Two oxetanocin A derivatives have been synthesized stereospecifically. The crystal structures of both derivatives and a synthetic intermediate are reported on herein. 10H9ClN4O, 3-[(6-chloro-7H-purin-7-yl)methyl]cyclobutan-1-one (I), and two N-7 and N-9 regioisomeric oxetanocin nucleoside analogs, C10H13ClN4O, 3-[methyl]cyclobutan-1-ol (II) and C10H11ClN4O, 3-[(6-chloro-9H-purin-9-yl)methyl]cyclobutan-1-ol (IV), are reported. The crystal structures of the nucleoside analogs confirmed the reduction of the N-7- and N-9-substituted cyclobutanones with LiAl(OtBu)3 to occur with facial selectivity, yielding cis-nucleosides analogs similar to those found in nature. Reduction of the purine ring of the N-7 cyclobutanone to a dihydropurine was observed for compound (II) but not for the purine ring of the N-9 cyclobutanone on formation of compound (IV). In the crystal of (I), molecules are linked by a weak Cl⋯O interaction, forming a 21 helix along [010]. The helices are linked by offset π–π interactions [intercentroid distance = 3.498 (1) Å], forming layers parallel to (101). In the crystal of (II), molecules are linked by pairs of O—H⋯N hydrogen bonds, forming inversion dimers with an R22(8) ring motif. The dimers are linked by O—H⋯N hydrogen bonds, forming chains along [001], which in turn are linked by C—H⋯π and offset π–π interactions [intercentroid distance = 3.509 (1) Å], forming slabs parallel to the ac plane. In the crystal of (IV), molecules are linked by O—H⋯N hydrogen bonds, forming chains along [101]. The chains are linked by C—H⋯N and C—H⋯O hydrogen bonds and C—H⋯π and offset π–π interactions [intercentroid distance = 3.364 (1) Å], forming a supramolecular framework.The crystal structures of an intermediate, C The mean plane of the cyclobutane ring (A = C2′–C5′) is inclined to the mean plane of the purine ring system (B = N1/N37N7/N9/C2/C4/C5/C6/C8) by 52.62 (11)°, while the torsion angle N7—C5′—C4′⋯C2′ is ca 125.4°.In compound (I)tert-butoxyaluminum hydride lead to the formation of the oxetanocin derivative compound (II)A) is inclined to the mean plane of the purine ring system (B) by 26.37 (15)°, while the torsion angle N7—C5′—C4′⋯C2′ is ca 120.0°. Atoms C6′ and C4′ are positionally disordered and were split giving a refined occupancy ratio for C6′:C6′B and C4′:C4′B of 0.858 (4):0.142 (4) 4) Fig. 2.trans positioning of the cyclobutanol unit, there are no intramolecular hydrogen bonds between the chlorine atom and the cyclobutanol or methylene connector as observed in compounds (I)A) is inclined to the mean plane of the purine ring system (B) by 71.20 (13)°, and the torsion angle N7—C5′—C4′⋯C2′ is ca 144.8°.In compound (IV)4 to a dihydropurine . This strained orientation is not observed for the N-9 ketone, hence the integrity of its purine ring is preserved.Reduction of the purine ring of the N-7 cyclobutanone to a dihydropurine was observed for compound (II)x + 1, y − z + 1 helix along [010], see Fig. 4CgB⋯CgBi = 3.498 (1) Å, CgB is the centroid of the purine ring system, α = 0.00 (5) Å, β = 21.6°, interplanar distance = 3.252 (1) Å, offset = 1.289 Å, symmetry code (i) −x + 2, −y + 1, −z + 1.In the crystal of (I)ac plane. In the crystal of (II)f Table 2. The dimf Table 2 and offsCgB⋯CgBvi = 3.534 (1) Å, CgB is the centroid of the purine ring system, α = 0.02 (10) Å, β = 17.8°, interplanar distance = 3.364 (1) Å, offset = 1.08 Å, symmetry code (vi) −x + 1, −y + 1, −z. ]In the crystal of (IV)et al., 2016viz. 6-chloro-9-(3-hydroxymethyl-3-hydroxycyclobutyl)purine cyclobutanol  Potassium carbonate (12.0 mmol) was added