Development and application of novel scaffolds in drug discovery
Boltjes, André
DOI:
10.33612/diss.98161351
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Publication date: 2019
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Boltjes, A. (2019). Development and application of novel scaffolds in drug discovery: the MCR approach. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.98161351
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Chapter 7
Diverse One-Pot Synthesis of
Adenine Mimetics for Biomedical
Applications
André Boltjes, Markella Konstantinidou, Angel J. Ruiz-Moreno, Li Gao, John de Boer, Marco A. Velasco-Velazquez and Alexander Dömling
Abstract
A series of unprecedented imidazo[1,2-a]pyrazin-8-amines are synthesized from simple and readily available building blocks. This scaffold is of high interest due to its similarity to adenine. Multiple di-aminopyrazines, -pyrimidines and –pyridazines were subjected to Groebke-Blackburn-Bienaymè reaction conditions to afford imidazo[1,2-a]pyrazines, pyrimidines and pyridazines respectively. Pyrazine-2,3-diamine, as amidine component furnished a clean and selective reaction resulting in an adenine mimetic library of over 20 compounds in a single step under mild conditions.
7
In classical drug discovery a protein target which is believed to have a positive influence on a disease course is pursued and antagonists or agonist are searched for. Small molecules against this drug target can be found for example by high throughput screening of large libraries. This is the most popular approach in drug discovery and leads to more or less hits depending on the target class. Often no hits are resulting from so called undruggable targets. However, from the initial hit to a useful lead compound to probe the biology of a target protein is a long and stony way involving much time and efforts to improve the hit compounds affinity, selectivity, stability etc. and finally biological activity. Chemists on the other hand often find a new and versatile reaction pathway to a novel class of scaffold. However, a priori there is no knowledge what biological space could correspond to this particular chemical space. Traditionally, examples of the new scaffolds could be screened in HTS to eventually find an active hit for a given target. In the human proteome, however, there are hundreds of thousands of potentially targetable proteins and thus it is unlikely that a target will be found for a given compound in a reasonable time even in a pharma company which runs multiple HTS per year. A solution to this dilemma is reverse or inverse docking which aims to find a target for a given small molecule based on computational screening of the full or part of the PDB.1-2
Figure 1. Adenine and a new synthetic pathway towards mimics. Left: Zoom into the
adenine substructure of substructure of nicotineamide-adenine-dinucleotide binding pocket of the enoyl-ACP reductase of Mycobacterium tuberculosis InhA (PDB ID 4TRO). Right: Previous and current work using GBB-3CR towards adenine derivatives.
One of the most successful target classes in modern medicine are kinases and more than 30 small molecule inhibitors were introduced into clinical practice.3
Kinases catalyze the phosphorylation of Ser, Thr, His and Tyr and use as cofactor ATP. Small molecule kinase inhibitor drugs mimic the adenosine part of ATP and fit into a deep and narrow binding site and form in the so-called hinge region a bifurcated hydrogen bond between a diglycine and the adenine-N1 and C6-NH2. Besides kinases many more protein use adenine as a cofactor. In figure 1 a typical interaction of an adenine substructure is shown forming the signature hydrogen bonding network.4 In the current version of the protein data bank (PDB) more
than 22,000 structures can be found containing the adenine substructure.5 Thus,
adenine mimics are highly sought-after substructures and synthesis of substituted heterocycles mimicking A are promising potentially bioactive compounds. Most current synthetic pathways for adenine mimicking small molecules suffer from a lengthy multistep synthesis.6-8 In 2011 Pirali described a 2-step route
component reaction (GBB-3CR) of 2-amino-3-chloropyrazine followed by a SnAr with ammonia.9-13 A similar approach reported by Ren et al. in 2015 is showing
a side by side comparison between the 2-amino-3-chloropyrazine substrate and N-2,4-dimethoxybenzyl (DMB) protected diaminopyrazine.14 Although
the yields improved by using DMB protected diaminopyrazine, probably due to the better nucleophilicity, again a two-step methodology was highlighted. These GBB-3CR amidine substrates are quite unreactive and need considerable activation by high temperature. The subsequent SnAr with ammonia also requires harsh conditions and both reactions are not compatible with many delicate substituents. Moreover, only substituted benzaldehydes and aryl carbaldehydes were reported to be used in this limited methodology. Therefore, we want to report here our design of a one-pot general, middle and high yielding synthesis to 2,3-di-substituted imidazo[1,2-a]pyrazin-8-amine as adenine mimics. Amidine containing aromatic 5- and 6-membered heterocycles are interesting building blocks in bioactive molecules, able to form bifurcated hydrogen bondings to -COOH and -OH of Asp, Glu and Tyr. Thus, we were investigating the behavior of corresponding diamine substituted pyrazines and pyridazines in the GBB-3CR with the target to find conditions for a selective mono reaction (Scheme 1). N N N N NH2 NH2 H2N NH2 GBB-3CR N N NH2 NH2 N N N R1 NH R2 N N N R1 NH R2 N N N R1 NH R2 NH2 H2N H2N + N N N R1 HN HN NH R1 R2 R2 Degradation Degradation
Scheme 1. Pyrazines, pyrimidines and pyridazines in the GBB-3CR.
Although these three amidines give yield to the expected products (confirmed by MS), the products from pyridazine-3,6-diamine degrade upon exposure to air during purification. Furthermore, the pyridazine-3,6-diamine product is low yielding, shows degradation and yields an additional bis-GBB-3CR product which degrades within a short amount of time as well, probably due to the easy oxidizable bridging double bond.
We found that the GBB-3CR products from pyrazine-2,3-diamine; 2,3-di-substituted imidazo[1,2-a]pyrazin-8-amines do not show degradation, therefore this amidine together with benzaldehyde and 2,6-dimethylphenyl isocyanide was used in an optimizing sequence in which various reaction conditions were evaluated as depicted in table 1.
