C-reactive protein (CRP) is a commonly used serum biomarker for detecting sepsis in neonates. After the onset of sepsis, serial measurements are necessary to monitor disease progression; therefore, a non-invasive detection method is beneficial for neonatal well-being. While some studies have shown a correlation between serum and salivary CRP levels in septic neonates, the causal link behind this correlation remains unclear. To investigate this relationship, CRP was examined in serum and saliva samples from 18 septic neonates and compared with saliva samples from 22 healthy neonates. While the measured blood and saliva concentrations of the septic neonates varied individually, a correlation of CRP levels between serum and saliva samples was observed over time. To clarify the presence of active transport of CRP across the blood–salivary barrier (BSB), transport studies were performed with CRP using in vitro models of oral mucosa and submandibular salivary gland epithelium. The results showed enhanced transport toward saliva in both models, supporting the clinical relevance for salivary CRP as a biomarker. Furthermore, CRP regulated the expression of the receptor for advanced glycation end products (RAGE) and the addition of soluble RAGE during the transport studies indicated a RAGE-dependent transport process for CRP from blood to saliva. Neonatal sepsis and other neonatal infections account for 0.88% of all disability adjusted life years worldwide, demonstrating similar percentages compared with motor vehicle road injuries 0.98%) and asthma 0.91%) [% [1]. Ne8% and asFor the detection of neonatal sepsis CRP measurement has a sensitivity of 74–98% and a specificity of 71–94% performed at least 12 h after the onset of symptoms . Serial Recent developments in salivary biomarker analysis favor the usage of saliva as a non-invasively collected diagnostic fluid. As the serum/salivary ratio of biomarker concentrations varies between biomarkers , sensitiWhile previous clinical studies with neonates indicated a positive correlation of salivary CRP with serum CRP concentrations ,17, a thSince local inflammation of the oral mucosa may contribute to elevated CRP levels in saliva ,24, diffThis prospective observational part was conducted at the Division of Neonatology, Pediatric Intensive Care and Neuropediatrics at the Medical University Vienna/General Hospital Vienna, a tertiary care academic center, consisting of two separated neonatal intensive care units , two separated neonatal intermediate care units , and 38 beds on the maternity ward with rooming-in care. This part of the study was approved by the Ethics Committee of the Medical University of Vienna  and was performed in two phases. Phase one focused on determining the salivary CRP values in healthy neonates. This was limited to randomly selected neonates born at ≥33 weeks of gestational age and a birthweight of ≥1500 g admitted to the maternity ward without chromosomal aberrations, congenital malformations or inborn metabolic disorders upon obtaining parental informed consent. Saliva sampling was performed at earliest 12 h after birth and earliest 30 min after feeding in clinically healthy neonates. Phase two consisted of evaluation of a correlation between CRP values in saliva and blood. Neonates between 23 and 42 weeks of gestational age admitted to the NICUs or NIMCUs with serum CRP measurements were randomly selected and parental informed consent was obtained. Saliva sampling was performed at the earliest timepoint within 10 h of serum CRP determination and at a minimum of 30 min after feeding without any residues of food present. This time point was chosen based on the time needed for significant changes in CRP values after onset of sepsis of more than 10–12 h and to allow for ideal implementation in the care of critically ill preterm and term infants .In all patients, saliva was collected using the PediaSAL device  especially developed for preterm and term infants. This device is designed as “pacifier” with a passive collecting system based on a sponge. This allows to obtain saliva samples of neonates enabling a non-invasive painless sampling with minimal stress. Due to different age and bodyweight of the included neonates, the sponge was used for collection to allow for optimum patient-oriented sampling.Saliva sampling was most efficient when performed 60–120 min after feeding. To maximize the amount of collected saliva, the following technique proved to be useful: The neonate’s head was placed to the left or right, independent of supine or prone position. A pacifier of according size was offered for 5 min prior to sampling. An increase in the saliva production was achieved by gently stroking the lips with the pacifier or the device. After at least five minutes with the pacifier, the sampling device was placed in the downward cheek of the neonate, where the produced saliva collected. If the amount of saliva was not sufficient, swapping to the pacifier for another 2 min and then back to the sampling device helped. This was sometimes repeated several times. After sampling, the samples were continuously cooled. Saliva was stored within 15 min at −20 °C and subsequently 2 h at −80 °C until transport to the laboratory. Transportation was performed under continuous cooling at −78 °C using dry ice.