Easy synthesis of new series of pteridine analogs: di-and tetra-hydropyrimido[4,5-d ]pyrimidines via 5-pyrimidinecarbaldehydes

Pyrimidine-5-carbaldehyde derivatives, of easy access, were selected as precursors for the synthesis of mimic pteridine derivatives 5,6-dihydropyrimido[4,5-d ]pyrimidines and 5,6,7,8-tetrahydropyrimido[4,5-d ]pyrimidines via 4-amino-(5-aminomethyl)pyrimidine intermediates. Straightforward procedures allow a quick access to these compounds. The dihydro derivatives were prepared means of a final cyclocondensation carried out with orthoesters, catalysed by acid and assisted by microwaves irradiation under solvent free conditions. The final cyclo-condensation with carbonyl compounds forming the tetrahydro derivatives was done under mild conditions, in which stereochemical induction was carried out on the building of this skeleton, and stereochemistry assignments corroborated by theoretical calculations.


Introduction
2][3][4] See Figure 1 for compounds with the pyrimido [4,5-d]pyrimidine scaffold that have been found to show pharmacological activity, such as antiplatelet, 5 tyrosine kinase inhibitor, [6][7][8] antifolate (I-II), 9 antibacterial (III-IV), 10,11 anti-inflammatory, 12 hepatoprotective (V) 13 and antiviral 14 properties.1][22][23][24] Microwave irradiation has been used to carry out a wide range of reactions in short times, with high yields and regioselectivity, and frequently without the need of solvents. 25ith the aim to tune up simple reaction methodologies, we have focused our strategy in the use of 4-aminopyrimidine-5-carbaldehydes as key precursors for the preparation of cited fused heterocyclic system (see Figure 2) because they have the appropriate spatial arrangement of atoms.We have previously reported some 4-aminopyrimidine-5-carbaldehydes and employed them for the preparation of pyridopyrimidine derivatives. 26

Figure 2
We here present the synthesis of a new series of 5,6-dihydro and 5,6,7,8-tetrahydro derivatives by a three step process starting from the corresponding 4-aminopyrimidines, via formation of 4-amino-(5-aminomethyl)pyrimidines as intermediates for further cyclocondensation with orthoesters and carbonyl compounds respectively.
In the tetrahydropyrimido [4,5-d]pyrimidines a stereogenic center is generated and the experimental conditions to induce stereoselectivity are discussed below.