7
Table 1. Optimizing conditions applied on the most promising amidine.
NC 1 2a 3a MeOH N N NH2 NH2 O N N N NH NH2 Catalyst + + 4a Reaction
Time r.t.a 60 °C 5% Sc(OTf)3b 20% Sc(OTf)3b
10 min 4 h 8 h 24 h 48 h --37% 49% 82% 82% MW (100 °C) 10% HClO4b 10% PTSAb -- 62% -- -- -- --42% 59% 64% 75% --20% 31% 62% 68% 39% 61% 75% 75% 16% 33% 46% 82% 14% 27% 44% 67%
a Standard protocol: amidine 1 (0.25 mmol, 1 eq.) and aldehyde 2 (0.25 mmol, 1 eq.) were
dissolved in 1 mL MeOH, after 5 minutes isocyanide 3 (0.25 mmol, 1 eq.) and Sc(OTf)3 (0.025 mmol, 0.1 eq.) were added and stirred at room temperature. b The percentages
described are mol-%. Analysis: 10 µL reaction mixture was diluted 20.000 times in MeOH. Each sample was analyzed via MS flow injection analysis, collecting signal only from the products [M+1]+ channel. The reported percentages are conversion, corrected to the actual
yield after column chromatography.
The reaction progress was monitored by quantitative MS analysis and 10 µL aliquots of reaction mixture were taken over time while varying temperature and catalysts. As expected at elevated temperature the rate of product formation increases, the amount of product in the room temperature reaction however, surpassed that of the heated reactions after 24 hours of stirring. Furthermore, TLC analysis is showing a cleaner reaction mixture in the rt experiment, explaining the higher conversion towards product. Assessment of three catalysts; scandium triflate (Sc(OTf)3, p-toluenesulfonic acid (PTSA) and HClO4(aq) and additionally catalyst load of scandium triflate; 5, 10 and 20 mol-% resulted in Sc(OTf)3 (10 mol-%) and perchloric acid HClO4 (10 mol-%) as good candidates, giving comparable conversion. Ease of handling and shorter reaction time made that we chose Sc(OTf)3 as optimal catalyst. Purification afforded N3
-(2,6-dimethylphenyl)-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4) in a yield of 44%. The concentration of the screening reaction was 0.25 M.
With respect to the two amino groups present in the amidine, the aldehyde was hypothesized to be scavenged through double Schiff-base formation. Two more dilute reactions C = 0.1 M were run, a 30 minute incubation of the amidine with aldehyde and slow addition of the aldehyde to the amidine respectively. Proper dilution generally results in mono substitution and to enforce our hypothesis, a third, higher concentrated reaction was performed at C = 0.5 M with a small excess of aldehyde (1.1 equiv.) and isocyanide (1,3 equiv.). Surprisingly dilution and slow addition of aldehyde lead to lower yields as compared to the more concentrated reaction with yields of 45, 24 and 82% respectively. When
benzaldehyde and 2,6-dimethylphenyl isocyanide were used, precipitation of the product was observed, resulting in a slurry in the 0.5 M concentration. To ensure proper mixing, the concentration was not further increased and kept at 0.5 M for all described reactions.
Figure 2. Top picture of fluorescent compound 4b spotted on a TLC plate. Bottom picture
showing a selection of pure products in solution <1 mg/mL in MeOH.
Scope and limitations for a large variety of substituents was assessed (Table 2). Both the aldehyde and isocyanide components give room to test compatibility of aromatic and aliphatic substituents. Moreover, additional functional groups attached to R1 or R2 such as esters, boronic acids, ethers and Boc protected amines were used to demonstrate the versatile character of this reaction. Interestingly the majority of the products exhibited fluorescence as observed while taking TLCs (figure 2). Overall there was no significant difference observed between the used aliphatic and aromatic isocyanides, while keeping the aldehyde (benzaldehyde) component constant. We were able to grow a crystal from compound 4e, confirming the proposed structure and not the possible inverse GBB-3CR product (Figure 2).
7
3.5 3.2 3.5 Figure 2. Crystal structures of some GBB-3CR products. Left: Crystal structure of 4a. Right: X-ray structure refinement unambiguously confirms formation of 4e and not the inverse GBB-3CR product 5. Hydrogen bonding of the exocyclic amine on C-3 underlines the importance of the adenine mimetic structure.The amino acid derived isocyanides (Gly and β-Ala) worked well, the additional carbon gave some improvement in yield of 60 and 78% for 4r and 4d respectively. Examples 4b and 4c with variations on the aldehyde component, having electron withdrawing groups gave significant lower yields. The yield was, however, observed to be higher when an aromatic isocyanide such as 2,6-dimethylphenyl isocyanide (3a) was used, indicated in example 4t. Glyoxylic acid was used as formaldehyde component and gave a poor yield of only 35% (4k). The Schiff base precipitated, which could explain the low yield. Heating would typically dissolve the Schiff base and should result in a better conversion. The reaction was therefore repeated in a microwave reactor at 100 °C for 10 minutes, resulting in a clear reaction mixture, but similar poor yield of 30% after purification, indicates that glyoxylic acid in combination with the pyrazine substrate (1) under these conditions is not optimal.
