CHAPTER ISynthesis o f 5-[n-(4,6-bis-alkylsulfanyl-lH-

pyrazolo[3,4-d]pyrimidin-l-yl)alkyl] -1 -alky 1-6- alkylsulfanyl-1,5-dihydropyrazolo[3,4-d]pyrimidin-

4-ones, their regioisomers and derivatives.

1.1 IntroductionMolecular organization and molecular interactions are the basis o f the functional properties o f most molecules, and a detailed understanding o f noncovalent chemistry is therefore fundamental to interpreting and predicting relationships between chemical structure and function. Molecular recognition processes are influenced by many different factors that make their study complicated. Progress requires a quantitative understanding o f these different factors. Among the noncovalent interactions that govern molecular recognition processes ranging from crystal growth to the formation o f protein-ligand, protein-protein complexes, apolar (hydrophobic) interactions, ion- ion, ion-dipole interactions, and H-bonding association have been the preferred subjects for a large number o f investigations during the past few decades.1 A wealth o f information on the nature and energetics o f these interactions has greatly enhanced our ability to use them for crystal structure prediction,2 crystal engineering,3 supramolecular synthesis,4 and structure based drug design.5 Some key functional group interactions, such as H-bonding, are relatively well understood. H-bonds are strong, single point interactions with a very well defined geometry, and the electrostatic forces between the donor hydrogen atom and the acceptor atom determine their magnitude. For weaker, less well defined interactions, the picture is not so clear. A thorough knowledge o f noncovalent interactions is crucial to the understanding o f biological complexity.One o f the less well understood but a significant weak interaction in nature is the arene interaction. Recent studies have provided new insight into the driving forces, stability and selectivity o f these interactions. These interactions, though modest in energy, play a crucial role in such diverse areas as protein folding, base-to-base stacking in DNA/RNA, drug-receptor interactions, host-guest binding in supramolecular assemblies, crystal engineering and other molecular recognition processes.1.1.1 The Nature and Geometry of Aromatic InteractionsArene interactions have been proposed to consist o f van der Waals, hydrophobic and electrostatic forces, however, contribution and magnitude o f each o f these components may vary from case to case and is a matter o f many investigations.6 This is further complicated by the fact that aromatic groups interact in one o f several geometries, depending on the nature o f the rings involved. Nonetheless, aromatic

[ 1 ]

interactions are intriguing molecular recognition elements as they are expected to be strong in water because o f the hydrophobic component o f the interaction yet, at the same time, the interaction should be selective if the electrostatic component is significant, thus providing the best features o f both hydrophobic interactions and hydrogen bonding.Several geometries are attractive, and have been proposed on the basis o f the electrostatic component o f the interaction (Fig. I).7 The electrostatic component has been proposed to arise from interactions o f the quadrupole moments o f the aromatic rings. Although benzene has no net dipole, it has an uneven distribution o f charge, with greater electron-density on the face o f the ring and reduced electron-density on the edge, which gives rise to the quadrupole moment. The edge-to-face geometry (Fig. la ), which can be considered a CH-7t interaction, is found in benzene in the solid state, and is commonly observed between aromatic residues in proteins. The offset stacked orientation (Fig. lb ) is also commonly found in proteins and is the

geometry o f base stacking in DNA. In this geometry, more surface area is buried, and the van der Waals and hydrophobic interactions are increased. This orientation appears to be more common when the electron density on the face o f one or both rings is reduced. A third possible geometry is the face-to-face stacked orientation (Fig. lc). This is commonly observed with donor-acceptor pairs and compounds that have opposite quadrupole moments, such that the interaction between the faces o f the rings is attractive. The benzene-perfluorobenzene interaction is an excellent example o f this type o f aromatic interaction, and has been calculated to provide -15.5 kJ/ mol in stability.8

F

Fig. 1 Geometries of aromatic interactions, (a) edge-to-face; (b) offset stacked; (c) face-to-face stacked.

[ 2 ]

The electronic structure theory has been applied to generate potential energy curves for the sandwich, T-shaped, and parallel-displaced configurations o f the simplest prototype o f aromatic J i - n interactions, the benzene dimer. These studies provide a better understanding o f how the strength o f n-n interactions varies with distance and orientation o f the rings and will assist in the development o f approximate methods capable o f modeling weakly bound tc- k systems.9Very recently, n-n interaction in pyridine dimer and trimer (Fig. 2) has been investigated in different geometries and orientations at the ab initio (HF, MP2) and DFT (B3LYP) levels o f theory using various basis sets (6-31G*, 6-31G**, 6- 311++G**) and corrected for basis set superposition error (BSSE).10

The calculated binding energy (100% BSSE corrected) o f the parallel-sandwich, anti- parallel-sandwich, parallel-displaced, anti-parallel-displaced, T-up and T-down geometries for pyridine dimer were found to be 1.53, 3.05, 2.39, 3.97, 1.91, 1.47 kcal/mol, respectively. The results showed the anti-parallel-displaced geometry to be the most stable. The binding energies for the trimer in parallel sandwich, anti-parallel

Antiparallel -displaqpd T-Up T-Down

Parallel-sandw ich A ntiparallel-sandw ich Parallel-d isplaced Fig. 2. Different geometries of pyridine dimer and trimer.

[ 3 ]

sandwich, and anti-parallel displaced geometry were found to be 3.18, 6.14, and 8.04 kcal/mol, respectively.The binding energy for the mixed dimers of substituted benzene and pyridine in a parallel-slipped geometry has been estimated by Mignon et al. to be in the range of 2.8-4.2 kcal/mol.111.1.2 Experimental Study o f n - n InteractionsExtensive work has focused on these interactions to determine their importance as a

izrrecognition element, including statistical analyses of the Protein Data Bank, mutation studies in proteins17 and protein—nucleic acid complexes18’19 and theoretical studies.6 Model systems have also proven to be an informative method of investigating the nature and significance of aromatic interactions as molecular recognition elements in biological and non-biological systems. Ferguson and Diederich studied the complexation of a series of 2,6-disubstituted naphthalene derivatives by cyclophanes in ^-methanol (Fig. 3). The interaction between host and guest was most favorable for guests with electron withdrawing substituents such as X = CO2H, NO2 and CN and least favorable for those with electron donating substituents such as X = CH2OH, NH2 and CH3.21

X = Y = CH2OH, NHZ, CH3, C02H, N02, CN Fig. 3 Cyclophane complexes used to study substituent effects by Diederich.

The cyclophane can be thought of as a donor host with four phenyl rings substituted with electron donating methoxy groups. The most stable complexes were formed with electron poor guests, and this suggests that electrostatic interactions are the major factor determining the stability of the complexes. No charge-transfer bands were observed in the UV-visible absorption spectra, indicating CT played no part in the stability of such complexes. This work demonstrated the importance of electronic complementarity in the complexation of aromatic guests.

[4 ]

The effect of solvent on aromatic interactions was also studied by Smithrud and Diederich using the complexation of pyrene by a different cyclophane (Fig. 4).22 The association constants were determined in 18 solvents of differing polarities. A linear

Fig. 4 Cyclophane complexes used to study solvent effects by Diederich.

relationship was obtained between the stability of the complexes and the solvent polarity described by the empirical Ex(30) parameter. This model describes the solvent properties, which appear to be most important in determining the strength of apolar host-guest complexation. Binding is strongest in polar solvents possessing low molecular polarizability and high cohesive factors. Solvents with high cohesive interactions interact more strongly with “like” bulk solvent than with the apolar surfaces of the host and guest molecules, so when complexation takes place, free energy is gained upon the release of surface-solvating molecules to bulk solvent. Thus water is the best solvent for apolar binding.Whitlock et al. designed a macrocyclic host to bind nitrophenol (Fig. 5).23’24 A combination of aromatic stacking interactions and hydrogen bonding was found to be responsible for tight binding. Use of a more flexible linker reduced the binding constant indicating the importance of pre-organization.

W

Fig. 5 Two views of the complex formed between Whitlock’s bicycle and p-nitrophenol.

[ 5 ]

Dougherty et al. used the system shown in Fig. 6 to examine the contributions of aromatic and ion-quadrupole interactions to complexation in aqueous media Hosts 1 and 2 have similar dimensions and comparable degrees of pre-organization. The rigid framework ensured the charged groups could not interact with the guests, and therefore any difference in binding could be ascribed to interactions with the spacer group. If the hydrophobic effect was dominant, then the cyclohexyl derivative should show the strongest binding (cyclohexyl is more hydrophobic than phenyl). The phenyl derivative should be a better host, if aromatic interactions are important. Both hosts were found to bind the electron deficient quinoline and isoquinoline units 3-7 more strongly than the electron rich indole units 8 and 9. This was evidence that electrostatics play an important role in the binding. The phenyl host 1 bound the charged guests 10 and U more strongly than the cyclohexyl derivative 2. Comparison of the results for isostructural guest pairs showed this enhancement was due to charge and not to steric or hydrophobic effects. The enhanced binding of cations 10 and 11 by the phenyl host 1 was therefore due to the interaction of the positive charge with the n -electrons: the cation-it effect. The directionality of the cation-jr effect was studied by Schwabacher and co-workers.26

2

t* 11Fig. 6 Dougherty’s phenyl host 1 (X-ray structure) and cyclohexyl host 2, which bind

guest molecules 3-11 in water.

[6 ]

The cationic 12 and anionic 13 hosts in Fig. 7 were designed to study the interaction o f charges with the edge o f a bound aromatic ring. Schneider and co-workers had previously shown enhanced binding o f aromatic guests by cationic cyclophanes over anionic analogs.27 Association constants for dihydroxynaphthalenes 14 and 15, tropolone 16 and acenaphthalene 17 are larger for the anionic host which implies that

OH

Fig. 7 Schwabacher’s cationic (12) and anionic (13) cyclophane complexes and aromatic guests 14-17.

the positive edge o f the guest interacts more favorably with the negative walls o f the anionic host. With the larger aromatic guests, tropolone 16 and especially acenaphthalene 17, where the edge of the aromatic ring is much closer to the charged junction, significant increases in association constants were observed.In 1987, Hamilton et a!, reported the synthesis o f a class o f thymine receptors which showed edge-to-face or stacked aromatic interactions depending on the electronic properties o f the substituents.28,29 Macrocycle 18 formed a 1 : 1 complex with 1-butyl- thymine 20 (K = 570 M '1 in chloroform). NMR studies indicated a stacked geometry that was confirmed by an X-ray crystal structure (Fig. 8a and 8c). MNDO calculations on 2,7-dimethoxynaphthalene-3,6-dicarboxylic acid and thymine indicated a precise alignment of five pairs o f oppositely charged atoms (Fig. 8d) which confirmed the importance o f complementary electrostatic interactions in face- to-face stacking. Tetraether macrocycle 19 bound more weakly (K = 138 M '1). MNDO calculations indicated a mismatch in the charge distributions for this system, and NMR spectroscopy and the X-ray crystal structure showed that an edge-to-face interaction is used to avoid stacking (Fig. 8b).Rebek and Nemeth designed a molecular cleft (21) to bind aromatic guests (Fig. 9).30 The binding o f 21 to heterocyclic diamines was studied using 'H NMR spectroscopy. For pyrazine 22, the binding constant was 1.4 * 103 M '1 in chloroform. Quinoxaline

[ 7 ]

23 showed a 15-fold enhancement in binding (K = 2.3 x 104 M'1), due to a stacking interaction with the anthracene group which was revealed by upfield shifts of the quinoxaline protons.

««

18: R = C02(CH2)2CH3 19: R = 0(CH2)3CH3

Fig. 8 Hamilton’s thymine receptors, (a) Ester substituents lead to a stacking interaction. (b)Alkoxy substituents prevent stacking, (c) The geometry of the stacking interaction in (a) (d) The alignment of

charges which leads to the attractive interaction in (a).

22

21 23Fig. 9 The complex formed between Rebek’s cleft 21 and pyrazine 22.

Rebek et al. later developed a synthetic system that can recognize adenine using Watson-Crick or Hogstein hydrogen bonding and aromatic interactions (Fig. 10). Kemp’s triacid formed the basis of the receptor which could be substituted with a variety of aromatic groups of varying size and electronic properties. The results of NMR binding experiments show that the phenyl and naphthalene systems show only a small increase in the association constant compared to the control methyl amide,

[ 8 ]

whereas anthracene shows a nearly six-fold increase in binding constant which corresponds to a stacking interaction of 4.2 kJ/mol.

Fig. 10 Complexes used to investigate stacking interactions with adenine.

Chen and Whitlock first defined molecular tweezers as synthetic receptors containing two complexing aromatic chromophores connected by a single spacer.32 Bisfunctional derivatives o f caffeine 29 showed an increase in association constant relative to simple caffeine derivatives when complexed with planar aromatic guests such as 2,6- dihydroxy benzoate and l,3-dihydroxy-2-naphthoate (Fig. 11). Since then, molecular

n,m = 1 ,2 29Fig. 11 Whitlock’s molecular tweezer.

tweezers have been the subject o f an extensive study by Zimmerman. In 1987, he described a molecular tweezer in which a rigid spacer enforced a syn co-facial arrangement o f two acridine chromophores as shown by the X-ray structure in Fig. 12a.33 The spacer holds the chromophores approximately 7 A apart, ideal for a planar aromatic guest. Complexation studies were carried out in chloroform solution by !H NMR spectroscopy, and the tweezer shown in Fig. 12a binds 2,4,7-trinitrofluoren-9- one (TNF) with an association constant o f 172 M"1. Large upfield shifts observed for the TNF resonances suggest the TNF carbonyl is directed towards the spacer. Both the mono- and di-acridine control compounds 34 and 35 showed association constants o f less than 5 M '1 with TNF, indicating both acridines are required and that the rigidity o f the spacer plays an important role (Fig. 12c). Electron donor-acceptor effects were probed using the tweezers 30-33 and the results indicate that as the electron density o f the host 7r-system increases, the association constant increases (Fig. 12b).34 The use

[ 9 ]

Fig. 12 (a) X-Ray structure of Zimmerman’s molecular tweezer, (b) The structures of tweezer derivatives 30-33. (c) Control compounds 34 and 35.

of donor solvents, THF and 1,4-dioxane, which solvate TNF better than chloroform greatly reduced the association constants. Zimmerman et al. covalently linked the tweezers to silica to construct chemically bonded stationary phases for HPLC.35’36 The retention times of several nitro-substituted polycylic aromatic hydrocarbons were measured. The HPLC chromatogram in Fig. 13 show, how increasingly electron-poor aromatics are retained longer on the column. With such good separation, this system was proposed as a potential tool for analyzing nitro-polyaromatic hydrocarbons, an important class of environmental pollutants. The tweezer motif is still being used by Zimmerman et al. to organize dendritic systems.

NO; * 3 ; HG-

Fig. 13 HPLC Chromatogram for a tweezer bound stationary phase compound

[ 10]

Nolte et al. used hydrogen bonding and aromatic interactions to design a series of molecular clips.38 Separation of the effects of hydrogen bonding and aromatic interactions on the binding of resorcinol derivatives was carried out by synthesizing a series of clips with different numbers of aromatic side-walls (Fig. 14).

<«> ie>Fig. 14 Nolte’s molecular clips and the complex formed with catechol.

The host with no walls can only bind resorcinol by hydrogen bonding (K = 25 M"1). With one cavity wall, the association constant increased to 65 M'1, but a second wall dramatically increased the association constant to 2600 M’1. Changing the methoxy substituent on the cavity walls to a methyl group and then to a hydrogen decreased the association constants, and the differences were attributed to a decrease in the strength of the aromatic stacking interaction. Naphthalene side-walls were introduced in an effort to increase the van der Wads contacts between the host and guest. However, this decreased the association constants, presumably due to an increase in the 7t - electron repulsion between host and guest, indicating it is electrostatics rather than van der Waals forces which play the pivotal role in determining the magnitudes of the aromatic stacking interactions here.A cleft type receptor for aromatic acids was reported by Crego et al. (Fig. 15).40 The

36Fig. 15 Crego’s clefts bind benzoic acid derivatives.

[ 11]

receptor 36 relies on stacking interactions and hydrogen bonding and binds a variety o f substituted aromatic acids and amides. Generally, the binding constants increase with increasing n-electron density on the guest. However, there are some anomalies (e. g. with 3,4-methylenedioxybenzoic acid) indicating that the system is more complicated.Moore et al. prepared hexakis(phenylacetylene) molecules (PAMs) with varying degrees o f electron withdrawing (ester) and donating (alkyl ether) substituents and studied their aggregation properties by 'H NMR in chloroform (Fig. 16a).41,42 The chemical shifts o f the aromatic protons depend strongly on concentration, and dimerization through aromatic stacking interactions was proposed to account for this. Compounds 37, 38 and 39 show dimerization constants o f 60, 18 and 26 M '1 respectively.Compounds 40 and 41 show no aggregation behavior. These results indicate that the aromatic substituents have a significant influence on the stacking interaction, tert- Butyl ester substituents prevent aggregation, indicating that the interaction is due to face-to-face stacking which is hindered by the bulky groups. Non-planar penta-kis and heptakis-(phenylacetylene) molecules also have reduced association constants. Tobe

R

37: R1'6 = C 0 2n-Bu38: R1'3'5 = C 02n-Bu, R2’4 6 = O-n-Bu39: R1'2'3 = C 02n-Bu, R4'56 = O-n-Bu40: R1 6 = O-n-Bu , 1-6 .

