Studies Toward the Synthesis of Caramboxin Analogues

Abstract

Intrigued by the recent discovery of caramboxin by Brazilian researchers, we present the results from our studies toward the racemic synthesis of caramboxin analogs through the ortho-carboxylation of 3,5-dimethoxy benzyl derivatives. Three different approaches were tested, and the route involving a Vilsmeier-Haack formylation followed by a Lindgren oxidation provide a potential intermediate for the synthesis of several caramboxin analogs.

Keywords:
caramboxin; star fruit; Vilsmeier-Haack reaction; Lindgren oxidation; Curtius rearrangement; Bischler-Napieralski reaction

Introduction

Originally from Asia, star fruit or carambola (Averrhoa carambola) is a star-shaped fruit popularly consumed and used as traditional medicine in tropical countries around the globe. The toxic effect of this fruit, which mechanism was unclear, involves not only neurotoxicity, but also nephrotoxicity even for people with normal renal function.¹ Recently, a review of publications from 2000 to 2014 related to the toxicity of carambola noted 27 deaths from 110 patients. In addition, the most commonly reported symptoms after consuming carambola are hiccups, vomiting and confusion.² Although several studies have suggested that oxalate (C₂O₄^2-) is the responsible for toxicity, in 2013, Brazilian researchers³ isolated a neurotoxin, caramboxin (1) (Figure 1), that can inhibit the GABAergic system (related to the central nervous system).

Figure 1
Caramboxin structure (1) and the tetrahydroisoquinolinic derivative 2 synthesized by Quintiliano and Silva.⁴

Compound 1 contains a phenylalanine skeleton, and the absolute configuration of the carbon α to the amino acid has only been inferred by comparison with the [α]_D signal of L-phenylalanine.³ So far, only the 2D chemical structure of 1 was confirmed by a computational study using DFT (density functional theory) calculations.⁵ In this case, the theoretical nuclear magnetic resonance (NMR) chemical shifts are in accordance with the experimental NMR measured in dimethyl sulfoxide (DMSO-d₆).

To the best of our knowledge, there are no reports dedicated to the total synthesis of 1. In 2012, Quintiliano and Silva⁴ reported a 10-step synthesis of the tetrahydroisoquinolinic derivative 2 starting from dimedone (Figure 1). Years later, an unsuccessful attempt to convert 2 to 1 was only reported in the PhD thesis of the same author.⁶

In view of the recent and the important discovery of caramboxin, studies toward the synthesis of derivatives of 1 could also be of great importance. The development of synthetic routes to obtain the core of caramboxin could contribute to a possible total synthesis of 1.

Structural analysis of 1 reveals an intriguing carboxyl group at the ortho position of the phenylalanine moiety, which we consider a significant synthetic challenge. Although a synthesis of (DL)-o-carboxy¹³C-phenylalanine starting from .-bromotoluene was reported,⁷ the required benzylic bromination of toluene could be difficult if an activated aromatic analog is applied. Thus, we investigated three synthetic pathways for the direct .-carboxylation of the aromatic ring to synthesize 3-methoxylated lactam 4 (Scheme 1). Hydrolysis of 4 could lead to the 3-methoxylated derivative of caramboxin (3).

Scheme 1
Three approaches (A, B, and C) explored in this work to prepare lactam 4.

The first pathway involves the Bischler-Napieralski (BN) cyclization of N-Boc-protected ester 7 (Scheme 1, route A). Kim and co-workers⁸ developed an in situ Friedel-Crafts-type intramolecular cyclization of N-Boc carbamates via isocyanate intermediate 5 using triflic anhydride (Tf₂O) and 4-(dimethylamino)pyridine (DMAP). In this case, however, N-Boc carbamates containing an α-ester group (compound 6) were not tested. Isocyanate intermediate 5 could also be generated in situ through a Curtius rearrangement of monohydrolyzed malonate 7 (Scheme 1, route B). This second pathway arose from the work reported by Judd et al.⁹ These authors reported a one-pot procedure to obtain dihydroisoquinolin-1-ones from activated dihydrocinnamic acids through a modified Curtius rearrangement in the presence of BF₃·OEt₂. However, although a protocol to transform monoester malonic acids into N-Boc carbamates has been reported,¹⁰ the direct conversion of compound 7 into isocyanate 5 in the presence of a monoester is still unknown.

Finally, the third route was proposed to prioritize the C–H functionalization step (Scheme 1, route C). Compound 4 could be obtained through an intramolecular alkylation of .-amide malonate diester 8 followed by a decarboxylation step. Selective oxidation of aldehyde 9 could lead to the acid precursor of amide 8. Interestingly, compound 9 had previously been synthesized by Danishefsky and co-workers¹¹ through the Vilsmeier-Haack formylation of 10.

Results and Discussion

Bischler-Napieralski pathway

The Bischler-Napieralski approach had begun with commercially available 3,5-dimethoxybenzoic acid 11 (Scheme 2). Reduction of 11 with LiAlH₄, followed by benzylic bromination assisted by PBr₃ in dioxane provided bromide 12 in almost quantitative yield over two steps.¹² To insert the stable enolate fragment, nucleophilic substitution of 12 with the carbanion formed from previously prepared .-Boc malonate 14 with Cs₂CO₃ gave desired alkylated product 15 in 80% isolated yield. Another alkylation protocol using microwave irradiation at high temperature was applied;¹³ however, in this case, we observed that the Boc group from 14 is heat sensitive, and a decrease in the isolated yield was observed.

Scheme 2
(a) LiAlH₄, THF, 0 ºC → rt, N₂, 1 h (99%); (b) PBr₃, dioxane, 40 ºC, N2, 1 h (97%); (c) Boc₂O, NaHCO₃, DMAP (cat.), dioxane/H₂O (2:1), rt, overnight (quantitative); (d) 14, Cs₂CO₃, CH₃CN, rt, N₂, 24 h (80%); (e) Tf₂O, 2-ClPy or DMAP, DCM, N₂, 0 ºC → rt, overnight.

With carbamate 15 in hand, two sets of cyclization conditions using Tf₂O in 2-chloropyridine (2-ClPy) or DMAP, according to Banwell's protocol, were tested.¹⁴ Even in the presence of the two methoxy groups, in both cases, NMR analysis showed no evidence of cyclization product in the aromatic region. The major product obtained in both cases was the free amine 16 with 85 and 40% yield using DMAP and 2-ClPy, respectively. Spyropoulos and Kokotos¹⁵ proposed the formation of an imino triflate intermediate when Tf₂O and .-Boc protected amino acids are mixed. We believe the formation of 16 might have occurred probably by the formation of the isocyanate followed by hydrolysis or by simple deprotection of the Boc through traces of trifluoromethanesulfonic acid.¹⁶

Although the cyclization of N-Boc amides with Tf₂O has been reported,¹⁷ no examples containing diesters groups was found. Identical conditions with hydrocinnamic acid derivatives afforded the desired lactam. However, no reports using malonic acid monoesters were found.¹⁸

In order to verify the influence of the Boc group, the same reaction was conducted using an NHAc group (17). In this case, the desired BN product (18) was obtained in 20% isolated yield. Thus, probably the lability of the Boc group and the purity of the triflic anhydride are compromising the success of the cyclization.