Table 2. Substrate scope for aldehydes and isocyanides. NC R2
+
1 2 3 MeOH, Sc(OTf)3 24h, rt N N NH2 NH2 R1 O N N N R1 NH R2 NH2 N N N NH NH2 4a (82%) N N N NH NH2 Cl BocHN 4b (20%) N N N NH NH2 NO2 4c (40%) N N N NH NH2 OMe 4e (96%) 4f (66%) 4g (75%) N N N NH NH2 N N N NH NH2 4i (54%) 4j (91%) 4k (35%) N N N NH S NH2 N N N NH NH2 N N N NH NH NH2 4m (96%) 4n (81%) 4o (78%) N N N NH NH2 N N N NH NH2 N N N NH NH2 4q (60%) 4r (60%) N N N NH NH2 N N N NH NH2 O O 4h (68%) 4d (78%) 4l (78%) 4p (81%) 4s (90%) N N N NH NH2 O O N N N NH NH2 BocHN N N N NH NH2 BPin N N N NH NH2 B HO OH N N N NH NH2 NHBoc 4a-t N N N NH NH2 Cl N N N NH NH2 OMe N N N NH NH2 O O 4t (75%) 4u (61%) 4v (72%)7
unprotected (4p) and its pinacol ester (4l) were well tolerated, with yields between 68 and 90%. During purification of reactions that produced 4e and 4u, minor bis product formation was observed. The electron donating groups on the used aromatic aldehydes for these products seem to slightly activate the remaining amidine in the mono product, thus allowing for a second GBB-3CR. About less than half of the performed reactions showed precipitation of the product. The reported yields of these reactions were yields after column chromatography. Repetition of these reactions and filtration of the products gave slightly lower to similar yields. The lower yield could be explained by the slight solubility of the product in the mother liquor, which is otherwise retrieved by column chromatography. To highlight the simplicity of this reaction we ran a multigram reaction of 2,3-diaminopyrazine, 2,6-dimethylphenylisocyanide and benzaldehyde to afford 1.97 g ( 6.0 mmol, 60% ) 4a after filtration in a single step as a brown solid.
Molecular docking
An adenine substructure-based search was performed in the PDB database in order to define structures that can bind adenine-related ligands. We selected only X-ray protein structures corresponding to the biological assembly 1 for the experiments, resulting in 13.864 structures. Structures retrieved from PDB were prepared for molecular docking experiments by removing water, ions, and solvent molecules using Mdtraj (26488642). Co-crystallized ligands containing adenine or adenine-substructures (adenine-scaffold) were saved separately from proteins to serve as references. For some proteins, more than one co-crystallized ligand was found, because of that, the number of references was 23.267. For proteins, hydrogens, missing atoms, and residues were added, and nonstandard residues were converted to their standard forms using PDBfixer (https://
github.com/pandegroup/pdbfixer). Prepared proteins were saved as receptors.
Nine adenine-mimetic ligands were designed through our original GBB one-pot synthesis of adenine-mimetic molecules. Ligands contain 2,3-dimethylimidazo[1,2-a]pyrazin-8-amine plus aliphatic, and/or aromatic modifications in R1 and/or R2 groups (see scheme at the top). Structures of designed ligands can be found in figure 3. For each ligand, hydrogens, charges and 3D coordinates were added to the molecules by OpenBabel (21982300).
N N N NH NH2 N N N NH NH2 N N N NH NH2 N N N NH NH2 N N N NH NH2 N N N NH NH2 N N N NH NH2 N N N NH NH2 N N N NH NH2 6 7 8 11 10 9 12 13 14
Figure 3. Ligands based on the synthesized GBB-3CR products used for docking. With
variations on the R1 and R2 positions from the aldehyde and isocyanide respectively.
The identification of potential targets for the designed ligands was performed by implementing an inverse docking approach considering the co-crystallized references. For dockings, the 3D coordinates of reference were used to define a docking cavity in the receptor. The cavity was established by selecting the receptor residues 8 Å far from the reference. Then, ligand and reference were aligned by an adenine-scaffold atom map pairing within the receptor cavity implementing the open source cheminformatics library RDKit (http://www.rdkit.org). After that, molecular docking between each ligand and receptor was performed utilizing GOLD from the Cambridge Crystallography Data Center (9126849). GOLD docking experiments were evaluated by ChemPLP score considering a penalization whether during docking adenine-mimetic from ligand moved more than 1.5 Å of the adenine-scaffold in reference. For the docking results, best pose of each docking was kept for further analysis. Nevertheless, poses with negative score values were not considered for analysis due they correspond to null predicted affinity between the ligand and the receptor or were highly penalized for lack of match between adenine-mimetic and adenine-scaffold.
7
Conclusions
In conclusion, we have developed a mild method to conveniently synthesize in a single step 2,3-di-substituted imidazo[1,2-a]pyrazin-8-amines through the GBB-3CR in overall good to excellent yields. This method is superior to previously reported methods in terms of number of steps and generality. The mild reaction conditions allowed a large variety of substituents, including functional groups, such as ester and BOC groups that would otherwise degrade or react (4b,
4d, 4h, 4l, 4r and 4s) when extended elevated temperatures would have been
applied as previously described. Functional group compatibility beyond very simple model substrates is key for an early industrial adaption and avoiding a synthetic bias in medicinal chemistry programs toward making molecules with less drug-like properties.15 In total 22 reactions were performed and those
reactions without exception were found to give the expected products. We are currently investigating the reaction scaffold to discovery novel adenine mimetics with biological activity, which will be reported in due course.
Experimental procedures and Spectral Data
Instruments employed: Grace Reveleris X2 flash chromatography apparatus, Advion CMS ExpressionL. Mass spectrometer, Bruker Avance DRX 500 MHz NMR spectrometer (B AV-500) equipped with Bruker Automatic Sample Changer (B ACS 60); High resolution mass spectra were recorded using a LTQ-Orbitrap-XL (Thermo) at a resolution of 60000@m/z400.NMR spectra were recorded in CDCl3 (with 0.03% TMS), DMSO-d6 at either 500 MHz (δH) or 125 MHz (δC); the coupling constants (J) are in Hz. Abbreviations: s (singlet); d (doublet); t (triplet); q (quartet); br (broad); dd (doublet of doublet), etc. The nomenclatures of all the compounds were derived by ChemDraw (CambridgeSoft). Flash chromatography separations were performed on Flashpure Normal-phase Silica Flash Columns (Silica Gel 40µm irregular) unless otherwise noted. Reagents were commercial grades and were used without any purification unless otherwise noted. Reagents were commercial grades and were used without any purification unless otherwise noted. Methanol, dichloromethane, ethyl acetate, petroleum ether 40 – 60, were purchased from commercial sources.
