N,N′-Di-Boc-2H-Isoindole-2-carboxamidine—First Guanidine-Substituted Isoindole

Synthesis of N,N′-Di-Boc-2H-isoindole-2-carboxamidine, the first representative of isoindoles containing guanidine functionality, was carried out. The cycloaddition reactivity of this new Diels–Alder heterodiene was studied and the title compound was employed as a cycloaddition delivery reagent for guanidine functionality. Higher reactivity was found in comparison with the corresponding pyrrole derivative. Substitution with fluorine or guanidine functionality does not change the reactivities of isoindoles, and these findings are in good accord with computational results.


Introduction
Guanidines are a class of nitrogen-containing molecules with very interesting physico-chemical properties, [1] especially very high basicity [2] and biological activity [3]. Hence, various aspects of guanidine chemistry were extensively studied computationally and experimentally, including their synthesis. The most viable synthetic routes towards polycyclic, complex organic molecules include the introduction of guanidine functionality at the later stages of multi-step synthesis, such as in tetrodotoxin synthesis [4,5]. One of the most efficient ways for the construction of polycyclic molecules is Diels-Alder (DA) cycloaddition; however, the cycloaddition approach to polycycles containing guanidine functionality has been rarely utilized. Diels-Alder cycloadditions, which involve diene or dienophile partners possessing guanidine functionality, were summarized in Figure 1 [6,7] which also depicts guanidine delivery cycloaddition reagents [8,9]. All of these are unsymmetrical diene molecules, and their cycloaddition reactions lead to the formation of unsymmetrical products.
For the purpose of our studies towards the synthesis of polycyclic molecules, symmetrical diene reagents were required, and, in view of earlier studies [10,11], pyrrole and isoindole N-carboxamidine derivatives 1 and 2 were selected. These molecules possess C 2v symmetry [12] and upon cycloaddition form guanidine functionality which is 'protected' in pyrrole moiety. Whereas pyrrole-2-carboxamidine 1 is a known compound, its DA cycloaddition properties were not reported. Only rhodium-catalyzed [4+3] cycloaddition was utilized to prepare tropane bicyclo [3.2.1] octane skeleton 3, where pyrrole acted as a dipolarophile partner (Figure 1) [13]. Corresponding isoindole-2-carboxamidines have not been synthetized previously.
The objective of this work is to prepare isoindole-2-carboxamidine 2, the first representative of an isoindole guanidine cycloaddition delivery reagent, and assess its cycloaddition properties experimentally and computationally. Our preliminary DFT computational study revealed that this approach is feasible and both pyrrole and isoindole dienes are predicted to have sufficient reactivity [14].

Cycloadditions of 1-Carboxamidine Pyrrole
The 1-(N′,N′-Di-Boc)pyrrole carboxamidine 1 was prepared according to the literature starting from 3-pyrroline [15,16]. Pyrrole 1 showed poor reactivity in cycloaddition reactions when acting as 1,3-diene (Scheme 1). For instance, the thermal reaction of 1 with N-methylmaleimide did not provide the expected cycloadduct 5. In order to increase its reactivity, an extremely high-pressure technique was employed [17]. The pressurization at 10 kbar, for 2 days, in dichloromethane resulted in the formation of 5 in 51 % yield. It was found that product 5 was unstable in CHCl3 solution (also in solid state) and quickly cycloreverses back to reactants. This behavior could explain the failure of thermal conditions. An alternative way to increase dienes' reactivity is to employ more reactive dienophiles. Thermal and high-pressure reactions with naphthoquinone did not provide conclusive evidence for the formation of a cycloadduct. Equally unsuccessful were reactions of mechanochemically solid state in-situ-generated imide 8 [18] in a ball mill, due to harsh conditions for the guanidine moiety of 1. Reaction with related anhydride 10 [19] also provided a complex reaction mixture.

Cycloadditions of 1-Carboxamidine Pyrrole
The 1-(N ,N -Di-Boc)pyrrole carboxamidine 1 was prepared according to the literature starting from 3-pyrroline [15,16]. Pyrrole 1 showed poor reactivity in cycloaddition reactions when acting as 1,3-diene (Scheme 1). For instance, the thermal reaction of 1 with N-methylmaleimide did not provide the expected cycloadduct 5. In order to increase its reactivity, an extremely high-pressure technique was employed [17]. The pressurization at 10 kbar, for 2 days, in dichloromethane resulted in the formation of 5 in 51% yield. It was found that product 5 was unstable in CHCl 3 solution (also in solid state) and quickly cycloreverses back to reactants. This behavior could explain the failure of thermal conditions. An alternative way to increase dienes' reactivity is to employ more reactive dienophiles. Thermal and high-pressure reactions with naphthoquinone did not provide conclusive evidence for the formation of a cycloadduct. Equally unsuccessful were reactions of mechanochemically solid state in-situ-generated imide 8 [18] in a ball mill, due to harsh conditions for the guanidine moiety of 1. Reaction with related anhydride 10 [19] also provided a complex reaction mixture.