to a solution of (3-oxocyclobutyl)methyl benzoate (10.0 mmol) in methanol (20 ml) and stirred for 1 h at room temperature. Saturated sodium bicarbonate (10.0 ml) was added and stirring continued for an additional 15 min. The solvent was evaporated under vacuum, followed by purification by flash column chromatography with ethyl acetate, resulting in 3-(hydroxymethyl)cyclobutan-1-one in 70% yield.3-(Hydroxymethyl)cyclobutan-1-one (1 mmol) was dissolved in 10 ml of dry dichloromethane and cooled to 195 K. Hunig’s base (3.2 mmol) was added, followed by trifluoromethanesulfonic anhydride (1 mmol) and the mixture was stirred for 10 min, cooled to 273 K and stirred to obtain the qualitative conversion to (3-oxocyclobutyl)methyl trifluoromethanesulfonate.H-purine (5.61 mmol), potassium hydroxide (5.61 mmol), tris[2-(2-methoxyethoxy)ethyl]amine (0.28 mmol), magnesium sulfate (2 g) and anhydrous acetonitrile (100 ml), which was then heated to 333 K for 5 h and cooled to room temperature. The product was purified using 5% methanol and 5% trimethylamine in chloroform, which yielded two UV-active compounds.The (3-oxocyclobutyl)methyl trifluoromethanesulfonate (5.61 mmol) was added to a mixture containing 6-chloro-7H-purin-9-yl)methyl]cyclobutan-1-one (III) and 37% of the N-7 alkylated derivative 3-[(6-chloro-7H-purin-7-yl)methyl]cyclobutan-1-one (I)The two UV-active compounds were separated using flash column chromatography with ethyl acetate, giving 51% of the N-9 alkylated derivative; 3-[methyl]cyclobutan-1-ol (II) 3-[(6-Chloro-7H-purin-7-yl)methyl]cyclobutan-1-one (I)tert-butoxyaluminum hydride was added. The mixture was cooled to room temperature and sodium borohydride (0.32 mmol) was added and the resulting mixture allowed to stir overnight. Methanol (2 ml) was added and the mixture allowed to stir overnight to convert the over-reduced 3-[(6-chloro-7H-purin-7-yl]methyl)cyclobutanone(I) to 3-[methyl]cyclobutan-1-ol (II)Synthesis ofcis-3-[(6-chloro-9H-purin-9-yl)methyl]cyclobutan-1-ol (IV) 3-[(6-Chloro-9H-purin-9-yl)methyl]cyclobutan-1-one (III) (0.21 mmol) was added to diethyl ether and cooled to 195 K and lithium tri-tert-butoxyaluminum hydride (0.32 mmol) was added. The reaction was allowed to warm to room temperature and left to stir overnight, which provided quantitative conversion to cis-3-[(6-chloro-9H-purin-9-yl)methyl]cyclobutan-1-ol (IV)Pale-yellow plate-like crystals of (I)Uiso(H) = 1.2Ueq(C). For compound (II)B and C4′:C4′B of 0.858 (4):0.142 (4). For the final refinement of compound (II)Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989019004432/zp2033sup1.cifCrystal structure: contains datablock(s) global, I, II, IV. DOI: 10.1107/S2056989019004432/zp2033Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019004432/zp2033IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989019004432/zp2033IIsup5.cmlSupporting information file. DOI: 1907200, 1907199, 1907198CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E. In the crystal, O—H⋯O hydrogen-bond interactions consolidate the crystal packing. A Hirshfeld surface analysis and fingerprint plots were used to further investigate the intermolecular interactions in the solid state.In the title Schiff base derivative carrying a 2-bromo-3-methylphenyl group, the conformation about the C=N bond is  14H12BrNO2, was synthesized by the condensation reaction of 2,3-dihydroxybenzaldehyde and 2-bromo-3-methylaniline. It crystallizes in the centrosymmetric triclinic space group PE. The dihedral angle between the planes of the 5-(2-bromo-3-methylphenyl ring and the catechol