above reactions establish two modes of cyclization that depend on the location of the acid in isatin. When the acid, 1a, is in the 7th position, rightward cyclization proceeds by the azide ion attacking the C-3 keto of isatin (Figure 2a, step-2), leading to the elimination of the nitrogen through cleavage (Figure 2a, step-3) and resulting in in situ generation of imidic acid (Figure 2a, step-4), which is in equilibrium with nitrile. Having an acid at the 7th position favors the nitrile, which is slowly hydrolyzed to the secondary amide by trapping stable secondary carbonium ions, whereas having an ester or fluoro at the 7th position (1g) of isatin favors the imidic acid at equilibrium. This scenario leads to instantaneous hydrolysis, giving a primary amide without reacting with the sec-carbonium ion. In the case of isatin-4-ester 1h, the alternative mode of cyclization results, where a leaving group (COOR) is placed in the 4th position. In this scenario, in situ generation of imidic acid (Figure 2b, step-4) leads to intramolecular nucleophilic substitution at the 4th position of isatin, giving the phthalimide 4a and the carbonium ion from alcohols (primary, secondary) that are not trapped by intermolecular imidic acid.

Figure 2.

Plausible mechanism of isatoic anhydride-8-secondary amide (3a) and phthalimide formation (4a).

Facile Synthesis of Clinically Useful Compounds Using IASA.

The real-world utility of IASA described above is illustrated using our method to achieve simple synthetic schemes for numerous published small-molecular inhibitors in high yield (Figure 1). These schemes are substantially more straightforward and environmentally friendly than reported methods. First, IASA can be easily converted to another isomeric set (having the same molecular formula but differing in arrangement) of quinazoline-N3-alkyl compounds that are intermediates in the synthesis of adenosine uptake inhibitors,^39,40 such as prazosin-4-substituted functionalized derivatives (Figures S5 and S6). We developed three efficient protocols. First, exposure to basic pH, followed by acidification for isolation purposes, gives the product 5a in the pure form in quantitative yields (Table 1, entry 1). Second, treating the compound with tertiary butoxide in DMSO gives an analogous species (Table 1, entry 2). This is also isolated using an acid workup. Third, simply heating at 100 °C for 1 h in DMSO can yield a range of products (Table 1, entries 3–8).

Table 1.

Optimization of the Reaction Condition for the Synthesis of Quinazolin-dione 3-N-Alkyl Derivatives

entry	R1	R2	reagent	temp (°C)	time (h)	yield (%)
1	H	isopropyl	NaOH, pH 12	RT	0.5	90, 5a
2	H	isobutyl	t-BuOK	RT	3	93, 5b
3	H	2-hexyl		100	3	89, 5c
4	H	4-heptyl		100	3	91, 5d
5	H	cyclopentyl		100	3	93, 5e
6	H	cyclohexyl		100	3	92, 5f
7	Br	cyclopentyl		100	3	94, 5g
8	H	H		100	3	94, 5h

Second, IASA enables a simple one-pot synthesis of quinazolin-dione-3-N-alkyl-8-sec-amide and related compounds. These compounds have shown activity against hepatitis C.²⁴ Similar to the reactions shown in Table 1, treatment of 3d with HBTU and isopropylamine yields the quinaozline-N3 hexyl-8-isopropyl amide (6) in 89% yield (Figure 3, Part-A).

Figure 3.

Conversion of IAA-sec-amide to useful intermediates.

Third, quinazolin-dione-2-alkyl-4,8-di-amides have shown activity against BRAF kinase,²⁵ sirtuin modulators (Figure S7), β-secretase (Figures S8–S11), and PI3K²⁶ and p70S6K.³⁸ An example synthetic scheme for this class of compound is shown in Figure S9, where functionalized 2-alkyl quinazoline-8-amide⁴¹ is synthesized by treating the 3c with benzimidine at 42 °C for 6 h. Upon execution of this reaction, an aqueous workup and acidification yielded the pure product⁴² 7 in 90% yield (Figure 3, Part-B).

As another example, quinazolin-dione-3-N-alkyl-8-amide has shown activity against PARG²⁷ (Figures S12 and S13). To generate this compound, the quinazoline-N-primary alkyl is synthesized by exchanging its N-secondary alkyl counterpart. In a one-pot reaction, 3a was treated with ammonium carbonate in DMSO at 40 °C to obtain the open-chain unsymmetrical amide.⁴³ Treatment of phenyl thio isocyanate in the presence of pyridine allowed isolation of quianzoline-3-phenyl-8-amide (8)⁴⁴ in 85% yield (Figure 3, Part-C). This example shows that cyclization is preferred in the case of a secondary amine (elimination) over the primary amide. This method is useful for reactions where the N3-primary alkyl or phenyl group cannot be synthesized from the corresponding isatoic anhydride-8-sec-amide.

Finally, quinazolin-dione-3-N-alkyl-8-acids have shown activity as sirtuin modulators²² and PARG inhibitors.²⁷ These can be generated by exchanging the N-3H-quinazolidinone-8-acid. In an exemplary reaction, quinazolin-dione-8-acid (5h)⁴⁵ was treated with 1 equiv of N-t-butyl-2-amino-benzimidazole in DMSO and heated.⁴⁵ This yielded the quinazolin-dione-N3-allkyl-8-acid (9) in 93% yield (Figure 4). This shows that cyclization prefers the secondary amide (elimination) over the acid, whereas N-3-alkyl quinazoline (5a) is unreactive under similar conditions. This reaction enables the synthesis of quinazolin-dione-N3-primary alkyl or phenyl acid derivatives,⁴⁶ which cannot be synthesized through IAA using the primary alcohol or the phenyl diazonium salt, respectively.

Figure 4.

Synthesis of quinazolin-dione-3-N-alkyl-8-acid.

Synthesis of Open-Chain Compounds from Isatoic Anhydride-8-sec-Amide.

It is also possible to convert the IASA into open-chain compounds (Table 2, entries 1 and 2). This can be accomplished by treatment with a base (Table 2, entries 3–5) or by heating at 50 °C with a catalytic amount of sulfuric acid (Table 2, entry 6). This considerably simplifies the process that would typically require solid-phase synthesis or use of protection and deprotection steps⁴⁷ for the synthesis of 3-hydroxyquinoline (Figures S16 and S17). A range of trifunctional anilines with secondary amides is feasible with these methods 10a–10f (Table 2, entries 1–6). Corresponding esters such as 11 (Table 2, entry 7) can also be prepared by treating with K₂CO₃ in DMSO⁴⁸ with alcohol.

Table 2.