Synthetic procedures and characterization for compound 4a-4v
General procedure for all GBB-3CR reactions: In a 4 mL vial Sc(OTf)3 (24.6 mg, 0.05 mmol) and 2,3-diaminopyrazine (55 mg, 0.5 mmol, 1 equiv.), were dissolved in methanol (1 mL). Then aldehyde (0.55 mmol, 1.1 equiv.) and isocyanide (0.65 mmol, 1,3 equiv.) were added and the reaction mixture was stirred for 48 h at room temperature. The solvent was removed under reduced pressure and the remainder was purified by flash chromatography with either 0 - 5% methanol in dichloromethane (method A) or 0 - 100% ethyl acetate in petroleum ether 40-60 (method B) to afford the product.
N3-(2,6-dimethylphenyl)-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4a) N N N NH NH2
Using benzaldehyde, 2,6-dimethylphenyl isocyanide and purification method B; 135 mg, 82% product was obtained as a pale yellow solid. 1H NMR (500 MHz, DMSO-d
6) δ 8.02 – 7.95 (m, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.26 – 7.19 (m, 2H), 7.16 (d, J = 4.6 Hz, 1H), 7.03 (d, J = 4.7 Hz, 1H), 6.95 (s, 2H), 6.89 (d, J = 7.5 Hz, 2H), 6.69 (t, J = 7.5 Hz, 1H), 1.90 (s, 6H), 1.22 (s, 1H). 13C NMR (126 MHz, DMSO) δ 150.1, 140.7, 134.9, 133.6, 129.4, 129.3, 128.1, 128.1, 127.7, 127.0, 126.7, 126.6, 126.2, 124.4, 120.7, 120.6, 106.8, 18.5, 18.4. HRMS (ESI) m/z calculated for C20H20N5 [M+H]+: 330,17132; found [M+H]+: 330,17130
tert-Butyl (2-((8-amino-2-(4-chlorophenyl)imidazo[1,2-a]pyrazin-3-yl)amino) ethyl)carbamate (4b) N N N NH NH2 Cl
BocHN Using 4-chlorobenzaldehyde, tert-butyl (2-isocyanoethyl)
carbamate and purification method A; 40.5 mg, 20% product was obtained as a yellow solid. 1H NMR (500
MHz, DMSO-d6) δ 8.16 – 8.09 (m, 2H), 7.59 (d, J = 4.3 Hz, 1H), 7.51 – 7.46 (m, 2H), 7.21 (s, 1H), 6.93 – 6.77 (m, 3H), 4.95 (t, J = 6.3 Hz, 1H), 3.04 (q, J = 6.3 Hz, 2H), 2.96 (q, J = 6.8, 6.4 Hz, 2H), 1.34 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 155.6, 150.1, 144.1, 133.0, 131.7, 131.4, 129.1, 128.4, 128.4, 128.1, 128.0, 127.8, 107.1, 107.0, 78.3, 77.7, 47.5, 40.7, 28.3, 28.2, 28.2. HRMS (ESI) m/z calculated for C19H24N6O2Cl [M+H]+: 403,16438; found [M+H]+: 403,16479 N3-(tert-butyl)-2-(4-nitrophenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4c) N N N NH NH2 NO2
Using p-nitro-benzaldehyde, t-butyl isocyanide and purification method B; 66 mg, 40% product was obtained as an orange solid. 1H NMR (500 MHz, DMSO-d
6) δ 8.48
(d, J = 9.0 Hz, 2H), 8.29 (d, J = 9.0 Hz, 2H), 7.67 (d, J = 4.6 Hz, 1H), 7.23 (d, J = 4.6 Hz, 1H), 6.91 (s, 2H), 4.84 (s, 1H), 1.03 (s, 9H). 13C NMR (126 MHz, DMSO) δ 150.3, 145.9,
141.8, 134.8, 130.7, 128.2, 128.0, 123.9, 123.4, 108.1, 56.2, 30.0. HRMS (ESI) m/z calculated for C16H19O2N6 [M+H]+: 327,1564; found [M+H]+: 327,15628
7
Methyl 3-((8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)amino)propanoate (4d) N N N NH NH2 OO Using benzaldehyde, methyl 3-isocyanopropanoate,
purification method A; 121mg, 78% product was obtained as a brown solid. 1H NMR (500 MHz, Chloroform-d) δ 7.92
(d, J = 6.8 Hz, 2H), 7.50 – 7.41 (m, 3H), 7.36 – 7.27 (m, 2H), 5.83 (s, 2H), 3.82 (t, J = 6.9 Hz, 1H), 3.67 (s, 3H), 3.29 (q, J = 6.1 Hz, 2H), 2.54 (t, J = 5.8 Hz, 2H). 13C NMR (126 MHz,
Chloroform-d) 13C NMR (126 MHz, Chloroform-d) δ 173.0,
149.9, 135.7, 133.7, 128.9, 128.2, 127.8, 127.6, 127.2, 107.9, 52.0, 43.7, 34.5. HRMS (ESI) m/z calculated for C16H18N5O2 [M+H]+: 312,14550; found [M+H]+: 312,14532
N3-(tert-butyl)-2-(4-methoxyphenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4e) N N N NH NH2 OMe
Using anisaldehyde, t-butyl isocyanide and purification method A; 148,8 mg, 96% product was obtained as an off-white solid. 1H NMR (500 MHz, DMSO-d
6) δ 8.15 – 7.99 (m, 2H), 7.62 (d, J = 4.6 Hz, 1H), 7.18 (d, J = 5.1 Hz, 1H), 7.02 – 6.92 (m, 2H), 6.75 (s, 2H), 4.56 (s, 1H), 3.78 (s, 3H), 0.98 (s, 9H). 13C NMR (126 MHz, DMSO-d 6) δ 158.4, 149.9, 137.5, 129.1, 128.9, 128.9, 128.7, 128.5, 127.5, 127.2, 125.5, 113.4, 113.3, 113.3, 108.2, 108.1, 55.6, 30.1, 30.1 ppm. HRMS (ESI) m/z calculated for C17H22N5O [M+H]+: 312,18237; found [M+H]+: 312,18189 2-Benzyl-N3-(tert-butyl)imidazo[1,2-a]pyrazine-3,8-diamine (4f) N N N NH NH2