Synthesis of Isoindoles and Their Reactivity
Further increase in the reactivity of pyrroles could be achieved by the addition of a benzene ring, i.e., to use isoindole derivatives as dienes. Synthesis of isoindole precursor 20 in four reaction steps is shown in Scheme 2. It follows the already established synthesis of 7-azabenzonorbornadiene 18, and subsequent guanylation with N,N -Di-Boc-1H-pyrazole-1-carboxamidine provided 20 in 62% yield. Alternatively, 20 could be prepared by in-situgenerated benzyne cycloaddition with pyrrole 1 (84%). Preparation of a nitro derivative of 20 was achieved by in-situ generation of 4-nitro benzyne from iodonium salt 22 [20] and its reaction with pyrrole 1, which provided cycloadduct 23 in 32% yield. These reactions show that the reactivity of pyrrole-1-carboxamidine 1 could be increased by the presence of a highly reactive dienophile such as arynes.

Synthesis of Isoindoles and Their Reactivity
Further increase in the reactivity of pyrroles could be achieved by the addition of a benzene ring, i.e., to use isoindole derivatives as dienes. Synthesis of isoindole precursor 20 in four reaction steps is shown in Scheme 2. It follows the already established synthesis of 7-azabenzonorbornadiene 18, and subsequent guanylation with N,N′-Di-Boc-1H-pyrazole-1-carboxamidine provided 20 in 62% yield. Alternatively, 20 could be prepared by in-situ-generated benzyne cycloaddition with pyrrole 1 (84%). Preparation of a nitro derivative of 20 was achieved by in-situ generation of 4-nitro benzyne from iodonium salt 22 [20] and its reaction with pyrrole 1, which provided cycloadduct 23 in 32% yield. These reactions show that the reactivity of pyrrole-1-carboxamidine 1 could be increased by the presence of a highly reactive dienophile such as arynes. Warrener's cycloaddition/elimination/cycloreversion method employing bis(2pyridyl)-sym-1,2,4,5-tetrazine 24 [21,22] was used for the generation of isoindole 2 (Scheme 3). The formation of 2 was confirmed by 1 H NMR spectroscopy, by spectrum recorded 30 min after the addition of 24 to a solution of 20 in an NMR tube ( Figure 2). The most characteristic signals which indicate the presence of 2 are a singlet of H1,3 appearing at δ 7.57, whereas aromatic multiplets of H4,7 and H5,6 are found at δ 7.36 and 6.85. However, trapping experiments offer indirect but more solid evidence of its formation. Warrener's cycloaddition/elimination/cycloreversion method employing bis(2-pyridyl)sym-1,2,4,5-tetrazine 24 [21,22] was used for the generation of isoindole 2 (Scheme 3). The formation of 2 was confirmed by 1 H NMR spectroscopy, by spectrum recorded 30 min after the addition of 24 to a solution of 20 in an NMR tube ( Figure 2). The most characteristic signals which indicate the presence of 2 are a singlet of H 1,3 appearing at δ 7.57, whereas aromatic multiplets of H 4,7 and H 5,6 are found at δ 7.36 and 6.85. However, trapping experiments offer indirect but more solid evidence of its formation. Warrener's cycloaddition/elimination/cycloreversion method employing bis(2pyridyl)-sym-1,2,4,5-tetrazine 24 [21,22] was used for the generation of isoindole 2 (Scheme 3). The formation of 2 was confirmed by 1 H NMR spectroscopy, by spectrum recorded 30 min after the addition of 24 to a solution of 20 in an NMR tube ( Figure 2). The most characteristic signals which indicate the presence of 2 are a singlet of H1,3 appearing at δ 7.57, whereas aromatic multiplets of H4,7 and H5,6 are found at δ 7.36 and 6.85. However, trapping experiments offer indirect but more solid evidence of its formation. Scheme 3. Generation of isoindole 2 using tetrazine method.   