Curtius rearrangement pathway

Since the BN pathway did not provide the desired cyclized product, we focused our efforts on the Curtius approach (Scheme 3). Thus, starting with the same bromide, 12, the alkylation with diethyl malonate under microwave irradiation¹³ followed by monohydrolysis with an equimolar amount of KOH provided monoester acid 7 in good overall yield. Deprotonation of the diethyl malonate with NaH gave the same diester 19, in only 48% isolated yield.

Scheme 3
(a) Diethyl malonate, K₂CO₃, MeCN, MW, 150 W, 130ºC, N₂, 3 × 10 min (77%); (b) KOH, EtOH, 30 ºC, N2, 30 min (67%); (c) (i) DPPA, TEA, toluene, reflux, N₂, 1.5 h; (ii) BF₃·Et₂O, 0 → 50 ºC, N₂, 5 h; (iii) 2 M NaOH, rt, 1 h (59%).

Different bases for the Curtius rearrangement using diphenylphosphoryl azide (DPPA) with compound 7 were tested.⁹ Among them, only triethylamine (TEA) provided corresponding isocyanate 5 in 59% isolated yield. However, the intramolecular S_EAr was not observed. Another attempt using a greater amount of BF₃ at higher temperature (90 ºC) only afforded a trace amount of recovered 5. Analogous to the BN approach, the failure of the reaction can be attributed to the presence of the ester.

Vilsmeier-Haack pathway

In view of the difficulty of the ortho-carboxylation of functionalized aromatics, likely due to chemoselectivity issues, we decided to prioritize the formylation in the beginning of the route through the Vilsmeier-Haack (VH) reaction of a single substrate. According to the literature, the VH reaction of benzylic alcohol 10 was achieved by Danishefsky and co-workers.¹¹ Following the same protocol, using freshly distilled POCl₃, we obtained highly functionalized chloride 9 in 90% isolated yield (Scheme 4). Due to the presence of the labile benzylic chloride, the oxidation of the benzaldehyde to the corresponding benzoic acid was carefully studied. The oxidation of the aldehyde is crucial for the synthesis of the amide. The results of the tested oxidation protocols are summarized in Table 1.

Scheme 4
(a) POCl₃, DMF, 0 → 75 ºC, N₂, 2.5 h (90%).

Table 1
Conditions tested for the oxidation of benzaldehyde 9

entry	Oxidizing agent	Scavenger	Solvent	Temperature / ºC	time / min	20:21:22 ratioa
1	Oxone®	-	DMF	rt	overnight	-
2	H2O2/AgNO3	-	MeCN	50	overnight	-
3	KMnO4	-	MeCN/H2O	rt	overnight	traces of 20
4	KMnO4	-	MeOH/H2O	rt	overnight	1:1:0
5	NaClO2	NH2SO3H/DMSO	THF/H2O	0	20	1:1:0
6	NaClO2	NH2SO3H/DMSO	THF/H2O	0	30	1.5:1:0
7	NaClO2	NH2SO3H/DMSO	THF/H2O	0	50	19:1:0
8	NaClO2	NH2SO3H/DMSO	THF/H2O	-20	50	4:1:0
9	NaClO2	NH2SO3H/DMSO	THF/H2O	0 to rt	overnight	only 22
10	NaClO2	NH2SO3H/DMSO	acetone	0	30	2.5:1:0
11	NaClO2	resorcinol/t-BuOH	THF/phosphate buffer	rt	60	only 21
12	NaClO2	DMSO	phosphate buffer	rt	120	only 21

• ^a
  Determined by LC-MS. DMSO: dimethyl sulfoxide; DMF: dimethylformamide; THF: tetrahydrofuran; rt: room temperature.

Depending on the oxidation protocol tested, compounds 20, 21 and 22 were obtained in different ratio. Initially, the protocols for benzaldehyde oxidation using Oxone®¹⁹ and H₂O₂/AgNO₃²⁰ did not provide any polar compounds by thin layer chromatography (TLC) analysis. Additionally, oxidation using KMnO₄ revealed only traces of compound 20 or a 1:1 mixture of 20 and lactone 21 (entry 4).²¹

Lindgren oxidation is one of the mildest protocols to oxidize benzaldehydes to the corresponding benzoic acids.²² This reaction uses sodium chlorite as the oxidant and is operationally simple. However, due to the formation of hypochlorite in situ, chlorination of the activated aromatic ring can be observed. The results of the Lindgren reaction, shown in Table 1 (entries 5 to 12), suggests that the reaction is dependent on the temperature, the reaction time and the careful addition of the chlorite. Extending the reaction time (entry 9) and adding the sodium chlorite in one portion led exclusively to chlorinated acid 22. Very slow addition of sodium chlorite at low temperature provided mixtures of 20 and 21 (entries 5 to 8). Reactions at higher temperatures (entries 11 and 12) gave lactone 21,²³ indicating that acid 20 is quite sensitive. Danishefsky and co-workers¹¹ reported a 7:1 ratio of 20 and 22, and no formation of lactone 21 was observed. In our case, a higher proportion of the acid was obtained (19:1); however, the isolated yield was only 16% (entry 7). Another protocol using acetone as the solvent gave similar results to those achieved in the THF/H₂O system (entry 10).²⁴ Even in under buffer conditions (entries 11 and 12), lactone 21 was also obtained.

In terms of isolated yield, we could not reproduce the Danishefsky protocol. Purification of 20 in the presence of lactone 21 by chromatographic methods (silica gel, alumina, preparative TLC, and preparative high performance liquid chromatography (HPLC)) and by other methods (acid-base extraction and recrystallization) failed in our hands. In all purification attempts, we observed the lactonization of 20.

The position of the chlorine atom on the aromatic ring in 22 was determined by two-dimensional NMR through analysis of the HMBC (heteronuclear multiple-bond correlation) spectra. Long distance heteronuclear coupling constants (ⁿJ_C-H, n ≥ 2)²⁵ of compound 22 are shown in Table 2.

Table 2
Signals in the 2D NMR HMBC spectrum of acid 22

δ (C)	δ (H)
	3.86 (10)	3.95 (11)	4.74 (9)	6.89 (3)	13.23 (8)
40.5 (9)
56.4 (11)
56.7 (10)
98.2 (3)
113.3 (1)			++	+
118.6 (5)			++	+
132.7 (6)			+
155.6 (4)	+			++
156.1 (2)		+		++
166.9 (7)

• NMR HMBC: nuclear magnetic resonance heteronuclear multiple bond correlation; +: low intensity; ++: high intensity.

According to Table 2, two-bond correlations between H(3)↔C(4) and H(3)↔C(2) and less intense three-bond correlations between H(3)↔C(1) and H(3)↔C(5) were observed. These correlations suggest the aromatic proton is located at C(3) and the chlorine atom at C(5) relative to the carboxyl group (C(1)).