Using 2-phenyl-acetaldehyde, t-butyl isocyanide and purification method B; 97 mg, 66% product was obtained as a brown solid.1H NMR (500 MHz, Chloroform-d) δ 7.47
(d, J = 4.2 Hz, 1H), 7.26 – 7.20 (m, 2H), 7.21 – 7.13 (m, 4H), 4.14 (s, 2H), 1.14 (s, 9H).13C NMR (126 MHz, CDCl
3) δ 149.1,
139.4, 139.0, 128.5, 128.4, 127.3, 126.2, 125.7, 108.8, 55.8, 33.7, 30.4. HRMS (ESI) m/z calculated for C17H22N5 [M+H]+:
296,18697; found [M+H]+: 296,18759 N3-(2,6-dimethylphenyl)-2-isobutylimidazo[1,2-a]pyrazine-3,8-diamine (4g) N N N NH NH2
Using isovaleraldehyde, 2,6-dimethylphenyl isocyanide and purification method A; 115.3 mg, 75% product was obtained as an off-white solid. 1H NMR (500 MHz, DMSO-d
6) δ 7.43 (d, J = 4.7 Hz, 1H), 7.21 (d, J = 4.7 Hz, 1H), 6.97 (d, J = 7.4 Hz, 2H), 6.78 (t, J = 7.5 Hz, 1H), 6.76 – 6.67 (m, 3H), 2.03 (d, J = 7.1 Hz, 2H), 1.92 (s, 6H), 1.85 – 1.74 (m, 1H), 0.71 (d, J = 6.6 Hz, 6H). 13C NMR (126 MHz, DMSO-d 6) δ 144.6, 138.2, 133.3, 131.5, 130.9, 129.7, 125.9, 122.9, 118.2, 109.8, 109.6, 34.7, 28.6, 28.5, 23.1, 23.0, 19.3, 19.2. HRMS (ESI) m/z calculated for C18H24N5 [M+H]+: 310,20262; found [M+H]+:
310,20236 tert-Butyl (2-((8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)amino)ethyl) carbamate (4h) N N N NH NH2
BocHN Using benzaldehyde, tert-butyl (2-isocyanoethyl)carbamate,
purification method A; 126 mg, 68% product was obtained as a yellow solid. 1H NMR (500 MHz, Chloroform-d) δ 7.84
(d, J = 7.6 Hz, 2H), 7.39 (t, J = 7.7 Hz, 2H), 7.35 – 7.25 (m, 2H), 7.18 (s, 1H), 6.09 (s, 2H), 5.13 (s, 1H), 3.60 (s, 1H), 3.22 – 3.00 (m, 4H), 1.42 (s, 9H). 13C NMR (126 MHz, Chloroform-d) δ
156.5, 149.9, 135.5, 133.6, 128.8, 128.8, 128.6, 127.7, 127.5, 127.2, 107.6, 79.7, 48.4, 41.0, 28.5. HRMS (ESI) m/z calculated for C19H25N6O2 [M+H]+: 369,20335; found [M+H]+: 369,20334 N3-(2,6-dimethylphenyl)-2-(thiophen-2-yl)imidazo[1,2-a]pyrazine-3,8-diamine (4i) N N N NH S NH2
Using thiophene-2-carbaldehyde, 2,6-dimethylphenyl isocyanide and purification method B; 91 mg, 54% product was obtained as a grey solid. 1H NMR (500 MHz, DMSO-d
6) δ 7.44 (dd, J = 5.1, 1.0 Hz, 1H), 7.19 (dd, J = 3.6, 1.1 Hz, 1H), 7.16 (d, J = 4.8 Hz, 2H), 7.06 (d, J = 4.6 Hz, 1H), 7.00 (dd, J = 5.0, 3.6 Hz, 1H), 6.93 (d, J = 7.5 Hz, 2H), 6.85 (s, 2H), 6.73 (t, J = 7.5 Hz, 1H), 1.90 (s, 6H). 13C NMR (126 MHz, DMSO) δ 149.8, 140.5, 136.6, 131.5, 129.3, 128.4, 127.6, 127.5, 126.3, 125.3, 123.9, 123.0, 120.8, 106.7, 18. 4, 18. 3. HRMS (ESI) m/z calculated for C18H18N5S [M+H]+:
336,12774; found [M+H]+: 336,12781 N3-(tert-butyl)-2-(1H-indol-3-yl)imidazo[1,2-a]pyrazine-3,8-diamine (4j) N N N NH NH NH2
Using indole-3-carboxaldehyde, t-butyl isocyanide and purification method A; 146,2 mg, 91% product was obtained as an off-white solid. 1H NMR (500 MHz, DMSO-d
6) δ 11.20 (d, J = 2.5 Hz, 1H), 8.52 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 2.6 Hz, 1H), 7.64 (d, J = 4.7 Hz, 1H), 7.42 – 7.37 (m, 1H), 7.19 (d, J = 4.7 Hz, 1H), 7.16 – 7.09 (m, 1H), 7.09 – 7.03 (m, 1H), 6.78 (s, 2H), 4.58 (s, 1H), 1.03 (s, 9H). 13C NMR (126 MHz, DMSO-d 6) δ 149.4, 135.9, 135.9, 128.4, 126.6, 126.6, 126.4, 124.9, 124.5, 124.5, 122.2, 122.2, 121.2, 119.0, 111.2, 109.3, 108.2, 108.1, 55.7, 30.2, 30.1 ppm. HRMS (ESI) m/z calculated for C18H21N6 [M+H]+: 397,23465; found [M+H]+: 397,23444
7
N3-(2,6-dimethylphenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4k) N N N NH NH2Using glyoxylic acid, 2,6-dimethylphenyl isocyanide and purification method A; 43.8 mg, 35% product was obtained as an orange solid. 1H NMR (500 MHz, DMSO-d
6) δ 7.53 (d, J = 4.7 Hz,
1H), 7.20 (d, J = 4.6 Hz, 1H), 7.08 – 7.02 (m, 3H), 6.93 (t, J = 7.5 Hz, 1H), 6.77 (s, 2H), 6.50 (s, 1H), 2.06 (s, 6H). 13C NMR (126 MHz,