Warrener's cycloaddition/elimination/cycloreversion method employing bis(2pyridyl)-sym-1,2,4,5-tetrazine 24 [21,22] was used for the generation of isoindole 2 (Scheme 3). The formation of 2 was confirmed by 1 H NMR spectroscopy, by spectrum recorded 30 min after the addition of 24 to a solution of 20 in an NMR tube ( Figure 2). The most characteristic signals which indicate the presence of 2 are a singlet of H1,3 appearing at δ 7.57, whereas aromatic multiplets of H4,7 and H5,6 are found at δ 7.36 and 6.85. However, trapping experiments offer indirect but more solid evidence of its formation. Scheme 3. Generation of isoindole 2 using tetrazine method.    Scheme 4 summarizes the cycloaddition properties of isoindole-2-carboxamidine 2. When 2 was generated in the presence of dienophiles N-methylmaleimide, dimethylacetylenedicarboxylate (DMAD), and benzoquinone, corresponding cycloadducts 26, 28, and 29 were obtained (in 91, 80 and 77% yields, respectively). In variance, norbornenes 10, 20, 30, and 31 did not react or afforded intractable mixtures, regardless of reaction conditions (thermal or high pressure). The endo-adduct 26 was solely formed, as shown by the single methyl resonance at δ 2.28 in the 1 H NMR spectrum (see Supplementary Materials), and the endo-configuration is proven by the shielding of the N-methyl protons by the ring current effect of the aromatic ring [22]. Furthermore, the exo-protons are multiplets, is characteristic of the endo-adducts of isoindoles [23,24]. This endo-stereospecificity is similar to N-benzyl-isoindole cycloaddition [25] and in variance with maleic anhydride reactions of isoindoles where exo/endo mixtures were formed, [24,26] whereas the outcome of cycloadditions of 2-substituted isoindoles with tolyl-maleimide was not specified [27].
An interesting feature of the 1 H NMR spectra of cycloaddition products 26 and 28 is the broadness of bridgehead signals at 20 • C. Recording the spectra at 50 • C led to the sharpening of the signal, whereas cooling down to 5 • C gives two sets of bridgehead signals, which are associated with nitrogen inversion [28]. The N-inversion barrier in 26 is estimated to be low, 13.5 kcal mol −1 in deuterated chloroform. A similar broadness of bridgehead protons was observed for pyrrole cycloadduct 5; however, this adduct is thermally unstable and quickly cycloreverses.
In continuation, the electronics of isoindoles were altered by fluorine substituents on the aromatic ring and positioning of the guanidine functionality. Tetrafluoro isoindole precursor 37 was prepared in 35% yield by mechanochemical guanylation [29] of the known 7-azabenzonorbornadiene 36 [30,31] (Scheme 5). It was found that fluorine substitution did not have noticeable effects on the cycloaddition reactivity of isoindole. When tetrafluoroisoindole 38 was trapped in a tetrazine reaction with 37, the endo-cycloadduct 39 was obtained in 11% yield, while, similarly to isoindole 2, tetrafluoro derivative 38 also did not react with its precursor 37. 29 were obtained (in 91, 80 and 77 % yields, respectively). In variance, norbornenes 10, 20, 30, and 31 did not react or afforded intractable mixtures, regardless of reaction conditions (thermal or high pressure). The endo-adduct 26 was solely formed, as shown by the single methyl resonance at δ2.28 in the 1 H NMR spectrum (see Supplementary Materials), and the endo-configuration is proven by the shielding of the N-methyl protons by the ring current effect of the aromatic ring [22]. Furthermore, the exo-protons are multiplets, is characteristic of the endo-adducts of isoindoles [23,24]. This endo-stereospecificity is similar to N-benzyl-isoindole cycloaddition [25] and in variance with maleic anhydride reactions of isoindoles where exo/endo mixtures were formed, [24,26] whereas the outcome of cycloadditions of 2-substituted isoindoles with tolyl-maleimide was not specified [27].