Next, the conversion of the acid to the amide was studied (Scheme 5). The diethyl aminomalonate hydrochloride (13) was chosen to guarantee the following intramolecular alkylation step. Thus, impure acid 20 and dichloride 22 were reacted with HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) in the presence of triethylamine in dichloromethane (DCM) at room temperature.²⁶ Desired amide 8 was obtained in low yield (7%) and the major compound was lactone 21, which was obtained in 66% isolated yield. As expected, 20 was found to be unstable in the presence of triethylamine. However, when dichlorinated acid 22 was submitted in the same conditions that were used for 20, we could obtain amide 23 in 68% yield. The presence of the chlorine atom on the aromatic ring in 23 makes the molecule less sensitive to lactonization. Based on the low yields with sensitive acid 20, we decided to continue the pathway using amide 23 for the next steps.

Scheme 5
(a) HBTU, TEA, DCM, rt, N₂, 2.5 h (7% for 8 and 66% for 21); (b) HBTU, TEA, DCM, rt, N2, 2.5 h (68%).

We considered the intramolecular cyclization of 23 to be the crucial step for this route (Scheme 6). Due to the successful use of Cs₂CO₃ in the BN pathway, we decided to subject dichloride 23 to similar conditions. A catalytic amount of KI was added to increase the reactivity of the benzylic chloride portion.²⁷ In this case, a mixture of lactam 24 and iodide 25 were obtained in low yields. However, compound 25 was recovered and reacted again with Cs₂CO₃ to furnish 24. The low yields in the first cyclization protocol can be attributed to the reaction being conducted at room temperature. A better conversion was observed when a higher temperature and microwave (MW) irradiation were used to recycle 25 to 24. The overall isolated yield of 24 was 27% over two steps.

Scheme 6
(a) Cs₂CO₃, KI (cat.), DMF, rt, 12 h (12% for 24 and 19% for 25); (b) Cs₂CO₃, MeCN, MW (150 W, 130 ºC, 10 min) (77%).

Lactam 24 possesses the majority of the functional groups present in caramboxin. Attempts to decarboxylate one of the esters and open the lactam ring in a one-pot fashion were carried out (Scheme 7).²⁸ In the presence of 6 mol L^-1 HCl at 130 ºC, several byproducts were obtained. Liquid chromatography-mass spectrometry (LC-MS) analysis did not provide any evidence of a possible hydrolysis product.

Scheme 7
(a) HCl 6 mol L^-1, MW (200 W, 130 ºC), 10 min.

The hydrolysis of amides is usually difficult. Thus, to avoid the δ-lactam opening, a portion of remaining acid 22 was esterified by CH₃I (Scheme 8). Corresponding methyl ester 27 was then submitted to the benzylic alkylation. In this case, we used protected diethyl acetamidomalonate 28, and corresponding product 29 was obtained in a higher yield (60%) than what was achieved with the intramolecular version (23 to 24). The hydrolysis of amide triester 29 was partially successful. In fact, the decarboxylation of only one of the malonate esters occurred, affording acetamide benzyl methyl ester 30 in quantitative yield. Unfortunately, further acid hydrolysis of 30 with longer reaction times and at higher temperatures gave complex mixtures of products.

Scheme 8
(a) CH₃I, Cs₂CO₃, DMF, rt, N₂, 30 min (59%); (b) KI (cat.), Cs₂CO₃, MeCN, rt, overnight (60%); (c) HCl 6 mol L^-1, CH₃COOH, MW (150 W, 90 ºC, 10 min) (94%).

As previously mentioned, on the basis of the high sensitivity of acid 20, we anticipated that the Vilsmeier-Haack product, stable aldehyde 9, could be converted to a less reactive benzonitrile analogue, which could later be converted to the corresponding acid by hydrolysis. Thus, the treatment of 9 with NaN₃ in POCl₃ generated nitrile 31 in reasonable yield (67%) (Scheme 9).²⁹ Alkylation of 31 using 28 was accomplished under similar conditions to those mentioned before affording the malonate 32. At this point, the hydrolysis of the latter compound was more carefully investigated.

Scheme 9
(a) NaN₃, POCl₃, 50 ºC, N₂, 1 h (67%); (b) KI, Cs₂CO₃, MeCN, rt, 48 h (78%); (c) NaOH 2 mol L_-1, ultrasonic bath, 80 ºC, 45 min (67%); (d) KOH 35%, EtOH, 80 ºC, N₂, 2.5 h (45%); (e) HCl 2 mol L_-1, dioxane, reflux, N₂, 48 h (12%).

Since the last hydrolyses were carried out in acidic media, we decided to test the reactions under basic conditions with two distinct protocols. With the first set of conditions using 2 mol L^-1 NaOH and an ultrasonic bath at 80 ºC,³⁰ we obtained only the corresponding dicarboxylate disodium salt 33. On the other hand, using KOH as the base in refluxing ethanol,³¹ we obtained only decarboxylation derivative 34. Interestingly, in the last attempt, when compound 33 was subjected to acidic hydrolysis (2 mol L^-1 HCl) for an extended period, we could obtain imino tetrahydroisoquinolinone 35 in low isolated yield (12%). In this case, the expected hydrolysis of the acetamido group and the malonate decarboxylation occurred; however, the nitrile group was attacked by the free amino group, and a similar reaction was reported before by Hamley and co-workers.³²

Conclusions

We presented three different approaches for the ortho-carboxylation of 3,5-dimethoxy benzyl derivatives toward the preparation of caramboxin analogs. We observed that the insertion of the carboxyl group early on in the synthesis helps avoid chemoselective issues. The Vilsmeier-Haack formylation was chosen due to the concomitant halogenation of the benzylic alcohol, which facilitates the malonate alkylation. For the synthesis of caramboxin, a study regarding the regioselectivity of the VH will be necessary. We also conclude that the “protection” of the carboxylate by esterification or by functional group interconversion seems to be more attractive than lactamization by an intramolecular alkylation. Lastly, the challenges presented by the final steps, mainly the hydrolysis of the amide and the esters, require more detailed study.

Experimental

General information

The reagents (Sigma-Aldrich®) and solvents (Synth®) were used without purification. POCl₃ was distillated before used. The TLC analysis was made in silica gel 60 with aluminum, 0.2 mm, with indicator for 254 nm, and compounds visualized using UV irradiation, ninhydrin or vanillin stains. Flash column chromatography was performed using silica gel 60 Å (35-70 μm) from Fluka Analytical. The gas chromatography-mass spectrometry (GC-MS) analysis was made in one ion trap, Varian 4000 from Federal University of ABC. The LC-MS analysis was made in quadrupole Agilent 6130 Infinity coupled to an Agilent 1260 HPLC system, from Federal University of ABC. The HRMS analysis was made in micro-TOF (time of flight) Bruker Daltonics from São Paulo University. The ¹H and ¹³C NMR were made on Varian (500 MHz) from Federal University of ABC and Varian AIII or Bruker DPX-300 (300 MHz) from São Paulo University. The solvents used were deuterated chloroform (CDCl₃) and deuterated dimethyl sulfoxide (DMSO-d₆). The melting point analyses were made on Büchi B-540 or EZ-Melt SRS-Stanford Research Systems from Federal University of ABC. The purification on HPLC was made in Waters coupling with UV-Vis detector model 2489, using a semipreparative column Phenomenex C18. The Microwave™ synthesis system were made on CEM Focused, model Discover, from Federal University of ABC.