Optimization of the Reaction Condition for the Synthesis of Various Substituted Anilines

entry	condition	R1	R2	R3	yield (%)
1	NaN3/NaOH, pH 8	isopropyl	H	COOH	80, 10a
2	NaN3/NaOH, pH 8	isobutyl	H	COOH	88, 10b
3	NaOH, pH 8	4-heptyl	H	COOH	80, 10c
4	NaOH, pH 8	cyclopentyl	H	COOH	88, 10d
5	NaOH, pH 8	cyclohexyl	H	COOH	73, 10e
6	H2SO4, 50 °C	cyclopentyl	Br	COOH	91, 10f
7	isopropanol, K2CO3	isopropyl	H	CO2 isopyl	78, 11
8	(NH4)2CO3, 50 °C	isopropyl	H	CONH2	88, 12
9	1.1 equiv Br2/H+	cyclopentyl	Br	COOH	93, 10f
10	2.2 equiv Br2/H+	isopropyl	Br	Br	91, 13

With our method, IASA provides an avenue for generation of hetero halide derivatives of anthranilamides, which have shown activity in fatty liver disease.^49,50 To obtain the nonsymmetrical amide 12, we heated in DMSO at 50 °C for 3 h (Table 2, entry 8) and then isolated the pure product at pH 3. 5-Bromo substituted anilines, 10f (Table 2, entry 9), can be obtained by IAA ring-opening. Mono bromination occurs with 1 equiv of bromine in sulfuric acid, whereas 2 equiv of bromine gives the dibrominated anthranilamide, 13 (Table 2, entry 10), and accompanying decarboxylation.⁵¹ The substituted aniline derivatives, 10a–10f, can undergo Sandmeyer reactions to give diversified intermediates, many of which act as active pharmaceutical intermediates to medicinal compounds.

In summary, these methods enable conversion of IASA to another isomeric set of fully substituted quinazoline-3-N-alkyl derivatives, including medicinal compounds. These reactions are highly scalable and facile. Although we have shown that this method can generate several classes of biologically active molecules, the full potential of this chemistry is yet to be explored. This simplified synthetic approach will enable further development of these scaffold classes, leading to the discovery of additional medicinal uses.

EXPERIMENTAL SECTION

Chemicals.

Starting materials, reagents, and solvents were purchased from commercial sources and used as received, unless stated otherwise. Isatin derivatives (1a–1i) were purchased from Enamine, a Sigma-Aldrich partner, and 2 series compounds have already been reported by us and used for the current study.²⁸ Melting points were determined using the μTherm°Cal₁₀ (Analab Scientific Pvt. Ltd.) melting point apparatus and are not corrected. Reaction progress was monitored by thin-layer chromatography on Merck’s silica plates. ¹H and ¹³C NMR spectra were recorded on Varian 400 MHz instruments using TMS as the internal standard. Mass spectrometry data were recorded on Shimadzu LCMS 2010 and ESI-QTOF mass spectrometers.

General Procedure for Isatin Derivatives Opening in the Presence of Alcohols.

First, 0.5 mmol of isatin derivatives, 40 mg (1.2 mmol) of NaN₃, and 2.0 mL of concentrated sulfuric acid were stirred until effervescence diminished (5–10 min); then, 2.0 equiv (1.0 mmol) of alcohol was added and stirred for 4 h at room temperature. This was poured into water, and a precipitate formed (in case the product is solid), it was filtered and allowed to dry overnight. If a precipitate is not formed, the product was extracted with diethyl ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator, yielding the product with 70–88% yield.

Methyl 2-Amino-3-carbamoylbenzoate⁵² (2a): Method A Following the General Procedure.

78 mg of yellow amorphous solid product, 80% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 8.02 (s, 2H, NH₂), 7.93 (s, 1H, NH), 7.89 (dd, J = 7.9, 1.6 Hz, 1H), 7.79 (dd, J = 7.7, 1.6 Hz, 1H), 7.34 (s, 1H, NH), 6.55 (t, J = 7.8 Hz, 1H), 3.78 (s, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 171.2, 168.0, 152.0, 135.1, 135.0, 116.6, 113.7, 110.9, 52.1. LC-MS (ESI) m/z: [M − H]⁺ calcd for C₉H₁₁N₂O₃ 195.1; found 195.2.

Method B: From Methyl 2,3-Dioxoindoline-7-carboxylate.

First, 102 mg (0.5 mmol) of methyl 2,3-dioxoindoline-7-carboxylate, 79 mg (0.6 mmol) of NaN₃, and 2.5 mL of concentrated sulfuric acid were stirred; after efferevescene eased (5 min), 2.0 equiv of alcohol was added and stirred for 4 h at room temperature. The product was extracted with ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator, yielding 90 mg of a yellow amorphous solid product in 93% yield.

Ethyl 2-Amino-3-carbamoylbenzoate³³ (2b).

87 mg, pale-yellow amorphous solid, 83% yield. ¹H NMR (400 MHz, DMSO-d_b): δ 8.01 (s, 2H), 7.92 (s, 1H),7.89 (dt, J = 8.1, 1.3 Hz, 1H), 7.78 (dt, J = 7.8, 1.3 Hz, 1H), 7.32 (s, 1H), 6.55 (td, J = 7.8, 1.0 Hz, 1H), 4.25 (qd, J = 7.1, 1.0 Hz, 2H), 1.29 (td, J = 7.1, 1.0 Hz, 3H). ¹H NMR (400 MHz, chloroform-d): δ 8.05 (dd, J = 7.9, 1.6 Hz, 1H), 8.00 (s, 2H, NH2), 7.55 (dd, J = 7.7, 1.6 Hz, 1H), 6.54 (t, J = 7.8 Hz, 1H), 5.83 (s, 2H, NH2), 4.33 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 171.2, 167.6, 152.0, 135.0, 116.6, 113.7, 111.1, 60.7, 14.6. ¹³C{H}NMR (101 MHz, chloroform-d): δ 171.3, 167.7, 152.3, 136.1, 133.4, 115.1, 113.4, 112.3, 60.6, 14.3. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₀H₁₃N₂O₃ m/z 209.0920; found 209.0926.

3-Cyano-2-(methylamino)benzoic Acid³⁵ (2d).

The product was extracted with ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator, yielding 65 mg of the product as a brown amorphous solid, 73% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 8.59 (s, 1H, NH), 8.04 (dd, J = 7.8, 1.7 Hz, 1H), 7.71 (dd, J = 7.7, 1.7 Hz, 1H), 6.66 (t, J = 7.7 Hz, 1H), 3.21 (s, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 169.6, 153.6, 141.8, 137.4, 120.4, 115.5, 113.7, 94.2, 32.3. LC-MS (ESI) m/z: [M − H]⁺ calcd for C₉H₇N₂sO₂ 175.1; found 175.1.

Methyl 3-Cyano-2-(methylamino)-benzoate²⁸ (2e).

The product was extracted with ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator, yielding 70 mg of the product as a yellow amorphous solid, 78% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 8.25 (q, J = 5.4 Hz, 1H, NH), 8.01 (dd, J = 7.9, 1.7 Hz, 1H), 7.73 (dd, J = 7.7, 1.7 Hz, 1H), 6.67 (t, J = 7.8 Hz, 1H), 3.80 (s, 3H), 3.21 (d, J = 5.4 Hz, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 167.7, 153.1, 142.1, 137.0, 120.2, 115.6, 113.0, 94.6, 52.7, 32.4. LC-MS (ESI) m/z: [M + H]⁺ calcd for C₁₀H₁₁N₂O₂ 191.1; found 191.1.

3-((2-Carbamoylphenyl)amino)-propanoic Acid³⁶ (2f).

It was poured into water to form a precipitate. This was collected by filtration, washed with water, and dried to obtain 94 mg of the product as a yellow amorphous solid, 90% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 8.13 (t, J = 5.7 Hz, 1H), 7.78 (s, 1H, NH), 7.56 (dd, J = 7.9, 1.6 Hz, 1H), 7.25 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.10 (s, 1H, NH), 6.66. ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 173.6, 172.0, 149.6, 133.3, 129.5, 114.8, 114.4, 111.4, 38.3, 34.0. LC-MS (ESI) m/z: [M − H]⁺ calcd for C₁₀H₁₁N₂O₃ 207.0; found 207.1.⁵³

2-Amino-3-fluorobenzamide³⁷ (2g).

