Study on the Alkylation Reactions of N(7)-Unsubstituted 1,3-Diazaoxindoles

The chemistry of the 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (1,3-diazaoxindole) compound family, possessing a drug-like scaffold, is unexplored. In this study, the alkylation reactions of N(7)-unsubstituted 5-isopropyl-1,3-diazaoxindoles bearing various substituents at the C(2) position have been investigated. The starting compounds were synthesized from the C(5)-unsubstituted parent compounds by condensation with acetone and subsequent catalytic reduction of the 5-isopropylidene moiety. Alkylation of the thus obtained 5-isopropyl derivatives with methyl iodide or benzyl bromide in the presence of a large excess of sodium hydroxide led to 5,7-disubstituted derivatives. Use of butyllithium as the base rendered alkylation in the C(5) position possible with reasonable selectivity, without affecting the N(7) atom. During the study on the alkylation reactions, some interesting by-products were also isolated and characterized.

In continuation of our studies on the monoalkylation at the C(3) position of N-unprotected oxindole (1) [14][15][16] to give 3-alkyloxindoles 9 and on the selective introduction of the second alkyl group, without N-alkylation to afford N-unprotected 3,3-dialkyloxindoles 10 (Scheme 2) [17], we now aimed to elaborate a method for the introduction of two different alkyl groups into the structurally related carbonyl-adjacent C (5) site of N-unprotected 1,3-diazaoxindoles 6.
An alternative approach for the preparation of N(7)-unsubstituted-C(5)-monoor C(5)-disubstituted 1,3-diazaoxindoles is also described [27][28][29][30][31]. Herein, the pyrrolo [2,3-d]pyrimidin-6-one scaffold is synthesized by a condensation of building blocks already containing the required substituents in the appropriate positions. This strategy is exemplified by the reaction sequence outlined in Scheme 7. All the above reactions led to the desired C(5)-alkyl or C(5)-dialkyl derivatives in low, moderate or undisclosed yields. The selectivity between the C(5) mono-and dialkyl products, and the formation of N(7)-alkylated by-products were not discussed.
In the reaction of amidines 20 with the corresponding substituted malonitriles 21, C(5)-disubstituted 1,3-diazaoxindoles 23 were obtained, obviously via pyrimidines 22. The common drawback of these reactions is that the C(5) substituents of the target compounds have to be present in the starting materials 21, thereby limiting the versatility of this approach.

Results and Discussion
In the present study, we aimed to elaborate a general procedure for the introduction of two different alkyl groups into the C(5) position of N(7)-unprotected 1,3-diazaoxindoles 6. We decided to use the isopropyl group as the first alkyl substituent in position C(5), in order to increase steric hindrance in the second alkylation reaction. 5-Isopropyl-1,3-diazaoxindoles 24 can be prepared by using methods described in the literature for C(3)-alkylation of oxindoles [32][33][34]. Condensation of 1,3-diazaoxindoles 6 with acetone either under basic or acidic conditions afforded 5-isopropylidene-1,3-diazaoxindoles 25, which were reduced by catalytic hydrogenation to give 5-isopropyl-1,3-diazaoxindoles 24 (Scheme 8).
Molecules 2017, 22, 846 5 of 22 In the reaction of amidines 20 with the corresponding substituted malonitriles 21, C(5)-disubstituted 1,3-diazaoxindoles 23 were obtained, obviously via pyrimidines 22. The common drawback of these reactions is that the C(5) substituents of the target compounds have to be present in the starting materials 21, thereby limiting the versatility of this approach.

Results and Discussion
In the present study, we aimed to elaborate a general procedure for the introduction of two different alkyl groups into the C(5) position of N(7)-unprotected 1,3-diazaoxindoles 6. We decided to use the isopropyl group as the first alkyl substituent in position C (5), in order to increase steric hindrance in the second alkylation reaction. 5-Isopropyl-1,3-diazaoxindoles 24 can be prepared by using methods described in the literature for C(3)-alkylation of oxindoles [32][33][34]. Condensation of 1,3-diazaoxindoles 6 with acetone either under basic or acidic conditions afforded 5-isopropylidene-1,3-diazaoxindoles 25, which were reduced by catalytic hydrogenation to give 5-isopropyl-1,3diazaoxindoles 24 (Scheme 8). In order to study the introduction of a second alkyl group into the C(5) position of 5-isopropyl-1,3-diazaoxindoles 24, first we treated 2-methyl derivative 24b with MeI (1.2 eq.) in the presence of sodium hydroxide (NaOH, 2.2 eq.) in DMF at room temperature (Scheme 9). In addition to C(5)-methylated product 26b, a substantial amount of C (5),N(7)-dimethylated compound 27b was also formed (besides unidentified minor products), as detected by the LC-MS analysis of the product mixture. After purification by flash chromatography, products 26b and 27b were isolated in 42 and 25% yields, respectively. This finding is in agreement with the experience gained with oxindoles [35][36][37][38]: sodium bases seem to be unsuitable for the high-yielding selective introduction of a second substituent into position C(5) also in the case of 1,3-diaza-derivatives 24. Scheme 9. Formation of a product mixture during the alkylation of 24b in the presence of NaOH.
Molecules 2017, 22, 846 5 of 22 In the reaction of amidines 20 with the corresponding substituted malonitriles 21, C(5)-disubstituted 1,3-diazaoxindoles 23 were obtained, obviously via pyrimidines 22. The common drawback of these reactions is that the C(5) substituents of the target compounds have to be present in the starting materials 21, thereby limiting the versatility of this approach.