Scheme 4. Cycloaddition reactions of isoindole 2.
An interesting feature of the 1 H NMR spectra of cycloaddition products 26 and 28 is the broadness of bridgehead signals at 20 °C. Recording the spectra at 50 °C led to the sharpening of the signal, whereas cooling down to 5 °C gives two sets of bridgehead signals, which are associated with nitrogen inversion [28]. The N-inversion barrier in 26 is estimated to be low, 13.5 kcal mol −1 in deuterated chloroform. A similar broadness of bridgehead protons was observed for pyrrole cycloadduct 5; however, this adduct is thermally unstable and quickly cycloreverses.
In continuation, the electronics of isoindoles were altered by fluorine substituents on the aromatic ring and positioning of the guanidine functionality. Tetrafluoro isoindole precursor 37 was prepared in 35% yield by mechanochemical guanylation [29] of the known 7-azabenzonorbornadiene 36 [30,31] (Scheme 5). It was found that fluorine substitution did not have noticeable effects on the cycloaddition reactivity of isoindole. When tetrafluoroisoindole 38 was trapped in a tetrazine reaction with 37, the endo-cycloadduct 39 was obtained in 11% yield, while, similarly to isoindole 2, tetrafluoro derivative 38 also did not react with its precursor 37. Until now, isoindoles substituted on an aromatic ring with the nitrogen atom have been known only with the nitro group, [32][33][34][35] and we prepared the first example of a guanidine aromatic-ring-substituted isoindole. The synthetic route for the introduction of guanidine functionality at position 5 of the isoindole ring in 45 is depicted in Scheme 6. In-situ-generated 4-nitro-benzyne was reacted with 1-benzyloxycarbonyl pyrrole 41 to afford the known cycloadduct 42 [23]. The nitro group was reduced by Al/Hg and the amine 43 was obtained in 66% yield. Guanylation in solution led to the formation of isoindole precursor 44 in 86% yield. This compound was treated with tetrazine 24 in chloroform and intermediate isoindole 45 was trapped as N-methylmaleimide cycloadduct 46 (66%). The change in the position of guanidine functionality and N-CBz substitution did not increase the cycloaddition reactivity of isoindole. Analogously to isoindoles 5 and 38, in the case of 45, a reaction with 44 as a dienophile was not observed. These results indicate the similar cycloaddition reactivity of all three investigated guanidine isoindoles. Until now, isoindoles substituted on an aromatic ring with the nitrogen atom have been known only with the nitro group, [32][33][34][35] and we prepared the first example of a guanidine aromatic-ring-substituted isoindole. The synthetic route for the introduction of guanidine functionality at position 5 of the isoindole ring in 45 is depicted in Scheme 6. Insitu-generated 4-nitro-benzyne was reacted with 1-benzyloxycarbonyl pyrrole 41 to afford the known cycloadduct 42 [23]. The nitro group was reduced by Al/Hg and the amine 43 was obtained in 66% yield. Guanylation in solution led to the formation of isoindole precursor 44 in 86% yield. This compound was treated with tetrazine 24 in chloroform and intermediate isoindole 45 was trapped as N-methylmaleimide cycloadduct 46 (66%). The change in the position of guanidine functionality and N-CBz substitution did not increase the cycloaddition reactivity of isoindole. Analogously to isoindoles 5 and 38, in the case of 45, a reaction with 44 as a dienophile was not observed. These results indicate the similar cycloaddition reactivity of all three investigated guanidine isoindoles. guanidine functionality at position 5 of the isoindole ring in 45 is depicted in Scheme 6. In-situ-generated 4-nitro-benzyne was reacted with 1-benzyloxycarbonyl pyrrole 41 to afford the known cycloadduct 42 [23]. The nitro group was reduced by Al/Hg and the amine 43 was obtained in 66% yield. Guanylation in solution led to the formation of isoindole precursor 44 in 86% yield. This compound was treated with tetrazine 24 in chloroform and intermediate isoindole 45 was trapped as N-methylmaleimide cycloadduct 46 (66%). The change in the position of guanidine functionality and N-CBz substitution did not increase the cycloaddition reactivity of isoindole. Analogously to isoindoles 5 and 38, in the case of 45, a reaction with 44 as a dienophile was not observed. These results indicate the similar cycloaddition reactivity of all three investigated guanidine isoindoles. Previous density functional theory (DFT) calculations B3LYP/6-31G(d) predict that activation energies (E a ) for reactions of pyrrole and isoindole-2-carboxamidine with DMAD are 32.37 and 23.17 kcal mol −1 , respectively, indicating that the amidine substitution decreases E a by 4-5 kcal mol −1 in comparison to parent unsubstituted dienes, whereas Boc protection of amidinopyrrole causes a further drop in E a by 2.5 kcal mol −1 . Now, these theoretical predictions are supplemented with the M062X/6-311+G** calculations [23] of the reaction of acetylene with pyrrole and isoindoles. All located transition states possess structures resembling the synchronous concerted mechanism of Diels-Alder reactions, such as the one illustrated in Figure 3. Computed activation-free energies (∆G } values) are given in Figure 3 and reveal similar predictions to the previously obtained B3LYP calculations. Firstly, N-substitution with amidine lowers ∆G } by 1.5-2.3 kcal mol −1 . The largest difference in ∆G } values was obtained when pyrrole was fused with a benzene ring in isoindoles, which is in qualitative accordance with published AM1 results [23]. The position of an amidine (guanidine) substituent and the addition of fluorine atoms has only a marginal effect on the ∆G } values, with differences in the reactivity of three experimentally studied isoindoles within 0.54 kcal mol −1 . These predictions are in full accordance with almost identical experimentally observed reactivities of three isoindoles. the reaction of acetylene with pyrrole and isoindoles. All located transition states possess structures resembling the synchronous concerted mechanism of Diels-Alder reactions, such as the one illustrated in Figure 3. Computed activation-free energies (ΔG ⧧ values) are given in Figure 3 and reveal similar predictions to the previously obtained B3LYP calculations. Firstly, N-substitution with amidine lowers ΔG ⧧ by 1.5-2.3 kcal mol −1 . The largest difference in ΔG ⧧ values was obtained when pyrrole was fused with a benzene ring in isoindoles, which is in qualitative accordance with published AM1 results [23]. The position of an amidine (guanidine) substituent and the addition of fluorine atoms has only a marginal effect on the ΔG ⧧ values, with differences in the reactivity of three experimentally studied isoindoles within 0.54 kcal mol −1 . These predictions are in full accordance with almost identical experimentally observed reactivities of three isoindoles.