42: R = C 0 2C8H17, X = CN 43: R = C 0 2C8H17, X = H

41: R = CH20-n-Bu

Fig. 16 (a) Moore’s macrocyclic phenylacetylene molecules (PAMs), (b) Tobe’s macrocyclic PAMs.

[ 1 2 ]

et al. designed a PAM system capable o f hetero-aggregation and binding metal ions (Fig. 16b).43 Compounds 42 and 43 form a 1 : 1 hetero-aggregate but 42 does not self­associate. The electron withdrawing cyano substituents appear to enhance aromatic stacking interactions in the hetero-aggregate.In 1990 Hunter and Sanders developed a simple electrostatic model, Fig. 17 and 18, where a set o f point charges is used to represent the electrostatic charge distribution in a pi-system. Useful general rules have been derived from this work for a qualitative understanding o f n-n interactions.44 The key feature o f the model is that it considers p electrons and the a bonding system separately, and reveals p -p repulsion and p-s attractions to be the governing factors in the interaction between two aromatic molecules. This model has been challenged recently by ab initio studies, which contend it should be sufficient to recognize solely the role o f molecular quadrupoles when describing aromatic interactions.45

> @ —(.*)

Fig. 17 An sp2 hybridized atom in a 7t-system.

As the hydrophobic effect has a significant influence on aromatic interactions, water preferring to interact with it self rather than with aromatic surfaces, Newcomb and Gellman carried out a series o f experiments to investigate this effect for two covalently tethered aromatic groups. A comparison o f the stacking tendencies o f hydrocarbon (phenyl and naphthyl) and heterocyclic (adenine) rings in aqueous solution was carried out using 'H NMR spectroscopy to study the conformational properties o f carboxylate derivatives 44-48 (Fig. 19).46 Large negative shifts o f the adenine protons o f 44 were observed compared with the control 47. This is indicative

Ar*ylt*r

— ®-© -(*)—Fig. 18 Electrostatic interactions between 7t-charge

distributions as a function o f orientation.

[ 1 3 ]

o f an intramolecular aromatic interaction in 44. In contrast, 45 and 48 have similar spectra which indicates that there is no intramolecular interaction for the di-naphthyl derivative. An X-ray structure o f a phenyl derivative 49 showed the phenyl rings splayed far apart. In 46, negative shifts on both rings indicate significant stacking. DMSO destroyed the interaction. If the intramolecular stacking were due solely to the hydrophobic effect, then 45 would exhibit a stacking interaction. The results are most consistent with the alignment o f partial positive and negative charges on neighbouring groups as the main force influencing the stacking interactions.

'O Na ^ Ov51 52

Fig. 19 Compounds used to probe intramolecular aromatic interactions in water.

Naphthyl units connected by a flexible linker were prepared to further probe hydrophobic collapse.47 The three atom linker previously used forced a near parallel arrangement, but the four atom linker in 50 allowed different approaches o f the aromatic moieties. An X-ray crystal structure o f 50 showed an edge-to-face arrangement o f the naphthyl rings and ]H NMR experiments showed that the naphthyl rings are in close proximity in aqueous solution. The chemical shift differences between 50 and 51 in benzene were very similar to those in water, which suggests that the hydrophobic effect have little influence on the folding o f this molecule.

[ 1 4 ]

Kollman el al. used a combination of modeling and NM R studies on similar compounds having indole system as arene moiety with different results.48 The possible geometries o f the indole derivative 52 were calculated theoretically (Fig. 20). The linker allows the molecule to adopt edge-to-face (IT5, IT7), offset stacked, face- to-face stacked and non-stacked conformations (IN’s). The calculations suggested that the edge-to-face and non-stacked conformations are the most stable in water. 1H NMR studies on 52 showed a larger population o f the edge-to-face stacked conformation in water than in DMSO at 22 °C.

i f u

INI

IN8

IN6

Fig. 20 Different conformations of 52

Proton NMR spectroscopy has been used by Jimenez-Barbero to investigate intramolecular interactions between different aromatic groups in a series o f di-esters 53-58 (Fig. 21).49 The 'H NMR spectrum o f the symmetrical di-esters 54 and 56 and corresponding control mono esters 53 and 55 are very similar, indicating there is no intramolecular interaction. In contrast, substantial upfield shifts (0.1 and 0.5 ppm) are observed in the resonance frequencies o f all aromatic protons in the anthracenyl and

[ 1 5 ]

3,5-dinitrophenyl groups o f the unsymmetrical di-ester, l-(9-anthracenecarbonyloxy)-3-[(3,5-dinitrobenzoyl)oxy]-2-methylpropane (57), relative to those for the aromatic

o 2n n o 2 o 2n

o 2n c h 3 n o 2 o 2n c h 353 54

57 58Fig. 21 Compounds used to probe intramolecular aromatic interactions.

protons o f the respective mono esters and symmetrical di-esters. A stacked intramolecular complex was proposed. If van der Waals interactions were dominant in the complex, the greatest effect would be in the symmetrical anthracene derivative, as it would provide the largest van der Waals contact. No charge transfer bands in the UV spectra were observed. Hence the interaction was attributed to electrostatic quadrupole interactions, as the quadrupole moments o f the dinitro-phenyl and anthracenyl groups have opposite signs. Stoddart et al. used aromatic stacking interactions to direct the synthesis and influence the properties o f large number o f catenanes, pseudorotaxanes and rotaxanes. A 1:1 complex was observed between di- />-phenylene-34-crown-10 and paraquat dication (Fig. 22a).50 The complex between a tetracationic ring based on paraquat and 1,4- dimethoxybenzene was also crystallized (Fig. 22b), and this revealed the aromatic stacking interactions that are responsible for complexation. The interactions in these complexes formed the basis o f the template directed synthesis o f a [2]catenane (Fig. 22c).51 This initial design led to higher order catenanes, the largest being a [7]catenane which was synthesized under high pressure.52 Pseudorotaxanes followed and stoppering the ends o f the thread o f the pseudorotaxane resulted in [2]rotaxanes. Again, higher order pseudorotaxanes and rotaxanes were synthesized and characterized.53

[ 1 6 ]

Fig. 22 Host-guest complexes fonned by the Stoddart macrocycles (a) and (b), and thecorresponding [2]catenane (c).

Itahara studied the intramolecular stacking interactions in polymethylene linked adenines, 9,9'-(a ,co-alkanediyl)bis[6-(dimethylamino)purine] (59) and 9,9'-(a ,co- alkanediyl)bis[6-(methylamino)purine] (60), using proton NMR spectroscopy. The N6-methyIation was found to increase the population of intermolecular aggregates in the buffer solution at pD 7*0 and had an additive effect on aggregation which was interpreted due to hydrophobic effect of the N6-methyl groups. The aggregation of 59 and 60 was found to depend on the length of the polymethylene chains.54

A relationship between the chemical shifts of adenine and xanthine ring protons of 7- [-(6-aminopurin-9-yl)aIkyl]-l,3-dimethylxanthines (61) and the number of carbons (ft = 2-10) in their polymethylene chains has been compared with that of l-[-(6- aminopurin-9-yl)alkyl]-3,7-dimethylxanthines (62) in the buffer solutions at pD 7.0,

1.0 and 13.0 and in organic solvents. The relationship of 61 is clearly distinct from that of 62. The concentration dependence and the effects of temperature on the

59: F?i = F?2 = Me 60: R, « Me, R2 = H 82

chemical shifts of 61 and 62 have also been investigated. While the upfield shifts of the ring protons of 61 and 62 in the buffer solutions at pD 7.0 and 13.0 are explained

[ 17]

in terms o f stacking interactions between adenine and xanthine rings, the results in the buffer solution at pD 1.0 may be due to cation- 7 1 interactions. On the basis o f these data, it can be assumed that the stacking interactions do not only consist of interactions between adenine and xanthine rings.551.1.3 Aromatic Interactions in Biological SystemsAromatic interactions are ubiquitous in nature. They are believed to provide stability to duplex DNA,56 they have been proposed to contribute to the unique properties o f thermophilic proteins,57 they may play a role in aggregation o f amyloid P in Alzheimer’s disease,58 and they are common motifs in bio-molecular recognition.In various complex chemical and biological systems, aromatic rings can be found at different orientations and distances from each other. Due to steric constraints imposed on the aromatic groups, these geometries might not correspond to the potential energy minima for n-n interactions. Nevertheless, the aromatic rings might still interact favorably enough to contribute significantly to the overall stability o f the system. In their original X-ray crystallographic study involving 34 high-resolution protein structures, Burley and Petsko analyzed the frequency o f aromatic pairs and their interaction geometry (distance and dihedral angle) and concluded that around 60 % o f aromatic side chains o f phenylalanine, tyrosine, and tryptophan were involved in arene interactions.59 Aromatic rings separated by distances ranging from 4.5 to 7 A and dihedral angles near 90° were found to be the most common. Pair-wise nonbonded potential energy calculations indicated that 54 % o f the aromatic interactions are attractive by l-^ k ca l/ mol. McGaughey, et al. extended the analysis to a larger sample o f proteins and suggested that the parallel-displaced geometry was a preferred orientation.60 Remarkably, none o f these two studies described the face-to- face geometry.In a study o f a larger database o f 52 proteins, Hunter, et al. examined the orientational preferences o f phenylalanine side chains in proteins using crytallographically derived atomic coordinates.61 They observed that these interacting pairs are found in a wide range o f T-shaped (edge-to-face) and parallel-displaced (offset-stacked) arrangements, but they are scattered over a wide variety o f conformational space with no strongly preferred single orientation. From the above discussion, it becomes clear that high-quality potential energy curves for prototype systems would be very helpful

[ 1 8 ]

in better understanding how n -it interactions depend on both the orientation and distance between the aromatic rings.Aromatic-aromatic interactions between phenylalanine side chains in peptides have been probed by the structure determination in crystals of three peptides: Boc-Val-Ala- Phe-Aib- Val-Ala-Phe-Aib-OMe, I; Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala- Phe-Aib-OMe, II; Boc-Aib-Ala-Phe-Aib-Phe-Ala-Val-Aib-OMe, HI (Fig. 23)“ X- ray diffraction studies reveal that all three peptides adopt helical conformations in the solid state with the Phe side chains projecting outward. Interhelix association in the crystals is promoted by Phe-Phe interactions. A total of 15 unique aromatic pairs have been characterized in the three independent crystal structures. The distances between the centroids of aromatic pair ranges from 5.11 to 6.86 A, while the distance of closest approach of ring carbon atoms ranges from 3.27 to 4.59 A. T-shaped and parallel- displaced arrangements of aromatic pairs were observed, in addition to several examples of inclined arrangements. The results support the view that the interaction potential for a pair of aromatic rings is relatively broad and rugged with several minima of similar energies, separated by small activation barriers.

I II IIIFig. 23 Molecular conformation in crystals of peptides I-III. All the hydrogen bonds

are shown as dotted lines.Nucleic acids play a central role in cellular metabolism and so are a common target for drugs designed to prevent cell replication. Aromatic stacking interactions play a pivotal role in drugs, which intercalate with DNA. Intercalation was first observed by Lerman when he studied the complex between DNA and acridine.63 A mechanism

[ 19]

was proposed whereby the acridine could fit between the base pairs of DNA without disrupting the hydrogen bonding motif. This process, however, causes a change in the physical characteristics of DNA as the helix unwinds, and the bases unstack to allow the intercalator in. This leads to an increase in length of the DNA and a disruption of the regular helical structure (Fig. 24). A variety of DNA intercalators have been found to reduce tumor growth in animals and man, and so these compounds are commonly used as anticancer agents.64

Fig. 24 The DNA double helix in the absence (a) and presence (b) of an intercalator (red).1.1.4 Aromatic Interactions of Heteroaromatic RingsHeteroaromatic rings are commonly found in biomolecules and in synthetic molecules that exert specific biological effects (e. g. drugs). Noncovalent interactions involving heteroaromatic units contribute > to the stability and specificity of macromolecular folding patterns and macromolecule-ligand interactions. Heterocycles engage in favorable interactions with one another65 and with hydrocarbon aromatic units66 in aqueous solution, but the origin of these favorable interactions remains unclear.67

Possible sources of heteroaromatic “stickiness” include the hydrophobic effect,68z ~a 7 / 4 T 1dispersion, polar interactions and “donor-acceptor” interactions. These

interaction modes are not exclusive of one another.Several studies of nucleic acids and synthetic model systems have attempted to address this. In a study of aromatic stacking in duplex DNA in which a wide range of aromatics were investigated, Kool et al.12 showed that polarizability and buried surface area correlate best with stacking of a 5'-dangling base, whereas the dipole moment and the hydrophobicity as measured by log P (the partition coefficient between octanol and water) do not correlate well. These results were interpreted as indicating that hydrophobic interactions provide the greatest contribution to stacking

[2 0 ]

interactions. The discrepancy between surface area and log P is explained by the fact that many o f these compounds, particularly the natural bases, can simultaneously bury surface area and interact with water through their polar groups on the edges o f the rings, resulting in the better correlation with buried surface area than log P. Quadrupole-quadrupole interactions were also proposed to be important for electron- poor rings such as 4-nitroindole. Rosemeyer and Seela73 have also shown that the polarizability o f the dangling base correlates with stability o f the duplex better than hydrophobicity as measured from RP-HPLC retention times, and have interpreted this as indicating that polarizability, rather than hydrophobicity, is the most important factor in stabilizing duplex DNA. A general difficulty with interpreting the results from dangling-base studies is that the geometry o f interaction in these systems typically is not known. For example, Richert et al™ found that duplex DNA with a 5'- quinolone cap was significantly stabilized relative to the uncapped sequence. However, detailed structural analysis o f this system indicates that the quinolone does not cap the terminal T:A base pair, but in fact disrupts the terminal pair in favor o f quinolone-adenine stacking against the penultimate G:C base pair. Gellman et al. have studied the role o f heteroatoms in aromatic interactions in their amide model system (Fig. 25, R = CH2C 0 2X).75 In this study they used a carefully designed molecular framework to examine how interactions between a phenyl unit and a naphthyl unit are affected as nitrogen atoms are introduced into one or both o f these units. In this system, the number and location o f nitrogen atoms in two aromatic rings, Ari and Ar2, were varied, and the effect on amide rotamer population was determined>in water. The £-rotamer places Ari and Ar2 in close proximity, and a crystal structure indicates that Ari and Ar2 interact in an offset-stacked geometry in this rotamer.

Fig. 25 Gellman’s system for measuring aromatic interactions in aqueous and organic solvents.