The product was extracted with ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator, yielding 72 mg of the product as a brown amorphous solid, 93% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 7.85 (s, 1H, NH), 7.36 (dd, J = 8.0, 1.4 Hz, 1H), 7.21 (s, 1H, NH), 7.16–7.06 (m, 1H), 6.48 (tdd, J = 8.0, 5.1, 1.0 Hz, 1H), 6.41 (s, 2H, NH2), ¹³C{1H} NMR (101 MHz, DMSO-d₆): δ 170.9 (d, J = 3.2 Hz), 151.6 (d, J = 237.5 Hz), 139.0 (d, J = 14.2 Hz), 124.7 (d, J = 3.1 Hz), 117.4 (d, J = 18.1 Hz), 116.5 (d, J = 4.6 Hz), 114.2 (d, J = 7.3 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆): δ −134.6, −134.6, −134.7, −134.7. LC-MS (ESI) m/z: [M + H]⁺ calcd for C₇H₈FN₂O 155.0; found 155.0.

2,4-Dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxylic Acid²⁸ (3).

First, 103 mg of IAA was suspended in 2 mL of sulfuric acid and 3.0 equiv of sodium nitrite was added and stirred at room temperature for 3 h. It was poured into water and extracted with diethyl ether, and the organic layers were combined and concentrated to get 93 mg of the essentially pure product as a yellow amorphous solid in 90% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 11.04 (s, 1H, NH), 8.32 (dd, J = 7.8, 1.6 Hz, 1H), 8.25–8.12 (m, 1H), 7.35 (t, J = 7.8 Hz, 1H). ¹H NMR (400 MHz, acetic acid-d₄): δ 8.29 (dt, J = 7.9, 1.5 Hz, 1H), 8.25 (dd, J = 7.9, 1.4 Hz, 1H), 7.39–7.32 (m, 1H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 168.6, 159.4, 146.4, 142.4, 138.8, 134.9, 123.5, 114.7, 112.3.

N-Isopropyl-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3a).

First, 1.0 g (5.5 mmol, 1.0 equiv) of 2,3-dioxoindoline-7-carboxylic acid (1a) was added to a 50 mL round-bottom flask equipped with a gas bubbler (the other end of the bubbler was dipped in a flask containing 100 mL of 1 N NaOH solution). The reaction mixture was cooled to 0 °C and spiked with 10 mL of concentrated sulfuric acid. After 10 min, 440 mg (6.3 mmol, 1.2 equiv) of sodium azide was added gradually over a period of 10 min; then, 2.0 equiv (11.0 mmol) of alcohol was added and stirred at 0 °C to RT for 1 h and left at room temperature for 2 h. The reaction was then combined with 100 mL of cold water to form a precipitate. The precipitate was recovered using a Buckner flask and dried at room temperature to obtain 1.06 g of the product (81.7%) as a pale-yellow amorphous solid in 80% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 11.85 (s, 1H, NH), 8.80 (d, J = 7.6 Hz, 1H, NH), 8.24 (dd, J = 7.9, 1.4 Hz, 1H), 8.08 (dd, J = 8.0, 1.3 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 4.11 (dq, J = 13.5, 6.7 Hz, 1H), 1.17 (d, J = 6.6 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 165.9, 159.7, 146.4, 141.6, 135.6, 132.9, 123.0, 117.4, 112.2, 41.8, 22.4. ¹H NMR (400 MHz, acetic acid-d₄): δ 8.21 (dd, J = 7.8, 1.4 Hz, 1H), 8.15 (dd, J = 7.8, 1.4 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 4.29 (h, J = 6.6 Hz, 1H), 1.28 (d, J = 6.6 Hz, 6H). HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₂H₁₃N₂O₄ m/z 249.0869; found 249.0880.

N-(sec-Butyl)-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3b).

We obtained 112 mg of the product as a pale-yellow amorphous solid in 85% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 11.84 (s, 1H, NH), 8.74 (d, J = 8.1 Hz, 1H, NH), 8.27 (dd, J = 7.9, 1.4 Hz, 1H), 8.09 (dd, J = 7.9, 1.3 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 3.94 (dh, J = 13.9, 6.8 Hz, 1H), 1.63–1.42 (m, 2H), 1.15 (d, J = 6.6 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 166.2, 159.7, 146.4, 141.6, 135.6, 132.9, 123.0, 117.4, 112.3, 47.2, 29.0, 20.3, 11.1. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₃H₁₅N₂O₄ m/z 263.1026; found 263.1027.

2,4-Dioxo-N-(pentan-3-yl)-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3c).

We obtained 114 mg of the product as a yellow amorphous solid in 83% yield. ¹H NMR (400 MHz, chloroform-d): δ 11.63 (d, J = 6.3 Hz, 1H, NH), 8.24 (dd, J = 8.0, 3.3 Hz, 1H), 7.86 (t, J = 7.8 Hz, 1H), 7.29 (dt, J = 8.2, 4.1 Hz, 1H), 6.12 (dd, J = 30.2, 8.7 Hz, 1H, NH), 4.32–3.91 (m, 1H), 1.55 (td, J = 7.6, 6.9, 4.5 Hz, 2H), 1.42 (p, J = 7.5 Hz, 1H), 1.27 (d, J = 6.6 Hz, 3H), 0.97 (td, J = 7.3, 4.2 Hz, 4H). ¹H NMR (400 MHz, DMSO-d₆): δ 11.85 (s, 1H, NH), 8.69 (dd, J = 40.2, 8.4 Hz, 1H, NH), 8.29 (dd, J = 17.0, 7.8 Hz, 1H), 8.10 (dd, J = 7.8, 3.1 Hz, 1H), 7.35 (td, J = 7.8, 5.1 Hz, 1H), 4.16–3.70 (m, 1H), 1.65–1.35 (m, 2H), 1.35–0.89 (m, 2H), 0.85 (dt, J = 12.1, 6.0 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 166.7, 166.1, 159.8, 146.4, 141.7, 141.7, 135.6, 135.5, 132.9, 132.9, 123.0, 117.4, 112.3, 52.9, 45.3, 38.2, 29.7, 27.1, 20.9, 19.5, 14.2, 11.0. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₁₇N₂O₄ m/z 277.1183; found 277.1176.

N-(Hexan-2-yl)-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3d).

We obtained 122 mg of the product as a yellow amorphous solid in 84% yield. ¹H NMR (400 MHz, chloroform-d): δ 11.65 (d, J = 6.3 Hz, 1H, NH), 8.23 (dd, J = 7.9, 2.1 Hz, 1H), 7.92–7.80 (m, 1H), 7.32–7.26 (m, 1H), 6.11 (dd, J = 35.1, 8.7 Hz, 1H, NH), 4.25–4.02 (m, 1H), 1.81–1.45 (m, 3H), 1.41–1.15 (m, 5H), 0.95 (dt, J = 14.2, 7.1 Hz, 4H). ¹H NMR (400 MHz, DMSO-d₆): δ 11.86 (d, J = 5.4 Hz, 1H, NH), 8.70 (dd, J = 39.7, 8.4 Hz, 1H, NH), 8.28 (dd, J = 13.3, 7.8 Hz, 1H), 8.10 (dd, J = 8.0, 2.5 Hz, 1H), 7.35 (td, J = 7.8, 3.2 Hz, 1H), 4.10–3.77 (m, 1H), 1.50 (dt, J = 14.4, 8.0 Hz, 2H), 1.32–1.12 (m, 4H), 0.85 (t, J = 6.4 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 166.6, 166.1, 159.8, 146.4, 141.7, 141.7, 135.6, 135.5, 133.0, 132.9, 123.0, 117.4, 117.2, 112.4, 112.3, 50.9, 45.6, 36.3, 35.8, 28.5, 27.6, 22.5, 20.9, 19.4, 14.4, 14.3, 11.0. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₅H₁₉N₂O₄ m/z 291.1339; found 291.1351.