(3,5-Dimethoxyphenyl)methanol (10, CAS 705-76-0)

Under N₂ atmosphere, a solution of LiAlH₄ (2.1041 g, 55.44 mmol) in anhydrous THF (100 mL) was added dropwise to a solution of 3,5-dimethoxybenzoic acid (11) (10.0 g, 54.9 mmol) in anhydrous THF (150 mL) at 0 ºC. After stirring for 20 min at 0 ºC and one hour at room temperature, the reaction was diluted with THF (90 mL) and water (4 mL) was added. The mixture was then filtered on celite, washed with ethyl acetate (4 × 65 mL) and concentrated under reduced pressure. The resulting clear yellow solid (9.14 g, 54.5 mmol, 99% yield) was applied to the next reaction without further purification. mp 50.2-51.1 ºC; ¹H NMR (300 MHz, CDCl₃) δ 1.90 (s, 1H, OH), 3.80 (s, 6H, Ar-OCH₃), 4.63 (s, 2H, Ar-CH₂), 6.39 (t, 1H, J 2.3 Hz, Ar-H), 6.53 (d, 2H, J 2.3 Hz, Ar-H); ¹³C NMR (126 MHz, CDCl₃) δ 55.4 (2C), 65.4, 99.7, 104.6 (2C), 143.4, 161.0 (2C); low resolution (LR)MS (EI (electron ionization)) m/z 168 (M⁺, 100), 151 (12), 139 (45), 109 (15). The spectra of compound 10 is in accordance with those previously reported.³³

3,5-Dimethoxybenzyl bromide (12, CAS 877-88-3)

Under N₂ atmosphere, phosphorus tribromide (618 μL, 6.58 mmol) was added dropwise to a solution containing (3,5-dimethoxyphenyl)methanol (10) (1.00 g, 5.98 mmol) in dioxane (8 mL). The solution was stirred at 40 ºC for 1 h. After cooling to room temperature the reaction was quenched with a saturated NaHCO₃ solution (28 mL) and the resulting aqueous phase was extracted with ethyl acetate (3 × 28 mL). The combined organic extracts were dried with Na₂SO₄, filtered and concentrated under reduced pressure. The resulting light yellow solid (1.38 g, 5.9 mmol, > 99% yield) was applied to the next reaction without further purification. mp 72.9-73.8 ºC; ¹H NMR (500 MHz, CDCl₃) δ 3.79 (s, 6H, Ar-OCH₃), 4.42 (s, 2H, Ar-CH₂), 6.39 (t, 1H, J 2.2 Hz, Ar-H), 6.54 (d, 2H, J 2.2 Hz, Ar-H); ¹³C NMR (126 MHz, CDCl₃) δ 33.6, 55.4 (2C), 100.6, 106.9 (2C), 139.7, 160.9 (2C); LRMS (EI) m/z 232 (M⁺, 30), 230 (27), 152 (15), 151 (100). The spectra of compound 12 is in accordance with those previously reported.³⁴

Diethyl 2-((tert-butoxycarbonyl)amino)malonate (14)

NaHCO₃ (462 mg, 5.5 mmol) was slowly added to a suspension of diethyl aminomalonate hydrochloride (1.0582 g, 5 mmol, 13) in water (7 mL) and dioxane (10 mL). The resulting solution was stirred for a few minutes at room temperature (rt) until a clear solution appeared. Next, DMAP (6.11 mg, 0.01 mmol) was added followed by a dropwise addition of a solution of Boc₂O (1.2004 g, 5.5 mmol) in dioxane (4 mL). The mixture was stirred at room temperature overnight. Then, the solution was concentrated under reduced pressure. The residue was suspended in ethyl acetate (25 mL) and then extracted with 5% aqueous KHSO₄ solution (20 mL), saturated aqueous NaHCO₃ solution (20 mL), water (15 mL) and brine (15 mL). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. 1.2803 g, 4.65 mmol (46% yield) of a light oil was obtained and applied for the next reaction without further purification. LRMS (ESI (electrospray ionization)) m/z 276.1 [M – CO₂]⁺, 100).

Diethyl 2-((tert-butoxycarbonyl)amino)-2-(3,5-dimethoxy­benzyl)malonate (15)

A solution of bromide 12 (231 mg, 1 mmol), diethyl (Boc-amino)malonate (275 mg, 1.1 mmol) and Cs₂CO₃ (325 mg, 1.1 mmol) in acetonitrile (4.5 mL) was stirred for 25 h at room temperature. The reaction was then diluted in 30 mL of ethyl acetate and extracted with water and brine. The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, using ethyl acetate/hexane (4:1) as the eluent, affording a white solid (326.2 mg, 0.77 mmol, 77% yield). mp 69.7-72.5 ºC; ¹H NMR (500 MHz, CDCl₃) δ 1.29 (t, 6H, J 7.1 Hz, CO₂CH₂CH₃), 1.47 (s, 9H, t-Bu), 3.55 (s, 2H, Ar-CH₂C), 3.74 (s, 6H, Ar-OCH₃), 4.17-4.26 (m, 2H, CO₂CH₂CH₃), 4.27-4.36 (m, 2H, CO₂CH₂CH₃), 5.79 (s, 1H, N-H), 6.20 (d, 2H, J 2.0 Hz, Ar-H), 6.34 (t, 1H, J 2.2 Hz, Ar-H); ¹³C NMR (126 MHz, CDCl₃) δ 14.0 (2C), 28.2 (3C), 38.5, 55.1 (2C), 62.5 (2C), 67.1, 80.1, 99.1, 108.1 (2C), 137.4, 153.8, 160.5 (2C), 167.6 (2C); LRMS (ESI) m/z 426.2 [M + H]⁺.

Diethyl 2-amino-2-(3,5-dimethoxybenzyl)malonate (16)

Triflic anhydride (1.05 mL, 1.05 mmol) was slowly added to a solution containing the carbamate 15 (91 mg, 0.21 mmol) and DMAP (78.2 mg, 0.64 mmol) in DCM (8 mL). The mixture was stirred for 18 h at 0 ºC, in N₂. The reaction was then diluted in 5 mL of DCM and washed with a saturated Na₂CO₃ solution (5 mL), then 20% citric acid solution (5 mL) and saturated Na₂CO₃ solution (5 mL). The combined organic phases was dried with anhydrous Na₂SO₄ and concentrated under pressure. The residue was purified by flash chromatography, on silica gel, using ethyl acetate/hexane (2:1) as the eluent; the amine 16 (58 mg, 0.178 mmol, 85% yield) was obtained as a light oil. ¹H NMR (500 MHz, CDCl₃) δ 1.29 (t, 6H, J 7.1 Hz, CO₂CH₂CH₃), 2.10 (s, 2H, NH₂), 3.28 (s, 2H, Ar-H), 3.75 (s, 6H, Ar-OCH₃), 4.25 (qd, 4H, J 7.1, 1.8 Hz, CO₂CH₂CH₃), 6.33 (d, 2H, J 2.3 Hz, Ar-H), 6.36 (t, 1H, J 2.3 Hz, Ar-H); ¹³C NMR (126 MHz, CDCl₃) δ 13.9 (2C), 41.1, 55.2 (2C), 62.0 (2C), 66.2, 99.1 (2C), 108.1 (2C), 137.0, 160.7 (2C), 170.8; LRMS (ESI) m/z 352.1 [M + H]⁺.