In this system, Gellman has found that, in general, an increase in the number o f heteroatoms in either or both Ari and Ar2 resulted in an increase in the £-rotamer,

X = Na, CH3 X = Na, CH3

Z R = H, CH2COOX E

[ 2 1 ]

presumably due to a favorable aromatic interaction. This argues for a significant polar component to the interaction. Nonetheless, when the experiments were performed in chloroform, very little effect was observed, indicating that aqueous solvent is required for significant interaction.Historically, polymethylene linkers, especially ‘trimethylene linker', have been used by Leonard et al. to study the intramolecular arene interactions.76 The main feature o f ‘trimethylene linker’ is that it provides a desired distance o f 3.4 A between the two terminal arene groups when folded. Browne and Leonard studied the interactions of bases found in nucleic acids in the absence o f complicating factors associated with hydrogen bonding or the usual carbohydrate and phosphodiester linkages.76 They have synthesized a series o f twelve dinucleotide analogs in which the bases are connected by a ‘trimethylene chain’, B-(CH2)3-B', where B and B' are 9-substituted adenine or guanine or 1-substituted cytosine, thymine or uracil residues (Fig. 26). These compounds were studied optically at concentrations low enough to preclude formation o f intermolecular complexes so that the perturbations associated with the 1:1 interaction o f a pair o f bases could be characterized, namely by UV spectra in aqueous solution at room temperature and by emission spectra in 1:1 ethylene-glycol- waterglass in the vicinity o f 77°K. In the series o f B-(CH2)3-B' the order o f interaction

O O

O

(?H2)3

n h 2 o63d: Ad-C3-Cy 63e: Ad-C3-Ur 63f. Gu-C3-Gu

Fig 26

[ 2 2 ]

in neutral aqueous solution is purine-purine > purine-pyrimidine > pyrimidine- pyrimidine as judged by hypochromism (decrease in integrated UV absorption intensity o f B-Cn-B' compared with equimolar B-(CH2)2-CH3 and B'-(CH2)2-CH3). The length o f polymethylene chain was also changed to C2 and C&. In a series o f 9,9'- polymethylene-bis(adenines) the order o f interaction deduced from hypochromism and emission studies was n = 3 > 2 & 6. Reduced interaction at n = 2 reflected the impossibility o f this molecule assuming folded, parallel-plane conformation which would allow maximal interaction as in Ad-C3-Ad. An entropy effect was probably responsible for decreased interaction in the n = 6 compound relative to n = 3.The orientation effect studies were done by Leonard and Ito by synthesizing six different trimethylene-bis(adenine) isomers having different positions o f attachment to the terminal adenines and therefore having differently oriented ring-axis permissible in their stacked conformations, in order to determine stacking interactions between two parallel adenine rings oriented at different axis angles toward each other (Fig. 27).77 The percent hypochromism ‘/ f for the long wavelength UV absorption

Fig. 27 dimers of adenine linked through different positions to study orientation effect on stacking.

band for each o f these compounds has been determined by comparison o f the UV spectrum o f trimethylene-bis-adenine in aqueous solution with the composite spectrum o f the two half molecules, the appropriate propyl-adenines. The IT

64a 64bNH2

HNH2

[ 2 3 ]

follows a dependence upon the folded conformations available to the individual trimethy lene-bis(aden ines).Seyama et al. in 1988, reported the relationship between the magnitude o f hypochromism and stacking geometry o f purine rings.78 Twelve purinophanes, (65a, 65c, 65d, 65f, 65g-65j) were synthesized in which two purine rings were fixed with different modes o f stacking by two or three polymethylene chains. X-Ray and/or 'H NMR analysis determined five kinds o f the stacking geometries o f the two component rings in the purinophanes. The interplanar distances vary from 3.2 to 6.6 A. All the purinophanes, except 65i and 65j, showed large hypochromism (decrease in integrated absorption intensity compared with corresponding dimeric linear compounds, 65m-65o). The largest value (47.6 % in H20 ) was observed for (65f), where the interplanar distance is quite short (around 3.20 A). The hypochromism values o f the purinophanes (65a-65j) in water were found to be 28.6, 26.3, 26.6, 47.6,23.0, 15.7, 8.5 and 8.3, respectively, and those o f linear dimeric compounds (65m- 65o) were 7.9, 12.2 and 12.9. The hypochromism values o f (65k and 651) could not be obtained in water because o f poor solubility. However, H values o f (65i and 65j) in ethanol are 29.9 and 26.1, respectively, and in 0.1 N HC1 it is 30.1 and 30.6. The hypochromism values o f the purinophanes (65a-65h, 65k and 651) were almost identical in four different media (ethanol, water, 0.1N HC1, and 0.1N NaOH). The effect o f separation between the rings on the magnitude o f hypochromism was obvious when the values o f homologs (65f) and (65g) or (65k) and (651) were compared, i. e., the smaller the interplanar distance, the larger the value o f*hypochromism. The bridge protons o f these purinophanes show complex multiplets in their NMR spectra in contrast to the first order coupling patterns o f acyclic reference compounds (Fig. 28). These results indicate that the conformations o f (65a-65h, 65k and 651) are almost frozen in the various media at room temperature.

65a: x = NH, m = 2, n = 3 65i: n = 365b: x = NH, m = 2, n = 4 65j: n = 465c: x = S, m = 2,n = 3 65d: x = S, m = 2, n = 4 65e: x = S, m = 3, n = 3

[ 2 4 ]

(65f): n = 2 (65g): n = 3

(65k) : n = 3 (651) : n = 4

X x

(65m): X = NHMe (65n) : X = SMe

(65o)

Fig. 28

Pyrazolo[3,4-</]pyrimidine core (which is isomeric with biologically important purine system) based compounds in which two such units are linked by ‘polymethylene linkers’ provide simple and easy to make flexible models for understanding the nature o f aromatic j c - j i interactions (APPI). The compound 66a has been reported as the first example (1995) based on pyrazolo[3,4-</|pyrimidine core to show intramolecular stacking in solution, as ’H NMR spectroscopy showed upfield chemical shift for 6- SMe protons when compared with its monomeric compound 70.79 Both intra- and intermolecular stacking were found in solid state by X-ray crystallography (Figs. 29 & 30).80 This intramolecular stacking due to arene interactions results in a unique*folded conformation: U-motif in which two pyrimidine moieties are partially overlapped while pyrazolo portion are away from each other. The robustness o f unusual conformation due to intramolecular %-n stacking interactions, resulting in a U-motif has been further demonstrated in many o f its analogs.81 The compound 67a (R = Me) derived from 66a and its regioisomers (68 and 69) and their various analogs also showed similar folded conformation in both solution and solid state.82 Recently, due to pioneering work o f Leonard76,77 on ‘trimethylene linker’ his name has been suggested for this linker.81 The characteristic 'H NMR chemical shifts o f compounds 66a, 67a, 68-74 are shown in Table I.

[ 2 5 ]

SR5 J U 3

SRN| ^ S v — 2 / NA X 'N N' X ARS^N, Ni I N ^ N ^ S RX (CH2)^

66a) n = 3, R = Me,b) n = 3, R = Et,c) n = 3, R = iPrd) n = 2, R = Me,e) n = 2, R = Et

V V >M e S ^ N / N| N ^ S M e

x (c h 2)^R = Me, Et, CH2Ph

67a:n = 3 67b:n = 4

M e S ^ N

Fig. 29

Fig. 29a ORTEP Diagram of 64 with atomic numbering Fig. 29b ORTEP diagram o f 64 showing partialscheme; Acta Crystallogr. 1995, C51, 2453. overlapping o f two pyrimidine rings.

[ 2 6 ]

Fig. 30 Packing diagram o f 66 showing interm olecular interaction between pyrazole and pyrimidine rings

Surprisingly, the compound 73 which is a positional isomer o f 6 6 a, differing in theposition o f connectivity o f the linker by one atom movement on one side (N-l to N-2)showed absence o f intramolecular stacking both in solution and solid state . 79,83

The symmetrical compound 74, another positional isomer o f 6 6 a in which the linkeris connected through N-2 positions o f both the aromatic units, also does not show anysignificant upfield shift for SMe protons indicating absence o f intramolecular stackingin solution .79 However, no X-ray crystallographic study on this compound (74) hasbeen reported, presumably, due to non availability o f single crystal suitable for X-raydiffraction to study the solid state conformation.

Table-I: 'H NMR Chemical shifts of SMe groups o f compounds 66-74.

S. No. R Chem. shift6 -SR 4-SR

6 6 a Me 2.45 2.6767a Me 2.29 -6 8 Me 2.27,2.38 -69 Me 2.42 -70 Me 2.64 2 . 6 8

71 Me 2.65 -72 Me 2 . 6 6 -73 Me 2.51,2.58 2.61,2.6374 Me 2.65 2 . 6 8

[ 2 7 ]

More interestingly, ethylene linker homolog (6 6 d) o f 6 6 a also showed comparable upfield shift for 6 -SMe protons (5 2.44 & 2.66) indicating isyn' conformation in solution, presumably, due to arene interactions. Similarly, ethyl analog o f 6 6 d (i.e., 6 6 e) also showed upfield shift for 6 -SEt protons as compared to its monomeric compound again indicating isyn' conformation in solution . 81 However, X-ray crystallographic studies on 6 6 e showed ‘antf conformation in solid state81 (Fig. 31). This is not surprising as in solid state, normally, the ‘anti’ conformation is the most

CIO

Fig. 31 ORTEP diagram of 66e with atomic numbering scheme; Tet. Lett 2001, 42, 7115.

N3 W

Fig. 32 Structure o f BBTA showing anti conformation

predominant conformation in the 1,2-disubstituted ethanes, e. g., the X-ray structure analysis o f l,2-bis(benzotriazol-l-yl)ethane (BBTA) have shown ‘anti’ conformation (Fig. 32). However, two ‘an//’ conformations [A (1) and A (2)] and two ‘gauche’ conformations [G (1) and G (2)] were proposed by theoretical calculations (Fig. 33). The relative energies for A (1)»A (2), G (1) and G (2) conformers were found to be0.00, 1.09, 1.52 and 2.07 kcal/mol, respectively .84

■■ • .W'

A(1)

»-$> a a • *-* *■o-*i «

G<1)

!.A1 o

-e% It

A(2)

! T * . > • »

y . .ho ^Q *>..

G(2)

Fig. 33 stable calculated geometries for BBTA

[ 2 8 ]

Theoretical calculations were carried out to determine the molecular geometries and the relative energies o f the conformations o f 1,2-diphenylethane. The computed data are indicative o f a small difference in the energies o f the two main conformations: the synclinal (gauche Ph/Ph) and the antiperiplanar (anti Ph/Ph) ones and the existence o f the equilibrium mixture o f the two conformations . 85

Experimental studies on the conformations o f 1,2-diphenylethane showed that the molecules crystallize in the antiperiplanar conformation . 86 The preponderance o f the same conformation (ratio about 2 :1 ) in solutions was also determined from the examination o f IR and Raman spectra o f 1,2-diphenylethane and its p, p'-dihalogeno-, dinitro- and dicyano-derivatives in a solid state and in non polar solvents . 87

In literature, there are very few examples of 1,2-diarylethanes possessing isyri' conformation . 88,89 For example, the X-ray crystal structure conformation for 2-[2- (17/-indol-3-yl)-l-methylethyl]-3-[3-(l-methylethoxyphenyl]-4(3//)-quinazolinone (75a) has been reported to have syn conformation around the central a bond o f the ethylene linker, 88 Very recently, another compound 2-[2-(4,6-dim ethylsulfanyl- l//-pyrazolo[3 ,4-< /]pyrim idin-l-y l)-ethy l]-2 //-ph thalazin-l-one (75b) has been synthesized by Avasthi et al. which showed ‘syrt’ conform ation both in solution and solid state by 'H NM R data and X-ray crystallographic studies (Fig. 34).89 Another sim ilar compound 3-[2-(4 ,6-dim ethylsulfanyl-l//-pyrazolo[3 ,4-d]pyrim idin-l-yl)ethyl]-3 //-quinazolin-4-one (75c) in which the phthalazinone moiety o f 75b is replaced by quinazolinone moiety was also synthesized, having sim ilar upfield shift for 6 -SMe protons ( 8 2.27 ppm) showing sim ilar ‘syn' conform ation . 89

75a 75b 75c

[ 2 9 ]

Fig. 34 M olecular structure o f 2-[2-(4 ,6-di-m ethylsulfanyl-l//-pyrazolo[3,4-</|pyrim idin-l- yl)-ethyl]-2 //-phthalazin-l-one 75b showing syn conformation.

The tetramethylene and pentamethylene homologs o f 6 6 a also showed upfield shifts for their 6 -SMe protons, but the shifts are not so significant as in ethylene and ‘propylene’ linker compounds .79 No X-ray crystallographic studies o f these analogs are reported. However, the tetramethylene homolog (67b, Fig. 29) o f 67a has shown fully extended conformation in solid state (Fig. 35 ) . 90

Fig. 35 Displacement ellipsoid plot (50% probability), showing the molecular structure o f 67b with atomic labelling scheme.

When one pyrazolo[3,4-cf] pyrimidine moiety o f 6 6 a was replaced by bicyclic phthalimido moiety to give new compound 76, intramolecular folding in solution still persisted as revealed by 'H NMR analysis ( 8 6 -SMe = 2.51 ppm). X-Ray crystallographic studies on 76 indeed confirmed folded conformation but due to C H -7t interactions between 6 -SMe group of pyrazolo[3,4-d]pyrimidine moiety and phenyl portion o f phthalimido moiety, and not because o f intramolecular %-n stacking . 91

Furthermore, related ‘trimethylene linker’ compound 77 having one pyrazolo[3,4-d] pyrimidine moiety and other a phenyl moiety did not show any intramolecular n-n stacking .92

These observations show not only the importance o f the length o f ‘linker’, but the position o f connectivity o f the linker, in addition to the nature o f the aromatic unit, for intramolecular n-n stacking, are also significant.

SMe O

MeS‘J

O 7776

1.2 Objective of StudyThe synthesis o f the compounds in this scheme was planned to study the effect of changing the position o f connectivity o f the linker in otherwise stacked pyrazolo [3,4-t/Jpyrimidine based compounds (i.e. when the two aromatic moieties were linked through N -l, N -l positions) to get better understanding o f orientation effect on intramolecular stacking. Since the linker position restricts number o f geometries available for the two aromatic units to interact with each other, connectivity at different positions may help in optimizing the proper geometry required to exhibit the most stable stacked conformation. This strategy gives new compounds in which linker occupies position between N -l o f one pyrazolo[3,4-c/]pyrimidine unit and N-5 o f other unit. The driving force for this connectivity was to achieve the conformation whereby the six-membered pyrimidine ring o f one pyrazolo[3,4-d]pyrimidine unit is expected to overlap the five-membered pyrazole ring o f the other unit and vice versa and to explore the stacking interactions in such systems, which is observed inter- molecularly in some o f the above mentioned stacked compounds. In addition, the compounds in which two pyrazolo[3,4-<i]pyrimidine moieties are connected through N-l o f one unit and C-4 o f the other, with an oxygen atom in place o f methylene group in the linker, are also studied.

1.3 Synthesis and CharacterizationThe synthesis o f these compounds was achieved according to the below mentioned general reaction scheme (Scheme I). The compounds synthesized were characterized on the basis o f different spectral techniques and chemical methods. The results were analyzed on the basis o f 'H NMR spectroscopy and X-ray crystallography. The starting materials 7893 and 7979 were synthesized using known procedures.

[ 3 1 ]

S. No. Comp No. n R Ri R2 r 31 80a 2 Me SMe SMe N-1-Me2 80b 2 Me Pyrr. SMe N-1-Me3 80c 2 Me SMe SEt N-1-Et4 80d 2 Me Pyrr. SEt N-1-Et5 80e 2 Me SMe SEt N-1-Me6 80f 2 Me SMe SMe N-1-Et7 80g 2 Me SMe S-iPr N-1-Et8 80h 2 Et SEt SMe N-1-Me9 80S 2 Et SEt S-iPr N-1-Et10 80j 2 Et SEt SMe N-2-Me11 80k-N-2 2 Et SEt SMe N-2-Me12 80I-N-2 2 Me SMe SMe N-1-Me13 80m-N-2 2 Me SMe S-iPr N-1-Et14 80n-N-2 2 ' Et SEt SMe N-1-Me15 80o 3 Me SMe SMe N-1-Me16 80p 3 Me SMe SEt N-1-Et17 80q 3 Me Pyrr. SEt N-1-Et18 80r 3 Me SMe SEt N-1-Me19 80s 4 Me SMe SMe N-1-Me20 80t 4 Me SMe SEt N-1-Et21 80u 4 Me Pyrr. SEt N-1-Et22 80v 5 Me SMe SMe N-1-Me23 80w 5 Me Pyrr. SMe N-1-Me24 80x 5 Me SMe SEt N-1-Et25 80y 10 Me SMe SEt N-1-Me26 81a 2 Me SMe SMe N-1-Me27 81b 2 Me Pyrr. SMe N-1-Me

[ 3 2 ]

28 81c 2 Me SMe SEt N-1-Et

29 81d 2 Me Pyrr. SEt N-1-Et

30 81 e 2 Me SMe SEt N-1-Me

31 81f 2 Me SMe SMe N-1-Et

32 81g 2 Me SMe S-iPr N-1-Et

33 81 h 2 Et SEt SMe N-1-Me

34 81 i 2 Et SEt S-iPr N-1-Et

35 81 j 2 Et SEt SMe N-2-Me

36 81k-N-2 2 Me SMe SMe N-1-Me

37 81 l-N-2 2 Me SMe S-iPr N-1-Et

38 81m-N-2 2 Et SEt SMe N-1-Me

39 81 n 3 Me SMe SMe N-1-Me

40 81o 3 Me Pyrr. SMe N-1-Me

41 81 p 3 Me SMe SEt N-1-Et

42 81q 3 Me Pyrr. SEt N-1-Et

43 81 r 3 Me SMe SEt N-1-Me

44 81s 4 Me SMe SMe N-1-Me

45 81t 4 Me SMe SEt N-1-Et

46 81 u 5 Me SMe SMe N-1-Me47 81v 5 Me Pyrr. SMe N-1-Me

48 81w 5 Me SMe SEt N-1-Et49 81x 5 Me Pyrr. SEt N-1-Et50 81y 10 Me SMe SEt N-1-Me

Note: All the compounds 80a-80j, 80o-80y, 81a-81j and 81n-81y are connected through N -l" position and the compounds 80k-N-2 to 80n-N-2 and 81k-N-2 to 81m-N-2 through N-2" position.