N-(Heptan-4-yl)-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3e).

We obtained 119 mg of the product as a brown gum in 78% yield. ¹H NMR (400 MHz, chloroform-d): δ 11.75 (s, 1H, NH), 8.19 (dd, J = 7.9, 2.7 Hz, 1H), 7.97 (q, J = 8.0, 6.7 Hz, 1H), 7.29 (ddd, J = 12.1, 7.7, 3.8 Hz, 1H), 6.46 (dd, J = 34.6, 8.6 Hz, 1H, NH), 4.24–3.98 (m, 1H), 1.57 (ddd, J = 21.6, 14.7, 7.1 Hz, 4H), 1.43–1.07 (m, 8H), 1.07–0.70 (m, 6H). ¹³C{H}NMR (151 MHz, chloroform-d): 166.8, 166.2, 159.8, 146.5, 141.8, 141.7, 135.6, 133.0, 123.1, 117.5, 117.4, 112.4, 112.4, 52.9, 45.4, 40.5, 40.4, 40.3, 40.1, 40.0, 39.9, 39.7, 39.6, 38.3, 29.8, 27.2, 20.9, 19.5, 14.3, 11.1. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₆H₂₁N₂O₄ m/z 305.1496; found 305.1497.

N-Cyclopentyl-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3f).

We obtained 121 mg of the product as a brown gum in 88% yield. ¹H NMR (400 MHz, chloroform-d): δ 11.62 (s, 1H, NH), 8.24 (dd, J = 7.9, 1.4 Hz, 1H), 7.82 (dd, J = 7.8, 1.4 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 6.23 (s, 1H, NH), 4.38 (h, J = 7.0 Hz, 1H), 2.25–2.00 (m, 2H), 1.90–1.62 (m, 4H), 1.51 (dq, J = 13.3, 6.6 Hz, 2H). ¹H NMR (400 MHz, DMSO-d₆): δ 11.82 (s, 1H), 8.84 (d, J = 7.1 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 4.24 (h, J = 7.2 Hz, 1H), 1.90 (td, J = 7.7, 3.9 Hz, 2H), 1.68 (dd, J = 7.5, 4.2 Hz, 2H), 1.54 (h, J = 5.9, 4.9 Hz, 4H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 166.4, 159.76, 146.4, 141.6, 135.8, 132.9, 123.0, 117.5, 112.2, 51.5, 32.3, 24.1. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₁₅N₂O₄ m/z 275.1026; found 275.1030.

N-Cyclohexyl-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3g).

We obtained 110 mg of the product as a brown amorphous solid in 76% yield. ¹H NMR (400 MHz, chloroform-d): δ 11.62 (s, 1H, NH), 8.24 (dd, J = 8.0, 1.4 Hz, 1H), 7.84 (dd, J = 7.8, 1.4 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 6.18 (d, J = 8.0 Hz, 1H), 3.97 (tdt, J = 11.2, 7.9, 4.0 Hz, 1H), 2.03 (dt, J = 12.3, 3.7 Hz, 2H), 1.79 (dt, J = 13.5, 3.8 Hz, 2H), 1.69 (dt, J = 13.0, 3.8 Hz, 1H), 1.53–1.35 (m, 2H), 1.35–1.14 (m, 4H). ¹H NMR (400 MHz, DMSO-d₆): δ 11.80 (s, 1H, NH), 8.78 (d, J = 7.8 Hz, 1H, NH), 8.25 (dd, J = 7.9, 1.4 Hz, 1H), 8.09 (dd, J = 7.9, 1.3 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 3.79 (pd, J = 7.3, 3.8 Hz, 1H), 1.84 (dd, J = 8.9, 4.3 Hz, 2H), 1.78–1.66 (m, 1H), 1.67–1.53 (m, 1H), 1.44–1.07 (m, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 165.8, 159.8, 146.4, 141.6, 135.7, 132.9, 123.0, 117.6, 112.2, 49.1, 39.3, 32.4, 25.6, 25.3. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₅H₁₇N₂O₄ m/z 289.1183; found 289.1187.

6-Bromo-N-cyclopentyl-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (3h).

We obtained 114 mg of the product as a brown gum in 70% yield. ¹H NMR (400 MHz, chloroform-d): δ 11.49 (s, 1H, NH), 8.33 (d, J = 2.1 Hz, 1H), 7.89 (d, J = 2.1 Hz, 1H), 6.24 (s, 1H, NH), 4.37 (h, J = 7.0 Hz, 1H), 2.13 (dd, J = 12.8, 6.1 Hz, 2H), 1.73 (ddt, J = 20.5, 16.5, 8.2 Hz, 4H), 1.53 (dt, J = 12.9, 6.5 Hz, 2H). ¹H NMR (400 MHz, DMSO-d₆): δ 11.74 (s, 1H, NH), 8.92 (d, J = 7.1 Hz, 1H, NH), 8.45 (d, J = 2.2 Hz, 1H), 8.17 (d, J = 2.2 Hz, 1H), 4.22 (q, J = 6.8 Hz, 1H), 1.90 (s, 2H), 1.68 (d, J = 11.9 Hz, 4H), 1.62–1.50 (m, 2H). ¹³C{H}NMR (151 MHz, chloroform-d): δ 164.5, 157.5, 145.1, 140.4, 136.4, 136.0, 119.3, 115.3, 113.5, 52.3, 33.0, 23.8. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₁₄BrN₂O₄ m/z 353.0131; found 353.0137.

2,4-Dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide²⁸ (3i).

We obtained 860 mg of the product as a pale-yellow amorphous solid (81%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.12 (s, 1H, NH), 8.59 (s, 1H, NH), 8.28 (dd, J = 7.9, 1.4 Hz, 1H), 8.11 (dd, J = 8.1, 1.5 Hz, 2H, 1 NH), 7.33 (t, J = 7.8 Hz, 1H). ¹H NMR (400 MHz, acetic acid-d₄): δ 8.29 (dt, J = 7.9, 1.5 Hz, 1H), 8.25 (dd, J = 7.9, 1.4 Hz, 1H), 7.39–7.32 (m, 1H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 169.4, 159.7, 146.4, 142.2, 136.1, 133.4, 123.0, 116.5, 112.4.

4-Aminoisoindoline-1,3-dione³⁸ (4a).

We obtained 80 mg of the product as a pale-yellow amorphous powder in 93% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 10.87 (s, 1H), 7.40 (dd, J = 8.4, 7.0 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 7.0 Hz, 1H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 171.5, 169.9, 146.9, 135.5, 133.9, 121.5, 110.8, 110.6.

4-Amino-3-carbamoylbenzoic Acid²⁸ (4b).