Results and Discussion
The key precursor 6-aminopyrimidin-4(3H)-one-5-carbaldehydes, 1a,b, were prepared by Vilsmeier-Haack formylation of 6-aminopyrimidin-4(3H)-ones VI, 26 and then reaction with various primary amines, such as aniline, benzylamine and cyclohexylamine, in order to get the required diamino intermediate 2 for further cyclocondensation via reaction with the carbaldehyde group.The standard reductive reagents and conditions were tested in order to afford compounds 2, but just the use of sodium triacetoxyborohydride in THF was effective to perform the one step amino reduction.The reaction workup is simple and straightforward, and compounds 2 were isolated by filtration in good to excellent yields (Scheme 1 and Table 1).Scheme 1. Synthetic route to pyrimidopyrimidines 3 from aminopyrimidines.From the spectroscopic data of compounds 2, the disappearance of signals corresponding to the carbaldehyde in 1, and the appearance of the derived methylene group and the signals which correspond to the amine residue, are clearly observed.In the case of 2a, the signal for 5-CH 2 is observed at 3.95 ppm (as a doublet, coupled with NH) and at 37.0 ppm, in the 1 H-and 13 C-NMR spectra respectively.
For the cyclocondensation of the diamine derivative 2, we first tried several acid derivatives such as acyl chlorides and anhydrides, and different conditions, but with no positive results.Finally we chose orthoesters, which provided the corresponding 5,6-dihydropyrimido [4,5-d]pyrimidin-4(3H)-ones 3, in satisfactory yields.
According to the literature, reactions with this kind of reagent need drastic conditions. 27,28An attempt to carry out these reactions by conventional heating gave decomposition of starting material because of the long reaction times required.To avoid conventional heating, we chose to assist the reaction with microwave irradiation, and we found that the reaction took place at 200 ºC over 10-15 minutes with orthoester in excess (three-and four-fold molar) and in the absence of solvent to yield the desired pyrimidopyrimidines 3, but not in good yields (< 45 %).This was probably due to the partial decomposition of the products under such conditions.From the different acid catalysts screened to optimize the reaction conditions, acetic acid was found to give the best results.After addition of acetic acid the reactions were greatly improved: the reaction time fell to 1 minute, the temperature for the reaction dropped to 150 ºC, and yields were considerably increased (Table 2).Nevertheless no useful results were obtained when the 6amino-5-(phenylaminomethyl)pyrimidin-4-one 2a was taken as starting material.Compounds 3 were fully characterized by spectroscopic and analytical data.From spectroscopic data of compounds 3, the formation of the new pyrimidine ring is clearly observed: in the 1 H-NMR spectra the signals for NH are absent and instead the proton or methyl attached to C(7) appears at 7.40-8.12ppm or around 2.0 ppm respectively; in the 13 C-NMR the C(7) appears at 152-164 ppm.
In the hope of improving the reaction efficiency, equimolar amounts of 6-amino-5-(benzylaminomethyl)pyrimidine 2b, ethyl orthoformate and acetic acid were tried; but in this case in addition to the cyclization reaction we found loss of the methoxy group at C(2), clearly indicated in the 1 H-NMR in which the NH of the lactam moiety was found (broad singlet at 10.90 ppm) for 3j (Scheme 2).Something similar has been reported in other acid reactions in the presence of a nucleophile such as ethanol, which is a byproduct of the cyclocondensation step. 26,29heme 2. Acetic acid in equimolar amount leads to demethylation and cyclization in the reaction of diaminopyrimidine 2b with ethyl orthoformate.
All the pyrimidopyrimidines 3 were fully characterized, the most characteristic NMR feature being the afore-mentioned disappearance of the NH signals of 2 and the appearance of a new signal for C(7)-H in the case of 3a,c,d,f,h or -CH 3 for 3b,e,g,i depending on whether ethyl orthoformate or methyl orthoacetate were used.
X-Ray diffraction analysis was carried out on the pyrimido [4,5-d]pyrimidine 3a, and its structure was unambiguously confirmed.The most relevant feature observed in its supramolecular structure is the π -π stacking interactions between phenyls which is reinforced by weak π -π interactions between pyrimidine rings.In order to extend the variety of hydropyrimido [4,5-d]pyrimidines available, the tetrahydro derivatives were prepared from the intermediates 2 and carbonyl compounds such as acetone and benzaldehyde in a similar fashion to those reported for the preparation of tetrahydroquinazolines. 30 Mild reaction conditions were used: EtOH as solvent, a temperature of 50 ºC, and p-toluenesulfonic acid as catalyst, which is needed because of the low nucleophilicity of the 6amino group in 2 (Scheme 3 and Table 3). 31 a Compound 4e was isolated from the reaction of 2c with acetone.b See Table 4.
The tetrahydropyrimidopyrimidines 4 were obtained in moderate to good yields and also fully characterized by spectroscopic and analytical data.From the spectroscopic data of compounds 4, the formation of the new tetrahydropyrimidine ring is clearly observed, for example in 1 H-NMR spectra; as in the case of compounds 3, the signals for NH largely disappear, there remaining just the N(8)-H, and substituents attached to C(7) are clearly observed, with the new C(7) appearing in the 13 C-NMR at 59-71 ppm.
The reaction between 2c and acetone afforded an unexpected product, which was characterized as 4e.Its 1 H-NMR spectrum shows a signal at 4.78 ppm corresponding to the methylene for C (7), which is coupled with the N(8)-H, but not those corresponding to the methyl groups expected from acetone.So the acetone is not involved in the reaction in this case, as was also confirmed by mass spectrometry.To prove this possibility 2c was reacted in ethanol with PTSA in the absence of acetone.Under these conditions compound 4e, together with the initial pyrimidine precursor VI and cyclohexylamine were obtained.Therefore, compound 2c is acting in this reaction both as a diamine and a formaldehyde-releasing agent, to render 4e, as shown in Scheme 4. This behavior can be explained by of the higher basicity of the dialkylamino residue in molecule 2c, compared to the other diamine derivatives 2, that in acid media leads to its partial hydrolysis that releases formaldehyde (like in acetals) which is then trapped by another molecule of 2c, forming compound 4e.Scheme 4. Compound 2c: decomposition in acid medium.
From the mass spectrometry analysis for the dihydro (3) and tetrahydro (4) pyrimido [4,5-d]pyrimidine derivatives, similar fragmentation patterns were observed, characterized by progressive loss of pendant substituents and ring opening.
A single crystal X-ray diffraction on the tetrahydropyrimido[4,5-d]pyrimidine derivative 4c was carried out (Figure 4), showing the predicted structure.Some considerations about the structure observed can be made: the puckering analysis shows a conformation of the saturated 6membered ring close to the twist-boat, with substituents at N(6) and C(7) in a axial disposition to minimize the steric interaction between them.When benzaldehyde is used as reagent, a stereogenic center is generated at C(7) (see Figure 5).In the first instance we observed that in order to induce stereoselectivity in this reaction, the presence of a chiral residue in the starting material was needed, and therefore the enantiomeric compounds 2g/2g' were prepared by reductive amination of 1b with (R)-and (S)methylbenzylamine.The stereoselectivity of the reaction of 2g/2g' with benzaldehyde was studied under several conditions.The conclusions of the chirality study are: slight importance of solvent (better results in acetonitrile than in ethanol), the necessity for a chiral auxiliary introduced in the molecule (R 1 ) (2g/2g') and also a chiral acid catalyst (camphorsulfonic acid).Without chiral acid catalyst the d.e. of the reaction was 4% in EtOH and 33% in acetonitrile, while it reaches 88% upon the addition of camphorsulfonic acid (Table 4).When the substituent is the isomer S and the acid is S too, the preferred configuration generated is S. Likewise, when starting material and catalyst are R, C( 7) is also R, and in both cases the diastereomeric excess obtained is around 88% (see Table 4).Analysis by NMR was essential to determine the stereochemistry in the 4g diasteromers and to calculate the diasteromeric excess for the reactions.NOESY experiments permitted assignment of 4g 1 as the major diastereoisomer (see figures 1S and 2S in supporting information).The NOESY (figure 1S) shows the correlation between C(7)-H with 1′-H for 4g 1 , but in the case of 4g 2 are observed between C(7)-H with 1′-CH 3 and N( 8)-H with 1′-H, which agrees with their optimized conformations.