Scheme I

General procedures:

Synthesis o f 5-[n-(4,6-dialkylsulfanyl-l//-pyrazolo[3,4-*/]pyrimidin-l-yl)aIkyl]-l- alkyl-6-alkylsulfanyl-l,5-dihydropyrazolo[3,4-</]pyriniidin-4-ones and their regioisomers.The synthesis o f these compounds was achieved by treating l-alkyl-6-alkyIsulfanyl-4- oxo-1,5-dihydropyrazolo[3,4-J]pyrimidine93 (78) with l-(n-bromoalkyl)-4,6-dialkyl- sulfanyl-l//-pyrazolo[3,4-</]pyrimidine79 (79) in DMF in presence o f anhydrous potassium carbonate at room temperature 50-90 h. The DMF was removed under reduced pressure and the residue extracted with water/chloroform system and chromatographed over silica gel to obtain the pure products.

[ 3 3 ]

Synthesis o f 5-[2-(4,6-diaIkylsulfanyl-2//-pyrazolo[3,4-*/]pyrimidin-2-yl)alkyl]- l/2-alkyl-6-alkylsulfanyl-l,5/2,5-dihydropyrazolo[3,4-</|pyrimidin-4-ones and their regioisomers.These compounds were synthesized using the similar procedure as used for synthesis o f 5-[n-(4,6 -dialky lsulfanyl-l//-pyrazolo[3,4-£/]pyrimidin-l-yl)a!kyl]-l-alky 1-6-alkyl sulfanyl-l,5-dihydropyrazolo[3,4-J]pyrimidin-4-ones and their regioisomers. Here l/2-alkyl-6-alkylsulfanyl-4-oxo-l,5/2,5-dihydropyrazolo[3,4-d]pyrimidine93 was treated with 2-(2-bromoethyl)-4,6 -dialkylsulfanyl-2//-pyrazolo[3,4-<f]pyrimidine.79

Reaction of 5-[n-(4,6-dialkylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)alkyl]-l- alkyI-6-alkyIsulfanyl-l,5-dihydropyrazolo[3,4-<fJpyrimidin-4-ones and their regioisomers with pyrrolidine.A solution o f 1.0 mmol o f 5-[n-(4,6-dialkylsulfanyl-l//-pyrazolo[3,4-t/]pyrimidin-l- yl)alkyl]-l-alkyl-6 -alkylsulfanyl-l ,5-dihydropyrazolo[3,4-t/]pyrimidin-4-ones (or their regioisomers) was refluxed with excess (25 eq) o f pyrrolidine on heating water bath for 8 h. The solvent was evaporated under reduced pressure and the residue extracted using water/chloroform system. The chloroform solution was dried over anhyd. sodium sulfate and chromatographed over silica gel to get the pure product.Representative procedure:Synthesis of 5-[2-(4,6-dimethylsulfanyl-lff-pyrazolo[3,4-rfJpyrimidin-l-yl)ethyl]- l-methyl-6-methylsulfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80a) and its regioisomer 4-[2-(4,6-dimethylsulfanyl-li/-pyrazolo[3,4-rf]pyrimidin-l- yl)ethoxy]-l-methyl-6-methylsulfanyl-l//-pyrazolo[3,4-*/] pyrimidine (81a).A solution o f 3.0 gm (15.3 mmol) o f l-methyl-6-methylsulfanyl-4-oxo-l,5- dihydropyrazolo[3,4-t/]pyrimidine in DMF was treated with 6.10 gm (19.1 mmol) o f l-(2-bromoethyl)-4,6-dimethylsulfanyl-l//-pyrazolo[3,4-<i]pyrimidine in presence o f anhydrous potassium carbonate at room temperature for 90 h. The DMF was removed under reduced pressure and the residue extracted with water/chloroform system. The organic layer was dried over anhydrous sodium sulfate and chromatographed over silica gel to give 3.57 gm (54 %) o f 80a and 2.22 gm (33 %) o f 81a. The characterization o f the compounds was done on the basis o f spectroscopic techniques. The mass spectrum (FAB MS) o f compound 80a showed a peak at m/z 435 (M +l). The 'H NMR spectrum showed three singlets o f three protons each at 8 2.35, 2.40 and

[ 3 4 ]

2.66 for three SMe groups. A three-proton singlet was found at 5 3.91 for methyl group at N -l position. Two two-proton triplets for two CH2 groups were observed at 5 4.53 and 4.78, having a coupling constant o f 5.4 Hz. The peaks for the protons at 3- positions appear as one-proton singlets at 8 7.90 and 7.94. The 13C NMR o f 80a could not be obtained due to its poor solubility. The Mass spectrum (FAB, MS) of compound 81a showed a peak at m/z 435 (M +l). The 'H NMR spectrum showed three singlets o f three protons each at 8 2.51, 2.59 and 2.68 for three SMe groups. A three-proton singlet for methyl group at N -l position was found at 8 3.98. Two two- proton triplets for NCH 2 and OCH2 groups were observed at 8 4.83 and 4.96, having a coupling constant o f 5.4 Hz. The peaks for the protons at 3-positions appear as one- proton singlets at 8 7.74 and 7.92. The 13C NMR o f 81a showed the peaks for three SMe carbons at 8 11.8, 14.1 and 14.2. The peak for methyl carbon at N-l-position was found at 8 33.8. Two methylenic carbons o f NCH 2 and OCH2 groups were observed at 8 46.0 and 64.5, respectively. The peaks at 8 131.2 and 132.1 correspond to two carbons at 3-positions. The peaks for the eight quaternary carbons o f the two ring systems appeared at 8 99.7, 109.5, 152.5, 155.5, 161.8, 164.9, 168.8 and 169.2. The proper assignment o f the two SMe groups was done on the basis o f chemical transformations. Thus, the peak at 8 2.66 was assigned to 4-SMe as this peak was found missing when a pyrrolidinyl group was introduced at this position. The corresponding compound 80b showed two SMe peaks at 8 2.35 and 2.48. Synthesis o f compound 80e in which the SMe group at 6 -position is replaced by SEt group showed peaks for SMe groups at 8 2.30 and 2.65, indicating the 6 "-position for SMe group appearing at 8 2.30. This is also confirmed by the synthesis o f 80h, in which two SMe groups at 4"- and 6 "- positions o f 80a were replaced by SEt groups, which showed the single SMe peak at 8 2.42.

1.4 Results and discussionThe symmetrical compounds 6 6 , 67a and 69 (Fig. 29) possess folded conformations whereas the dissymmetrical compound 73 and the symmetrical compound 74 show open conformation. The compounds 80 and 81 can be viewed as the hybrids o f the compounds 6 6 & 67a and 6 6 & 69, respectively, all o f which show intramolecular stacking interactions. However, in new compounds (80 and 81) the position o f linker is N -l o f one pyrazolo[3,4-<f]pyrimidine moiety and N-5 or C-4 o f the other. In addition, the linker between N -l/C-4 is having an extra -O- atom in the linker.

[ 3 5 ]

The *H NMR spectrum o f the ethylene linker compound 80a (Table II) showed unusual upfield chemical shift for both 6 '- and 6 "-SMe protons ( 8 2.40 and 2.35 ppm) as compared with the corresponding monomeric compounds 70 and 71 (5 2.64 and 2.65, respectively - Table I). This 'H NMR data is very comparable to 75b {‘syrf conformation by X-ray crystallography) and 75c, mentioned earlier. The structure o f 80a is very similar to that o f 75c, differing only in the remote (away from stacking region) phenyl ring o f quinazolinone unit and pyrazole ring in 80a. Above 'H NMR data analysis, supports predominance o f unusual ‘syn’ conformation for 80a in solution due to intramolecular stacking interactions. The compound 80b in which one of the SMe groups is replaced by the pyrrolidinyl group, synthesized for proper characterization o f the SMe groups o f compound 80a, also showed comparable upfield shift for its two SMe groups indicating favored ‘syrt conformation due to intramolecular arene interactions.

Table II: Important !H NMR chemical shifts o f compounds 80 and 81Comp

No.

5 values (ppm) Comp

No.

5 values (ppm)

r2 Ri R r 2 Ri R

80a 2.40 2.66 2.35 81a 2.59 2.68 2.5180b 2.48 3.74 2.35 81b 2.59 3.76 2.48

80c 1.14,2.95 2.64 2.25 81c 1.42,3.18 2.68 2.4980d 1.22,3.05 3.74 2.28 81d 1.42,3.17 3.76 2.4780e 1.16,2.98 2.65 2.30 81e 1.43,3.18 2.68 2.5180f 2.39 2.66 2.32 81f 2.59 2.68 2.5080g 3.72,1.20 2.64 2.22 81g 3.96,1.45 2.68 2.4980h 2.42 3.31,1.41 2.95,1.31 81h 2.59 3.33,1.43 3.16,1.3780i 3.73,1.20 3.30,1.39 2.82,1.25 81 i 3.97,1.46 3.33,~ 3.09,1.3780j 2.48 3.31,1.41 3.08,1.36 81 j 2.59 3.33,1.43 3.06,1.36

80k-N-2 2.58 3.32,1.43 3.24,1.41 81k-N-2 2.61 2.67 2.6280I-N-2 2.56 2.67 2.64 811-N-2 3.99,1.46 2.66 2.62

80m-N-2 3.93,1.34 2.66 2.63 81m-N-2 2.61 3.32,1.43 3.22,1.4180n-N-2 2.56 3.32,1.43 3.24,1.42 81 n 2.55 2.67 2.50

80o 2.55 2.69 2.60 81o 2.56 3.75 2.4880p 1.36,3.15 2.69 2.60 81p 1.39, 3.14 2.66 2.4980 q 1.38,3.17 3.78 2.55 81 q 1.40,3.15 3.75 2.4880r 1.37,3.16 2.69 2.60 81 r 1.40,3.14 2.67 2.5180s 2.59 2.68 2.61 81s 2.60 2.68 2.6080t -,3.20 2.69 2.62 81t -,3.18 2.69 2.62

[ 3 6 ]

80u 1.41,3.21 3.77 2.56 81 u 2.61 2.69 2.60

80v 2.61 2.69 2.61 81v 2.59 3.76 2.55

80w 2.55 3.76 2.62 81w -,3.19 2.69 2.61

80x 1.45,3.23 2.69 2.61 81x -,3.19 3.77 2.56

80y 1.45,3.26 2.68 2.63 81y -,3.21 2.68 2.63

All the analogs, 80b-80i, o f 80a, in which SMe groups at different positions have been replaced by SEt, S-iPr and pyrrolidinyl groups show similar results supporting the above inference. The upfield shift of two methylsulfanyl groups in different region (6 '/6 "-SMe) o f 80a indicate that the two pyrazolo[3,4-</]pyrimidine units remain more or less parallel to each other with part o f pyrimidine rings overlapped. Thus, the below mentioned folded conformation is proposed for compound 80a (Fig. 35).

SMe

i .MeS

MeS

Fig. 36 Proposed conformational equilibrium for 80aThe compound 801, in which the two pyrazolo[3,4-c/]pyrimidinyl units are linked through N-2" and N-5' positions did not show any considerable upfield chemical shift for any SMe protons indicating absence of intramolecular stacking. Similar results were observed for its Et, iso-Pr analogs, 80m and 80n. The compound 80j, which is the isomer o f 80h with methyl-'group at N-2' position instead o f this substituent at N -l' position in 80h, also showed upfield shift for 6 -SMe protons indicating ‘syn’ conformation as in 80h. The compound 80k which is also isomeric to 80h, in which one pyrazolo[3,4-</]pyrimidine possess methyl substituent at N-2' position and the two pyrazolo[3,4-t/]pyrimidine units joined between N-2" & N-5' positions showed the chemical shift values almost similar to those in the corresponding monomeric compounds, indicating no intramolecular stacking due to arene interactions. Thus, the ethylene linker compounds 80I-80n in which the linker is joined through N-5' and N- 2 " positions do not show any significant change in chemical shifts indicating no intramolecular stacking interaction.The compound 80o, which is the ‘propylene linker ’ homolog o f 80a (ethylene linker) showed much less upfield shift (5 2.55 ppm) for its 6 '-SMe group when compared

[ 3 7 ]

with the monomeric compound 70. However, no other group/proton showed any significant shift, indicating a predominance o f open conformation. The upfield shift o f 6 '-SR group is, probably due to weak C H -7t interaction between C-H o f SCH3/CH2 at 6 ’- position and the adjacent pyrazolo[3,4-d]pyrimidinyl ring system.The compound 81a formed as a co-product along with 80a, which has one O atom in addition to ethylene linker (effectively, a three-atom linker between N -l" and C-4') showed considerable upfield chemical shift for its 6 "-SMe group ( 8 2.51 ppm). Interestingly, this chemical shift is similar to another ‘propylene linker’ compound76, known to have folded conformation due to C H -7t interaction .91 Another related compound 1 -phenoxybuty 1-4,6 -dimethylsulfany 1-1 //-pyrazolo[3,4-t/] pyrimidine,having a ‘tetramethylene linker’ with an additional O atom between pyrazolo[3,4-c/] pyrimidine and phenyl ring has been reported to possess open conformation in solution ( 8 2.61 ppm) and solid state. 94 These results suggest a folded conformation for 81a in such a way that 6 "-SMe group falls in the anisotropic region o f the adjacent aromatic moiety, due probably to CH-71 interaction. Similar results were found in all o f its analogs. The higher homolog o f 81a, i. e., 81n, with ‘propylene linker’ having an additional O atom between C-4' of one moiety and N -l" o f the other (effectively a four-atom linker), also showed considerable upfield shift for both its 6 '- and 6 "-SMe groups ( 8 2.55 and 2.50 ppm) which suggests that the two aromatic rings lie more or less parallel to each other. However, the upfield shift is less significant as compared to earlier reported compounds having folded conformation in solution and solid state. This indicates an equilibrium mixture o f folded and open conformations for 81 n. Similar observation was found with all o f its analogs.The proton NMR spectral analysis o f ‘tetramethylene’ and ‘pentamethylene’ linker homologs did not show any significant shift for their protons when compared with the corresponding monomeric compounds indicating the absence o f intramolecular stacking similar to that in 67b.In conclusion, above discussion clearly shows that the dimeric compounds in which the two aromatic units are linked through N -l" & N-5' positions show folded conformations due to intramolecular arene interactions, especially in ethylene linker compounds. The interaction was found to decrease rapidly with the increase in the length o f the linker. However, changing the position o f the linker from N -l" to N-2" dramatically reduced the intramolecular arene interaction. The change in the position

[ 3 8 ]

of the substituents from N -l' to N-2' did not affect the interaction significantly, as it is away from the stacking region (Fig. 36).The X-ray crystallographic studies o f 80a and its regioisomer 81a have been done. The X-ray crystallography o f 80a has revealed an anti conformation with two molecules forming a dimer due to CH—N interaction between H-3 o f one molecule and N-2 o f the other and vice versa. The X-ray crystallography o f 81a has also shown an open conformation.Variable temperature !H NMR studies were done with two ethylene linker compounds (80g & 80e) and one ‘decamethylene linker’ compound (80y) used as a control. The characteristic chemical shifts are summarized below in Table III.

Table III: The characteristic chemical shifts of 80g, 80e and 80y at different temperatures.

Comp.

No.

Temp.

CC)

Chemical shift

6’-S-iPr/Et 6"-SMe 4"-SMe H-3 N'Et/Me

80g 50.2 3.74, 1.22 2.26 2.64 7.89, 7.95 4.26, 1.4719.2 3.72, 1.20 2.21 2.64 7.92, 7.98 4.27, 1.470.0 3.70, 1.18 2.18 2.64 7.94, 8.00 4.28, 1.47

-24.8 3.68, 1.16 2.14 2.65 7.96, 8.03 4.28, 1.4780e 50.1 3.00, 1.19 2.32 2.65 7.88, 7.93 3.88

19.3 2.97, 1.16 2.29 2.65 7.90, 7.96 3.890.1 2.96, 1.14 2.28 2.65 7.92, 7.99 3.90

-24.8 2.94, 1.12 2.25 2.65 7.94, 8.01 3.9180y 50.3 3.26, 1.45 2.62 2.68 7.89, 7.94 3.91

19.1 3.26, 1.45 2.63 2.69 7.91, 7.97 3.92-0.1 3.26, 145 2.64 2.69 7.93, 7.99 3.93-24.9 3.26, 1.26 2.64 2.70 7.95, 8.01 3.95

The analysis o f the chemical shifts o f 80g show gradual increase in the 5 values o f 6- alkylsulfanyl group o f both pyrazolo[3,4-cf|pyrimidme units with the increase in temperature (plot 1). These results indicate that the equilibrium shifts towards the increase in the population o f lsyn’ conformers as the temperature is decreased, due to decrease in rotational energy for the C-C bond between carbon atoms o f the ethylene linker, thereby increasing the population o f the more stable ‘syn’ conformers. Similar results were observed for 80e also. No such change is observed for the chemical shifts o f other protons. Similar studies o f the compound (80y) having ‘decamethylene linker’ did not show any significant shift for any o f its protons/groups. These studies

[ 3 9 ]

clearly indicate that the ethylene linker compounds 80g and 80e are having an equilibrium mixture of ''syn’ and ‘'anti' conformations at room temperature.