Results and Discussion
In the present study, we aimed to elaborate a general procedure for the introduction of two different alkyl groups into the C(5) position of N(7)-unprotected 1,3-diazaoxindoles 6. We decided to use the isopropyl group as the first alkyl substituent in position C (5), in order to increase steric hindrance in the second alkylation reaction. 5-Isopropyl-1,3-diazaoxindoles 24 can be prepared by using methods described in the literature for C(3)-alkylation of oxindoles [32][33][34]. Condensation of 1,3-diazaoxindoles 6 with acetone either under basic or acidic conditions afforded 5-isopropylidene-1,3-diazaoxindoles 25, which were reduced by catalytic hydrogenation to give 5-isopropyl-1,3diazaoxindoles 24 (Scheme 8). In order to study the introduction of a second alkyl group into the C(5) position of 5-isopropyl-1,3-diazaoxindoles 24, first we treated 2-methyl derivative 24b with MeI (1.2 eq.) in the presence of sodium hydroxide (NaOH, 2.2 eq.) in DMF at room temperature (Scheme 9). In addition to C(5)-methylated product 26b, a substantial amount of C (5),N(7)-dimethylated compound 27b was also formed (besides unidentified minor products), as detected by the LC-MS analysis of the product mixture. After purification by flash chromatography, products 26b and 27b were isolated in 42 and 25% yields, respectively. This finding is in agreement with the experience gained with oxindoles [35][36][37][38]: sodium bases seem to be unsuitable for the high-yielding selective introduction of a second substituent into position C(5) also in the case of 1,3-diaza-derivatives 24. Scheme 9. Formation of a product mixture during the alkylation of 24b in the presence of NaOH.
First we carried out reactions using a large excess of both BuLi and the alkylating agent in order to obtain a full picture of the regiochemistry of the alkylations. Methylation of compounds 24a-c with BuLi (3.0 eq.) and MeI (3.0 eq.) was performed as follows (Scheme 11): (i) a solution of compounds 24a-c in THF was added dropwise to the solution of BuLi in hexanes and THF −78 °C, and the reaction mixture was stirred at −78 °C for further 30 min; (ii) the reaction mixture was allowed to warm to −20 °C; (iii) a solution of MeI in THF was added dropwise at −20 °C; (iv) the mixture was allowed to warm to room temperature; (v) then it was stirred at this temperature for further 1.5-2 h. This protocol led to a mixture of 5-isopropyl-5-methyl-(compounds 26a-c) and 5-isopropyl-5,7-dimethyl-1,3-diazaoxindoles 27a-c. The yields of isolated products 26a-c and 27a-c after work-up and chromatographic purification can be found in Table 2 indicating that, despite the high excess of BuLi and MeI, the selectivity of the alkylation was quite high in the case of the 2-unsubstituted congener 24a (entry 1), while it was poor in the case of the 2-methyl (24b) and 2-phenyl (24c) derivatives (entries 2-3). For R 1 , R 2 , X, and yields, see Table 1 Scheme 10. 5,7-Dialkylation of 5-isopropyl-1,3-diazaoxindoles 24a-c in the presence of NaOH.
As demonstrated by the reactions carried out using NaOH at our laboratory and by the procedures described in the literature using sodium, potassium or cesium bases for the deprotonation of 1,3-diazaoxindoles, selective alkylation at the C(5) position could not be achieved. Therefore, we continued our efforts by applying BuLi as the base in the alkylation reactions, which proved to be the optimal base in the analogous regioselective C(3)-alkylation reactions of N-unprotected oxindoles, particularly for the introduction of a second substituent into the carbonyl-adjacent C(3) position of N-unprotected 3-monoalkyloxindoles 9 (Scheme 2) [17,39].
First we carried out reactions using a large excess of both BuLi and the alkylating agent in order to obtain a full picture of the regiochemistry of the alkylations. Methylation of compounds 24a-c with BuLi (3.0 eq.) and MeI (3.0 eq.) was performed as follows (Scheme 11): (i) a solution of compounds 24a-c in THF was added dropwise to the solution of BuLi in hexanes and THF −78 • C, and the reaction mixture was stirred at −78 • C for further 30 min; (ii) the reaction mixture was allowed to warm to −20 • C; (iii) a solution of MeI in THF was added dropwise at −20 • C; (iv) the mixture was allowed to warm to room temperature; (v) then it was stirred at this temperature for further 1.5-2 h. This protocol led to a mixture of 5-isopropyl-5-methyl-(compounds 26a-c) and 5-isopropyl-5,7-dimethyl-1,3-diazaoxindoles 27a-c. The yields of isolated products 26a-c and 27a-c after work-up and chromatographic purification can be found in Table 2 indicating that, despite the high excess of BuLi and MeI, the selectivity of the alkylation was quite high in the case of the 2-unsubstituted congener 24a (entry 1), while it was poor in the case of the 2-methyl (24b) and 2-phenyl (24c) derivatives (entries 2-3).  Table 2 Scheme 11. Methylation of 5-isopropyl-1,3-diazaoxindoles 24a-c in the presence of a large excess of BuLi and MeI.
When deprotonation of 2-methyl derivative 24b with BuLi (3.0 eq.) was performed at −20 °C (instead of −78 °C), and the reaction mixture was allowed to warm to room temperature, finally MeI (3.0 eq.) was added at this temperature (instead of −20 °C), methylation of the 2-methyl moiety was also observed resulting in 2-ethyl derivative 28 ( Figure 2) as a minor product in 11% yield after flash chromatography, besides 26b (30%) and 27b (17%). A similar reactivity of the methyl moiety of 2-methylpyrimidines was observed by other research groups [40,41]. As the consequence of an insufficient pre-inertization with argon, 5-hydroxy byproducts 29a-c ( Figure 2) were also isolated in some cases during our preliminary experiments. The targeted synthesis of the 5-hydroxy derivatives 29a-c was carried out with BuLi (2.5 eq.) without alkylating agent and under non-inert conditions with good yields (Scheme 12). The presence of the hydroxy moiety in products 29a-c renders further functionalizations possible thereby making these compounds valuable synthetic building blocks. An analogous oxidative side reaction via the corresponding hydroperoxide is also described in the oxindole family [42], and the corresponding 3-hydroxy derivatives in the oxindole series were also synthesised at our laboratory [17]. In order to achieve full conversion of the starting materials 24, to maximize the amount of monomethyl products 26 and to minimize that of dimethyl byproducts 27, various conditions were explored, including the excess of BuLi and MeI, different deprotonation temperatures and the temperature of MeI addition (Scheme 13). It was found that significantly different protocols had to be used for starting materials 24a, 24b and 24c. Deprotonation with BuLi leading to the formation of the corresponding dianions was performed at −78 °C in each case, although with different amounts of BuLi (2.4-3.0 eq.). Then, contrary to our earlier observations with 3-monoalkyloxindoles (9) where When deprotonation of 2-methyl derivative 24b with BuLi (3.0 eq.) was performed at −20 • C (instead of −78 • C), and the reaction mixture was allowed to warm to room temperature, finally MeI (3.0 eq.) was added at this temperature (instead of −20 • C), methylation of the 2-methyl moiety was also observed resulting in 2-ethyl derivative 28 ( Figure 2) as a minor product in 11% yield after flash chromatography, besides 26b (30%) and 27b (17%). A similar reactivity of the methyl moiety of 2-methylpyrimidines was observed by other research groups [40,41].  Table 2 Scheme 11. Methylation of 5-isopropyl-1,3-diazaoxindoles 24a-c in the presence of a large excess of BuLi and MeI.
When deprotonation of 2-methyl derivative 24b with BuLi (3.0 eq.) was performed at −20 °C (instead of −78 °C), and the reaction mixture was allowed to warm to room temperature, finally MeI (3.0 eq.) was added at this temperature (instead of −20 °C), methylation of the 2-methyl moiety was also observed resulting in 2-ethyl derivative 28 ( Figure 2) as a minor product in 11% yield after flash chromatography, besides 26b (30%) and 27b (17%). A similar reactivity of the methyl moiety of 2-methylpyrimidines was observed by other research groups [40,41]. As the consequence of an insufficient pre-inertization with argon, 5-hydroxy byproducts 29a-c ( Figure 2) were also isolated in some cases during our preliminary experiments. The targeted synthesis of the 5-hydroxy derivatives 29a-c was carried out with BuLi (2.5 eq.) without alkylating agent and under non-inert conditions with good yields (Scheme 12). The presence of the hydroxy moiety in products 29a-c renders further functionalizations possible thereby making these compounds valuable synthetic building blocks. An analogous oxidative side reaction via the corresponding hydroperoxide is also described in the oxindole family [42], and the corresponding 3-hydroxy derivatives in the oxindole series were also synthesised at our laboratory [17]. In order to achieve full conversion of the starting materials 24, to maximize the amount of monomethyl products 26 and to minimize that of dimethyl byproducts 27, various conditions were explored, including the excess of BuLi and MeI, different deprotonation temperatures and the temperature of MeI addition (Scheme 13). It was found that significantly different protocols had to be used for starting materials 24a, 24b and 24c. Deprotonation with BuLi leading to the formation of the corresponding dianions was performed at −78 °C in each case, although with different amounts of BuLi (2.4-3.0 eq.). Then, contrary to our earlier observations with 3-monoalkyloxindoles (9) where As the consequence of an insufficient pre-inertization with argon, 5-hydroxy byproducts 29a-c ( Figure 2) were also isolated in some cases during our preliminary experiments. The targeted synthesis of the 5-hydroxy derivatives 29a-c was carried out with BuLi (2.5 eq.) without alkylating agent and under non-inert conditions with good yields (Scheme 12). The presence of the hydroxy moiety in products 29a-c renders further functionalizations possible thereby making these compounds valuable synthetic building blocks. An analogous oxidative side reaction via the corresponding hydroperoxide is also described in the oxindole family [42], and the corresponding 3-hydroxy derivatives in the oxindole series were also synthesised at our laboratory [17].  Table 2 Scheme 11. Methylation of 5-isopropyl-1,3-diazaoxindoles 24a-c in the presence of a large excess of BuLi and MeI.