Diethyl 2-(3,5-dimethoxybenzyl)malonate (19, CAS 5859-68-7)

A mixture of bromide 12 (334 mg, 1.44 mmol), diethyl manolate (263 μL, 1.73 mmol), Cs₂CO₃ (562 mg, 1.73 mmol) and CH₃CN (13 mL) was added to an Ace sealed tube. Three cycles of irradiation at 150 W, with maximum temperature of 130 ºC for 10 min were done. Next, the reaction was diluted in ethyl acetate (35 mL) and washed with water (20 mL) and brine (20 mL). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. An oil 19 (344.5 mg, 1.11 mmol, 77% yield) was obtained and submitted to the next reaction without further purification. ¹H NMR (500 MHz, CDCl₃) δ 1.23 (t, 6H, J 7.1 Hz, CO₂CH₂CH₃), 3.16 (d, 2H, J 7.8 Hz, Ar-CH₂CH), 3.63 (t, 1H, J 7.8 Hz, Ar-CH₂CH), 3.76 (s, 6H, Ar-OCH₃), 4.18 (dq, 4H, J 10.7, 7.1, 7.1, 3.6, 3.4 Hz, CO₂CH₂CH₃), 6.32 (s, 1H, J 2.2 Hz, Ar-H), 6.36 (d, 2H, J 2.2 Hz, Ar-H); ¹³C NMR (126 MHz, CDCl₃) δ 14.0 (2C), 34.9, 53.7, 55.3 (2C), 61.5, 98.8, 106.8 (2C), 140.3, 160.8 (2C), 168.9 (2C); LRMS (EI) m/z 310 (M⁺, 70), 265 (8), 237 (100), 219 (16), 192 (60), 165 (12), 136 (6), 105 (4), 91 (4). The spectra of compound 19 are in accordance with those previously reported.³⁵

2-(3,5-Dimethoxybenzyl)-3-ethoxy-3-oxopropanoic acid (7)

To a solution of malonate 19 (173 mg, 0.56 mmol) in ethanol (3 mL), a solution of KOH (32 mg, 0.57 mmol) in water (0.65 mL) was slowly added. The reaction was stirred for 30 min at 30 ºC. Then, a 5% aqueous HCl solution (3 mL) was slowly added. The mixture was diluted in water (5 mL) and extracted with dichloromethane (3 × 5 mL). The organic phases was combined and neutralized with saturated aqueous NaHCO₃ solution (10 mL). The remaining aqueous phase was acidified with 2 M HCl solution and extracted with more DCM (3 × 5 mL). All the organic phases was combined, dried with anhydrous Na₂SO₄ and concentrated under reduced pressure, affording white solid (116.8 mg, 0.41 mmol, 73% yield). The crude product was applied to the next reaction without further purification. ¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, 3H, J 7.1 Hz, CO₂CH₂CH₃), 3.18 (d, 2H, J 7.7 Hz, Ar-CH₂CH), 3.70 (t, 1H, J 7.7 Hz, Ar-CH₂CH), 3.76 (s, 6H, Ar-OCH₃), 4.19 (q, 2H, J 7.1 Hz, CO₂CH₂CH₃), 6.34 (t, 1H, J 2.2 Hz, Ar-H), 6.37 (d, 2H, J 2.2 Hz, Ar-H), 9.08 (s, 1H, COOH); ¹³C NMR (75 MHz, CDCl₃) δ 13.9, 34.8, 53.3, 55.2 (2C), 61.8, 98.9, 106.7 (2C), 139.7, 160.8 (2C), 168.6, 174.0; LRMS (EI) m/z 238 [M – CO₂]⁺, 85), 193 (20), 165 (100).

Ethyl 3-(3,5-dimethoxyphenyl)-2-isocyanatopropanoate (5)

Diphenyl phosphoryl azide (86 μL, 0.4 mmol) was added to a solution containing 2-(3,5-dimethoxybenzyl)-3-ethoxy-3-oxopropanoic acid (115 mg, 0.4 mmol, 7) and triethylamine (55 μL, 0.4 mmol) in toluene (4 mL). The mixture was stirred for 90 min at 90 ºC. Then, the reaction was cooled at 0 ºC and BF₃.OEt₂ (200 μL) was added. After, stirring at 50 ºC for 5 h in N₂, the mixture was cooled at rt and then a 2 M NaOH solution was added (at pH = 10). Next, after extraction with EtOAc (3 × 10 mL) and brine (10 mL) the combined organic phases was dried with anhydrous Na₂SO₄ and concentrated under pressure. The residue was purified by flash chromatography, on silica gel, using ethyl acetate/hexane (4:1 to 1:1) as the eluent; the isocyanate 5 (20.6 mg, 0.07 mmol, 19% yield) was obtained as a light oil. ¹H NMR (300 MHz, CDCl₃) δ 1.25 (t, 3H, CO₂CH₂CH₃), 3.02 (d, 2H, J 5.8 Hz, Ar-CH₂CH), 3.74 (s, 6H, Ar-OCH₃), 4.13-4.18 (m, 2H, CO₂CH₂CH₃), 4.66-4.77 (m, 1H, Ar-CH₂CH), 6.27 (d, 2H, J 2.2 Hz, O-Ar-H), 6.30-6.36 (m, 1H, p-Ar-H); LRMS (EI) m/z 279 (M⁺, 100).

2-(Chloromethyl)-4,6-dimethoxybenzaldehyde (9, CAS 166322-67-4)

First, a solution of a freshly distilled POCl₃ (4.44 mL, 47.6 mmol) in anhydrous DMF (7.2 mL) at 0 ºC in N₂ was prepared. To this solution, a solution of (3,5-dimethoxyphenyl)methanol (10) (2.0 g, 11.9 mmol) in anhydrous DMF (5.1 mL) was slowly added and the resulting mixture was stirred for 2 h at 75 ºC in N₂. The reaction was cooled to room temperature and poured into ice water (180 mL). The pH was adjusted to 7 by addition of 2 M NaOH solution and the mixture was stirred for additional 1.5 h. The green precipitate was filtered, washed with cooled water and was dried under reduced pressure. The solid was diluted in acetone, and the solution was filtered in celite and the filtrate was concentrated under reduced pressure. Finally, the solid was purified by flash chromatography on silica gel, eluting with a gradient of ethyl acetate/hexane (4:1 to 1:1), providing a white solid (2.286 g, 10.7 mmol, 90% yield). mp 105.5-106.5 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.90 (s, 6H, Ar-OCH₃), 5.05 (s, 2H, Ar-CH₂), 6.44 (d, 1H, J 2.4 Hz, Ar-H), 6.76 (d, 1H, J 2.9 Hz, Ar-H), 10.46 (s, 1H, Ar-CHO); ¹³C NMR (75 MHz, CDCl₃) δ 44.9, 55.7, 56.0, 97.7, 107.6, 115.9, 142.4, 165.0, 165.3, 189.9; LRMS (ESI) m/z 215.0 ([M + H]⁺, 100), 217.0; HRMS (TOF) m/z, observed: 237.0278; C₁₀H₁₁ClO₃Na [M + Na]⁺ requires: 237.0294. The spectra of compound 9 is in accordance with those previously reported.³⁶