Theoretical calculations
To support the experimental results, theoretical calculations have been done on compound 4g 1-4 .An exhaustive conformational study has been carried out using the Multidimensional Conformational Analysis (MDCA). 32A combination of semiempirical (PM6) and DFT (B3LYP/6-31G(d)) computations were employed in order to obtain the energetically preferred conformations.A detailed explanation about the conformational search is given in the experimental section.From the low-energy conformations obtained for both diasteromers R/R and R/S we calculated the shielding tensors using the GIAO approach 33,34 from the program Gaussian 03. 35Our study also includes the calculation of 13 C-NMR chemical shift for the diasteroisomers (R/R; R/S; S/R and S/S) by using the gauge independent atomic orbital (GIAO) approximation.
Observing the curves obtained in figure 6, it is evident that the correlation obtained for the R/R enantiomer is excellent indicating that such assignment for 4a-g is correct.Figures 6a and 6b give the correlations obtained for the experimentally obtained chemical shifts versus calculated chemical shifts obtained for R/R and R/S, respectively.It is pertinent to remark that the theoretical calculations reported here are in agreement with the experimental results obtained from X-ray measurements.Figure 3S (supporting information) gives in a comparative fashion the conformations obtained from X-ray and from theoretical calculations.The very similar spatial ordering obtained for both forms can be appreciated very well in this figure.

Antimicrobial activity
It should be noted that the synthesized compounds have a certain structural similarity with compounds IV and V shown in Figure 1 which have been reported as antibacterial agents Thus, we decide to test these compounds against different pathogenic fungi and bacteria.Minimum Inhibitory Concentration (MIC) of each compound was determined by using broth microdilution techniques according to the guidelines of the National Committee for Clinical Laboratory Standards for yeasts (M27-A3) and for filamentous fungi (M 38-A2). 36] Most of the compounds described herein were evaluated against Candida albicans, C. neoformans, Staphylococcus aureus methicillin-sensitive ATCC 29213, Staphylococcus aureus methicillin-resistant ATCC 43300, Escherichia coli ATCC 25922, LM 1 -Escherichia coli LM 2 , , Pseudomonas aeruginosa ATCC 27853, PI-Yersinia enterocolítica, MI-Salmonella enteritidis and Salmonella sp (LM).Unfortunately, none of the test compounds displayed antifungal nor antibacterial activity at the highest concentration tested (250 μg/mL).Other bioassays are in progress and will be the subject of another report in the near future.

Conclusions
We have developed the synthesis of a new series of 5,6-dihydro-and 5,6,7,8tetrahydropyrimido [4,5-d]pyrimidin-4(3H)-ones starting from the corresponding 4-aminopyrimidines, via formation of 4-amino-(5-aminomethyl)pyrimidines as intermediates.The cyclocondensation was achieved using orthoesters under MW irradiation and solvent-free conditions.This latter reaction was optimized by acid catalyst to get the desired products in just 1 minute in good yields.This methodology is straightforward and the isolation of desired products is simple.These compounds have an attractive skeleton from a biological point of view, and they are being tested with the aim to find potential pharmacological applications.