The product was extracted with diethyl ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator, yielding 68 mg of the product as a yellow gum in 75% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 12.37 (s, 1H, OH), 8.14 (d, J = 2.0 Hz, 1H), 7.96 (s, 1H, NH), 7.66 (dd, J = 8.7, 1.9 Hz, 1H), 7.21 (s, 2H, NH₂), 7.14 (s, 1H, NH), 6.68 (d, J = 8.6 Hz, 1H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 171.2, 167.6, 154.2, 133.3, 131.9, 116.5, 116.0, 113.3. LC-MS (ESI) (m/z): [M − H]⁺ calcd for C₈H₇N₂O₃ 179.1; found 179.2.

Synthesis of Quinazoline-N-3-sec-alkyl-8-acid.

General Procedure for Quinazoline-N-3-sec-alkyl-8-acid, Method A.

First, 95 mg (0.5 mmol) of 2,3-dioxoindoline-7-carboxylic acid, 40 mg (1.2 mmol) of NaN₃, and 1.0 mL of concentrated sulfuric acid were stirred until effervescence diminished (5–10 min); then, 2.0 equiv (1.0 mmol) of alcohol was added and stirred for 2 h at room temperature. A 1.0 N NaOH solution was added dropwise, until reaching pH 12. After 5 min of stirring, a clear solution was obtained. After stirring for an additional 15 min, it was acidified to pH 3. The product was recovered by filtration, washed with water, and dried to obtain the product. If the precipitate was not formed, the product was extracted with diethyl ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator to obtain the product.

General Procedure for Quinazoline-N-3-sec-alkyl-8-acid, Method B.

First, 0.1 mmol of isatoic 8-sec-amide (IASA) in DMSO was combined with 0.11 mmol (1.2 equiv) of potassium tert-butoxide and stirred at room temperature overnight. The reaction mixture was poured into water and acidified to pH 2 to form a precipitate. The precipitate was recovered by filtration using a Buckner flask and dried at room temperature to obtain the product. If the precipitate was not formed, the product was extracted with diethyl ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator to obtain the product.

General Procedure for Quinazoline-N-3-sec-alkyl-8-acid, Method C.

First, 0.1 mmol of isatoic 8-sec-amide (IASA) in 0.6 mL of DMSO was heated at 100 °C (heating mantle) for 1 h. The reaction mixture was poured into water and acidified to pH 2 to form a precipitate. This was recovered by filtration using a Buckner flask and dried at room temperature to obtain the product. If the precipitate was not formed, the product was extracted with diethyl ether, washed with water, dried over MgSO₄, and filtered. The solution was concentrated using a rotary evaporator to obtain the product.

3-Isopropyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5a).

Following Method A, 2.0 mmol (500 mg) of IAA (2a) was suspended in water and cooled to 0 °C. A 0.5 N NaOH solution was added dropwise, until reaching pH 12. After 5 min of stirring, a clear solution was obtained. After stirring for an additional 15 min, it was acidified to pH 3. The product was recovered by filtration, washed with water, and dried to obtain 453 mg of the product in 90% yield as a pale-yellow amorphous solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.92 (s, 1H, NH), 8.22 (ddd, J = 23.2, 7.8, 1.6 Hz, 2H), 7.29 (t, J = 7.8 Hz, 1H), 5.10 (p, J = 6.9 Hz, 1H), 1.42 (d, J = 6.9 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 168.9, 161.9, 149.5, 140.6, 137.3, 133.7, 122.4, 116.1, 113.8, 45.2, 19.6. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₂H₁₃N₂O₄ 249.0869; found 249.0868.

3-(sec-Butyl)-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5b).

Following Method B, we obtained 24.4 mg of the product as a yellow gum (93%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.90 (s, 1H, NH), 8.25 (dd, J = 7.8, 1.5 Hz, 1H), 8.19 (dd, J = 8.0, 1.5 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 4.86 (dt, J = 9.0, 6.7 Hz, 1H), 2.03 (ddd, J = 13.5, 8.8, 7.2 Hz, 1H), 1.75 (dp, J = 14.2, 7.3 Hz, 1H), 1.40 (d, J = 6.9 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 168.9, 162.4, 149.6, 140.7, 137.4, 133.8, 122.5, 115.8, 113.9, 51.2, 26.0, 17.9, 11.6. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₃H₁₅N₂O₄ m/z 263.1026; found 263.1026.

3-(Hexan-2-yl)-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5c).

Following Method C, we obtained 25.9 mg of the product as a yellow gum (89%). ¹H NMR (400 MHz, chloroform-d): δ 11.11 (s, 1H, NH), 8.35 (ddt, J = 9.6, 7.8, 1.7 Hz, 2H), 7.34–7.20 (m, 1H), 5.26–4.72 (m, 1H), 2.14 (dtd, J = 14.6, 9.6, 5.9 Hz, 1H), 1.91–1.63 (m, 1H), 1.65–1.06 (m, 4H), 0.88 (ddd, J = 19.2, 9.7, 5.0 Hz, 6H). ¹H NMR (400 MHz, DMSO-d₆): δ 10.89 (s, 1H, NH), 8.26 (ddd, J = 7.7, 3.7, 1.6 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.29 (td, J = 7.8, 3.7 Hz, 1H), 5.03–4.58 (m, 1H), 2.16–1.95 (m, 1H), 1.78–1.43 (m, 1H), 1.44–1.01 (m, 4H), 0.90–0.62 (m, 6H). ¹³C{H}NMR (101 MHz, chloroform-d): δ 169.4, 160.5, 151.4, 140.4, 137.3, 137.3, 134.5, 122.2, 113.4, 113.4, 39.9, 33.8, 32.8, 29.1, 24.9, 22.5, 20.0, 17.9, 14.0, 13.9, 11.2. ¹³C NMR (101 MHz, DMSO-d₆) δ 170.5, 159.7, 149.5, 142.3, 137.4, 133.8, 122.6, 122.5, 113.9, 50.7, 32.6, 28.9, 23.00, 19.8, 17.2, 15.1, 11.8. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₅H₁₉N₂O₄ m/z 291.1339; found 291.1346.

3-(Heptan-4-yl)-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5d).

Following Method C, we obtained 27.8 mg of the product as a yellow amorphous solid (91%). ¹H NMR (400 MHz, chloroform-d): δ 11.16 (s, 1H, NH), 8.41–8.30 (m, 2H), 7.32–7.23 (m, 1H), 5.26–4.75 (m, 1H), 2.16 (d, J = 14.3 Hz, 1H), 1.89–1.73 (m, 1H), 1.52 (d, J = 6.9 Hz, 1H), 1.28 (ddt, J = 14.9, 11.3, 7.1 Hz, 4H), 1.21 (s, 2H), 0.86 (td, J = 7.3, 4.0 Hz, 4H). ¹H NMR (400 MHz, DMSO-d₆): δ 11.20 (s, 1H, NH), 8.25 (ddd, J = 7.7, 3.6, 1.6 Hz, 1H), 8.17 (d, J = 7.5 Hz, 1H), 7.33–7.23 (m, 1H), 5.10–4.61 (m, 1H), 2.17–1.95 (m, 1H), 1.68 (ddt, J = 30.2, 22.9, 9.5 Hz, 2H), 1.41 (dd, J = 10.2, 6.9 Hz, 1H), 1.18 (tdd, J = 24.2, 10.7, 5.6 Hz, 4H), 0.80 (ddt, J = 14.8, 11.5, 6.3 Hz, 6H). ¹³C{H}NMR (151 MHz, chloroform-d): δ 169.9, 140.5, 137.4, 137.4, 122.4, 113.6, 113.5, 33.2, 31.7, 29.1, 26.7, 22.7, 22.7, 20.1, 18.1, 14.2, 14.1, 14.0, 11.3, HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₆H₂₁N₂O₄ m/z 305.1496; found 305.1521.