ring is 2.80 (17)°. In the crystal, O—H⋯O hydrogen-bond interactions consolidate the crystal packing.The title compound, C Schiff PZ = 4 (Z′ = 2). The two crystallographically independent molecules have nearly the same geometrical parameters and the primary difference between them is the rotational orientation of H2 and H4A. The discussion will therefore be limited to that of the molecule containing O1. The molecular structure is constructed from two individually planar rings. The whole molecule is approximately planar, with a maximum deviation of 0.117 (3) Å from planarity for the hydroxyl O1 atom of the catechol ring. The dihedral angle between the two benzene ring planes is 2.80 (17)°. The methyl C1 atom deviates from the plane of the C2–C7 benzene ring by 0.039 (2) Å while C9 deviates from the plane of the C9–C14 benzene ring by 0.024 (3) Å. The C8—N1—C7—C6 and C14— C9—C8—N1 torsion angles are −1.6 (5) and −1.1 (5)°, respectively. The planar molecular conformation of each molecule is stabilized by an intramolecular O—H⋯N hydrogen bond  and Cg3⋯Cg4, where Cg1, Cg2, Cg3 and Cg4 are the centroids of the C2–C7, C9–C14, C16–C21 and C23–C28 rings, respectively.In the crystal, the Schiff base units are linked by O—H⋯O and C—H⋯O hydrogen bonds -N-(2-bromophenyl)-1-phenylmethanimine skeleton yielded nine hits. The N1—C8 bond in the title structure is the same length within standard uncertainties as those in the structures of 2-bromo-N-salicylideneaniline -1-(2-fluorophenyl)methanimine -methyl]-4-bromophenol naphthaldimine -2-bromoaniline  plot with de and di for the title compound is shown in Fig. 3de = di = 1.5 Å; Fig. 4Hirshfeld surface analysis of the title compound was performed utilizing the A mixture of 2,3-dihydroxybenzaldehyde  and 2-bromo-3-methylaniline  was stirred with ethanol (30 mL) at 377 K for 5 h, affording the title compound . Single crystals suitable for X-ray measurements were obtained by recrystallization from ethanol at room temperature.Uiso(H) = 1.5Ueq(O). The C-bound H atoms were positioned geometrically and refined using a riding model: C—H = 0.93 Å with Uiso(H) = 1.2Ueq(C) for aromatic H atoms and C—H = 0.96 Å with Uiso(H) = 1.5Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019015718/mw2151sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019015718/mw2151Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019015718/mw2151Isup3.cmlSupporting information file. DOI: 1967023CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The asymmetric unit of the title solvate comprises a half molecule of each component as both species are disposed about a centre of inversion. In the crystal, two-dimensional arrays are formed by amide-N—H⋯N(pyridyl) hydrogen bonds, which are connected into a three-dimensional architecture by C—H⋯π(benzene and pyridyl) interactions with benzene acting as the acceptor and donor, respectively. 14H14N4O2·C6H6 , comprises a half molecule of each constituent as each is disposed about a centre of inversion. In the oxalamide molecule, the central C2N2O2 atoms are planar (r.m.s. deviation = 0.0006 Å). An intramolecular amide-N—H⋯O(amide) hydrogen bond is evident, which gives rise to an S(5) loop. Overall, the molecule adopts an antiperiplanar disposition of the pyridyl rings, and an orthogonal relationship is evident between the central plane and each terminal pyridyl ring [dihedral angle = 86.89 (3)°]. In the crystal, supramolecular layers parallel to (10via methylene-C—H⋯π(benzene) and benzene-C—H⋯π(pyridyl) interactions. The specified contacts are indicated in an analysis of the