2-(Chloromethyl)-4,6-dimethoxybenzoic acid (20)

To a solution of 2-(chloro­methyl)-4,6-dimethoxy­benzaldehyde 9 (500 mg, 2.33 mmol) in acetone (25 mL) and DMSO (10 mL) at 0 ºC, a solution of sulfamic acid (384.5 mg, 3.96 mmol) in water (7.5 mL) was added. The reaction was stirred for 1 min on N₂ atmosphere. To this solution, a solution of NClO₂ (326.5 mg, 3.61 mmol) in water (17 mL) was slowly added. After stirring for 30 min at 0 ºC the reaction was extracted with ethyl acetate (3 × 15 mL). The combined organic phases were washed with water (15 mL) and brine (15 mL), then dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. A yellow solid (347 mg) of a mixture of the acid 20 with the lactone 21 (1:1 ratio) was obtained. LRMS (ESI) m/z 231.0 ([M + H]⁺, 100), 233.0 (34) for acid 20 and 195.1 (M⁺, 100) for lactone 21.

3-Chloro-2-(chloromethyl)-4,6-dimethoxybenzoic acid (22)

To a solution of 2-(chloro­methyl)-4,6-dimethoxy­benzaldehyde (9) (100 mg, 0.47 mmol) in THF (5 mL) and DMSO (0.3 mL) at 0 ºC, a solution of sulfamic acid (153.8 mg, 1.58 mmol) in water (2.5 mL) was added. The reaction was stirred for 1 min on N₂ atmosphere. The NaClO₂ (136.0 mg, 1.50 mmol) was fastly added and stirred for 1 h at 0 ºC and overnight at rt. The reaction was diluted with ethyl acetate (25 mL) and extracted with saturated aqueous NH₄Cl solution (40 mL). The aqueous phase was extracted with more ethyl acetate (15 mL). The combined organic phases were washed with brine, dried with anhydrous MgSO₄ and concentrated under reduced pressure, providing a white solid (134.0 mg, in a quantitative yield). mp 143 ºC; ¹H NMR (500 MHz, DMSO-d₆) δ 3.86 (s, 3H, Ar-OCH₃), 3.94 (s, 3H, Ar-OCH₃), 4.74 (s, 2H, Ar-CH₂), 6.89 (s, 1H, Ar-H), 13.23 (br s, 1H, Ar-CO₂H); ¹³C NMR (126 MHz, DMSO-d₆) δ 40.5, 56.4, 56.7, 98.2, 109.5, 113.3, 118.6, 132.7, 155.6, 156.2, 166.9; heteronuclear multiple quantum correlation (HMQC, 500 MHz, DMSO-d₆) H6.89 ↔C113.3, H6.89↔C118.6, H6.89↔C155.6, H6.89↔C156.3, H4.74↔C113.3, H4.74↔C118.5, H4.74↔C132.7, H3.95↔C156.1, H3.86↔C155.7; LRMS (ESI) m/z 267.0 (65), 265.0 ([M + H]⁺, 100), 247.0 (24), 229.0 (35), 117.1 (20); HRMS (TOF) m/z, observed: 286.9845; C₁₀H₁₀Cl₂O₄Na [M + Na]⁺ requires: 286.9853.

Diethyl 2-(3-chloro-2-(chloromethyl)-4,6-dimethoxy­benzamido)malonate (23)

To a solution of acid 22 (200 mg, 0.75 mmol), trietilamine (420 mL, 2.6 mmol) in DCM (10 mL), HBTU (363 mg, 0.96 mmol) was added. After stirring for 15 min at room temperature, a solution of diethyl aminomalonate hydrochloride (203 mg, 0.95 mmol) in DCM (10 mL) was added dropwise, and the resulting mixture was stirred for additional 2.5 h at rt. The reaction was diluted in DCM (50 mL) and washed with 5% aqueous citric acid solution, saturated aqueous NaHCO₃ solution and water. The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by two consecutive flash chromatography columns, on silica gel: the first was eluted with ethyl acetate/hexane (1:1) and second was eluted with DCM/hexane (3:1), giving the compound 23 as a white solid (205 mg, 0.47 mmol, 63% yield). mp 152-156 ºC; ¹H NMR (500 MHz, CDCl₃) δ 1.33 (t, 6H, J 4.4 Hz, CO₂CH₂CH₃), 3.87 (s, 3H, Ar-OCH₃), 3.95 (s, 3H, Ar-OCH₃), 4.32 (m, 4H, CO₂CH₂CH₃), 4.87 (s, 2H, Ar-CH₂), 5.34 (d, 1H, J 3.8 Hz, -C.-NH-), 6.52 (s, 1H, -CH-N.-), 7.12 (d, 1H, J 3.9 Hz, Ar-H); ¹³C NMR (126 MHz, CDCl₃) δ 14.1, 40.5, 56.4, 56.5, 57.0, 62.8, 96.7, 115.8, 118.5, 135.5, 156.3, 165.3, 166.0; LRMS (ESI) m/z 424.1 (75), 422.1 ([M + H]⁺, 100), 249.0 (20), 247.0 (M⁺ – NHCH(CO₂Et)₂, 40); HRMS (TOF) m/z, observed: 246.9920; C₁₀H₉Cl₂O₃ [M – NHCH(CO₂Et)₂]⁺ requires: 246.9928.

Diethyl 5-chloro-6,8-dimethoxy-1-oxo-1,2-dihydro­isoquinoline-3,3(4H)-dicarboxylate (24)

Method A

A solution containing 23 (100 mg, 0.26 mmol), KI (102 mg, 0.62 mmol) and Cs₂CO₃ (201 mg, 0.62 mmol) in CH₃CN (5 mL) was stirred for 12 h at room temperature. The reaction was diluted with ethyl acetate (20 mL), washed with water (20 mL) and brine (20 mL). The organic phase was dried with anhydrous Na₂SO₄ and concentrated. The solid was purified by flash chromatography, on silica gel, with ethyl acetate/hexane (9:1) as the eluent; the cyclized product 24 (11 mg, 0.03 mmol, 11% yield) and iodinated precursor 25 (25 mg, 0.05 mmol, 19% yield) were obtained.