Temp (°C)Plot 1 Variation in chemical shifts of 6"-SMe protons of 80g, 80e and 80y with temperature.

1.5 Biological ActivityPyrazolo[3,4-</]pyrimidines constitute one of the important class of biologically active compounds. These compounds show significant activity as adenosine receptor antagonists95 and display antibacterial,96 anti-inflammatory,97 antihistaminic,98 antileukemia,99 immunosuppressive,100 antiviral,101 anticonvulsant,102 platelet aggregation inhibitoiy activity103 and treating a chemokine mediated disease such as

• ' 104psoriasis.Among the compounds synthesized in this scheme 80a, 80b, 80, 80d, 80e, 80f, 80g, 801, 80o, 80p, 80q, 8©r, 80s, 80v, 80w, 81a, 81b, 81c, 81d, 81e, 81f, 81g, 81i, 81n, 81o, 81u, and 81v were submitted for various biological activities. The results obtained are shown below.Anti-inflammatory activityThe anti-inflammatory activity was done in collaboration with Dr. Rakesh Shukla, Division of Pharmacology, Central Drug Research Institute, Lucknow.The compounds 80a, 81a, 81b, 80c, 81c, 80d, 81d, 80p, 80s, 80v, 80w, 81u and 81v were tested for anti-inflammatory activity. The compounds were tested at a dose of 100 mg/kg body weight. None of the compounds tested showed significant activity.

[ 4 0 ]

Antibacterial and anti-fungal activityThe anti-fungal and antibacterial activity was done in collaboration with Dr. P. K. Shukla, Division o f Fermentation Technology, Central Drug Research Institute, Lucknow.The compounds 80b, 80e, 80f, 80g, 80i, 80o, 80q, 80r, 81b, 81e, 81f, 81g, 81i, 81n, and 81o were tested for antibacterial and anti-fungal activities. Maximum concentration tested for fungi and bacteria was 50 fig/ml. No significant activity was found in any compound.Anti-tubercular activityThe anti-tubercular activity was done in collaboration with Dr. Sudhir Sinha, Division o f Drug Target Discovery and Development, Central Drug Research Institute, Lucknow.The compounds 80b, 80e, 80f, 80g, 80i, 80o, 80q, 80r, 81b, 81e, 81f, 81g, 81i, 81n, and 81o were also tested for anti-tubercular activity. The compounds were tested at 25 mcg/ml concentration and the percentage inhibition criteria was > 90 %. No significant activity was found in any compound.

1.6 ExperimentalMelting points were performed on Buchi 530 (oil bath type) melting point apparatus and are uncorrected. 'H NMR and 13C NMR spectra were recorded on Bruker WM- 200 (200 MHz and 50 MHz, respectively) spectrometer in CDCI3 or stated otherwise. Infra red spectral analysis Was done using Perkin-Elmer Ac-1 spectrophotometer (KBr). Mass Spectra were done using Fast Atomic Bombardment spectrometer. Microanalysis (C, H, N) were performed on a Carlo Erba EA-1108 elemental analyzer and were within ± 0.4% o f the theoretical values. TLCs were recorded on 3x7 cm precoated silica gel plates. Analytical grade reagents were used as such without further purification.

5-[2-(4,6-DimethyIsulfanyl-ltf-pyrazolo[3,4-</]pyrimidin-l-yl)-ethyl]-l-methyl-6- methylsulfanyl-l,5-dihydropyrazolo[3,4-tf]pyrimidin-4-one (80a).Yield 54 %; mp 236-238 °C; MS (FAB) m/z 435 (M +l); 'H NMR: 5 2.35 (s, 3H, SMe), 2.40 (s, 3H, SMe), 2.66 (s, 3H, SMe), 3.91 (s, 3H, NMe), 4.53 (t, J = 5.4 Hz,

[ 4 1 ]

2H, NCH2), 4.78 (t, J = 5.4 Hz, 2H, NCH2), 7.90 (s, IH, H-3), 7.94 (s, IH, H-3); Anal, calcd.; C: 44.22, H: 4.17, N: 25.79; Found: C: 44.02, H: 4.35, N: 26.13.4-[2-(4,6-DimethyIsulfanyl-l//-pyrazolo[3,4-*/] pyrimidin-l-yl)ethoxy ]-l-methyl-6-methylsulfanyl-lflr-pyrazolo[3,4-</]pyrimidine (81a).Yield 33 %; mp 174-176 °C; MS (FAB) m/z 435 (M +l); 'H NMR: 5 2.51 (s, 3H, SMe), 2.59 (s, 3H, SMe), 2.68 (s, 3H, SMe), 3.98 (s, 3H, NMe), 4.83 (t, J = 5.4 Hz, 2H, NCH2), 4.96 (t, J = 5.4 Hz, 2H, OCH2), 7.74 (s, IH, H-3), 7.92 (s, IH, H-3); 13C NMR: 5 11.8, 14.1, 14.2, 33.8, 46.0, 64.5, 99.7, 109.5, 131.2, 132.1, 152.5, 155.5,161.8, 164.9, 168.8, 169.2; Anal, calcd.; C: 44.22, H: 4.17, N: 25.79; Found: C: 43.88, H: 4.45, N: 26.27.5-[2-(6-M ethylsulfanyl-4-(pyrrolidin-l-yl)-l//-pyrazolo[3,4-*/|pyriniidin-l-yl)- ethyI]-l-methyl-6-methylsulfanyl-l,5-dihydropyrazolo[3,4-*/]pyrimidin-4-one (80b).Yield 70 %; mp 234-236 °C; MS (FAB) m/z 458 (M +l); IR (KBr): 1708 c m 1; ]H NMR: 8 1.97-2.13 (m, 4H, 2CH2), 2.35 (s, 3H, SMe), 2.48 (s, 3H, SMe), 3.74 (t, J =6.6 Hz, 4H, 2NCH2), 3.91 (s, 3H, NMe), 4.54 (t, J = 5.2 Hz, 2H, NCH2), 4.72 (t, J =5.2 Hz, 2H, NCH2), 7.78 (s, IH, H-3), 7.92 (s, IH, H-3); Anal, calcd.; C: 49.87, H:5.07, N: 27.55; Found: C: 49.55, H: 4.65, N: 26.97.4-[2-(6-Methylsulfanyl-4-(pyrrolidin-l-yl)-liy-pyrazolo[3,4-</]pyrimidin-l-yl)- ethoxy]-l-methyl-6-methylsulfanyl-l//-pyrazoIo[3,4-*/| pyrimidine (81b).Yield 66 %; mp 200-202 °C;*MS (FAB) m/z 458 (M +l); ‘H NMR: 8 1.96-2.17 (m, 4H, 2CH2), 2.49 (s, 3H, SMe), 2.59 (s, 3H, SMe), 3.77 (t, J = 6.6 Hz, 4H, 2NCH2),3.98 (s, 3H, NMe), 4.78 (t, J = 5.4 Hz, 2H, NCH2), 4.96 (t, J = 5.4 Hz, 2H, OCH2), 7.78 (s, IH, H-3), 7.80 (s, IH, H-3); ,3C NMR: 8 14.0, 14.2, 24.3, 25.9, 33.8, 45.7,47.4, 48.1, 64.6, 99.1, 99.9, 131.3, 133.3, 154.2, 155.1, 155.6, 162.0, 169.0, 169.2; Anal, calcd.; C: 49.87, H: 5.07, N: 27.55; Found: C: 49.70, H: 5.43, N: 27.92.5-[2-(4,6-DimethylsuIfanyl-l/7-pyrazolo[3,4-</]pyrimidin-l-yl)ethyl]-l-ethyl-6- ethylsuVfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80c).Yield 48.5 %; mp 164-66 °C; MS (FAB) m/z 463 (M +l); 'H NMR: 8 1.14 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.47 (t, J = 7.2 Hz, 3H, NCH2CH3), 2.25 (s, 3H, SMe), 2.64 (s, 3H, SMe), 2.95 (q, J = 7.3 Hz, 2H, SCH2), 4.28 (q, J = 7.2 Hz, 2H, NCH2), 4.49 (t, J

[ 4 2 ]

= 5.2 Hz, 2H, NCH2), 4.77 (t, J = 5.2 Hz, 2H, NCH2), 7.91 (s, 1H, H-3), 7.98 (s, 1H, H-3); 13C NMR: 8 11.7, 13.5, 13.6, 14.8, 26.8, 42.2, 44.3, 45.3, 102.4, 109.6, 132.1,134.8, 150.0, 152.7, 157.7, 160.2, 164.7, 169.0; Anal, calcd.; C: 46.73, H: 4.79, N: 24.22; Found: C: 46.52, H: 5.12, N: 24.92.4-[2-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-*/]pyrimidin-l-yl)ethoxy]-l-ethyl-6- ethylsuIfanyI-l//-pyrazolo[3,4-</Jpyrimidine (81c).Yield 42.5 %; mp 138-140 °C; MS (FAB) m/z 463 (M +l); 'H NMR: 8 1.42 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.47 (t, J = 7.3 Hz, 3H, NCH2CH3), 2.49 (s, 3H, SMe), 2.68 (s, 3H, SMe), 3.18 (q, J = 7.4 Hz, 2H, SCH2), 4.39 (q, J = 7.3 Hz, 2H, NCH2), 4.83 (t, J = 5.0 Hz, 2H, NCH2), 4.96 (t, J = 5.2 Hz, 2H, OCH2), 7.73 (s, 1H, H-3), 7.92 (s, 1H, H-3); ,3C NMR: 8 11.8, 14.1, 14.5, 14.7, 25.4, 42.2, 46.1, 64.5, 100.0, 109.5, 131.1,132.2, 152.6, 155.0, 162.0, 164.9, 168.5, 168.9; Anal, calcd.; C: 46.73, H: 4.79, N: 24.22; Found: C: 46.43, H: 5.19, N: 24.68.5-[2-(6-MethyIsuIfanyl-4-(pyrrolidin-l-yl)-l//-pyrazolo[3,4-*/|pyrimidin-l-yl)- ethyl]-l-ethyl-6-ethyIsulfanyl-l,5-dihydropyrazolo[3,4-*/]pyrimidin-4-one (80d).Yield 60 %; mp 194-196 °C; MS (FAB) m/z 486 (M +l); 'H NMR: 8 1.22 (t, J = 7.3 Hz, 3H, SCH2 CH3), 1.48 (t, J = 7.2 Hz, 3H, NCH2CH3), 1.97-2.13 (m, 4H, 2CH2),2.28 (s, 3H, SMe), 3.05 (q, J = 7.3 Hz, 2H, SCH2), 3.74 (t, J = 6.5 Hz, 4H, 2NCH2),4.29 (q, J = 7.2 Hz, 2H, NCH2), 4.51 (t, / = 5.3 Hz, 2H, NCH2), 4.72 (t, J = 5.3 Hz, 2H, NCH2), 7.79 (s, 1H, H-3), 7.95 (s, 1H, H-3); 13C NMR: 8 13.5, 13.6, 14.8, 24.3,25.9, 26.8, 42.1, 43.9, 44.9, 47.4, 48.0, 99.1, 102.4, 133.2, 134.8, 150.0, 154.1, 155.1,157.7, 160.3, 169.1; Anal, calcd.; C: 51.94, H: 5.60, N: 25.96; Found: C: 52.32, H: 5.40, N: 25.72.4-[2-(6-MethylsulfanyI-4-(pyrrolidin-l-yl)-l//-pyrazolo[3,4-</jpyrimidin-l-yl)- ethoxy]-l-ethyl-6-ethylsulfanyl-l//-pyrazolo|3,4-</] pyrimidine (81d).Yield 72 %; mp 162-164 °C; MS (FAB) m/z 486 (M +l); lH NMR: 8 1.42 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.47 (t, J = 7.2 Hz, 3H, NCH 2CH3), 1.98-2.14 (m, 4H, 2CH2), 2.47 (s, 3H, SMe), 3.17 (q, J = 7.4 Hz, 2H, SCH2), 3.76 (t, J = 6 . 6 Hz, 4H, 2NCH2),4.38 (q, J = 7.2 Hz, 2H, NCH2), 4.78 (t, J = 5.3 Hz, 2H, NCH2), 4.94 (t, J = 5.3 Hz, 2H, NCH2), 7.78 (s, 1H, H-3), 7.81 (s, 1H, H-3); 13C NMR: 8 14.0, 14.5, 14.7, 24.4,25.4, 25.9, 42.1, 45.7, 47.4, 48.1, 64.6, 99.1, 100.0, 131.3, 133.3, 154.2, 155.0, 155.2,

[ 4 3 ]

162.0, 168.5, 169.1; Anal, calcd.; C: 51.94, H: 5.60, N: 25.96; Found: C: 52.14, H: 5.48, N: 25.68.5-[2-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-*/|pyrimidin-l-yl)ethyl]-6-ethyl- sulfanyl-l-methyl-l,5-dihydropyrazoIo[3,4-rf]pyrimidin-4-one (80e).Yield 33 %; mp 174-176 °C; MS (FAB) m/z 449 (M +l); IR (KBr): 1700 cm '1; 'H NMR: 8 1.16 (t, J = 7.3 Hz, 3H, SCH2CH3), 2.30 (s, 3H, SMe), 2.65 (s, 3H, SMe),2.98 (q, J = 7.3 Hz, 2H, SCH2), 3.89 (s, 3H, NMe), 4.49 (t, J = 5.2 Hz, 2H, NCH2), 4.77 (t, J = 5.2 Hz, 2H, NCH2), 7.90 (s, 1H, H-3), 7.96 (s, 1H, H-3); Anal, calcd.; C: 45.52, H: 4.49, N: 24.98; Found: C: 45.13, H: 5.07, N: 25.73.4-[2-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)ethoxy]-6-ethyl- suIfanyl-l-methyl-l//-pyrazolo[3,4-*/]pyrimidine (81e).Yield 30 %; mp 144-146 °C; MS (FAB) m/z 449 (M +l); 'H NMR: 5 1.43 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.51 (s, 3H, SMe), 2.68 (s, 3H, SMe), 3.18 (q, J = 7.4 Hz, 2H, SCH2), 3.97 (s, 3H, NMe), 4.83 (t, J = 5.0 Hz, 2H, NCH2), 4.96 (t, 5.0 Hz, 2H,OCH2), 7.73 (s, 1H, H-3), 7.92 (s, 1H, H-3); 13C NMR: 5 11.7, 14.1, 14.4, 25.3, 33.7,46.0, 64.4, 99.8, 109.4, 131.1, 132.1, 152.5, 155.5, 161.8, 164.8, 168.8; Anal, calcd.; C: 45.52, H: 4.49, N: 24.98; Found: C: 44.83, H: 4.65, N: 25.12.5-[2-(4,6-Diinethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)ethyl]-l-ethyl-6- methylsuIfanyl-l,5-dihydropyrazolo[3,4-</Jpyrimidin-4-one (80f).Yield 43 %; mp 172-174 °C; MS (FAB) m/z 449 (M +l); 'H NMR: 5 1.48 (t, J = 7.2 Hz, 3H, NCH 2CH3), 2.32 (s, 3Ff, SMe), 2.39 (s, 3H, SMe), 2.66 (s, 3H, SMe), 4.29 (q, J = 7.2 Hz, 2H, SCH2), 4.53 (t, J = 5.4 Hz, 2H, NCH2), 4.77 (t, J = 5.4 Hz, 2H, NCH2), 7.91 (s, 1H, H-3), 7.96 (s, 1H, H-3); 13C NMR: 5 11.7, 13.7, 14.7, 15.3, 42.1,44.0, 45.1, 102.2, 109.4, 132.1, 134.8, 149.8, 152.5, 157.5, 160.7, 164.7, 169.0; Anal, calcd.; C: 45.52, H: 4.49, N: 24.98; Found: C: 45.39, H: 4.67, N: 25.52.4-[2-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyriniidin-l-yl)ethoxy]-l-ethyl-6- methylsulfanyl-l//-pyrazolo[3,4-rf]pyrimidine (81f).Yield 37 %; mp 148-150 °C; MS (FAB) m/z 449 (M +l); 'H NMR: 6 1.47 (t, J = 7.2 Hz, 3H, NCH 2CH3), 2.50 (s, 3H, SMe), 2.59 (s, 3H, SMe), 2.68 (s, 3H, SMe), 4.39 (q, J = 7.2 Hz, 2H, NCH2), 4.83 (t, J = 5.2 Hz, 2H, NCH2), 4.96 (t, J = 5.2 Hz, 2H, OCH2), 7.74 (s, 1H, H-3), 7.92 (s, 1H, H-3); l3C NMR: 8 11.8, 14.1, 14.2, 14.6, 42.1,

[ 4 4 ]

46.0, 64.4, 99.2, 109.4, 131.1, 132.1, 152.5, 154.9, 161.8, 164.9, 168.9; Anal, calcd.; C: 45.52, H: 4.49, N: 24.98; Found: C: 45.05, H: 4.87, N: 25.42.5-[2-(4,6-Dimethylsulfanyl-l/T-pyrazolo[3,4-</jpyrimidin-l-yl)ethyl]-l-ethyl-6-iso- propylsulfanyl-l,5-dihydropyrazolo[3,4-d \pvrimidin-4-one (80g).