3-Cyclopentyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5e).

Following Method C, we obtained 25.6 mg of the product as a pale-yellow amorphous solid (93%). ¹H NMR (400 MHz, chloroform-d): δ 11.14 (s, 1H, NH), 8.38 (dd, J = 7.8, 1.5 Hz, 1H), 8.32 (dd, J = 7.8, 1.7 Hz, 1H), 7.30–7.25 (m, 1H, chloroform-d), 5.42 (p, J = 8.7 Hz, 1H), 2.19 (dq, J = 15.1, 7.3 Hz, 2H), 2.00 (qd, J = 11.7, 11.2, 7.1 Hz, 2H), 1.94–1.80 (m, 2H), 1.69–1.55 (m, 2H). ¹H NMR (400 MHz, DMSO-d₆): δ 10.91 (s, 1H, NH), 8.24 (dd, J = 7.7, 1.6 Hz, 1H), 8.18 (dd, J = 7.9, 1.6 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 5.30–5.15 (m, 1H), 2.11–1.97 (m, 2H), 1.92–1.81 (m, 2H), 1.81–1.69 (m, 2H), 1.54 (qdd, J = 6.9, 5.8, 4.9, 2.9 Hz, 2H). ¹³C{H}NMR (101 MHz, chloroform-d): δ 169.5, 161.9, 150.8, 140.2, 137.2, 134.6, 122.2, 116.3, 113.2, 53.5, 28.4, 25.8. ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 168.8, 161.9, 149.5, 140.4, 137.3, 133.7, 122.5, 115.9, 113.8, 52.7, 28.5, 25.8. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₁₅N₂O₄ m/z 275.1026; found 275.1033.

3-Cyclohexyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5f).

Following Method C, we obtained 26.5 mg of the product as a yellow gum (92%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.86 (s, 1H, NH), 8.24 (dd, J = 7.8, 1.6 Hz, 1H), 8.17 (dd, J = 7.8, 1.6 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 4.69 (tt, J = 12.2, 3.7 Hz, 1H), 2.33 (qd, J = 12.5, 3.6 Hz, 2H), 1.82–1.71 (m, 2H), 1.65–1.56 (m, 3H), 1.37–1.03 (m, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 168.9, 162.0, 149.6, 140.6, 137.3, 133.8, 122.4, 116.00, 113.8, 53.6, 28.7, 26.3, 25.5. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₅H₁₇N₂O₄ m/z 289.1183; found 289.1191.

6-Bromo-3-cyclopentyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5g).

Following Method C, we obtained 33 mg of the product as a brown gum (94%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.81 (s, 1H, NH), 8.25 (d, J = 2.4 Hz, 1H), 8.22 (d, J = 2.5 Hz, 1H), 5.20 (p, J = 8.7 Hz, 1H), 2.00 (dt, J = 15.0, 7.3 Hz, 2H), 1.90–1.69 (m, 4H), 1.52 (q, J = 5.9 Hz, 2H). ¹³C{H}NMR (151 MHz, DMSO-d₆): δ 167.7, 167.6, 161.6, 161.0, 149.5, 149.3, 141.0, 139.5, 139.2, 139.0, 135.3, 134.9, 118.2, 118.0, 116.7, 116.3, 113.6, 113.6, 53.0, 40.2, 28.4, 25.9. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₁₄BrN₂O₄ m/z 353.0131; found 353.0137.

2,4-Dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (5h).

Following Method C, we obtained 19.4 mg of the product as a yellow amorphous powder (94%). ¹H NMR (400 MHz, DMSO-d₆): δ 11.64 (s, 1H, NH), 10.85 (s, 1H, NH), 8.27 (dd, J = 7.8, 1.6 Hz, 1H), 8.17 (dd, J = 7.8, 1.6 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 169.1, 162.5, 149.6, 142.0, 137.5, 133.2, 122.3, 116.2, 114.3. LC-MS (ESI) m/z: [M − H]⁺ calcd for C₉H₅N₂O₄ (m/z): 205.1; found 205.1.¹²

3-(Hexan-2-yl)-N-isopropyl-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxamide (6).

First, 0.1 mmol of the compound 3d in 0.6 mL of DMSO was heated at 100 °C for 1 h. The progress of the reaction was monitored by GC. After cooling to room temperature, 1.2 equiv of each of HBTU (46 mg) and isopropylamine (14 mg) were added and stirred at room temperature for 6 h, extracted with dichloromethane, dried, and obtained essentially 29.4 mg of the pure product as a yellow oil (89% yield). ¹H NMR (400 MHz, chloroform-d): δ 11.19 (s, 1H, NH), 8.34–8.19 (m, 1H), 7.72 (dt, J = 7.8, 1.7 Hz, 1H), 7.19–7.13 (m, 1H), 6.08 (d, J = 7.7 Hz, 1H, NH), 5.19–4.77 (m, 1H), 4.27 (dp, J = 13.7, 6.9 Hz, 1H), 2.23–2.08 (m, 1H), 1.88–1.45 (m, 4H), 1.29 (d, J = 6.5 Hz, 8H), 0.91–0.82 (m, 5H). ¹³C{H}NMR (151 MHz, chloroform-d): δ 166.0, 139.8, 131.6, 131.5, 121.4, 116.8, 116.8, 42.2, 32.8, 29.1, 22.7, 22.5, 20.0, 18.0, 14.0, 14.0, 11.2. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₈H₂₆N₃O₃ m/z 332.1968; found 332.1971.

4-Oxo-N-(pentan-3-yl)-2-phenyl-1,4-dihydroquinazoline-8-carboxamide⁴² (7).

An equimolar mixture of the compound 3c (0.1 mmol) and benzimidine (0.1 mmol) was combined in DMSO and heated at 42 °C (heating mantle) for 6 h. The progress of the reaction was monitored by GC. The solution was poured into water and acidified with HCl to pH 2. The product was recovered with filtration and dried to obtain 30 mg as a yellow amorphous solid (90% yield). ¹H NMR (400 MHz, chloroform-d): δ 11.66 (s, 1H, NH), 10.50 (dd, J = 27.2, 8.3 Hz, 1H, NH), 8.90 (ddd, J = 7.6, 4.3, 1.7 Hz, 1H), 8.47 (dt, J = 7.9, 2.2 Hz, 1H), 8.15–8.04 (m, 2H), 7.65 (tdd, J = 10.7, 6.6, 2.8 Hz, 4H), 4.42–4.04 (m, 1H), 1.90–1.40 (m, 4H), 1.40–0.88 (m, 6H). ¹³C{H}NMR (101 MHz, chloroform-d) δ 164.1, 163.5, 152.0, 146.6, 138.5, 138.4, 132.6, 132.3, 129.9, 129.4, 129.4, 128.9, 128.6, 127.3, 127.3, 126.9, 120.9, 52.5, 45.5, 39.5, 27.7, 21.3, 19.5, 14.0, 10.5. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₂₀H₂₂N₃O₂ m/z 336.1706; found 336.1713.