When deprotonation of 2-methyl derivative 24b with BuLi (3.0 eq.) was performed at −20 °C (instead of −78 °C), and the reaction mixture was allowed to warm to room temperature, finally MeI (3.0 eq.) was added at this temperature (instead of −20 °C), methylation of the 2-methyl moiety was also observed resulting in 2-ethyl derivative 28 ( Figure 2) as a minor product in 11% yield after flash chromatography, besides 26b (30%) and 27b (17%). A similar reactivity of the methyl moiety of 2-methylpyrimidines was observed by other research groups [40,41]. As the consequence of an insufficient pre-inertization with argon, 5-hydroxy byproducts 29a-c ( Figure 2) were also isolated in some cases during our preliminary experiments. The targeted synthesis of the 5-hydroxy derivatives 29a-c was carried out with BuLi (2.5 eq.) without alkylating agent and under non-inert conditions with good yields (Scheme 12). The presence of the hydroxy moiety in products 29a-c renders further functionalizations possible thereby making these compounds valuable synthetic building blocks. An analogous oxidative side reaction via the corresponding hydroperoxide is also described in the oxindole family [42], and the corresponding 3-hydroxy derivatives in the oxindole series were also synthesised at our laboratory [17]. In order to achieve full conversion of the starting materials 24, to maximize the amount of monomethyl products 26 and to minimize that of dimethyl byproducts 27, various conditions were explored, including the excess of BuLi and MeI, different deprotonation temperatures and the temperature of MeI addition (Scheme 13). It was found that significantly different protocols had to be used for starting materials 24a, 24b and 24c. Deprotonation with BuLi leading to the formation of the corresponding dianions was performed at −78 °C in each case, although with different amounts of BuLi (2.4-3.0 eq.). Then, contrary to our earlier observations with 3-monoalkyloxindoles (9) where In order to achieve full conversion of the starting materials 24, to maximize the amount of monomethyl products 26 and to minimize that of dimethyl byproducts 27, various conditions were explored, including the excess of BuLi and MeI, different deprotonation temperatures and the temperature of MeI addition (Scheme 13). It was found that significantly different protocols had to be used for starting materials 24a, 24b and 24c. Deprotonation with BuLi leading to the formation of the corresponding dianions was performed at −78 • C in each case, although with different amounts of BuLi (2.4-3.0 eq.). Then, contrary to our earlier observations with 3-monoalkyloxindoles (9) where MeI was also added at −78 • C [17], for 1,3-diazaoxindoles 24a-c we found that addition of MeI had to be carried out at −20 • C so that starting materials 24a-c are fully consumed. Finally, the reaction mixture was allowed to warm to ambient temperature. When using 2-phenyl derivative 24c as the starting material, a significant amount (19%) of 5,7-dimethyl derivative 27c was formed even with 1.4 eq. of MeI, therefore the reaction was finally performed with a lower excess (1.1 eq.) of the alkylating agent. Using the optimized conditions, C(5)-methylated products 26a-c were obtained in good yields (65-73%). MeI was also added at −78 °C [17], for 1,3-diazaoxindoles 24a-c we found that addition of MeI had to be carried out at −20 °C so that starting materials 24a-c are fully consumed. Finally, the reaction mixture was allowed to warm to ambient temperature. When using 2-phenyl derivative 24c as the starting material, a significant amount (19%) of 5,7-dimethyl derivative 27c was formed even with 1.4 eq. of MeI, therefore the reaction was finally performed with a lower excess (1.1 eq.) of the alkylating agent. Using the optimized conditions, C(5)-methylated products 26a-c were obtained in good yields (65-73%). Next, alkylations with BnBr were attempted. When the deprotonation of 2-unsubstituted (24a) and 2-methyl-1,3-diazaoxindole (24b) with BuLi (3.0 eq.) was performed at −20 °C, then the addition of BnBr (3.0 eq.) at room temperature, surprising results were observed. After the work-up of the reaction mixture by flash chromatography, in addition to C(5)-benzylated derivatives 26d,e, N(3),C(5)dibenzylated compounds 30a,b and N(3),C (5),N(7)-tribenzylated quaternary bromide salts 31a,b were also isolated (Scheme 14), with a product distribution quite different in the two reactions (compare entries 1 and 2 in Table 3) and also substantially different from those of the alkylations performed with 3.0 eq. MeI (Scheme 11). It is also noteworthy that benzylation of the 2-methyl moiety of 24b was not detected. Tribenzylated quaternary bromide salts 31a,b were also prepared by benzylation of 5,7-dibenzyl-1,3-diazaoxindoles 27d,e with a high excess of BnBr (3.8 eq.) in acetonitrile at ambient temperature (Scheme 15). After long reaction times (27-116 h), quaternary salts 31a and 31b were obtained in good yields (86% and 71%, respectively). Next, alkylations with BnBr were attempted. When the deprotonation of 2-unsubstituted (24a) and 2-methyl-1,3-diazaoxindole (24b) with BuLi (3.0 eq.) was performed at −20 • C, then the addition of BnBr (3.0 eq.) at room temperature, surprising results were observed. After the work-up of the reaction mixture by flash chromatography, in addition to C(5)-benzylated derivatives 26d,e, N(3),C(5)-dibenzylated compounds 30a,b and N(3),C(5),N(7)-tribenzylated quaternary bromide salts 31a,b were also isolated (Scheme 14), with a product distribution quite different in the two reactions (compare entries 1 and 2 in Table 3) and also substantially different from those of the alkylations performed with 3.0 eq. MeI (Scheme 11). It is also noteworthy that benzylation of the 2-methyl moiety of 24b was not detected. MeI was also added at −78 °C [17], for 1,3-diazaoxindoles 24a-c we found that addition of MeI had to be carried out at −20 °C so that starting materials 24a-c are fully consumed. Finally, the reaction mixture was allowed to warm to ambient temperature. When using 2-phenyl derivative 24c as the starting material, a significant amount (19%) of 5,7-dimethyl derivative 27c was formed even with 1.4 eq. of MeI, therefore the reaction was finally performed with a lower excess (1.1 eq.) of the alkylating agent. Using the optimized conditions, C(5)-methylated products 26a-c were obtained in good yields (65-73%).
Next, alkylations with BnBr were attempted. When the deprotonation of 2-unsubstituted (24a) and 2-methyl-1,3-diazaoxindole (24b) with BuLi (3.0 eq.) was performed at −20 °C, then the addition of BnBr (3.0 eq.) at room temperature, surprising results were observed. After the work-up of the reaction mixture by flash chromatography, in addition to C(5)-benzylated derivatives 26d,e, N(3),C(5)dibenzylated compounds 30a,b and N(3),C (5),N(7)-tribenzylated quaternary bromide salts 31a,b were also isolated (Scheme 14), with a product distribution quite different in the two reactions (compare entries 1 and 2 in Table 3) and also substantially different from those of the alkylations performed with 3.0 eq. MeI (Scheme 11). It is also noteworthy that benzylation of the 2-methyl moiety of 24b was not detected. Table 3. Isolated products of the benzylation reactions of 5-isopropyl-1,3-diazaoxindoles 24a,b in the presence of a large excess of BuLi and BnBr. Tribenzylated quaternary bromide salts 31a,b were also prepared by benzylation of 5,7-dibenzyl-1,3-diazaoxindoles 27d,e with a high excess of BnBr (3.8 eq.) in acetonitrile at ambient temperature (Scheme 15). After long reaction times (27-116 h), quaternary salts 31a and 31b were obtained in good yields (86% and 71%, respectively).  Tribenzylated quaternary bromide salts 31a,b were also prepared by benzylation of 5,7-dibenzyl-1,3-diazaoxindoles 27d,e with a high excess of BnBr (3.8 eq.) in acetonitrile at ambient The structure of tribenzyl derivative 31a was also proven by single-crystal X-ray diffraction ( Figure 3 and Supplementary Materials). A detailed analysis of the 1 H-NMR and IR signals of 31a, compared to those of 5,7-dibenzyl (27d) and 3,5-dibenzyl (30a) derivatives, showed that the electron distribution of 31a is unambiguously closer to the framed resonance structure ( Figure 4). The strong downfield shift of C(2)-H and C(4)-H signals in 31a in comparison to 27d and 30a indicate that the positive charge is mainly located in the pyrimidine ring. The N(3)-CH2 signal of 31a is also shifted by ca. 0.5 ppm when compared to the analogous signal in 30a, due to the positive charge of the N(3) atom in 31a, while the N(7)-CH2 signal remains practically unchanged when compared to 27d. The presence of a positively charged nitrogen atom in the pyrrole ring (as it is the case in the resonance structure of 31a on the right hand side) would shift the IR signal of the carbonyl moiety of 31a to a frequency higher than the carbonyl signal detected in 27d (1741 cm −1 ).  The structure of tribenzyl derivative 31a was also proven by single-crystal X-ray diffraction ( Figure 3 and Supplementary Materials). A detailed analysis of the 1 H-NMR and IR signals of 31a, compared to those of 5,7-dibenzyl (27d) and 3,5-dibenzyl (30a) derivatives, showed that the electron distribution of 31a is unambiguously closer to the framed resonance structure ( Figure 4). The strong downfield shift of C(2)-H and C(4)-H signals in 31a in comparison to 27d and 30a indicate that the positive charge is mainly located in the pyrimidine ring. The N(3)-CH 2 signal of 31a is also shifted by ca. 0.5 ppm when compared to the analogous signal in 30a, due to the positive charge of the N(3) atom in 31a, while the N(7)-CH 2 signal remains practically unchanged when compared to 27d. The presence of a positively charged nitrogen atom in the pyrrole ring (as it is the case in the resonance structure of 31a on the right hand side) would shift the IR signal of the carbonyl moiety of 31a to a frequency higher than the carbonyl signal detected in 27d (1741 cm −1 ). The structure of tribenzyl derivative 31a was also proven by single-crystal X-ray diffraction ( Figure 3 and Supplementary Materials). A detailed analysis of the 1 H-NMR and IR signals of 31a, compared to those of 5,7-dibenzyl (27d) and 3,5-dibenzyl (30a) derivatives, showed that the electron distribution of 31a is unambiguously closer to the framed resonance structure ( Figure 4). The strong downfield shift of C(2)-H and C(4)-H signals in 31a in comparison to 27d and 30a indicate that the positive charge is mainly located in the pyrimidine ring. The N(3)-CH2 signal of 31a is also shifted by ca. 0.5 ppm when compared to the analogous signal in 30a, due to the positive charge of the N(3) atom in 31a, while the N(7)-CH2 signal remains practically unchanged when compared to 27d. The presence of a positively charged nitrogen atom in the pyrrole ring (as it is the case in the resonance structure of 31a on the right hand side) would shift the IR signal of the carbonyl moiety of 31a to a frequency higher than the carbonyl signal detected in 27d (1741 cm −1 ).   The structure of tribenzyl derivative 31a was also proven by single-crystal X-ray diffraction ( Figure 3 and Supplementary Materials). A detailed analysis of the 1 H-NMR and IR signals of 31a, compared to those of 5,7-dibenzyl (27d) and 3,5-dibenzyl (30a) derivatives, showed that the electron distribution of 31a is unambiguously closer to the framed resonance structure ( Figure 4). The strong downfield shift of C(2)-H and C(4)-H signals in 31a in comparison to 27d and 30a indicate that the positive charge is mainly located in the pyrimidine ring. The N(3)-CH2 signal of 31a is also shifted by ca. 0.5 ppm when compared to the analogous signal in 30a, due to the positive charge of the N(3) atom in 31a, while the N(7)-CH2 signal remains practically unchanged when compared to 27d. The presence of a positively charged nitrogen atom in the pyrrole ring (as it is the case in the resonance structure of 31a on the right hand side) would shift the IR signal of the carbonyl moiety of 31a to a frequency higher than the carbonyl signal detected in 27d (1741 cm −1 ).   As the next step, the optimal conditions of C(5)-monobenzylation were sought. Similarly to the methylation reactions described above (Scheme 13), different protocols had to be elaborated for the benzylation of starting materials 24a, 24b and 24c (Scheme 16). BnBr was added at −20 • C in an excess of 1.4-3.0 eq. Use of lower excesses led in each case to significant amounts of the starting materials in the crude product, probably due to the higher steric hindrance of BnBr compared to MeI. By using the optimized conditions, C(5)-benzylated products 26d-f were obtained in acceptable yields (45-64%). As the next step, the optimal conditions of C(5)-monobenzylation were sought. Similarly to the methylation reactions described above (Scheme 13), different protocols had to be elaborated for the benzylation of starting materials 24a, 24b and 24c (Scheme 16). BnBr was added at −20 °C in an excess of 1.4-3.0 eq. Use of lower excesses led in each case to significant amounts of the starting materials in the crude product, probably due to the higher steric hindrance of BnBr compared to MeI. By using the optimized conditions, C(5)-benzylated products 26d-f were obtained in acceptable yields (45-64%).  (7)-unsubstituted 5-isopropyl-1,3-diazaoxindoles 24 is an even more challenging task than the analogous selective C(3)-alkylation of oxindoles.