General
Solvents and chemicals were obtained from Tokyo Chemical Industry (Tokyo, Japan)

General
Solvents and chemicals were obtained from Tokyo Chemical Industry (Tokyo, Japan) and Sigma Aldrich (Burlington, VT, USA). Kemika (Zagreb, Croatia), Sigma Aldrich, and VWR Chemicals (Radnor, PA, USA) supplied the solvents, which were used without further purification, unless otherwise stated. The NMR spectra were recorded on Bruker Avance 300 MHz and Bruker Avance 600 MHz spectrometers in deuterated solvents. Chemical shifts (δ) are given in ppm using tetramethylsilane (TMS) as an internal standard, whereas coupling constants (J) are expressed in Hertz (Hz). The following abbreviations were used to describe multiplicity in the 1 H spectra: (s) singlet; (d) doublet; (dd) doublet of doublets; (t) triplet; (m) multiplet; (brs) broad signal. Fourier Transform Infrared Attenuated Total Reflection PerkinElmer UATR Two Spectrometer (range 400-4000 cm −1 ) was used to record infrared spectra (FTIR-ATR). Milling reactions were carried out in Retsch MM400 vibrational mill (frequency 30 Hz), using stainless steel (SS) vials (10 mL) and one 12 mm size SS milling ball. High-pressure reactions were performed in Teflon vials (V = 1.5 mL) using a high-pressure-piston cylinder apparatus (Unipress, Polish Academy of Sciences), and pentane as a pressure-transmitting liquid. Thin-layer chromatography (TLC) was performed on silica-gel plates (silica gel 60 F 254 , Merck), whereas silica gel (Silica gel 60, 0.063-0.200 mm, Merck, Darmstadt, Germany) was used for column chromatography. High-resolution mass spectra (HRMS) were recorded on Agilent 6550 Series Accurate-Mass-Quadrupole Time-of-Flight (Q-TOF) Agilent 1290 Infinity II instrument.

Synthesis of 42
Under argon, dry toluene (11 mL) was added to Cbz-pyrrole 41 (200 mg, 1.0 mmol) and iodonium salt 22 (539 mg, 1.0 mmol). LiHDMS solution in toluene (1.0 mL, 1.0 mmol, 1 M) was added dropwise and the resulting mixture was stirred for 1 h at room temperature. The reaction was quenched with saturated NH 4 Cl solution (40 mL) and extracted with EtOAc (3x30 mL); combined extracts were dried with Na 2 SO 4 and evaporated. The crude mixture was purified by radial chromatography using CH 2 Cl 2 and gradually increasing polarity with MeOH. Product 42 was isolated as a viscous yellow oil (128 mg, 40%). 1

Synthesis of 43
Azabenzonorbornadiene 42 (89 mg, 0.28 mmol) was dissolved in THF/H 2 O mixture (40 mL, 10% H 2 O) and heated to 60 • C. Aluminium amalgam was prepared by immersing aluminium foil (400 mg) in a solution of HgCl 2 (500 mg) in water (50 mL) for 1 min, followed by washing in ethanol (50 mL) and diethyl ether (50 mL). Amalgam was added to the solution and the mixture was continuously heated for 1 h, filtered through Celite and washed with THF. The filtrate was evaporated and purified by radial chromatography Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27248954/s1, 1 H and 13 C NMR and IR spectra for the synthesized compounds.