DMSO-d6) δ 150.3, 140.0, 131.5, 131.4, 128.7, 127.7, 127.4, 123.7, 118.3, 118.2, 107.1, 106.9, 18.0, 17.9. HRMS (ESI) m/z calculated for C14H16N5 [M+H]+: 254,14002; found [M+H]+: 254,13988 N3-(tert-butyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) imidazo[1,2-a]pyrazine-3,8-diamine (4l) N N N NH NH2 BPin Using 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzaldehyde, t-butyl isocyanide and purification method B; 160 mg, 78% product was obtained as a white solid. 1H NMR (500 MHz, DMSO-d 6) δ 8.20 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 4.7 Hz, 1H), 7.19 (d, J = 4.7 Hz, 1H), 6.80 (s, 2H), 4.66 (s, 1H), 1.30 (s, 12H), 0.99 (s, 9H). 13C NMR (126 MHz, CDCl 3) δ 150.1, 137.9, 136.8, 134.2, 134.0, 128.9, 127.5, 126.9, 126.8, 108.1, 108.0, 83.6, 55.9, 30.0, 24.8, 24.7. HRMS (ESI) m/z calculated for C22H31O2N5B [M+H]+: 408,25653; found [M+H]+: 408,25641
N3-(tert-butyl)-2-(naphthalen-1-yl)imidazo[1,2-a]pyrazine-3,8-diamine (4m) N N N NH NH2
Using 1-naphtaldehyde, t-butyl isocyanide and purification method B; 160 mg, 96% product was obtained as a brown solid. 1H NMR (500 MHz, DMSO-d 6) δ 8.22 (dd, J = 7.9, 1.6 Hz, 1H), 8.00 – 7.89 (m, 2H), 7.77 (dd, J = 7.1, 1.2 Hz, 2H), 7.64 – 7.53 (m, 1H), 7.50 (td, J = 8.0, 1.4 Hz, 3H), 7.20 (s, 2H), 4.40 (s, 1H), 0.73 (s, 9H).13C NMR (126 MHz, DMSO) δ 133.4, 132.2, 131.7, 128.4, 128.3, 128.1, 128.03, 128.0, 126.5, 126.0, 125.7, 125.2, 125.1, 54.9, 29.6. HRMS (ESI) m/z calculated for C20H22N5 [M+H]+: 332,18697; found [M+H]+: 332,18698
N3-pentyl-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4n) N N N NH NH2
Using benzaldehyde, 1-pentyl isocyanide, purification method A; 119 mg, 81% product was obtained as a brown solid. 1H NMR (500 MHz, Chloroform-d) δ 7.87 (d, J = 6.9 Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.40 (d, J = 4.4 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 6.00 (s, 2H), 3.25 (s, 1H), 3.04 (t, J = 7.1 Hz, 2H), 1.56 (p, J = 7.2 Hz, 2H), 1.37 – 1.28 (m, 4H), 0.87 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, Chloroform-d) δ 149.5, 135.5, 133.7, 129.6, 128.9, 128.2, 127.9, 127.2, 126.2, 108.1, 48.7, 30.5, 29.2, 22.6, 14.1. HRMS (ESI) m/z calculated for C17H22N5 [M+H]+: 296,18697; found [M+H]+: 296,18680
N3-benzyl-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4o) N N N NH NH2
Using benzaldehyde, benzyl isocyanide, purification method A; 123 mg, 78% product was obtained as a brown solid. 1H
NMR (500 MHz, Chloroform-d) δ 7.86 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 7.7 Hz, 2H), 7.35 – 7.25 (m, 7H), 7.19 (d, J = 4.6 Hz, 1H), 6.10 (s, 2H), 4.17 (d, J = 4.4 Hz, 2H), 3.60 (s, 1H). 13C NMR (126
MHz, Chloroform-d) δ 150.0, 138.8, 135.6, 133.7, 128.8, 128.8, 128.7, 128.2, 128.1, 127.8, 127.7, 127.1, 107.7, 52.5. HRMS (ESI) m/z calculated for C19H18N5 [M+H]+: 316,15567; found [M+H]+: 316,15555
(3-(8-Amino-3-(tert-butylamino)imidazo[1,2-a]pyrazin-2-yl)phenyl)boronic acid (4p) N N N NH NH2 B HO OH
Using tert-butyl (3-formylphenyl)carbamate, t-butyl isocyanide and purification method A, adapted for the polar character of the boronic acid moiety, by adding 5 minutes to the run going up to 10% MeOH in DCM; 132,2 mg, 81% product was obtained as a pale yellow solid. 1H NMR (500 MHz, DMSO-d 6) δ 8.49 (s, 1H), 8.19 – 8.13 (m, 1H), 8.04 (s, 2H), 7.74 – 7.69 (m, 1H), 7.67 (d, J = 4.8 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 4.8 Hz, 1H), 7.04 (s, 2H), 4.63 (s, 1H), 0.97 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 149.4, 138.4, 133.9, 133.8, 133.7, 133.7, 133.1, 133.0, 129.5, 128.4, 127.1, 127.0, 126.8, 125.7, 108.4, 108.2, 55.8, 30.0, 30.0, 30.0 ppm. HRMS (ESI) m/z calculated for C16H21BN5O2 [M+H]+: 326,17828; found [M+H]+: 326,17838
7
N3-(tert-butyl)-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4q) N N N NH NH2Using benzaldehyde, t-butyl isocyanide, purification method B; 84 mg, 60% product was obtained as an off-white solid. 1H