General Information
Compounds 24a-c, 25a-c, 26a-f, 27a-f, 28, 29a-c, 30a,b, and 31a,b are new and characterized below. All melting points were determined on an OptiMelt Automated Melting Point System by Stanford Research Systems (Sunnyvale, CA, USA). IR spectra were obtained on a Bruker ALPHA FT-IR spectrometer (Bruker, Billerica, MA, USA). NMR spectra were recorded either on a Bruker AVANCE III 400 ( 1 H and 13 C frequencies were 400 and 100 MHz, respectively) or a Bruker AVANCE III HD 600 ( 1 H and 13 C frequencies were 600 and 150 MHz, respectively) spectrometer. CDCl3, DMSO-d6 or TFA-d was used as the solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) and coupling constants (J) are given in ppm and in Hz, respectively. Some representatives of compound families 26-31 were also investigated using special NMR measurements ( 1 H-1 H COSY, 1 H-13 C HSQC, 1 H-13 C HMBC, selective NOESY). High-resolution mass spectra (HRMS spectra) were recorded either on a Micromass GCT (Waters, Milford, MA, USA) with EI (direct inlet) or on a Bruker Q-TOF MAXIS Impact mass spectrometer coupled to a Dionex Ultimate 3000 RS HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) with a diode array detector. The reactions were followed by analytical thin layer chromatography on silica gel 60 F254 (Merck, Darmstadt, Germany) and by HPLC-MS on a Shimadzu LC-20 HPLC equipment (Kyoto, Japan) with an SPD-M20A diode-array detector coupled with a LCMS-2020 mass spectrometer. All unspecified reagents were purchased from commercial sources.