%Yield 38.5 %; mp 146-148 °C; MS (FAB) m/z 477 (M +l); Vi.NMR: 8 1.20 (d, J =6.8 Hz, 6H, CHMe2), 1.47 (t, J = 7.2 Hz, 3H, NCH2CH3), 2.22 (s, 3H, SMe), 2.64 (s, 3H, SMe), 3.72 (sept., J = 6.8 Hz, IH, CH), 4.27 (q, J = 7.2 Hz, 2H, NCH2), 4.46 (t, J = 5.0 Hz, 2H, NCH2), 4.77 (t, J = 5.0 Hz, 2H, NCH2), 7.92 (s, IH, H-3), 7.98 (s, IH, H-3); 13C NMR: 8 11.7, 13.6, 14.9, 22.1, 38.2, 42.2, 44.4, 45.4, 102.4, 109.7, 132.0,134.8, 150.0, 152.7, 157.7, 160.2, 164.6, 169.0; Anal, calcd.; C: 47.88, H: 5.08, N: 23.49; Found: C: 47.67, H: 5.17, N: 24.03.4-[2-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)ethoxy]-l-ethyl-6- isopropylsulfanyl-l//-pyrazolo[3,4-</]pyrimidine (81g).Yield 31 %; mp 136-138 °C; MS (FAB) m/z 477 (M +l); 'H NMR: 8 1.44-1.50 (m, 9H, 3Me), 2.49 (s, 3H, SMe), 2.68 (s, 3H, SMe), 3.96 (sept., J = 6.8 Hz, IH, CH),4.38 (q, J = 7.2 Hz, 2H, NCH2), 4.83 (t, J = 4.9 Hz, 2H, NCH2), 4.95 (t, J = 4.9 Hz, 2H, OCH2), 7.73 (s, IH, H-3), 7.92 (s, IH, H-3); !3C NMR: 8 11.8, 14.1, 14.7, 22.8,35.9, 46.0, 64.5, 99.9, 109.4, 131.1, 132.1, 152.5, 154.9, 161.8, 164.9, 168.6, 168.8; Anal, calcd.; C: 47.88, H: 5.08, N: 23.49; Found: C: 47.73, H: 5.23, N: 24.22.5-[2-(4,6-DiethylsulfanyI-l/7-pyrazoIo[3,4-</]pyriinidin-l-yl)ethyI]-l-niethyl-6- methylsulfanyl-l,5-dihydropxrazolo[3,4-rflpyrimidin-4-one (80h).Yield 21 %; mp 162-164 °C; MS (FAB) m/z 463 (M +l); 'H NMR: 8 1.31 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.41 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.42 (s, 3H, SMe), 2.95 (q, J = 7.4 Hz, 2H, SCH2), 3.31 (q, J = 7.4 Hz, 2H, SCH2), 3.91 (s, 3H, NMe), 4.53 (t, J =5.3 Hz, 2H, NCH2), 4.76 (t, J = 5.3 Hz, 2H, NCH2), 7.88 (s, IH, H-3), 7.95 (s, IH, H- 3); l3C NMR: 8 14.3, 14.4, 15.3, 23.3, 25.0, 33.7, 43.8, 44.9, 102.2, 109.6, 132.2,134.8, 150.5, 152.6, 157.4, 160.9, 164.5, 168.4; Anal, calcd.: C: 46.73, H: 4.79, N:24.22, Found: C: 47.30, H: 5.10, N: 23.90.

4-[2-(4,6-Dicthylsulfanyl-l/f-pyrazolo[3,4-</]pyrimidin-l-yl)ethoxy]-l-methyl-6- methylsulfanyl-l//-pyrazolo[3,4-rf]pyrimidine (81h).

[ 4 5 ]

Yield 26 %; mp 124-126 °C; MS (FAB) m/z 462 (M+); 'H NMR: 5 1.38 (t, J = 7.4 Hz, 3H, SCH2CH 3), 1.43 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.59 (s, 3H, SMe), 3.10 (q, J = 7.4 Hz, 2H, SCH2), 3.33 (q, J = 7.4 Hz, 2H, SCH2), 3.98 (s, 3H, NMe), 4.82 (t, J =5.3 Hz, 2H, NCH2), 4.97 (t, J = 5.3 Hz, 2H, OCH2), 7.75 (s, 1H, H-3), 7.89 (s, 1H, H- 3); 13C NMR: 5 14.2, 14.4, 14.5, 23.3, 25.3, 33.8, 45.9, 64.5, 99.7, 109.5, 131.1,132.1, 152.5, 155.5, 161.7, 164.6, 168.3, 169.1; Anal, calcd.: C: 46.73, H: 4.79, N:24.22, Found: C: 46.50, H: 4.60, N: 23.60.5-l2-(4,6-DiethylsulfanyI-l//-pyrazolo[3,4-tf]pyrimidin-l-yl)ethyl]-l-ethyl-6-iso- propylsuIfanyl-l,5-dihydropyrazo!o[3,4-rf]pyrimidin-4-one (80i).Yield 43% ; mp 112-114 °C; MS (FAB) m/z 505 (M +l); 'H N M R :5 1.20 (d, J = 6 . 8

Hz, 6 H, CHMe2), 1.25 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.39 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.47 (t, J = 7.2 Hz, 3H, NCH2CH3), 2.82 (q, J = 7.3 Hz, 2H, SCH2), 3.30 (q, J = 7.3 Hz, 2H, SCH2), 3.73 (sept., J = 6 . 8 Hz, 1H, CH), 4.27 (q, J = 7.2 Hz, 2H, NCH2), 4.46 (t, J = 5.0 Hz, 2H, NCH2), 4.75 (t, J = 5.0 Hz, 2H, NCH2), 7.89 (s, 1H, H-3), 7.99 (s, 1H, H-3); 13C NMR: 5 14.2, 14.5, 14.9, 22.1, 23.3, 24.8, 38.1, 42.1,44.3, 45.3, 102.4, 109.7, 132.0, 134.7, 150.0, 152.7, 157.7, 160.1, 164.4, 168.4; Anal, calcd.: C: 49.98, H: 5.59, N: 22.20; Found: C: 49.76, H: 5.44, N: 22.56.4-[2-(4,6-DiethyIsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)ethoxy]-l-ethyl-6-iso- propylsulfanyl-l//-pyrazolo[3,4-</] pyrimidine (81i).Yield 31.5 %; mp 122-124 °C; MS (FAB) m/z 505 (M +l); ’H NMR: 8 1.35-1.49 (m, 15H, 5Me), 3.09 (q, J = 7.3 Hz, 2H, SCH2), 3.33 (q, J = 7.3 Hz, 2H, SCH2), 3.97 (sept., J = 6 . 8 Hz, 1H, CH), 4.38 (q, J = 7.2 Hz, 2H, NCH2), 4.82 (t, J = 5.1 Hz, 2H, NCH2), 4.94 (t, J = 5.1 Hz, 2H, OCH2), 7.74 (s, 1H, H-3), 7.90 (s, 1H, H-3); 13C NMR: 5 14.4, 14.5, 14.7, 22.8, 23.3, 25.3, 35.8, 42.1, 46.0, 64.4, 99.8, 109.5, 131.1,132.1, 152.5, 154.9, 161.8, 164.7, 168.3, 168.5; Anal, calcd.: C: 49.98, H: 5.59, N: 22.20; Found: C: 49.68, H: 5.70, N: 22.46.5-[2-(4,6-DiethyIsuIfanyl-l//-pyrazolo[3,4-*/|pyrimidin-l-yl)ethyl]-2-methyl-6- methylsulfanyl-2,5-dihydropyrazolo[3,4-rf]pyrimidin-4-one (80j).Yield 21 %; mp 188-190 °C; MS (FAB) m/z 463 (M +l); 'H NMR: 5 1.36 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.41 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.48 (s, 3H, SMe), 3.08 (q, J = 7.4 Hz, 2H, SCH2), 3.31 (q, J = 7.4 Hz, 2H, SCH2), 4.02 (s, 3H, NMe), 4.53 (t, J =5.8 Hz, 2H, NCH2), 4.75 (t, J = 5.8 Hz, 2H, NCH2), 7.85 (s, 1H, H-3), 7.92 (s, 1H, H-

[ 4 6 ]

3); 13C NMR: 8 14.3, 14.4, 15.4, 23.3, 25.1, 40.1, 43.3, 44.7, 104.3, 109.5, 128.6,132.0, 152.4, 157.8, 158.4, 159.5, 164.4, 168.3; Anal, calcd.; C: 46.73, H: 4.79, N: 24.22; Found: C: 46.56, H: 4.68, N: 24.66.4-[2-(4,6-Diethylsulfanyl-l//-pyrazolo[3,4-*/]pyrimidin-l-yl)ethoxy]-2-methyl-6- methylsuIfanyl-2//-pyrazoIo[3,4-*/]pyrimidine (81j).Yield 25 %; mp 160-162 °C; MS (FAB) m/z 463 (M +l); 'H NMR: 8 1.36 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.43 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.59 (s, 3H, SMe), 3.06 (q, J = 7.4 Hz, 2H, SCH2), 3.33 (q, J = 7.4 Hz, 2H, SCH2), 4.08 (s, 3H, NMe), 4.80 (t, J =4.9 Hz, 2H, NCH2), 4.94 (t, J = 5.0 Hz, 2H, NCH2), 7.70 (s, 1H, H-3), 7.89 (s, 1H, H- 3); 13C NMR: 8 14.1, 14.4, 23.3, 25.2, 40.4, 46.0, 64.4, 100.7, 109.5, 124.1, 132.0,152.5, 162.5, 164.7, 168.2, 168.6; Anal, calcd.; C: 46.73, H: 4.79, N: 24.22; Found: C: 46.44, H: 4.66, N: 24.54.5-[2-(4,6-Diethylsulfanyl-2//-pyrazoIo[3,4-</]pyrimidin-2-yl)ethyl]-2-methyl-6- methylsulfanyl-2,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80k-N-2).Yield 05 %; mp 198-200 °C; MS (FAB) m/z 463 (M +l); !H NMR: 8 1.41 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.43 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.58 (s, 3H, SMe), 3.24 (q, J = 7.4 Hz, 2H, SCH2), 3.32 (q, J = 7.4 Hz, 2H, SCH2), 4.05 (s, 3H, NMe), 4.64 (s, 4H, 2NCH2), 7.88 (s, 1H, H-3), 7.98 (s, 1H, H-3).

5-[2-(4,6-Dimethylsulfanyl-2iy-pyrazolo[3,4-</]pyrimidin-2-yl)ethyl]-l-inethyl-6- methylsulfanyl-l,5-dihydropyrazolo[3,4-</|pyrimidin-4-one (801-N-2).Yield 17 %; mp > 240 °C; M StFA B ) m/z 435 (M +l); IR (KBr): 1691 c m 1; ]H NMR: 8 2.56 (s, 3H, SMe), 2.63 (s, 3H, SMe), 2.67 (s, 3H, SMe), 3.95 (s, 3H, NMe), 4.65 (s, 4H, 2NCH2), 7.91 (s, 1H, H-3), 8.00 (s, 1H, H-3); Anal, calcd.; C: 44.22, H: 4.17, N: 25.79; Found: C: 43.96, H: 4.42, N: 26.24.4-[2-(4,6-DimethylsuIfanyl-2//-pyrazolo[3,4-</]pyrimidin-2-yl)ethoxy 1-1-methyl-6-methylsulfanyl-l//-pyrazolo[3,4-</|pyrimidine (81k-N-2).Yield 13 %; mp 210-212 °C; MS (FAB) m/z 435 (M +l); 'H NMR: 8 2.61 (s, 3H, SMe), 2.62 (s, 3H, SMe), 2.67 (s, 3H, SMe), 4.00 (s, 3H, NMe), 4.76 (t, J = 5.1 Hz, 2H, NCH2), 5.06 (t, J = 5.1 Hz, 2H, OCH2), 7.82 (s, 1H, H-3), 7.98 (s, 1H, H-3); Anal, calcd.; C: 44.22, H: 4.17, N: 25.79; Found: C: 44.08, H: 4.38, N: 26.20.

[ 4 7 ]

5-[2-(4,6-Dimethylsulfanyl-2//-pyrazolo[3,4-</]pyrimidin-2-yl)ethyl]-l-ethyl-6-iso- propylsuIfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80m-N-2).Yield 31 %; mp 132-134 °C; MS (FAB) m/z 477 (M +l); ]H NMR: 5 1.34 (d, J = 6.8 Hz, 6H, 2Me), 1.49 ( t , J = 1.2 Hz, 3H, NCH2CH3), 2.63 (s, 3H, SMe), 2.66 (s, 3H, SMe), 3.93 (sept., J = 6.8 Hz, IH, CH), 4.32 (q, 7.2 Hz, 2H, NCH2), 4.62 (s, 4H,2NCH2), 7.89 (s, IH, H-3), 8.00 (s, IH, H-3); 13C NMR: 8 11.8, 14.1, 14.8, 22.2,38.6,42.2, 44.1, 51.0, 102.2, 109.3, 124.3, 134.7, 149.9, 157.5, 158.9, 160.5, 166.3, 168.7; Anal, calcd.: C: 47.88, H: 5.08, N: 23.51, Found: C: 48.14, H: 4.488, N: 23.34.4-[2-(4,6-DimethylsulfanyI-2//-pyrazolo[3,4-i/]pyrimidin-2-yl)ethoxy]-l-ethyl-6- isopropylsulfanyl-l/7-pyrazolo [3,4-d \ pyrimidine (81 l-N-2).Yield 17.5 %; mp 130-132 °C; MS (FAB) m/z A ll (M +l); 'H NMR: 8 1.46 (d, J =7.0 Hz, 6H, 2Me), 1.49 (t, J = 7.2 Hz, 3H, NCH2CH3 ), 2.62 (s, 3H, SMe), 2.66 (s, 3H, SMe), 3.99 (sept., J = 6.8 Hz, IH, CH), 4.40 (q, J = 7.2 Hz, 2H, NCH2), 4.76 (t, J = 5.0 Hz, 2H, NCH2), 5.03 (t, J = 5.0 Hz, 2H, OCH2), 7.82 (s,lH , H-3), 7.98 (s, IH, H-3); 13C NMR (CDC13, 50 MHz): 8 11.8, 14.2, 14.8, 22.8, 36.0, 42.3, 52.9, 64.4,99.7, 109.2, 124.5, 130.8, 155.1, 159.0, 161.5, 166.3, 168.7, 168.8; Anal, calcd.: C:47.88, H: 5.08, N: 23.51, Found: C: 47.72, H: 5.14, N: 23.72.5-[2-(4,6-Diethylsulfanyl-2H-pyrazolo[3,4-rf]pyrimidin-2-yl)ethyl]-l-methyl-6- methylsulfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80n-N-2).Yield 14.5 %; mp 148-150 °C; MS (FAB) m/z 463 (M +l); 'H NMR: 8 1.42 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.43 (t, Ji= 7.3 Hz, 3H, SCH2CH3), 2.56 (s, 3H, SMe), 3.24 (q, J = 7.3 Hz, 2H, SCH2), 3.32 (q, J = 7.3 Hz, 2H, SCH2), 3.95 (s, 3H, NMe), 4.65 (s, 4H, 2NCH2), 7.89 (s, IH, H-3), 8.00 (s, IH, H-3); Anal, calcd.; C: 46.73, H: 4.79, N: 24.22; Found: C: 46.48, H: 4.92, N: 24.56.