4-Oxo-3-phenyl-2-thioxo-1,2,3,4-tetrahydroquinazoline-8-carboxamide⁴⁴ (8).

A suspension of 0.1 mmol of the compound 3a and ammonium carbonate (0.4 mmol) in DMSO was heated (heating mantle) at 50 °C for 6 h. The progress of the reaction was monitored by GC. Upon consumption of IAA, 1.0 mmol of PhNCS and 1.0 mmol of pyridine were added and maintained at 50 °C for about 6 h. The reaction mixture was added to water and acidified with HCl to pH 2 to form a precipitate, filtered, and dried to obtain 25 mg of the product as a yellow amorphous solid (85% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (d, J = 7.8 Hz, 1H, NH), 7.83 (s, 1H, NH), 7.63 (t, J = 7.0 Hz, 3H, NH2), 7.54 (d, J = 7.6 Hz, 1H), 7.20 (s, 1H, NH), 6.50 (t, J = 7.7 Hz, 1H), 4.13–3.90 (m, J = 6.8 Hz, 1H), 1.12 (d, J = 6.6 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 171.5, 168.2, 150.4, 132.2, 132.1, 118.1, 115.9, 113.3, 41.0, 22.7. LC-MS (ESI) m/z: [M − 1]⁺ calcd for C₁₅H₁₀N₃O₂S m/z 296.0; found 296.1.

3-(1-(tert-Butyl)-1H-benzo[d]imidazol-2-yl)-2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic Acid (9).

An equimolar mixture of quinazoline-8-acid, 5h (0.05 mmol), and N-t-butyl-benzimidine-8-amine (0.05 mmol) was combined in DMSO and heated at 100 °C (heating mantle) for 3 h. The progress of the reaction was monitored by GC. After completion, the solution was poured into water and acidified with HCl to pH 2. The product was recovered with filtration and dried to obtain 17.6 mg as a yellow amorphous solid (93% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 12.70 (s, 1H, NH), 11.39 (s, 1H, NH), 8.29 (dd, J = 7.6, 1.6 Hz, 1H), 8.00 (dd, J = 7.8, 1.6 Hz, 1H), 7.89 (s, 2H), 7.70 (d, J = 8.3 Hz, 1H), 7.29 (dd, J = 7.9, 1.3 Hz, 1H), 7.17 (dt, J = 14.7, 7.5 Hz, 2H), 7.07 (td, J = 7.8, 7.3, 1.4 Hz, 1H), 1.78 (s, 9H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 170.6, 163.3, 152.2, 149.8, 141.9, 137.5, 131.7, 130.4, 122.9, 121.8, 121.5, 115.5, 114.6, 112.7, 60.1, 29.4. HRMS (ESI-TOF) m/z: [M-t-butyl + Na]⁺ calcd for C₁₆H₁₀N₄O₄Na m/z 345.0599; found 345.0610.

Synthesis of Substituted Aniline.

General Procedure for Substituted Aniline, Method A.

A total of 96 mg (0.5 mmol, 1.0 equiv) of 2,3-dioxoindoline-7-carboxylic acid (1a) was added dropwise to a 25 mL round-bottom flask equipped with a gas bubbler and containing 100 mL of 1 N NaOH. The solution was cooled to 0 °C and spiked with 2 mL of concentrated sulfuric acid. After 10 min, 43 mg (1.2 equiv) of sodium azide was added. After effervescence ceased, alcohols were added. The solution was stirred at 0 °C to RT for 1 h and left at room temperature for 3 h. A precipitate formed after adding the reaction to 100 mL of cold water. The NaOH solution was used to adjust to pH 8. The product was recovered by filtration, washed with water, and dried at room temperature to get 80–84% of the products.

General Procedure for Substituted Aniline, Method B.

The isatoic anhydride-8-sec-amide was suspended in water, and slowly the pH was raised to 8 using 1 N NaOH solution; a clear solution was obtained after stirring for 15 min. The product was extracted with diethyl ether and the organic layer was combined; upon concentration on a rota evaporator, we obtained the product essentially in the pure form in 73–81% yield.

2-Amino-3-(isopropylcarbamoyl)-benzoic Acid (10a).

A total of 500 mg (2.75 mmol, 1.0 equiv) of 2,3-dioxoindoline-7-carboxylic acid (1a) was added dropwise to a 50 mL round-bottom flask equipped with a gas bubbler (the other end of the bubbler was dipped in a flask containing 100 mL of 1 N NaOH solution). The reaction mixture was cooled to 0 °C and spiked with 10 mL of concentrated sulfuric acid. After 10 min, 210 mg (6.3 mmol, 1.2 equiv) of sodium azide was added gradually over a period of 10 min; then, 2.0 equiv of (11.0 mmol) alcohol was added and stirred at 0 °C to RT for 1 h and left at room temperature for 2 h. A precipitate formed after adding the reaction to 100 mL of cold water. The NaOH solution was used to adjust to pH 8. The product was recovered by filtration, washed with water, and dried at room temperature to get 500 mg of a pale-yellow solid in 81.3% as a yellow amorphous solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.16 (d, J = 7.7 Hz, 1H, NH), 7.86 (dd, J = 7.9, 1.6 Hz, 1H), 7.65 (dd, J = 7.6, 1.7 Hz, 1H), 6.53 (t, J = 7.7 Hz, 1H), 4.04 (dp, J = 7.7, 6.5 Hz, 1H), 1.13 (d, J = 6.6 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 169.8, 168.0, 151.6, 134.9, 134.2, 118.2, 113.6, 111.5, 41.1, 22.7. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₁H₁₅N₂O₃ m/z 223.1077; found 223.1078.

2-Amino-3-(sec-butylcarbamoyl)-benzoic Acid (10b).

Following Method A, we obtained 99 mg of a pale-yellow amorphous powder in 88%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.10 (d, J = 8.2 Hz, 1H, NH), 7.91–7.81 (m, 1H), 7.66 (dt, J = 7.6, 1.2 Hz, 1H), 6.62–6.48 (m, 1H), 3.87 (p, J = 7.1 Hz, 1H), 2.48 (p, J = 1.8 Hz, 1H), 1.61–1.34 (m, 2H), 1.10 (d, J = 6.6 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 169.8, 168.2, 151.5, 134.9, 134.1, 118.4, 113.6, 111.6, 46.5, 29.2, 20.6, 11.2. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₂H₁₇N₂O₃ m/z 237.1233; found 237.1234.

2-Amino-3-(heptan-4-ylcarbamoyl)-benzoic Acid (10c).

Following Method B, we obtained 22.3 mg of a pale-yellow gum in 80%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.04 (dd, J = 39.1, 8.5 Hz, 1H, NH), 7.86 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 6.5 Hz, 1H), 6.54 (td, J = 7.7, 3.6 Hz, 1H), 4.01–3.74 (m, 1H), 2.06 (dt, J = 8.7, 4.8 Hz, 1H), 1.44 (tt, J = 17.7, 13.7, 6.4 Hz, 3H), 1.23 (d, J = 13.8 Hz, 4H), 0.83 (h, J = 10.5, 8.9 Hz, 6H). ¹³C{H}NMR (151 MHz, DMSO-d₆): δ 169.8, 168.6, 168.2, 151.3, 134.8, 134.0, 124.6, 118.7, 118.5, 114.0, 111.7, 50.5, 48.3, 44.9, 37.1, 36.2, 34.1, 31.6, 28.5, 27.8, 26.0, 22.5, 21.1, 19.4, 14.4, 14.3, 14.3, 11.0. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₅H₂₃N₂O₃ m/z 279.1703; found 279.1707.