General Procedure I for the Synthesis of Compounds 25a-c
A mixture of the corresponding 1,3-diazaoxindole (6a, 6b or 6c), pyrrolidine (0.60 eq.) and acetone (2.0 eq.) in toluene was stirred at reflux temperature for 2-4 h. The water was removed with a Dean-Stark apparatus. After the reaction was complete, the volatile components were removed in vacuo at 60 °C and the residue was triturated thoroughly with hexane. The precipitate was filtered off, washed with hexane four times and dried.
The different behavior of the 2-H (24a), 2-Me (24b) and 2-Ph (24c) derivatives observed in the C(5)-alkylation reactions can be attributed first of all to the different character of the C(2) substituents, on the other hand to the different alkylating agents, giving rise to various side reactions. Besides the 5,7-dialkylated 1,3-diazaoxindoles 27, the formation of C(2)-ethyl derivative 28 and products 30a,b and 31a,b benzylated at the N(3) position was observed, indicating that the selective alkylation at the C(5) position of N(7)-unsubstituted 5-isopropyl-1,3-diazaoxindoles 24 is an even more challenging task than the analogous selective C(3)-alkylation of oxindoles.

General Information
Compounds 24a-c, 25a-c, 26a-f, 27a-f, 28, 29a-c, 30a,b, and 31a,b are new and characterized below. All melting points were determined on an OptiMelt Automated Melting Point System by Stanford Research Systems (Sunnyvale, CA, USA). IR spectra were obtained on a Bruker ALPHA FT-IR spectrometer (Bruker, Billerica, MA, USA). NMR spectra were recorded either on a Bruker AVANCE III 400 ( 1 H and 13 C frequencies were 400 and 100 MHz, respectively) or a Bruker AVANCE III HD 600 ( 1 H and 13 C frequencies were 600 and 150 MHz, respectively) spectrometer. CDCl 3 , DMSO-d 6 or TFA-d was used as the solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) and coupling constants (J) are given in ppm and in Hz, respectively. Some representatives of compound families 26-31 were also investigated using special NMR measurements ( 1 H-1 H COSY, 1 H-13 C HSQC, 1 H-13 C HMBC, selective NOESY). High-resolution mass spectra (HRMS spectra) were recorded either on a Micromass GCT (Waters, Milford, MA, USA) with EI (direct inlet) or on a Bruker Q-TOF MAXIS Impact mass spectrometer coupled to a Dionex Ultimate 3000 RS HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) with a diode array detector. The reactions were followed by analytical thin layer chromatography on silica gel 60 F 254 (Merck, Darmstadt, Germany) and by HPLC-MS on a Shimadzu LC-20 HPLC equipment (Kyoto, Japan) with an SPD-M20A diode-array detector coupled with a LCMS-2020 mass spectrometer. All unspecified reagents were purchased from commercial sources.