Method B

25 (200 mg, 0.5 mmol), Cs₂CO₃ (401 mg, 1.0 mmol) and CH₃CN (5 mL) were added to an Ace sealed tube. The tube was irradiated at 200 W, with maximum temperature of 130 ºC for 10 min. Then, the reaction was diluted in ethyl acetate (20 mL), washed with water (20 mL) and brine (20 mL). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure affording the cyclized product 24 (150 mg, 0.39 mmol, 77% yield).

mp 188.9-194.4 ºC; ¹H NMR (500 MHz, CDCl₃) δ 1.25 (t, 6H, J 7.0 Hz, CO₂CH₂CH₃), 3.61 (s, 2H, Ar-CH₂), 3.95 (s, 3H, Ar-OCH₃), 3.97 (s, 3H, Ar-OCH₃), 4.19-4.27 (m, 4H, CO₂CH₂CH₃), 6.48 (s, 1H, Ar-H), 6.58 (s, 1H, NH); ¹³C NMR (126 MHz, CDCl₃) δ 13.9 (2C), 32.3, 56.2, 56.5, 63.0 (2C), 64.1, 76.8, 77.3, 95.8, 109.7, 112.8, 136.7, 158.9, 160.7, 162.6, 167.3 (2C); LRMS (ESI) m/z 388.1 (30), 386.1 ([M + H]⁺, 100); HRMS (TOF) m/z, observed: 408.0823; C₁₇H₂₀ClNO₇Na [M + Na]⁺ requires: 408.0826.

Methyl 3-chloro-2-(chloromethyl)-4,6-dimethoxybenzoate (27)

A solution containing acid 22 (200 mg, 0.87 mmol), CH₃I (82 μL, 1.31 mmol) and Cs₂CO₃ (339 mg, 1.04 mmol) in DMF (2.5 mL) was stirred for 30 min at rt. The reaction was diluted in DCM (15 mL) and extracted with saturated aqueous NaHCO₃ solution (20 mL) and brine (20 mL). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude solid was purified by flash chromatography on silica gel with ethyl acetate/hexane (2:1 to 1:1) as the eluent, providing a solid (141 mg, 0.51 mmol, 59% yield). ¹H NMR (500 MHz, CDCl₃) δ 3.88 (s, 3H, Ar-CO₂CH₃), 3.95 (s, 6H, Ar-OCH₃), 4.75 (s, 2H, Ar-CH₂-Cl), 6.53 (s, 1H, Ar-H); LRMS (ESI) m/z 281.0 (70), 279.0 ([M + H]⁺, 100); HRMS (TOF) m/z, observed: 301.0009; C₁₁H₁₂Cl₂O₄Na [M + Na]⁺ requires: 301.0010.

Diethyl 2-acetamido-2-(2-chloro-3,5-dimethoxy-6-(methoxy­carbonyl)benzyl)malonate (29)

A solution of methyl 3-chloro-6-(chloromethyl)-2,4-dimethoxybenzoate (115 mg, 0.47 mmol, 27), diethyl acetamidomalonate (102 mg, 0.47 mmol, 28), Cs₂CO₃ (368 mg, 1.13 mmol) and KI (187 mg, 1.13 mmol) in acetonitrile (5 mL) was stirred for 3 days at rt. The reaction was diluted with ethyl acetate (40 mL) and washed with water (10 mL) and brine (10 mL). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude solid was purified by flash chromatography on silica gel, using ethyl acetate/hexane (4:1 to 1:1) as the eluent, giving a white solid (134.0 mg, 60% yield). mp 169.6-173.0 ºC; ¹H NMR (500 MHz, CDCl₃) δ 1.24 (t, 6H, J 7.1 Hz, CO₂CH₂CH₃), 1.99 (s, 3H, NHAc), 3.83 (s, 3H, Ar-OCH₃), 3.84 (s, 3H, Ar-OCH₃), 3.90 (s, 3H, Ar-CO₂CH₃), 3.96 (s, 2H, Ar-CH₂-C), 4.15 (dq, 2H, J 10.8, 7.2 Hz, CO₂CH₂CH₃), 4.26 (dq, 2H, J 10.8, 7.2 Hz, CO₂CH₂CH₃), 6.44 (s, 1H, N.Ac), 6.47 (s, 1H, Ar-H); LRMS (EI) m/z 424 (M⁺ – Cl^-, 50), 314 (37), 312 (100), 244 (19), 238 (25); HRMS (TOF) m/z, observed: 482.1179; C₂₀H₂₆ClNO₉Na [M + Na]⁺ requires: 482.1193.

2-Acetamido-3-(2-chloro-3,5-dimethoxy-6-(methoxy­carbonyl)phenyl)propanoic acid (30)

To a solution of the diester 29 (51 mg, 0.1 mmol) in acetic acid (2 mL), 6 mol L^-1 HCl solution (2 mL) was added. The flask was irradiated with 150 W at 90 ºC for 10 min. The crude mixture was concentrated under reduced pressure and purified by flash chromatography on silica gel, using DCM/MeOH (9:1 to 4:1) as the eluent, providing a solid (41 mg, 10.7 mmol, quantitative). ¹H NMR (500 MHz, DMSO-d₆) δ 1.68 (s, 3H, NHAc), 3.30 (d, 1H, J 3.2 Hz, Ar-CH₂CH), 3.32 (d, 1H, J 3.4 Hz, Ar-CH₂CH), 3.80 (s, 6H, Ar-OCH₃), 3.89 (s, 3H, Ar-CO₂CH₃), 4.35-4.42 (m, 1H, Ar-CH₂CH), 6.71 (s, 1H, Ar-H), 7.22 (br s, 1H, N.Ac), 8.33 (s, 1H, CO₂H); LRMS (ESI) m/z 362.0 (20), 360.1 ([M + H]⁺, 100); HRMS (TOF) m/z, observed: 382.0661; C₁₅H₁₈ClNO₇Na [M]⁺ requires: 382.0669.

2-(Chloromethyl)-4,6-dimethoxybenzonitrile (31)

A solution containing 2-(chloromethyl)-4,6-dimethoxy­benzaldehyde (107.6 mg, 0.5 mmol, 9) and NaN₃ (33 mg, 0.5 mmol) in POCl₃ (233 μL, 2.5 mmol) was stirred overnight at 50 ºC in N₂. The mixture was concentrated under reduced pressure and the crude product was purified by flash chromatography on silica gel, using ethyl acetate/hexane (2:1) as the eluent, providing a red solid (65 mg, 0.31 mmol, 62% yield). mp 112.4-114.0 ºC; ¹H NMR (500 MHz, CDCl₃) δ 3.89 (s, 3H, Ar-OCH₃), 3.92 (s, 3H, Ar-OCH₃), 4.68 (s, 2H, Ar-CH₂), 6.43 (d, 1H, J 2.2 Hz, Ar-H), 6.67 (d, 1H, J 2.2 Hz, Ar-H); ¹³C NMR (126 MHz, CDCl₃) δ 43.4, 55.8, 56.2, 94.0, 98.2, 106.7, 110.0, 114.8, 143.7, 163.3, 164.3; LRMS (EI) m/z 213 (32), 211 (M⁺, 100), 176 (72), 146 (20); HRMS (TOF) m/z, observed: 234.0273; C₁₀H₁₀ClNO₂Na [M + Na]⁺ requires: 234.0297.