NMR (500 MHz, DMSO-d6) δ 8.16 – 8.11 (m, 2H), 7.63 (d, J = 4.7 Hz, 1H), 7.40 (t, J = 7.7 Hz, 2H), 7.32 – 7.24 (m, 1H), 7.19 (d, J = 4.7 Hz, 1H), 6.76 (s, 2H), 4.62 (s, 1H), 0.98 (s, 9H). 13C NMR
(126 MHz, DMSO-d6) δ 150.0, 137.4, 135.0, 128.7, 127.9, 127.6, 127.4, 127.0, 126.2, 108.0, 55.7, 30.0. HRMS (ESI) m/z calculated for C16H20N5 [M+H]+: 282,17132; found [M+H]+: 282,17093 Methyl (8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)glycinate (4r) N N N NH NH2
O O Using benzaldehyde, methyl 2-isocyanoacetate and purification method B; 90 mg, 60% product was obtained as a grey solid. 1H
NMR (500 MHz, DMSO-d6) δ 8.05 (d, J = 7.1 Hz, 2H), 7.71 (d, J = 4.7 Hz, 1H), 7.44 (t, J = 7.7 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 7.19 (d, J = 4.7 Hz, 1H), 6.75 (s, 2H), 5.50 (t, J = 6.4 Hz, 1H), 3.78 (d, J = 6.4 Hz, 2H), 3.52 (s, 3H). 13C NMR (126 MHz, DMSO) δ 171.9,
150.0, 134.1, 131.9, 128.4, 127.8, 127.2, 126.9, 126.4, 107.8, 51.6, 48.2. HRMS (ESI) m/z calculated for C15H16N5O2 [M+H]+: 298,12985; found [M+H]+:
298,12955 tert-butyl (3-(8-amino-3-(tert-butylamino)imidazo[1,2-a]pyrazin-2-yl)phenyl) carbamate (4s) N N N NH NH2 NHBoc
Using tert-butyl (3-formylphenyl)carbamate, t-butyl isocyanide and purification method B; 180 mg, 90% product was obtained as a brown solid.1H NMR (500
MHz, Chloroform-d) δ 7.94 (b, 1H), 7.56 (b, 1H), 7.52 (d,
J = 7.0 Hz, 1H), 7.36 – 7.30 (m, 2H), 6.66 (b, 1H), 6.06 (s,
1H), 3.15 (b, 3H), 1.53 (s, 9H), 1.04 (s, 9H).13C NMR (126
MHz, CDCl3) δ 152.8, 149.5, 139.4, 138.7, 135.0, 129.0, 126.8, 125.9, 122.5, 118.1, 117.7, 108.9, 80.5, 56.6, 30.2, 28.3.
HRMS (ESI) m/z calculated for C21H29O2N6 [M+H]+: 397,23465; found [M+H]+:
2-(4-Chlorophenyl)-N3 -(2,6-dimethylphenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4t) N N N NH NH2 Cl
Using 4-chlorobenzaldehyde, 2,6-Dimethylphenyl isocyanide, purification method A; 1mmol scale, 221mg, 61% product was obtained as solid. 1H NMR (500 MHz,
DMSO-d6) δ 7.99 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.6 Hz, 2H), 7.25 (s, 1H), 7.14 (d, J = 4.6 Hz, 1H), 6.96 (d, J = 4.7 Hz, 1H), 6.94 – 6.88 (m, 4H), 6.71 (t, J = 7.4 Hz, 1H), 1.88 (s, 6H). 13C
NMR (126 MHz, DMSO-d6) δ 150.2, 140.5, 133.5, 132.5, 131.6, 129.3, 128.4, 128.3, 128.2, 127.8, 126.4, 124.7, 120.9, 106.7, 18.5, 18.3. HRMS (ESI) m/z calculated for C20H19ClN5[ M+H]+: 364,13235; found [M+H]+: 364,13220
N3 -(2,6-dimethylphenyl)-2-(4-methoxyphenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4u) N N N NH NH2 OMe
Using 4-methoxybenzaldehyde, 2,6-dimethylphenyl isocyanide, purification method A; 1mmol scale 270mg, 75 % product was obtained as solid. 1H NMR (500 MHz,
Chloroform-d) δ 7.99 (d, J = 8.9 Hz, 2H), 7.15 (d, J = 4.7 Hz, 1H), 6.99 (d, J = 7.5 Hz, 2H), 6.94 – 6.88 (m, 3H), 6.83 (t, J = 7.5 Hz, 1H), 5.71 (s, 2H), 3.82 (s, 3H), 3.48 (s, 1H), 2.02 (s, 6H). 13C NMR (126 MHz, Chloroform-d) δ 159.4, 149.5, 140.0, 136.9, 129.9, 128.4,
128.2, 127.9, 126.3, 126.0, 123.0, 121.9, 114.1, 108.1, 55.4, 18.6. HRMS (ESI) m/z calculated for C21H22N5O [M+H]+: 360,18189; found [M+H]+: 360,18146
N3-(benzo[d][1,3]dioxol-5-ylmethyl)-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4v) N N N NH NH2 O O
Using benzaldehyde, piperonyl isocyanide, purification method A; 130 mg, 72.2% product was obtained as a yellow solid. 1H NMR (500 MHz, Chloroform-d) δ 7.82 (d, J = 7.1 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.35 – 7.27 (m, 2H), 7.20 (d, J = 4.8 Hz, 1H), 6.74 (s, 1H), 6.67 (t, J = 6.4 Hz, 2H), 6.08 (s, 2H), 5.90 (s, 2H), 4.04 (s, 2H), 3.57 (s, 1H), 3.44 (s, 1H). 13C NMR (126 MHz, Chloroform-d) δ 149.9, 147.9, 147.1, 135.7, 133.7, 132.6, 128.8, 128.1, 127.7, 127.5, 127.1, 121.5, 108.6, 108.3, 107.7, 101.1, 52.3. HRMS (ESI) m/z calculated for C20H18N5O2 [M+H]+: 360,14550; found [M+H]+:
7
Exemplary 1H-NMR and 13C-NMR of typical GBB-3CR products.