calculated Hirshfeld surfaces. The energy of stabilization provided by the conventional hydrogen bonding  is just over double that by the C—H⋯π contacts (dispersion forces).The asymmetric unit of the title 1:1 solvate, C The first of these arises from amide-N—H⋯O(amide) hydrogen bonding between the amide groups, on both sides of the 3LH2 molecule, through ten-membered amide synthons {⋯HNC2O}2 . Molecules of nLH2 also featured prominently in early, systematic studies of halogen bonding. An illustrative example is found in the 1:1 co-crystal formed between 3LH2 and 1,4-di-iodobuta-1,3-diyne, I—C≡C—C≡C—C—I  hydrogen bonding and these are linked into a two-dimensional array via amide-N–H⋯N(pyridyl) hydrogen bonds  hydrogen bonds to generate a two-dimensional array. In this context, and in the context of recent work on 4LH2 in co-crystals via an analysis of the calculated Hirshfeld surfaces as well as a computational chemistry study.With a combination of centrally located amide and terminal pyridyl functional groups, the isomeric molecules related to the title compound of the general formula  Å to either side of the plane. An intramolecular amide-N—H⋯O(amide)i hydrogen bond, occurring between the symmetry related amide groups, gives rise to an S(5) loop, Table 1x, 1 − y, − z. The crystallographic symmetry also implies an antiperiplanar disposition of the pyridyl rings. The dihedral angle between the central plane and terminal pyridyl ring is 86.89 (3)°, indicating an orthogonal relationship.The title co-crystal (I)a), with dimensions defined by O10⋯O10 and N8⋯N8 separations of 9.6770 (11) and 12.3255 (11) Å, respectively. The other notable contacts in the crystal are of the type C—H⋯π, Table 1i.e. two (as acceptor) and two (as donor), such interactions, Fig. 2b). The side-on view of Fig. 2b) shown in Fig. 2c) indicates the amide-N—H and pyridyl-N project in all directions around the five-molecule aggregate. Indeed, it is the C—H⋯π interactions that connect the layers into a three-dimensional architecture, Fig. 2d).The geometric parameters characterizing the interatomic contacts identified in the crystal of (I)Mercury dnorm map of 4LH2 displays several red spots, that range from intense to weak, which reflect the interactions identified in the crystal  interactions, Fig. 4a), with both indicative of contact distances shorter than the respective sum of the van der Waals radii. Reflecting the relatively long separation, the benzene-C11—H11⋯π(pyridyl) interaction is reflected as only a white spot as the contact distance is only just within the sum of van der Waals radii, as shown in Fig. 4b).The Hirshfeld surface mapped over B⋯π(benzene) contact is indeed electrostatic in nature as revealed by the distinct blue (i.e. electropositive) and red (i.e. electronegative) colour scheme on the surface of the contact points, Fig. 5a). In contrast, the benzene-C11—H11⋯π(pyridyl) contact displays pale colouration around the contact zone suggesting that the interaction could be attributed to weak dispersion forces, Fig. 5b).The C—H⋯π interactions were subjected to electrostatic potential mapping for verification purposes. The result shows that the methylene-C7—H7a)–(e). As shown in the overall fingerprint plot in Fig. 6a), (I)4LH2 molecule, therefore indicating that the intermolecular interactions in (I)4LH2 molecules. Decomposition of the overall fingerprint plots of (I)di + de ∼2.42 Å), H⋯C/C⋯H , H⋯O/O⋯H , H⋯N/N⋯H  and other contacts (0.8%). Except for the H⋯H contacts, to differing extents, the remaining major contacts are shorter than the