4-[2-(4,6-Diethylsulfanyl-2i/-pyrazolo[3,4-rf]pyrimidin-2-yl)ethoxy|-l-methyl-6- methylsulfanyl-l/7-pyrazolo[3,4-</lpyrimidine (81m-N-2).Yield 8.5 %; mp 118-120 °C; MS (FAB) m/z 463 (M +l); ]H NMR: 8 1.41 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.43 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.61 (s, 3H, SMe), 3.22 (q, J = 7.4 Hz, 2H, SCH2), 3.32 (q, J = 7.4 Hz, 2H, SCH2), 4.00 (s, 3H, NMe), 4.76 (t, J =5.0 Hz, 2H, NCH2), 5.05 (t,J = 5.0 Hz, 2H, OCH2), 7.83 (s, IH, H-3), 7.95 (s, IH, H- 3); 13C NMR: 8 14.2, 14.3, 14.4, 23.5, 25.1, 33.9, 52.9, 64.5, 99.6, 109.3, 124.4,

[ 4 8 ]

131.0, 155.7, 159.0, 161.5, 166.1, 168.3, 169.4; Anal, calcd.; C: 46.73, H: 4.79, N: 24.22; Found: C: 46.98, H: 4.64, N: 24.04.5-[3-(4,6-DimethylsuIfanyI-l//-pyrazolo[3,4-</]pyrimidin-l-yl)propyl|-l-methyl-6-methylsuIfanyl-l,5-dihydro-l//-pyrazolo[3,4-d]pyrimidin-4-one (80o).Yield 48 %; mp 180-182 °C; MS (FAB) m/z 449 (M +l); 'H NMR: 8 2.37 (m, 2H,CH2), 2.55 (s, 3H, SMe), 2.60 (s, 3H, SMe), 2.69 (s, 3H, SMe), 3.91 (s, 3H, NMe),4.17 (t, J = 7.6 Hz, 2H, NCH2), 4.50 (t, J = 6.6 Hz, 2H, NCH2), 7.93 (s, 1H, H-3), 7.95 (s, 1H, H-3); 13C NMR: 8 11.7, 14.2, 15.2, 27.8, 33.6, 41.5, 44.5, 102.2, 109.3,131.6, 136.8, 150.5, 151.8, 157.4, 160.9, 164.8, 168.7; Anal, calcd.: C: 45.50, H: 4.46, N: 25.00, Found: C: 45.38, H: 4.84, N: 25.85.4-[3-(4,6-DimethylsuIfanyl-l//-pyrazolo[3,4-*/]pyrimidin-l-yl)propoxy]-l-methyI-6-methylsulfanyl-l//-pyrazolo [3,4-*/] pyrimidine (81 n).Yield 34 %; mp 170-172 °C; MS (FAB) m/z 449 (M +l); 'H NMR: 8 2.47 (m, 2H,CH2), 2.51 (s, 3H, SMe), 2.55 (s, 3H, SMe), 2.67 (s, 3H, SMe), 3.99 (s, 3H, NMe),4.53-4.62 (m, 4H, NCH2 + OCH2), 7.78 (s, 1H, H-3), 7.92 (s, 1H, H-3); ,3C NMR: 811.8, 14.1, 14.2, 28.5,33.8, 43.9, 64.0, 99.7, 109.5, 131.1, 131.7, 151.9, 155.5, 162.1,164.8, 168.6, 169.2; Anal, calcd.: C: 45.50, H: 4.46, N: 25.00, Found: C: 45.34, H: 4.62, N: 25.56.4-[3-(6-Methylsulfanyl-4-(pyrrolidin-l-yl)-l//-pyrazolo[3,4-*/]pyrimidin-l-yl)- propoxy]-l-methyl-6-methylsulfanyl-l/y-pyrazolo[3,4-</]pyrimidine (81o).Yield 76 %; mp 160-162 °C;*MS (FAB) m/z 472 (M +l); 'H NMR: 8 1.99-2.14 (m, 4H, 2CH2), 2.44 (m, 2H, CH2), 2.48 (s, 3H, SMe), 2.56 (s, 3H, SMe), 3.75 (t, J = 6.7 Hz, 4H, 2NCH2), 3.99 (s, 3H, NMe), 4.50-4.59 (m, 4H, NCH2+OCH2), 7.80 (s, 2H, 2H-3); ,3C NMR: 8 14.0, 14.2, 24.3, 25.9, 28.6, 33.8, 43.8, 47.4, 48.0, 64.4, 99.1,99.8, 131.2, 132.8, 154.2, 154.6, 155.6, 162.2, 168.9, 169.3; Anal, calcd.; C: 50.94, H: 5.34, N: 26.73; Found: C: 50.76, H: 5.52, N: 26.94.5-[3-(4,6-DimethyIsulfanyl-l//-pyrazolo[3,4-r/lpyrimidin-l-yl)propyl]-l-ethyl-6- ethylsulfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80p).Yield 45 %; mp 114-116 °C; MS (FAB) m/z 477 (M +l); IR (KBr): 1698 cm '1; 'H NMR: 8 1.37 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.47 (t, J = 7.3 Hz, 3H, NCH2CH3), 2.37 (m, 2H, CH2), 2.60 (s, 3H, SMe), 2.69 (s, 3H, SMe), 3.15 (q, J = 7.4 Hz, 2H, SCH2),

[ 4 9 ]

4.15 (t, J = 7.6 Hz 2H, NCH2), 4.30 (q, J = 7.2 Hz, 2H, NCH2), 4.50 (t, J = 6.6 Hz, 2H, NCH2), 7.93 (s, 1H, H-3), 7.95 (s, 1H, H-3); 13C NMR: 5 11.7, 13.4, 14.2, 14.8,26.8, 27.9, 41.4, 42.1, 44.6, 102.4, 109.4, 131.6, 134.7, 149.9, 151.9, 157.6, 160.2,164.8, 168.7; Anal, calcd.: C: 47.88, H: 5.08, N: 23.51, Found: C: 47.62, H: 4.94, N: 23.84.4-[3-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-*/]pyrimidin-l-yl)propoxy]-l-ethyl-6- ethylsulfanyl-l//-pyrazolo[3,4-rf|pyrimidine (81p).Yield 31 %; mp 80-82 °C; MS (FAB) m/z 477 (M +l); 'H NMR: 5 1.40 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.49 (t, J = 7.2 Hz, 3H, NCH2CH3), 2.46 (m, 2H, CH2), 2.50 (s, 3H, SMe), 2.67 (s, 3H, SMe), 3.14 (q, J = 7.3 Hz, 2H, SCH2), 4.40 (q, J = 7.2 Hz, 2H, NCH2), 4.54 (t, J = 5.8 Hz, 2H, NCH2), 4.58 (t, J = 6.4 Hz, 2H, OCH2), 7.78 (s, 1H, H-3), 7.93 (s, 1H, H-3); 13C NMR: 8 11.7, 14.1, 14.4, 14.7, 25.4, 28.6, 42.1, 44.0,64.0, 99.9, 109.4, 131.0, 131.7, 152.0, 155.0, 162.2, 164.8, 169.0, 169.1; Anal, calcd.: C: 47.88, H: 5.08, N: 23.51, Found: C: 47.54, H: 5.16, N: 23.76.5-[3-(6-Methylsulfanyl-4-(pyrrolidin-l-yl)pyrazolo[3,4-</]pyrimidin-l-yl)propyl]- l-ethyl-6-ethyIsuIfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80q).Yield 67.5 %; mp 150-152 °C; MS (FAB) m/z 501 (M+2); 'H NMR: 8 1.38 (t, J = 7.4 Hz, 3H, SCH2CH3), 1.47 (t, J = 7.2 Hz, 3H, NCH2CH3), 2.02 (m, 4H, 2CH2), 2.34 (m, 2H, CH2), 2.55 (s, 3H, SMe), 3.17 (q, J = 7.4 Hz, 2H, SCH2), 378 (t, J = 6.1 Hz, 4H, 2NCH2), 4.16 (t, J = 7.7 Hz, 2H, NCH2), 4.30 (q, J = 7.2 Hz, 2H, NCH2), 4.45 (t, J = 6.7 Hz, 2H, NCH2), 7.83 (s, lH^H-3), 7.95 (s, 1H, H-3); 13C NMR: 8 13.5, 14.1, 14.8,24.4, 26.0, 26.9, 28.1, 41.7, 42.1, 44.5, 47.4, 48.1, 99.1, 102.6, 132.8, 134.8, 150.0,154.4, 154.6, 157.6, 160.5, 168.9; Anal, calcd.; C: 52.88, H: 5.85, N: 25.23; Found: C: 53.12, H: 6.04, N: 25.52.

4-[3-(6-Methylsulfanyl-4-(pyrroIidin-l-yl)pyrazolo[3,4-*/]pyrimidin-l-yl)pro- poxy]-l-ethyl-6-ethylsulfanyl-l//-pyrazolo[3,4-*/] pyrimidine (81q).Yield 73 %; mp 120-122 °C; MS (FAB) m/z 500 (M +l); 'H NMR: 8 1.40 (t, J = 1.4 Hz, 3H, SCH2CH3), 1.49 (t, J = 7.2 Hz, 3H, NCH2CH3), 1.99-2.14 (m, 4H, 2CH2), 2.44 (m, 2H, CH2), 2.48 (s, 3H, SMe), 3.15 (q, J = 7.3 Hz, 2H, SCH2), 3.75 (t, J = 6.6 Hz, 4H, 2NCH2), 4.40 (q, J = 1.2 Hz, 2H, NCH2), 4.54 (t, J = 6.3 Hz, 4H, NCH2+OCH2), 7.81 (s, 2H, 2H-3); l3C NMR: 8 13.5, 14.1, 14.8, 24.4, 26.0, 26.9,28.1, 41.7, 42.1, 44.5, 47.4, 48.1, 99.1, 102.6, 132.8, 134.8, 150.0, 154.4, 154.6,

[ 5 0 ]

157.6, 160.5, 168.9; Anal, calcd.; C: 52.88, H: 5.85, N: 25.23; Found: C: 52.76, H:6.06, N: 25.44.5-[3-(4,6-Dimethylsulfanyl-l//-pyrazoIo[3,4-*/]pyrimidin-l-yl)propyl]-6-ethyl- sulfanyl-l-methyl-l,5-dihydropyrazolo[3,4-rf]pyriinidin-4-one (80r).Yield 51 %; mp 132-134 °C; MS (FAB) m/z 463 (M +l); lH NMR: 5 1.37 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.36 (m, 2H, CH2), 2.60 (s, 3H, SMe), 2.69 (s, 3H, SMe), 3.16 (q, J = 7.4 Hz, 2H, SCH2), 3.90 (s, 3H, NMe), 4.15 (t, J = 7.6 Hz, 2H, NCH2), 4.50 (t, J =6 . 6 Hz, 2H ,N CH 2), 7.94 (s, IH, H-3), 7.95 (s, IH, H-3); 13C NMR: 8 11.7, 13.3, 14.2,26.8, 27.8, 33.5, 41.4, 44.5, 102.2, 109.3, 131.5, 134.7, 150.5, 151.8, 157.4, 160.4,164.7, 168.6; Anal, calcd.: C: 46.73, H: 4.79, N: 24.22, Found: C: 46.56, H: 4.75, N: 24.42.4-[3-(4,6-DimethyIsulfanyl-l//-pyrazolo[3,4-</]pyriinidin-l-yl)propoxy]-6-ethyl- sulfanyl-l-methyl-l//-pyrazolo[3,4-*/|pyrimidine (81r).Yield 37 %; mp 132-134 °C; MS (FAB) m/z 463 (M +l); 'H NMR: 8 1.40 (t, J = 7.4 Hz, 3H, SCH2CH3), 2.46 (m, 2H, CH2), 2.51 (s, 3H, SMe), 2.67 (s, 3H, SMe), 3.14 (q, J = 7.4 Hz, 2H, SCH2), 3.99 (s, 3H, NMe), 4.54 (t, J = 6.0 Hz, 2H, OCH2), 4.59 (t, J =6.4 Hz, 2H, NCH2), 7.78 (s, IH, H-3), 7.92 (s, IH, H-3); 13C NMR: 8 11.7, 14.1, 14.4,25.3, 28.5, 33.7, 43.9, 64.0, 99.7, 109.2, 131.0, 131.6, 151.8, 155.4, 162.1, 164.7,168.5, 168.8; Anal, calcd.: C: 46.73, H: 4.79, N: 24.22, Found: C: 46.65, H: 4.88, N: 24.56.5-[4-(4,6-DimethylsuIfanyI-l//-pyrazolo[3,4-</]pyrimidin-l-y!)butyl]-l-methyI-6- methylsulfanyl-l,5-dihydropyrazolo[3,4-tf]pyrimidin-4-one (80s).Yield 45 %; mp 140-142 °C; MS (FAB) m/z 463 (M +l); IR (KBr): 1685 cm '1; ’H NMR: 8 1.73 (m, 2H, CH2), 2.03 (m, 2H, CH2), 2.59 (s, 3H, SMe), 2.61 (s, 3H, SMe),2.68 (s, 3H, SMe), 3.91 (s, 3H, NMe), 4.11 (t, J = 7.5 Hz, 2H, NCH2), 4.43 (t, J = 6 . 8

Hz, 2H, NCH2), 7.91 (s, IH, H-3), 7.95 (s, IH, H-3); 13C NMR: 8 11.8, 14.3, 15.2,25.1, 26.6, 33.7, 43.2, 46.3, 102.3, 109.3, 131.5, 134.9, 150.5, 151.8, 157.5, 161.0,164.8, 168.6; Anal, calcd.: C: 46.73, H: 4.79, N: 24.22, Found: C: 46.60, H: 4.70, N: 24.50.4-[4-(4,6-Dimethylsulfanyl-17/-pyrazolo[3,4-rf]pyrimidin-l-yl)butoxy]-l-methyl-6-methylsulfanyl-lA/-pyrazolo[3,4-*/|pyrimidine (81s).

[ 5 1 ]

Yield 29 %; mp 150-152 °C; MS (FAB) m/z 463 (M +l); *H NMR: 5 1.81 (m, 2H, CH2), 2.13 (m, 2H, CH2), 2.60 (s, 3H, SMe), 2.62 (s, 3H, SMe), 2.68 (s, 3H, SMe),4.00 (s, 3H, NMe), 4.47 (t, J = 6.8 Hz, 2H, NCH2), 4.56 (t, J = 6.4 Hz, 2H, NCH2), 7.87 (s, 1H, H-3), 7.91 (s, 1H, H-3); 13C NMR: 11.8, 14.2, 14.3, 25.8, 25.9, 33.8, 46.4,66.2, 99.8, 109.3, 131.2, 131.5, 151.8, 155.6, 162.4, 164.9, 168.6, 169.3; Anal, calcd.: C: 46.73, H: 4.79, N: 24.22, Found: C: 46.52, H: 4.65, N: 24.42.5-[4-(4,6-DimethylsulfanyI-l//-pyrazolo[3,4-*/]pyrimidin-l-yl)butyl]-l-ethyl-6- ethylsulfanyl-l,5-dihydropyrazoIo[3,4-rf]pyrimidin-4-one (80t).Yield 40 %; mp 120-122 °C; MS (FAB) m/z 491 (M +l); IR (KBr): 1695 c m 1; 'H NMR: 8 1.41 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.48 (t, J = 7.2 Hz, 3H, NCH2CH3), 1.72 (m, 2H, CH2), 2.04 (m, 2H, CH2), 2.62 (s, 3H, SMe), 2.68 (s, 3H, SMe), 3.20 (q, J =7.3 Hz, 2H, SCH2), 4.09 (t, J = 7.6 Hz, 2H, NCH2), 4.31 (q, J = 1.2 Hz, 2H, NCH2), 4.43 (q, J = 6.8 Hz, 2H, NCH2), 7.91 (s, 1H, H-3), 7.96 (s, 1H, H-3); ,3C NMR: 811.7, 13.6, 14.3, 14.8, 25.1, 26.5, 26.9, 42.1, 43.1, 46.2, 102.5, 109.3, 131.5, 134.7,149.9, 151.8, 157.7, 160.3, 164.7, 168.5; Anal, calcd.: C: 48.96, H: 5.34, N: 22.84, Found: C: 48.74, H: 5.52, N: 22.98.4-[4-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)butoxy]-l-ethyl-6- ethylsulfanyl-l//-pyrazolo[3,4-</] pyrimidine (81t).Yield 28 %; mp 98-100 °C; MS (FAB) m/z 491 (M +l); ]H NMR: 8 1.42 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.49 (t, J = 1.2 Hz, 3H, NCH2CH3), 1.81 (m, 2H, CH2), 2.13 (m, 2H, CH2), 2.62 (s, 3H, SMe), 2;69 (s, 3H, SMe), 3.18 (q, J = 7.3 Hz, 2H, SCH2), 4.35- 4.57 (m, 6H, 2NCH2 + OCH2), 7.88 (s, 1H, H-3), 7.92 (s, 1H, H-3); 13C NMR: 8 11.8,14.3, 14.5, 14.7, 25.4, 25.8, 25.9, 42.1, 46.4, 66.1, 100.0, 109.3, 131.1, 131.5, 151.9,155.0, 162.5, 164.9, 168.6; Anal, calcd.: C: 48.96, H: 5.34, N: 22.84, Found: C: 48.82, H: 5.50, N: 23.12.