2-Benzyl-N3-(tert-butyl)imidazo[1,2-a]pyrazine-3,8-diamine (4f) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 f1 (ppm) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 8.74 0.66 2.00 2.20 6.18 1.03 1.16 2.56 4.17 5.74 7.18 7.19 7.20 7.20 7.21 7.21 7.21 7.22 7.22 7.23 7.24 7.25 7.26 7.26 CDCl3 7.27 7.27 7.28 7.28 7.28 7.29 7.29 7.29 7.49 7.50 N N N NH2 NH C H3 CH3 CH3 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 f1 (ppm) -5000 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 55000 60000 65000 70000 75000 80000 85000 90000 30.57 34.04 55.89 76.91 CDCl3 77.16 CDCl3 77.41 CDCl3 109.16 126.37 127.08 127.22 128.63 128.71 128.71 129.18 139.34 139.42 149.44 N N N NH2 NH C H3 CH3 CH3
N3 -(tert-butyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4l) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 f1 (ppm) -200 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 A (s) 4.66 B (s) 1.30 C (s) 0.99 D (d) 8.20 E (d) 7.70 F (d) 7.63 G (d) 7.19 H (s) 6.80 9.10 12.34 1.00 1.91 0.99 0.96 2.04 1.98 0.99 1.30 2.49 2.49 2.50 2.50 2.50 3.33 4.66 6.80 7.18 7.19 7.63 7.64 7.69 7.71 8.19 8.21 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 f1 (ppm) 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 24.71 24.79 30.04 30.07 39.02 39.19 39.35 39.44 39.52 39.61 39.69 39.78 39.85 39.94 40.02 40.11 55.85 83.58 108.03 108.11 126.80 126.85 127.52 128.87 134.03 134.15 136.82 137.92 150.11
7
Single Crystal X-Ray Structure Determination
Crystallographic data were collected on the«Xcalibur-3» diffractometer (graphite monochromated MoKα radiation, CCD detector, ω-scanning, 2Θmax = 60°). The structure was solved by direct method using SHELXTL package.16 Positions of the hydrogen atoms were located from electron density
difference maps and refined by “riding” model with Uiso = nUeq (n= 1.5 for methyl groups and n=1.2 for other hydrogen atoms) of the carrier atom.
Figure 4. Molecular structure of compound 4e according to X-ray diffraction data.
Thermal ellipsoids are shown at the 50 % probability level.
The colourless crystals of 4e (C17H21N5O) are triclinic. At 293 K a = 6.0032(5), b = 12.429(2), c = 12.840(2) Å, α = 118.34(2)°, β = 90.432(9)°, γ = 103.477(9)°, V = 812.2(2) Ǻ3, M
r = 311.39, Z = 2, space group P
1
, dcalc= 1.273 g/сm3, µm(MoKαa)= 0.084 mm-1, F(000) = 332. Intensities of 8230 reflections (4732 independent,
Rint=0.068) Full-matrix least-squares refinement against F2 in anisotropic
approximation for non-hydrogen atoms using 4585 reflections was converged to wR2 = 0.154 (R1 = 0.068 for 1638 reflections with F>4σ(F), S = 0.813).
Figure 5. Molecular structure of compound 4a according to X-ray diffraction data. Thermal
ellipsoids are shown at the 50 % probability level.
The colourless crystals of 4a (C20H19N5) are monoclinic. At 293 K a = 20.174(3), b = 5.9755(8), c = 29.439(4) Å, β = 104.96(1)°, V = 3428.7(8) Ǻ3, M
r = 329.40, Z
= 8, space group C2/c, dcalc= 1.276 g/сm3, µm(MoKα
a) = 0.079 mm-1, F(000)
= 1392. Intensities of 11965 reflections (3010 independent, Rint=0.112) were measured on the«Xcalibur-3» diffractometer (graphite monochromated MoKα radiation, CCD detector, ω-scanning, 2Θmax = 50°). The structure was solved by direct method using SHELXTL package. Positions of the hydrogen atoms were located from electron density difference maps and refined by “riding” model with Uiso = nUeq (n= 1.5 for methyl group and n=1.2 for other hydrogen atoms) of the carrier atom. Full-matrix least-squares refinement against F2 in
anisotropic approximation for non-hydrogen atoms using 2995 reflections was converged to wR2 = 0.141 (R1 = 0.066 for 1453 reflections with F>4σ(F), S = 0.943).
7
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