General Procedure I for the Synthesis of Compounds 25a-c
A mixture of the corresponding 1,3-diazaoxindole (6a, 6b or 6c), pyrrolidine (0.60 eq.) and acetone (2.0 eq.) in toluene was stirred at reflux temperature for 2-4 h. The water was removed with a Dean-Stark apparatus. After the reaction was complete, the volatile components were removed in vacuo at 60 • C and the residue was triturated thoroughly with hexane. The precipitate was filtered off, washed with hexane four times and dried.

General Procedure II for the Synthesis of Compounds 25a-c
A mixture of the corresponding 1,3-diazaoxindole (6a, 6b or 6c), acetic acid (24 eq.) and acetone (46 eq.) was stirred at reflux temperature under argon atmosphere (Ar) for 12-48 h. After the reaction was complete, the volatile components were removed in vacuo at 40 • C and the residue was triturated thoroughly with diethyl ether (DEE) or water. The precipitate was filtered off and dried.

General Procedure III for the Synthesis of Compounds 24a-c
Diazaoxindole derivatives 25a-c were hydrogenated at 60 °C under atmospheric pressure i DMF using activated palladium on charcoal (Pd/C, 10% Pd) catalyst for 12 h. After the reaction wa completed, the catalyst was filtered off and washed with DMF three times. The filtrate and th washings were combined and the solvent was removed at 60 °C under reduced pressure. The residu was triturated thoroughly with water, the precipitate was filtered off and dried. [2,3-d]pyrimidin-6-one (24a). This compound was prepare according to General Procedure III using 25a (500 mg, 2.85 mmol) and Pd/C catalyst (140 mg) in DM (50 mL). The crude product was recrystallized from toluene-DMF to give 25a (389 mg, 77%) a ] + 175.0746; found 175.0762. Method B: This compound was also prepared according to General Procedure II using 6a (3.0 g, 22.2 mmol), acetic acid (30 mL, 524 mmol), acetone (75 mL, 59.25 g, 1022 mmol). The crude product was recrystallized from DMF to give 25a (3.34 g, 86%) as pale brown crystals. Spectral data were identical with those of the product obtained using General Procedure I.