corresponding sum of van der Waals radii for H⋯C (∼2.90 Å), H⋯O (∼2.72 Å) and H⋯N (∼2.75 Å).The two-dimensional fingerprint plots were generated for overall (I)4LH2 molecule exhibits at similar distribution of the major contacts compared to overall (I)4LH2 these are either inclined towards the external or internal contacts presumably due to interaction with the solvent benzene molecule. For instance, the H⋯C/C⋯H contact in the individual 4LH2 molecule comprises 9.9% -H⋯C- and 14.6% -C⋯H- contacts as compared to 12.0 and 14.6% for the equivalent contacts in overall (I)c). Similar observations pertain for the H⋯O/ O⋯H and H⋯N/ N⋯H interactions, Fig. 6d)–(e).The individual i.e. 20.5% for -H⋯C- and 21.4% for -C⋯H- contacts. In addition, the solvent molecules are sustained in the molecular architecture through minor contributions from H⋯O (5.6%) and H⋯N (5.9%) contacts, respectively. These interactions are at distances of ∼2.52 Å (H⋯H), ∼2.92 Å (H⋯C/C⋯H), ∼2.98 Å (H⋯O) and ∼2.79 Å (H⋯N), which are greater than the corresponding sum of van der Waals radii, indicating the identified C—H⋯π(benzene and pyridyl) interactions can largely be considered as localized interactions.As for the benzene molecule, an irregular fingerprint profile is noted with the distribution dominated by H⋯H (46.4%) and H⋯C/ C⋯H (41.9%) surface contacts. The latter are almost equally distributed between the internal and external contacts, Crystal Explorer 17 based on the procedures as described previously  of −38.1 kJ mol−1, Table 2B⋯π(benzene) and benzene-C11—H11⋯π(pyridyl) contacts with a very similar Eint values of −18.9 and −16.9 kJ mol−1, respectively, despite the dnorm contact distance being significantly greater for the latter. The calculation results reveal that the repulsion energy is greater in methylene-C7—H7B⋯π(benzene) compared with the benzene-C11—H11⋯π(pyridyl) contact, which contributes to the slight variation in their Eint values. In short, the N—H⋯N interaction is stabilized largely by electrostatic forces while the C—H⋯π interactions are stabilized largely by dispersion forces. Overall, the crystal of (I)a)–(c).The calculation of interaction energy was performed using 4LH2 available in the literature ; molecular inversions were enabled during calculation. The result shows that out of the 20 molecules in the cluster, only one 4LH2 molecule in each polymorph resembled the reference packing in (I)a) and (b). The result clearly demonstrates the influence of solvent molecule upon the molecular packing in (I)Calculations were also performed to compare the molecular packing similarity of (I)4LH2 exhibit a close similarity in the distribution of molecular contacts as judged from the percentage contribution of the corresponding contacts on the Hirshfeld surface. The maximum variation in the distribution of H⋯H, H⋯C/C⋯H, H⋯O/O⋯H and H⋯N/N⋯H contacts ranged from 7.1, 4.9, 2.2 and 3.8%, respectively among the three crystals.Finally, and referring to Fig. 9Chemical Context, there are two polymorphs available for 4LH2 CH2N(H)C(=S)C(=S)N(H)CH2(C5H4N-n), for n = 2, 3 and 4 oxalamide, was prepared in accordance with the literature procedure −1): 3322 ν(N—H), 3141–2804 ν(C—H), 1696–1661 ν(C=O), 1563–1515 ν(C=C), 1414 ν(C—N), 794 δ(C=C).The precursor, Uiso(H) set to 1.2Ueq(C). The nitrogen-bound H atom was located from difference-Fourier maps and refined with N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989019009551/hb7835sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019009551/hb7835Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019009551/hb7835Isup3.cmlSupporting