2-Amino-3-(cyclopentylcarbamoyl)-benzoic Acid (10d).

Following Method B, we obtained 22 mg of a pale-yellow amorphous solid in 88%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.23 (d, J = 7.2 Hz, 1H, NH), 7.85 (dd, J = 7.9, 1.6 Hz, 1H), 7.65 (dd, J = 7.6, 1.7 Hz, 1H), 6.53 (td, J = 7.7, 1.3 Hz, 1H), 4.17 (h, J = 6.8 Hz, 1H), 1.85 (dp, J = 13.7, 4.9, 4.3 Hz, 2H), 1.67 (q, J = 7.3 Hz, 2H), 1.60–1.40 (m, 4H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 169.8, 168.5, 151.6, 134.9, 134.4, 118.2, 113.6, 111.5, 51.0, 32.5, 24.1. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₃H₁₇N₂O₃ m/z 249.1234; found 249.1247.

2-Amino-3-(cyclohexylcarbamoyl)-benzoic Acid (10e).

Following Method B, we obtained 19 mg of a pale-yellow gum in 73%. ¹H NMR (400 MHz, DMSO-d₆): δ 7.81 (dd, J = 7.9, 1.6 Hz, 1H), 7.59 (dd, J = 7.6, 1.6 Hz, 1H), 6.58 (t, J = 7.8 Hz, 1H), 3.60 (s, 1H), 1.73–1.43 (m, 6H), 1.27–1.09 (m, 4H). ¹³C{H}NMR (151 MHz, DMSO-d₆): 169.9, 168.5, 151.6, 134.9, 134.4, 118.2, 113.6, 111.5, 51.1, 32.5, 24.1, 22.7. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₁₉N₂O₃ m/z 263.1390; found 263.1399.

2-Amino-5-bromo-3-(cyclopentylcarbamoyl)-benzoic Acid (10f).

Following Method C, we obtained 29.7 mg of a pale-white amorphous solid in 91%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.40 (d, J = 7.1 Hz, 1H, NH), 7.91 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 2.5 Hz, 1H), 4.16 (q, J = 6.8 Hz, 1H), 1.85 (dddd, J = 11.9, 9.6, 6.7, 3.7 Hz, 2H), 1.66 (pd, J = 7.1, 5.8, 3.5 Hz, 2H), 1.57–1.43 (m, 4H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 168.6, 167.0, 150.6, 136.5, 136.2, 120.1, 113.4, 103.9, 51.2, 32.5, 24.1. ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 170.5, 169.7, 151.5, 135.4, 134.8, 119.0, 114.5, 112.9, 50.7, 44.2, 37.8, 37.2, 33.4, 32.1, 28.9, 23.00, 19.8, 17.2, 15.1, 11.8. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₃H₁₆BrN₂O₃ m/z 327.0339; found 327.0335.

2-Amino-5-bromo-3-(cyclopentylcarbamoyl)-benzoic Acid (10f): From Bromination of Isatoic Anhydride-8-sec-Amide.

A total of 27.4 mg of the compound 3f was dissolved in 1.0 mL of concentrated sulfuric acid at room temperature. A 1.1 equiv (18.0 mg) of liquid bromine was added to the solution at room temperature. This was stirred while heating (heating mantle) at 100 °C until the bromine was completely dissolved (10–15 min). After cooling to room temperature and adding to cold water, an orange-yellow precipitate formed and was recovered by filtration and washed with additional water. We obtained 29.0 mg of the product (90% yield).

Isopropyl 2-Amino-3-(isopropylcarbamoyl)-benzoate (11).

A total of 0.1 mmol of the compound 3a in 1.0 mL of DMSO and 0.2 mmol of isopropyl alcohol were combined with 0.2 mmol (28 mg) of K₂CO₃ and stirred at room temperature (12 h). After complete disappearance of the IAA peak by GC, the reaction mixture was added to water and extracted with dichloromethane. This was further washed with water and dried over MgSO₄, and we recovered 20.5 mg of the product as a yellow amorphous solid by filtration. 78% yield.^{54 1}H NMR (400 MHz, chloroform-d): δ 7.97 (dd, J = 8.0, 1.5 Hz, 1H), 7.71 (s, 2H, NH2), 7.42 (dd, J = 7.6, 1.6 Hz, 1H), 6.51 (t, J = 7.8 Hz, 1H), 5.84 (d, J = 8.0 Hz, 1H, NH), 5.19 (p, J = 6.3 Hz, 1H), 4.22 (ddt, J = 13.1, 8.0, 6.7 Hz, 1H), 1.34 (d, J = 6.3 Hz, 6H), 1.24 (d, J = 6.5 Hz, 6H). ¹³C{H}NMR (101 MHz, chloroform-d): δ 168.3, 167.3, 151.5, 134.9, 132.2, 118.0, 113.6, 112.4, 77.3, 77.0, 76.7, 67.9, 41.6, 22.8, 22.0. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₂₁N₂O₃ m/z 265.1547; found 265.1555.

2-Amino-N-isopropylisophthalamide (12).

A suspension of 0.1 mmol of the compound 3a and ammonium carbonate (0.4 mmol) in DMSO was heated (heating mantle) at 50 °C for 6 h. Water was added to form a precipitate. The precipitate was recovered by filtration, washed with water, and dried at room temperature; we obtained 19.5 mg of the product as a yellow amorphous solid in 88% yield. ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (d, J = 7.8 Hz, 1H, NH), 7.83 (s, 1H, NH), 7.63 (t, J = 7.0 Hz, 3H, NH2), 7.54 (d, J = 7.6 Hz, 1H), 7.20 (s, 1H, NH), 6.50 (t, J = 7.7 Hz, 1H), 4.13–3.90 (m, J = 6.8 Hz, 1H), 1.12 (d, J = 6.6 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 171.5, 168.2, 150.4, 132.2, 132.1, 118.0, 116.0, 113.3, 41.0, 22.7. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₁H₁₆N₂O₃ m/z 222.1237; found 222.1238.

2-Amino-3,5-dibromo-N-isopropylbenzamide (13).

A total of 20.6 mg of the compound 3a was dissolved in 1.0 mL of concentrated sulfuric acid at room temperature. A 2.2 equiv (35.0 mg) of liquid bromine was added to this solution at room temperature. This was stirred at 100 °C (heating mantle) until the bromine completely dissolved in sulfuric acid (10–15 min). The mixture was cooled to room temperature and added to cold water until a pale-brown precipitate formed. The precipitate was recovered by filtration, washed with additional water, and dried to give 30 mg of the product as a pale-brown amorphous solid (91% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 8.32 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H), 7.67 (d, J = 2.3 Hz, 1H), 6.53 (s, 2H), 4.09–3.96 (m, 1H), 1.12 (d, J = 6.6 Hz, 6H). ¹³C{H}NMR (101 MHz, DMSO-d₆): δ 166.4, 145.8, 136.6, 130.6, 118.7, 110.6, 105.6, 41.4, 22.6. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₀H₁₃Br₂N₂O m/z 334.9389; found 334.9391.