General Procedure III for the Synthesis of Compounds 24a-c
Diazaoxindole derivatives 25a-c were hydrogenated at 60 • C under atmospheric pressure in DMF using activated palladium on charcoal (Pd/C, 10% Pd) catalyst for 12 h. After the reaction was completed, the catalyst was filtered off and washed with DMF three times. The filtrate and the washings were combined and the solvent was removed at 60 • C under reduced pressure. The residue was triturated thoroughly with water, the precipitate was filtered off and dried.  3-diazaoxindole (6a, 6b or 6c), acetic acid (24 eq.) and acetone (46 eq.) was stirred at reflux temperature under argon atmosphere (Ar) for 12-48 h. After the reaction was complete, the volatile components were removed in vacuo at 40 °C and the residue was triturated thoroughly with diethyl ether (DEE) or water. The precipitate was filtered off and dried. [2,3-d]pyrimidin-6-one (25a). Method A: This compound was prepared according to General Procedure I using 6a (500 mg, 3.7 mmol), pyrrolidine (0.18 mL, 0.16 g, 2.2 mmol), acetone (0.54 mL, 0.43 g, 7.4 mmol) in toluene (10 mL This compound was also prepared according to General Procedure II using 6a (3.0 g, 22.2 mmol), acetic acid (30 mL, 524 mmol), acetone (75 mL, 59.25 g, 1022 mmol). The crude product was recrystallized from DMF to give 25a (3.34 g, 86%) as pale brown crystals. Spectral data were identical with those of the product obtained using General Procedure I.  29.04 mmol), acetone (4.09 mL, 3.23 g, 55.66 mmol). The product 25b was obtained as yellow crystals (98 mg, 42%). Spectral data were identical with those of the product obtained using General Procedure I. This compound was also prepared according to General Procedure II using 6c (120 mg, 0.57 mmol), acetic acid (0.78 mL, 0.82 g, 13.70 mmol), acetone (1.90 mL, 1.50 g, 26.20 mmol). The product 25c was obtained as orange crystals (89 mg, 62%). Spectral data were identical with those of the product obtained using General Procedure I.

General Procedure III for the Synthesis of Compounds 24a-c
Diazaoxindole derivatives 25a-c were hydrogenated at 60 °C under atmospheric pressure in DMF using activated palladium on charcoal (Pd/C, 10% Pd) catalyst for 12 h. After the reaction was completed, the catalyst was filtered off and washed with DMF three times. The filtrate and the washings were combined and the solvent was removed at 60 °C under reduced pressure. The residue was triturated thoroughly with water, the precipitate was filtered off and dried. [2,3-d]pyrimidin-6-one (24a). This compound was prepared according to General Procedure III using 25a (500 mg, 2.85 mmol) and Pd/C catalyst (140 mg) in DMF (50 mL). The crude product was recrystallized from toluene-DMF to give 25a (389 mg, 77%) as ] + 177.0902; found 177.0910.

General Procedure III for the Synthesis of Compounds 24a-c
Diazaoxindole derivatives 25a-c were hydrogenated at 60 °C under atmospheric pressure in DMF using activated palladium on charcoal (Pd/C, 10% Pd) catalyst for 12 h. After the reaction was completed, the catalyst was filtered off and washed with DMF three times. The filtrate and the washings were combined and the solvent was removed at 60 °C under reduced pressure. The residue was triturated thoroughly with water, the precipitate was filtered off and dried.
. This compound was prepared according to General Procedure III using 25a (500 mg, 2.85 mmol) and Pd/C catalyst (140 mg) in DMF (50 mL). The crude product was recrystallized from toluene-DMF to give 25a (389 mg, 77%) as

General Procedure II for the Synthesis of Compounds 25a-c
A mixture of the corresponding 1,3-diazaoxindole (6a, 6b or 6c), acetic acid (24 eq.) and acetone (46 eq.) was stirred at reflux temperature under argon atmosphere (Ar) for 12-48 h. After the reaction was complete, the volatile components were removed in vacuo at 40 °C and the residue was triturated thoroughly with diethyl ether (DEE) or water. The precipitate was filtered off and dried.

General Procedure IV for the Synthesis of Compounds 27a-f
To a mixture of NaOH (2.5 eq.) in DMF, the solution of 5-isopropyl-1,3-diazaoxindoles 24a-c in DMF was added dropwise at room temperature, under Ar atmosphere and it was stirred for 30 min. Then the solution of the appropriate alkyl halide (2.5 eq.) in DMF was added dropwise at 15-20 • C. The stirring was continued for further 1 h. The reaction mixture was poured onto ice-water. The precipitate was filtered off or the aqueous layer was extracted with dichloromethane (DCM, 3 × 15 mL). The combined organic layer was washed with brine (5 × 50 mL), dried over anhydrous MgSO 4 , filtered and the solvent was removed in vacuo at 40 • C. Purification of the crude products was performed as described below. 5,7-Dimethyl-5-(1-methylethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (27a). This compound was prepared according to General Procedure IV using 24a (275 mg, 1.55 mmol) in DMF (4 mL), NaOH (155 mg, 3.88 mmol) in DMF (2 mL) and MeI (0.24 mL, 0.55 g, 3.88 mmol) in DMF (1 mL). The residual oil was purified by gradient elution column chromatography using hexane and EtOAc as the eluents. The product was triturated in hexane (3 mL

General Procedure II for the Synthesis of Compounds 25a-c
A mixture of the corresponding 1,3-diazaoxindole (6a, 6b or 6c), acetic acid (24 eq.) and acetone (46 eq.) was stirred at reflux temperature under argon atmosphere (Ar) for 12-48 h. After the reaction was complete, the volatile components were removed in vacuo at 40 °C and the residue was triturated thoroughly with diethyl ether (DEE) or water. The precipitate was filtered off and dried. This compound was also prepared according to General Procedure II using 6a (3.0 g, 22.2 mmol), acetic acid (30 mL, 524 mmol), acetone (75 mL, 59.25 g, 1022 mmol). The crude product was recrystallized from DMF to give 25a (3.34 g, 86%) as pale brown crystals. Spectral data were identical with those of the product obtained using General Procedure I.

General Procedure III for the Synthesis of Compounds 24a-c
Diazaoxindole derivatives 25a-c were hydrogenated at 60 °C under atmospheric pressure in DMF using activated palladium on charcoal (Pd/C, 10% Pd) catalyst for 12 h. After the reaction was completed, the catalyst was filtered off and washed with DMF three times. The filtrate and the washings were combined and the solvent was removed at 60 °C under reduced pressure. The residue was triturated thoroughly with water, the precipitate was filtered off and dried. (1-Methylethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (24a). This compound was prepared according to General Procedure III using 25a (500 mg, 2.85 mmol) and Pd/C catalyst (140 mg) in DMF (50 mL). The crude product was recrystallized from toluene-DMF to give 25a (389 mg, 77%) as ] + 205.1215; found 205.1223.