Diethyl 2-acetamido-2-(2-cyano-3,5-dimethoxybenzyl)malonate (32)

A solution of methyl 2-(chloromethyl)-4,6-dimethoxy­benzonitrile (384.5 mg, 1.8 mmol, 31), diethyl acetamidemalonate (475 mg, 2.19 mmol, 28), Cs₂CO₃ (712 mg, 2.19 mmol) and KI (364 mg, 2.19 mmol) in acetonitrile (7 mL) was stirred for overnight at rt. The mixture was diluted with ethyl acetate (15 mL) and quenched with water (10 mL) and Na₂SO₃. The organic phase was separated, extracted with aqueous 2 M hydrochloridric acid solution (7 mL) and brine (10 mL). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude solid was purified by flash chromatography on silica gel, using ethyl acetate/hexane (1:1) as the eluent, giving a white solid (548.5 mg, 1.4 mmol, 78% yield). mp 168.0-171.9 ºC; ¹H NMR (500 MHz, CDCl₃) δ 1.29 (t, 6H, J 7.1 Hz, CO₂CH₂CH₃), 2.05 (s, 3H, NHAc), 3.79 (s, 2H, Ar-CH₂), 3.80 (s, 3H, Ar-OCH₃), 3.87 (s, 3H, OCH₃), 4.23-4.37 (m, 4H, CO₂CH₂CH₃), 6.28 (d, 1H, J 2.2 Hz, Ar-H), 6.36 (d, 1H, J 2.2 Hz, Ar-H), 6.59 (s, 1H, N.Ac); ¹³C NMR (126 MHz, CDCl₃) δ 13.9 (2C), 23.1, 36.6, 55.6, 56.0, 63.0 (2C), 66.3, 76.8, 77.0, 77.3, 95.8, 97.0, 108.7, 115.5, 142.2, 163.3, 163.6, 167.3 (2C), 169.4; LRMS (ESI) m/z 393.1 (M⁺, 100); HRMS (TOF) m/z, observed: 415.1453; C₁₉H₂₄N₂O₇Na [M + Na]⁺ requires: 415.1481.

Sodium 2-acetamido-2-(2-cyano-3,5-dimethoxybenzyl)malonate (33)

A mixture containing nitrile 32 (100 mg, 0.26 mmol) and 2 M NaOH solution (10 mL) was placed in an ultrasonic bath at 80 ºC for 45 min, under N₂ atmosphere. The reaction was then treated with 2 M HCl solution at pH 2 and frozen for 2 days. After thawing, the precipitate was filtered by vacuum and water phase was removed in high vacuum affording a white solid, the disodium salt 33 (43 mg, 0.14 mmol, 54% yield). mp 157.9-187.1 ºC; ¹H NMR (500 MHz, DMSO-d₆) δ 1.92 (s, 3H, NHAc), 3.51 (s, 2H, Ar-CH₂), 3.82 (s, 3H, Ar-OCH₃), 3.87 (s, 3H, Ar-OCH₃), 6.38 (d, 1H, J 2.2 Hz, Ar-H), 6.62 (d, 1H, J 2.0 Hz, Ar-H), 7.91 (s, 1H, N.Ac); ¹³C NMR (126 MHz, DMSO-d₆) δ 22.4, 36.1, 39.0, 39.2, 39.3, 39.7, 39.8, 40.0, 55.6, 55.8, 56.3, 66.4, 95.1, 97.2, 108.2, 115.3, 142.7, 162.9, 163.3, 168.5 (2C), 169.3; LRMS (ESI) m/z 383.1 [M + 2H]⁺, 40), 337.1 [M – 2Na]⁺, 100), 117.1 (22).

2-Acetamido-3-(2-cyano-3,5-dimethoxyphenyl)propanoic acid (34)

To a solution of nitrile 32 (82 mg, 0.21 mmol) in ethanol (2 mL) 35% KOH solution (2 mL) was added and the mixture was stirred for 2.5 h at 80 ºC. Then the reaction was diluted in water (15 mL) and extracted with diethyl ether (2 × 20 mL). The aqueous phase was acidified with 2 M HCl solution until pH 2 and extracted with DCM (3 × 15 mL). The organic phase was washed with water, dried with anhydrous Na₂SO₄ and concentrated under reduced pressure, providing a white solid, the acid 34 (28 mg, 0.095 mmol, 45% yield); without further purification. ¹H NMR (500 MHz, DMSO-d₆) δ 1.77 (s, 3H, NHAc), 2.93 (dd, 1H, J 14.0, 9.9 Hz, Ar-CH₂CH), 3.18 (dd, 1H, J 14.0, 5.1 Hz, Ar-CH₂CH), 3.83 (s, 3H, Ar-OCH₃), 3.88 (s, 3H, Ar-OCH₃), 4.51 (dt, 1H, J 4.9, 3.3 Hz, Ar-CH₂CH) 6.60 (d, 1H, J 2.2 Hz, Ar-H), 6.61 (d, 1H, J 2.2 Hz, Ar-H), 8.27 (d, 1H, J 8.4 Hz, N.Ac), 12.81 (br s, 1H, CO₂H); ¹³C NMR (126 MHz, DMSO-d₆) δ 22.3, 35.5, 51.9, 55.8, 56.3, 93.3, 96.9, 108.2, 115.6, 144.2, 162.9, 163.7, 169.2, 172.6; LRMS (ESI) m/z 293.1 (M⁺, 100); HRMS (TOF) m/z, observed: 315.0944; C₁₄H₁₆N₂O₅Na [M + Na]⁺ requires: 315.0956.

1-Imino-6,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (35)

A solution of nitrile 32 (82 mg, 0.2 mmol) in 2 M HCl (1 mL) and dioxane (1.2 mL) was refluxed (at 115 ºC) for 48 h under N₂ atmosphere. The solvent was removed under reduced pressure and the residue was lyophilized, furnishing 72 mg of brown solid. The crude product was purified by semi preparative HPLC using a C18 column, providing a white solid 35 (6 mg, 0.024 mmol, 12% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 3.20 (m, 1H, Ar-CH₂CH), 3.29 (m 1H, Ar-CH₂CH), 3.88 (s, 3H, Ar-OCH₃), 3.95 (s, 3H, Ar-OCH₃), 4.48 (td, 1H, J 6.4, 3.6 Hz, Ar-CH₂CH), 6.68 (d, 1H, J 2.3 Hz, Ar-H), 6.74 (d, 1H, J 2.3 Hz, Ar-H), 8.71 (s, 1H, C=NH), 8.81 (s, 1H, CO₂H), 8.94 (d, 1H, J 3.3 Hz, CHNH); ¹³C NMR (126 MHz, DMSO-d₆) δ 35.8, 44.2, 44.4, 44.5, 44.7, 44.9, 45.1, 45.2, 55.9, 61.3, 61.8, 103.1, 106.9, 112.1, 146.1, 163.3, 166.5, 170.5, 176.5; LRMS (ESI) m/z 252.1 (15), 251.1 (M⁺, 100), 207.1 (16), 169.1 (55); HRMS (TOF) m/z, observed: 249.0867; C₁₂H₁₃N₂O₄ [M – H]^- requires: 249.0875.

• Supplementary Information
  Supplementary data (¹H and ¹³C NMR spectra) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

We express our gratitude to FAPESP (grant No. 2014/25659-8) for financial support. Oliveira Filho thanks UFABC for the fellowship. We also would like to thank Prof Dr Rodrigo Luiz Oliveira Rodrigues Cunha for providing compound 28.

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Publication Dates

• Publication in this collection
  Mar 2019

History

• Received
  13 June 2018
• Accepted
  09 Oct 2018