information file. DOI: 1938031CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mature RNA (miRNeasy Mini kit) was reverse transcribed using RT2 First strand cDNA synthesis kit. The cDNA was used on the real‐time RT2 Profiler PCR Array  in combination with RT2 SYBR® Green qPCR Mastermix (Roche LightCycler® 480 Instrument). Threshold cycle (CT) values (excel file) were uploaded onto the data analysis centre web portal (http://www.qiagen.com/geneglobe). CT values were normalized based on a Manual Selection of reference genes. The fold change/regulation (2^(‐ΔΔCT)) was calculated using ΔΔCT method .The isolated hVFs were grouped into fibroblasts‐less differentiated (HF‐LD) and fibroblasts‐highly differentiated (HF‐HD) based on their α‐SMA expression (immunoblot), compared to the control hVFs Figure A,B. Poly2.2TFAP2A—F:5′‐GACCTCTCGATCCACTCCTTAC‐3′ R: 5′‐GAGACGGCATTGCTGTTGGACT‐3′; β‐2‐microglobulin (B2M)‐ F: 5′‐CCACTGAAAAAGATGAGTATGCCT‐3′ and R: 5′‐CCAATCCAAATGCGGCATCTTCA‐3′. The following PrimeTime qPCR mouse primer assays were used: α‐SMA (Mm.PT.58.16320644); COL1A1 (Mm.PT.58.7562513); COL2A1 (Mm.PT.58.5206680); COL3A1 (Mm.PT.58.13848686), TGFBR1 (Mm.PT.58.28402453), TGFBR2 (Mm.PT.58.6358355) and B2M (Mm.PT.39a.22214835). The cycling conditions were 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 s, 1 minute at 60°C, and 72°C for 40 s. Melt curve analysis was performed by an additional dissociation step of 1 cycle at 95°C for 5 s followed by 65°C for 1 min and ramping data collection to 97°C. Relative expression values (ΔCt) were obtained by normalizing Ct values (Roche Lightcycler 480 Software v1.5.1.62) of the tested genes with that of B2M.Total RNA was isolated from hVFs (miRNeasy Mini kit) and reverse transcribed (miScript RT II kit) with the supplied HiFlex buffer. qPCR was performed on the LightCycler 480 Instrument II, using the Power SYBR Green PCR Master Mix and 10 ng diluted cDNA per well. The following human primers were used: 2.3Creative Biogene, Shirley, NY.The TFAP2A knockout cell line with NIH/3T3 fibroblasts (TFAP2A‐KO) was established using CRISPR/CAS9 technology through 2.42 with DMEM media (10% BCS) and incubated at 37°C under 5%CO2. Following 24 hours, hVFs were either treated with TGF‐β1 (5 ng/mL), angiotensin II (100 nM) or kept as control in DMEM media (2.5% BCS). After 48‐72 hours, the fibroblasts/myofibroblasts were rinsed with Dulbecco's PBS and assayed.Fibroblasts from wild‐type or TFAP2A‐KO groups were plated at 4000 cells/cm2.5Standard western protocols were followed2.6t − t0]/{[log(Nt)‐log(N0)]/log(2)}, where t0 refers time , t represents time (second count), N0 refers count at time t0, and Nt represents count at time t.Both WT and KO fibroblasts were plated as stated before in triplicate (per time‐point) in 6‐well plates and counted by Cellometer Auto 2000  at 24, 48, and 72 hours post‐plating. Doubling time was calculated by [3TFAP2A expression, along with ELK1, was decreased with decrease in differentiation as visualized in the heat map   1A1  is increased in TFAP2A‐KO fibroblasts  compared to the wild‐type  and highly differentiated (HF‐HD) based on their α‐SMA expression, compared to control hVFs as shown in Figure There is no conflict of interest.GRR initiated, designed, executed, analysed the study and wrote the manuscript; SE executed the real‐time PCR and PCR array; CW and PH implemented the cell culture, immunoblotting and proliferation assays; FXD, LE, FR and AJ interpreted data and proof‐read the manuscript. Click here for additional data file.