General Procedure V for the Synthesis of Compounds 29a-c
To a mixture of BuLi in hexane (1.50 mL, 3.75 mmol, 2.5 eq., 2.5 M) and THF (3 mL), the solution of the appropriate 2-substituted 5-isopropyl-1,3-diazaoxindole 24a-c (1.50 mmol) in THF (8 mL) was added dropwise at −78 • C, under argon atmosphere. After the addition was complete, the acetone-dry ice bath was removed, the reaction mixture was allowed to warm to room temperature and the apparatus was opened to the air. The stirring was continued for further 2 days. The mixture was quenched with saturated NH 4 Cl solution (10 mL), water (5 mL) was added, then the layers were separated and the aqueous layer was extracted with EtOAc (3 × 5 mL). The combined organic layer was dried over anhydrous MgSO 4 , filtered and the solvent was removed in vacuo at 40 • C. The residue crystallized upon treatment with hexane/DIPE = 3/1 (7 mL), then it was filtered off, washed with hexane/DIPE = 3/1 (2 × 2 mL) and dried. To a mixture of BuLi in hexane (3.80 mL, 9.44 mmol, 3.3 eq., 2.5 M) and THF (3 mL), the solution of 5-(1-methylethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (24a, 500 mg, 2.86 mmol) in THF (8 mL) was added dropwise at −78 • C under Ar atmosphere. The mixture was stirred for 30 min, then the temperature was allowed to warm to -20 • C and MeI (0.59 mL, 1.33 g, 9.44 mmol, 3.3 eq.) in THF (1 mL) was added dropwise. The stirring was continued for further 1.5 h. The mixture was quenched with saturated NH 4 Cl solution (10 mL), and water (5 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 × 15 mL). The combined organic layer was washed with brine (20 mL), dried over anhydrous MgSO 4 , filtered and the solvent was removed in vacuo at 40 • C. The remaining yellow oil was purified by gradient elution column chromatography using DCM and DCM/MeOH = 9/1 as the eluents. The product was triturated in hexane (2 mL), washed with DIPE (2 × 1 mL) and dried to give 29a (50 mg, 9%) as colorless crystals. Spectral data were identical with those of the product obtained using Method A.

General Procedure II for the Synthesis of Compounds 25a-c
A mixture of the corresponding 1,3-diazaoxindole (6a, 6b or 6c), acetic acid (24 eq.) and acetone (46 eq.) was stirred at reflux temperature under argon atmosphere (Ar) for 12-48 h. After the reaction was complete, the volatile components were removed in vacuo at 40 °C and the residue was triturated thoroughly with diethyl ether (DEE) or water. The precipitate was filtered off and dried. [2,3-d]pyrimidin-6-one (25a). Method A: This compound was prepared according to General Procedure I using 6a (500 mg, 3.7 mmol), pyrrolidine (0.18 mL, 0.16 g, 2.2 mmol), acetone (0.54 mL, 0.43 g, 7.4 mmol) in toluene (10 mL This compound was also prepared according to General Procedure II using 6a (3.0 g, 22.2 mmol), acetic acid (30 mL, 524 mmol), acetone (75 mL, 59.25 g, 1022 mmol). The crude product was recrystallized from DMF to give 25a (3.34 g, 86%) as pale brown crystals. Spectral data were identical with those of the product obtained using General Procedure I. [2,3-d]pyrimidin-6-one (25b). Method A: This compound was prepared according to General Procedure I using 6b (15.00 g, 100 mmol), pyrrolidine (4.96 mL, 4.27 g, 60 mmol), acetone (14.68 mL, 11.60 g, 200 mmol) in toluene (150 mL). The crude product was recrystallized from DMF to give 25b (16. 29.04 mmol), acetone (4.09 mL, 3.23 g, 55.66 mmol). The product 25b was obtained as yellow crystals (98 mg, 42%). Spectral data were identical with those of the product obtained using General Procedure I. ] + 207.1008; found 207.1026. Method B: To a mixture of BuLi in hexane (4.13 mL, 6.60 mmol, 2.2 eq., 1.6 M) and THF (5 mL), the solution of 2-methyl-5-(1-methylethyl)-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (24b, 574 mg, 3.00 mmol) in THF (14 mL) was added dropwise at −78 • C under Ar atmosphere. The mixture was stirred for 30 min, then MeI (0.41 mL, 0.94 g, 6.60 mmol, 2.2 eq.) in THF (1 mL) was added dropwise. The temperature was allowed to warm to room temperature and stirring was continued for further 5 h. The mixture was quenched with saturated NH 4 Cl solution (10 mL), and water (5 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 × 15 mL). The combined organic layer was washed with brine (30 mL), dried over anhydrous MgSO 4 , filtered and the solvent was removed in vacuo at 40 • C. The remaining orange oil was purified by gradient elution column chromatography using hexane and EtOAc as the eluents. The product was triturated in hexane (2 mL), washed with DIPE (2 × 1 mL) and dried to give 29b (100 mg, 16%) as colorless crystals. Spectral data were identical with those of the product obtained using Method A. General Procedure II for the Synthesis of Compounds 25a-c A mixture of the corresponding 1,3-diazaoxindole (6a, 6b or 6c), acetic acid (24 eq.) and acetone eq.) was stirred at reflux temperature under argon atmosphere (Ar) for 12-48 h. After the reaction complete, the volatile components were removed in vacuo at 40 °C and the residue was triturated oughly with diethyl ether (DEE) or water. The precipitate was filtered off and dried.

General Procedure III for the Synthesis of Compounds 24a-c
Diazaoxindole derivatives 25a-c were hydrogenated at 60 °C under atmospheric pressure in F using activated palladium on charcoal (Pd/C, 10% Pd) catalyst for 12 h. After the reaction was pleted, the catalyst was filtered off and washed with DMF three times. The filtrate and the hings were combined and the solvent was removed at 60 °C under reduced pressure. The residue triturated thoroughly with water, the precipitate was filtered off and dried.