5-[4-(6-Methylsulfanyl-4-(pyrrolidin-l-yl)pyrazolo[3,4-rf]pyrimidin-l-yl)butyl]-l- ethyl-6-ethylsulfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80u).Yield 83 %; mp 168-170 °C; MS (FAB) m/z 514 (M +l); ’H NMR: 8 1.41 (t, J = 1.4 Hz, 3H, SCH2CH3), 1.48 (t, J = 1.2 Hz, 3H, NCH2CH3), 1.72 (m, 2H, CH2), 1.98-2.05 (m, 6H, 3CH2), 2.56 (s, 3H, SMe), 3.21 (q, J = 7.2 Hz, 2H, SCH2), 3.77 (t, J = 6.8 Hz, 4H, 2NCH2), 4.09 (t, J = 7.6 Hz, 2H, NCH2), 4.34 (q, J = 1.2 Hz, 2H, NCH2), 4.38 (t, J = 6.8 Hz, 2H, NCH2), 7.80 (s, 1H, H-3), 7.95 (s, 1H, H-3); l3C NMR: 8 14.1, 14.6,

[ 5 2 ]

14.7, 23.0, 24.4, 25.4, 25.9, 28.2, 29.0, 42.1, 46.3, 47.4, 48.1, 66.8, 99.0, 100.1, 131.2,132.6, 154.3, 154.5, 155.0, 162.6, 168.7; Anal, calcd.; C: 53.78, H: 6.08, N: 24.54; Found: C: 53.54, H: 6.22, N: 24.94.5-[5-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)pentyl]-l-methyl-6- methylsulfanyl-l,5-dihydropyrazolo[3,4-</]pyrimidin-4-one (80v).Yield 41 %; mp 114-116 °C; MS (FAB) m/z 477 (M +l); IR (KBr): 1694 cm’1; 'H NMR: 8 1.40 (m, 2H, CH2), 1.75 (m, 2H, CH2), 1.99 (m, 2H, CH2), 2.61 (s, 6H, 2SMe), 2.69 (s, 3H, SMe), 3.93 (s, 3H, NMe), 4.04 (t, J = 7.7 Hz, 2H, NCH2), 4.39 (t, J = 6.8 Hz, 2H, NCH2), 7.91 (s, 1H, H-3), 7.96 (s, 1H, H-3); Anal, calcd.: C: 47.88, H: 5.08, N: 23.51, Found: C: 47.76, H: 5.14, N: 23.72.4-[5-(4,6-DimethylsulfanyI-l//-pyrazolo[3,4-*/]pyrimidin-l-yI)pentoxy]-l-methyI-6-methylsulfanyl-l//-pyrazolo[3,4-</]pyrimidine (81u).Yield 32.5 %; mp 112-114 °C; MS (FAB) m/z 477 (M +l); 'H NMR: 8 1.45 (m, 2H, CH2), 1.88 (m, 2H, CH2), 2.01 (m, 2H, CH2), 2.60 (s, 3H, SMe), 2.61 (s, 3H, SMe),2.69 (s, 3H, SMe), 4.00 (s, 3H, NMe), 4.41 (t, J = 7.0 Hz, 2H, NCH2), 4.49 (t, J = 6.6 Hz, 2H, OCH2), 7.85 (s, 1H, H-3), 7.91 (s, 1H, H-3); 13C NMR: 11.8, 14.2, 14.3, 22.9,28.1, 28.9, 33.8, 46.6, 66.6, 99.8, 109.3, 131.2, 131.4, 151.7, 155.5, 162.4, 164.8,168.4, 169.3; Anal, calcd.: C: 47.88, H: 5.08, N: 23.51, Found: C: 48.12, H: 5.18, N: 23.25.5-[5-(6-MethyIsulfanyl-4-(pyrrolidin-l-yl)pyrazolo[3,4-</]pyrimidin-l-yl)pentyl]- l-methyl-6-methylsuIfanyl-l,5'dihydropyrazolo[3,4-</]pyrimidin-4-one (80w).Yield 71 %; mp 178-180 °C; MS (FAB) m/z 500 (M +l); IR (KBr): 1694 cm '1; 'H NMR: 8 1.39 (m, 2H, CH2), 1.75 (m, 2H, CH2), 1.90-2.22 (m, 6H, 3CH2), 2.55 (s, 3H, SMe), 2.62 (s, 3H, SMe), 3.76 (t, J = 6.6 Hz, 4H, 2NCH2), 3.92 (s, 3H, NMe), 4.04 (t, J = 7.7 Hz, 2H, NCH2), 4.34 (t, J = 6.8 Hz, 2H, NCH2), 7.79 (s, 1H, H-3), 7.95 (s, 1H, H-3); 13C NMR: 8 14.1, 15.2, 23.8, 24.3, 25.8, 27.4, 28.8, 33.6, 43.6, 46.2, 47.3, 48.0,98.8, 102.3, 132.5, 134.7, 150.5, 154.2, 154.3, 157.4, 161.0, 168.6; Anal, calcd.; C:52.88, H: 5.85, N: 25.23; Found: C: 53.22, H: 5.76, N: 25.02.

4-[5-(6-MethylsuIfanyl-4-(pyrrolidin-l-yI)pyrazolo[3,4-</]pyrimidin-l-yl)- pentoxy]-l-methyl-6-methylsuIfanyl-l//-pyrazolo[3,4-</]pyrimidine (81v).

[ 5 3 ]

Yield 72 %; mp 122-124 °C; MS (FAB) m/z 500 (M +l); 'H NMR: 5 1.47 (m, 2H, CH2), 1.84-2.10 (m, 8H, 4CH2), 2.55 (s, 3H, SMe), 2.59 (s, 3H, SMe), 3.76 (t, J = 6.5 Hz, 4H, 2NCH2), 3.99 (s, 3H, NMe), 4.36 (t, J = 6.8 Hz, 2H, OCH2), 4.48 (t, J = 6.5 Hz, 2H, NCH2), 7.78 (s, IH, H-3), 7.85 (s, IH, H-3); 13C NMR: 14.2, 22.9, 24.3, 25.9,28.1, 28.9, 33.8, 46.3, 47.4, 48.0, 66.8, 98.9, 99.9, 131.2, 132.5, 154.3, 154.4, 155.6,162.5, 168.7, 169.3; Anal, calcd.; C: 52.88, H: 5.85, N: 25.23; Found: C: 53.14, H: 5.98, N: 25.45.5-[5-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)pentyl]-l-ethyl-6- ethylsulfanyl-l,5-dihydropyrazolo[3,4-</Jpyrimidin-4-one (80x).Yield 45 %; mp 100-102 °C; MS (FAB) m/z 505 (M +l); IR (KBr): 1686 c m 1; ‘H NMR: 5 1.40-1.54 (m, 8H, SCH2CH3 + NCH2CH3 + CH2), 1.76 (m, 2H, CH2), 2.00 (m, 2H, CH2), 2.61 (s, 3H, SMe), 2.69 (s, 3H, SMe), 3.23 (q, J = 7.3 Hz, 2H, SCH2),4.04 (t, J = 7.6 Hz, 2H, NCH2), 4.35 (q, J = 7.2 Hz, 2H, NCH2), 4.39 (t, J = 7.0 Hz, 2H, NCH2), 7.91 (s, IH, H-3), 7.96 (s, IH, H-3); l3C NMR: 5 11.7, 13.7, 14.3, 14.8,23.9, 26.9, 27.5, 28.9, 42.1, 43.6, 46.6, 102.8, 109.3, 131.4, 134.7, 150.0, 151.8,157.6, 160.4, 164.7, 168.5; Anal, calcd.: C: 49.98, H: 5.59, N: 22.20, Found: C: 49.38, H: 5.73, N: 21.85.4-[5-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)pentoxy]-l-ethyI-6- ethylsulfanyl-lJJ-pyrazolo[3,4-</]pyrimidine (81w).Yield 40 %; mp 68-70 °C; MS (FAB) m/z 505 (M +l); ’H NMR: 8 1.39-1.53 (m, 8H, 2SCH2CH3 + CH2), 1.88 (m, 2H, CH2), 2.02 (m, 2H, CH2), 2.61 (s, 3H, SMe), 2.69 (s, 3H, SMe), 3.19 (q, J = 7.3 Hz, 2H, SCH2), 4.35-4.51 (m, 6H, 3NCH2), 7.85 (s, IH, H- 3), 7.91 (s, IH, H-3); l3C N M R : 5 11.8, 14.3, 14.6, 14.7, 23.0, 25.4, 28.2, 29.0, 42.2,46.6, 66.6, 100.1, 109.4, 131.1, 131.4, 151.8, 155.1, 162.6, 164.8, 168.5, 168.7; Anal, calcd.: C: 49.98, H: 5.59, N: 22.20, Found: C: 49.72, H: 5.42, N: 22.54.4-[5-(6-Methylsulfanyl-4-(pyrrolidin-l-yl)pyrazolo[3,4-*/]pyrimidin-l-yl)- pentoxy]-l-ethyl-6-ethylsulfanyl-l/J-pyrazolo[3,4-</]pyrimidine (81x).Yield 70 %; mp 106-108 °C; MS (FAB) m/z 528 (M +l); ’H NMR: 5 1.42 (t, J = 7.3 Hz, 3H, SCH2CH3), 1.49 (t, J = 7.2 Hz, 3H, NCH2CH3), 1.84-2.03 (m, 8H, 4CH2) 2.56 (s, 3H, SMe), 3.19 (q, J = 7.3 Hz, 2H, SCH2), 3.78 (t, J = 6.7 Hz, 4H, 2NCH2), 4.33-4.51 (m, 6H, 2NCH2+OCH2), 7.80 (s, IH, H-3), 7.86 (s, IH, H-3); l3C NMR: 514.1, 14.6, 14.7, 23.0, 24.4, 25.4, 25.9, 28.2, 29.0, 42.1, 46.3, 47.4, 48.0, 66.7, 98.9,

[ 5 4 ]

100.1, 131.2, 132.6, 154.3, 154.4, 155.0, 162.6, 168.7; Anal, calcd.; C: 54.62, H: 6.30, N: 23.89; Found: C: 54.34, H: 6.10, N: 23.54.5-[10-(4,6-Dimethylsulfanyl-l//-pyrazolo[3,4-</]pyrimidin-l-yl)decyl|-6-ethyl- sulfanyl-l-methyl-l,5-dihydropyrazolo[3,4-*/]pyrimidin-4-one (80y).Yield 52 %; mp 80-82 °C; MS (FAB) m/z 561 (M +l); 'H NMR: 5 1.28 (brs, 12H, 6CH2), 1.45 (t, 3H, J = 7.3 Hz, SCH2CH3), 1.70 (m, 2H, CH2), 1.90 (m, 2H, CH2), 2.63 (s, 3H, SMe), 2.68 (s, 3H, SMe), 3.26 (q, J = 7.3 Hz, 2H, SCH2), 3.92 (s, 3H, NCH3), 4.05 (t, J = 7.9 Hz, 2H, NCH2), 4.36 (t, J = 7.0 Hz, 2H, NCH2), 7.91 (s, IH, H-3), 7.97 (s, IH, H-3); l3C NMR: 5 11.7, 13.6, 14.2, 26.4, 26.7, 26.8, 27.9, 28.9,29.0, 29.2, 33.5, 43.9, 46.8, 102.5, 109.2, 131.2, 134.7, 150.6, 151.6, 157.6, 160.6,164.6, 168.3.4-[10-(4,6-Dimethylsulfanyl-l//r-pyrazolo[3,4-*/]pyrimidin-l-yl)decyloxy]-6-ethyl- sulfanyl-l-methyl-l//-pyrazolo[3,4-</]pyrimidine (81y).Yield 43 %; mp 66-68 °C; MS (FAB) m/z 561 (M +l); 'H NMR: 5 1.28 (brs, 12H, 6CH2), 1.44 (t, 3H, J = 7.4 Hz, SCH2CH3), 1.77-1.94 (m, 4H, 2CH2), 2.63 (s, 3H, SMe), 2.68 (s, 3H, SMe), 3.21 (q, J = 7.4 Hz, 2H, SCH2), 3.99 (s, 3H, NCH3), 4.36 (t, J = 7.0 Hz, 2H, NCH2), 4.50 (t, J = 6.6 Hz, 2H, NCH2), 7.89 (s, IH, H-3), 7.91 (s, IH, H-3); 13C NMR: 5 11.7, 14.2, 14.5, 25.4, 25.8, 26.4, 28.6, 28.9, 29.3, 33.7, 46.8, 67.1,100.0, 109.2, 131.2, 151.6, 155.5, 162.6, 164.6, 168.3, 168.9.Synthesis o f l-methyl-6-methylsulfanyl-5-phenethyl/phenylpropyI-l,5-dihydro- pyrazolo[3,4-</]pyrimidin-4-one»and their regioisomers.These compounds were synthesized by replacing one pyrazolo[3,4-t/]pyrimidinyl moiety o f compounds 80a, 81a, 80o and 81n by a phenyl ring to study the effect on intramolecular 7t-7i interactions. The synthesis o f these compounds was achieved by treating l-methyl-6-methylsulfanyl-4-oxo-l,5-dihydropyrazolo[3,4-c/]pyrimidine with 1 -[(2-bromoethyl)benzene/(3-bromopropyl)benzene] (Scheme II) in DMF in presence o f anhydrous potassium carbonate at room temperature for 20 h. The DMF was removed under reduced pressure and the residue extracted with water/chloroform system and chromatographed over silica gel to obtain the pure products.The 'H NMR spectra o f all these compounds didn’t show any significant variation in chemical shift o f any proton/group showing no intramolecular stacking.

[ 5 5 ]

o

a X ) * 0 - (cH2)n-1M N '------ '

D M F /K ,C O ,Br -----------------------r.t., 20 h

83, 85

Scheme II

l-Methyl-6-methylsulfanyl-5-phenethyl-l,5-dihydropyrazolo[3,4-*/]pyrimidin-4- one (83).Yield 17 %; mp 160-162 °C; MS (FAB) m/z 301 (M +l); ]H NMR: 8 2.67 (s, 3H, SMe), 3.01 (t, J = 8.4 Hz, 2H, CH2), 3.95 (s, 3H, NMe), 4.29 (t, J = 8.4 Hz, 2H, NCH2), 7.25-7.35 (m, 5H, ArH), 8.00 (s, 1H, H-3); 13C NMR: 8 15.2, 33.6, 34.0, 45.4,102.3, 126.6, 128.5, 128.7, 134.7, 137.6, 150.5, 157.3, 160.9; Anal. Calcd.: C: 59.98, H: 5.37, N: 18.65; Found: C: 60.80, H: 5.90, N: 18.60.l-Methyl-6-methylsulfanyl-4-phenethyloxy-l//-pyrazolo[3,4-(/] pyrimidine (84).Yield 40 %; mp 84-86 °C; MS (FAB) m/z 301 (M +l); 'H NMR: 8 2.61 (s, 3H, SMe),3.15 (t, J = 7.2 Hz, 2H, CH2> 4 .0 0 (s, 3H, NMe), 4.73 (t, J = 7.2 Hz, 2H, NCH2), 7.23-7.33 (m, 5H, ArH), 7.88 (s, 1H, H-3); l3C NMR: 8 14.2, 33.7, 35.1, 67.4, 99.8,126.5, 128.4, 128.9, 131.1, 137.6, 155.5, 162.2, 169.3. Anal. Calcd.: C: 59.98, H: 5.37, N: 18.65, Found: C: 60.70, H: 5.60, N: 18.60.l-Ethyl-6-methylsulfanyl-5-(3-phenylpropyl)-l,5-dihydropyrazolo[3,4-</]pyrimi- din-4-one (85).Yield 57 %; mp 154-156 °C; MS (FAB) m/z 315 (M +l); 'H NMR: 8 1.49 (t, J = 1.2 Hz, 3H, CH3), 2.08 (quint., J = 7.8 Hz, 2H, CH2), 2.61 (s, 3H, SMe), 2.75 (t, J = 1.1 Hz, 2H, CH2), 4.13 (t, J = 7.8 Hz, 2H, NCH2), 4.32 (q, J = 7.2 Hz, 2H, NCH2), 7.18-7.29 (m, 5H, ArH), 7.98 (s, 1H, H-3); 13C NMR: 8 14.6, 15.2, 29.1, 32.9, 42.0, 43.5,

[ 5 6 ]

102.3, 125.9, 128.1, 128.2, 134.6, 140.6, 149.8, 157.5, 160.8; Anal, calcd.; C: 61.12, H: 5.77, N: 17.82; Found: C: 60.86, H: 5.54, N: 17.44.l-Ethyl-6-methyIsulfanyl-4-(3-phenylpropoxy-l//-pyrazolo[3,4-f/] pyrimidine (86).Yield 36 %; mp oil; MS (FAB) m/z 315 (M +l); 'H NMR: 5 1.50 (t, J = 7.2 Hz, 3H, CH3), 2.13-2.24 (m, 2H, CH2), 2.59 (s, 3H, SMe), 2.81 (t, J = 7.5 Hz, 2H, CH2), 4.42 (q, J = 7.2 Hz, 2H, NCH2), 4.55 (t, J = 6.4 Hz, 2H, NCH2), 7.20-7.34 (m, 5H, ArH), 7.90 (s, 1H, H-3).

[ 5 7 ]

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