Experimental Section
General.Melting points were determined on a Barstead Electrothermal 9100 melting point apparatus.IR spectra were recorded in KBr disks on a Bruker TENSOR 27 spectrophotometer at "Centro de Instrumentación Científico-Técnica (CICT) at Universidad de Jaén". 1 H and 13 C NMR spectra were recorded on a Bruker Avance 400 spectrophotometer (CICT) operating at 400 MHz and 100 MHz respectively, using CDCl 3 and DMSO-d 6 as solvents and tetramethylsilane as internal standard; the carbon type described p (primary), s (secondary), t (tertiary) and q (quaternary) at 13 C-NMR was done using DEPT-135 and 2D-NMR experiments.Mass spectra were run on a Shimadzu-GCMS 2010-DI-2010 spectrometer (equipped with a direct inlet probe) operating at 70 eV.High resolution mass spectra were run on a Waters Micromass AutoSpec-Ultima spectrometer equipped with a direct inlet probe operating at 70 eV.Silica gel aluminum plates (Merck 60 F 254 ) were used for analytical TLC.The microwave-assisted reactions were carried out in a 10 ml glass sealed vial for focused mono-mode microwaves oven (Discover, by CEM Corp.) working in standard mode by controlling the target temperature.The starting amines and orthoesters were purchased from Aldrich, Fluka and Acros (analytical reagent grades) and were used without further purification.

Methods of calculations Multidimensional conformational analysis (MDCA).
From the concepts of MDCA 32 we performed a systematic conformational search for compounds 4g 1-4.In such analysis the following variables were taken into account: (i) The three torsional angles 1, 2 and 3, which determine the different conformations.
(MDCA predicts a multiplicity of 3 for the torsional angle 1 and a multiplicity of 2 for the torsional angles 2 and 3).
(ii) The two possible spatial orderings for the N atom of ring B (up or down)(multiplicity 2) (iii) The two possible spatial orderings, axial or equatorial, for the alkyl-phenyl substituent (ring D) (multiplicity 2).
(iv) The different configurational isomers due to both chiral centers (see figure 4) (multiplicity 2 for each chiral center).
Considering the above variables the conformational potential energy hypersurface (CPEH) might be raised as follow: E=E(1,2,3,up/down,ax/eq,R/S,R/S) Equation 1 might be also expressed in function of the multiplicities as follows: Equation 2 indicates that in theory there are 192 different forms for compounds 4g 1-4 .However, our preliminary calculations indicate that the torsional angles 2 and 3 adopt only two almost perpendicular conformations.Considering that rings C and D are symmetrical, they give nonidentical but indistinguishable forms (energetically degenerate).Thus, Equation 2 might be reduced to Equation 3.
On the basis of Equation 3 MDCA predicts 48 different forms.All these different spatial orderings were analyzed in our study.
Computations.An extensive search for the low energy conformations on the PEHS was carried out by using a systematic search in connection with semiempirical PM6 calculations.Next to determine the minima on the PEHS, fully relaxed DFT optimizations were performed.Correlation effects were included using density functional theory (DFT) with the Beke-Lee-Yang Parr (B3LYP) [39][40][41] functional and the 6-31 G(d) basis set.Minima were characterized through harmonic frequency analysis employing B3LYP/6-31 G(d) calculations.GIAO [33][34] (gauge independent atomic orbital) computations were carried out using B3LYP/6-31G(d) calculations.All the calculations reported here were performed using the Gaussian 03 Program. 35rom the 48 potentially available conformations of compound 4g, only 41 conformers were obtained for these molecules; seven conformations were annihilated during the optimization process.Annihilation means that they do not converge or finally converge to one of the other 41 conformers.It should be noted that the synthesis of compound 4g 1-4 was carried out from a particular enantiomer in the first chiral center C(1′), so that the result of the synthesis can only give rise to two stereoisomers R/R and R/S or S/S and S/R (C(1′)/C( 7)).This assumption was supported by the theoretical calculations performed here.Considering an energetic window of 6 Kcal/mol just eight conformers were obtained for both diasteromers (R/R and R/S) and their equivalents (S/S and S/R) (see Table 1S and Figure 4S in supporting information for angles analyzed in this study).All these conformations were optimized at DFT level using B3LYP/6/31G(d) calculations.These conformers and their relative energies are summarized in Table 1S.Figure 3S shows a spatial view of the lowest-energy conformation obtained for compound 4g 2 .
From the low-energy conformations obtained for both diasteromers R/R and R/S we calculated the shielding tensors using the GIAO approach 33,34 from the program Gaussian 03. 35From the tensors we obtain the value of calculated chemical shift (calc) for each C atom of each isomer, using the following expression (Eq.4): where b and m values were obtained from the linear correlation between  exp and the sum of I (isotropy) with A (asymmetry).]

Figure 6 .
Figure 6.Chemical shifts correlations: (a) assigned to R/R vs calculated for R/R, and (b) assigned to R/R vs calculated for R/S

Table 1 .
Data for the synthesis of 2a-g shown in Scheme 1

Table 2 .
Data for the reaction of 2 with orthoesters to products 3a-i with AcOH as catalyst (see