® 250/350 and VITROS® 5600 System  or using the Cobas 6000 Chemistry Analyzer .Determination of CRP concentration in serum was performed by the Department of Laboratory Medicine of the Medical University Vienna, Vienna, Austria using the VITROS® Systems was performed with VITROS® CRP slides  and VITROS® Chemistry Products Calibrator Kit 7 . For detection based on an HRP conjugated monoclonal anti-CRP antibody a drop of sample was applied on the slides and measured at wavelength 670 nm upon preparation according to the manufacturer’s instructions. Measurement was performed at a concentration range of 5–90 µg/mL with a limit of detection determined as 2.72 µg/mL by the manufacturer.Analysis of CRP using the VITROSFor detection of serum CRP concentrations with Cobas 6000 Chemistry Analyzer the CRP-hs reagent kit  was used according to the manufacturer’s instruction. Serum was separated from the cells within one hour of collection and stored at −70 °C until measurement. Samples were thawed at room temperature, homogenized, and centrifuged at 2000 rcf for 10 min prior to applying 100 µL for measurement using the c501 module based on a photometric and ion-selective electrode determination. For quality controls an internal pooled serum sample and Precipath U Plus Control  was used. Detection limit was determined as 0.15 µg/mL for an analytical measurement range up to 20 µg/mL according to the manufacturer.CRP concentration in saliva was determined using ELISAs by the Molecular Diagnostic Unit at AIT, the Austrian Institute of Technology, Vienna, Austria, and is described in detail in ®, 690175) in Dulbecco’s Modified Eagle Medium , supplemented with 10% Fetal Calf Serum  and 1% Penicillin/Streptomycin  at 37 °C, 5% CO2, 95% air atmosphere, and 95% humidity. Media change was performed every 2–3 days and cells were propagated weekly at a cell seeding concentration of 9.33 × 103/cm2. For transport studies TR146 were seeded at a cell density of 4.29 × 104/cm2 in 300 µL DMEM supplemented with 10% FCS and 1% Pen/Strep (DMEM media) on 24-well ThinCerts  with 900 µL DMEM media provided on the basolateral compartments in 24-well plates . To isolate protein, TR146 were seeded at a cell density of 4.29 × 104/cm2 in 2 mL DMEM media on 6-well ThinCerts  with 3.5 mL DMEM media provided on the basolateral side. As soon as cells reached confluency on day 5 or 8 the cultivation condition was switched from submerged to airlift and basolateral DMEM media was changed to DMEM media supplemented with 1% Human Keratinocytes Growth Supplements . Media change was performed every 2–3 days for four weeks. Cells were used from passage 13–38 for the experiments. The human buccal carcinoma cell line, TR146, was purchased from Sigma-Aldrich  and cultured in T25 flasks  was cultivated in T25 flasks in McCoy’s 5A , supplemented with 10% FCS and 1% Pen/Strep, termed as McCoy media, at 37 °C, 5% COclone B2 . Cells wOn the day of experiment, cell layers of TR146 were washed twice on the apical (300 µL) and basolateral (900 µL) side with Hank’s Balanced Salt Solution , while cell layers of the B2 clone were washed twice with basal McCoy media (McCoy’s 5A without supplements). TEER was measured in both models , sRAGE was dissolved in PBS  to 100 µg/mL and subsequently diluted 1:20 in HBSS or basal McCoy containing 1% TRIS, 5% PBS, and 10 µg/mL CRP shortly before the experiment. Media composition was adapted accordingly for all samples and controls during transport studies. Samples on the apical and basolateral side were collected after 24 h and stored at 4 °C until quantification with ELISA. Cell layers of two 24-well ThinCert were lysed with 350 µL RA1 buffer  supplemented with 1% ß—Mercaptoethanol and pooled as one biological sample for RNA isolation.Transparent high-binding 96-well plates  were coated with 100 µL 2 µg/mL Human CRP antibody  in PBS  overnight at 4 °C under orbital shaking conditions sealed with aluminum foil. Prior to usage on the next day, each well was washed three times with 300 µL PBS containing 0.05% Tween 20  and blocked with 300 µL/well carbonate-bicarbonate buffer  containing 1% BSA  for 1.5 h at room temperature under orbital shaking conditions sealed with aluminum foil. Consecutively, wells were washed three times with 300 µL/well 0.05% Tween 20/PBS prior to applying the samples.TM Ultra TMB-ELISA substrate solution  was added and the plate was incubated in the dark for 20 min at room temperature.Samples from the transport studies of the receiving compartment were diluted 1:50; applied CRP or CRP/sRAGE stock solutions 1:2000 in HBSS or basal McCoy containing 1% TRIS for transport studies with CRP and in HBSS or McCoy containing 1% TRIS and 5% PBS for transport studies with CRP and sRAGE. A calibration curve  with Human CRP Standard Antigen  was prepared in the respective HBSS or basal McCoy composition used for the dilution of the samples. The diluted samples and the samples for the calibration curve were additionally diluted 1:2 in PBS containing 1% BSA and 20 mM EDTA  in a non-binding 96-well plate  and applied as duplicates with 100 µL/well on the previously coated high binding 96-well plate. The plate was incubated for 1 hour at room temperature sealed with aluminium foil under orbital shaking conditions. Afterwards, wells were washed three times with 300 µL/well 0.05% Tween 20/PBS following an incubation step with 100 µL/well of 30 ng/mL detection antibody  diluted in PBS containing 1% BSA (1% BSA/PBS) for 1 h at room temperature sealed with aluminum foil under shaking conditions. After further washing steps, applying 300 µL/well 0.05% Tween 20/PBS for three times, 100 µL/well streptavidin-HRP , diluted 1:200 in 1% BSA/PBS, was added and incubated for 20 min under shaking conditions sealed with aluminum foil at room temperature. After washing three times with 0.05% Tween 20/PBS, 100 µL/well 1-Step® 2300 Multimode Plate Reader . Permeability coefficient was calculated as apparent permeability values (Papp) in [cm/s] using the formula shown below (Equation (1)) with crec as a measured concentration in the receiving side in µg/mL, Vrec the volume on the receiving compartment in mL, A as the area in cm2, c0 as the stock solution in µg/mL, and t as duration in seconds.To stop the reaction 50 µL/well of 1 M HCl  was added and absorbance was measured at 450 nm with the EnSpire® 2300 Multimode Plate Reader  at 450 nm as described above. Due to the small saliva volumes of neonates in the range of 30–100 µL, the ELISA method from TM Sybr Green Kit . Primer sequences are shown in For RNA isolation of cell lysates, the NucleoSpin RNA Kit  was used according to the manufacturer’s instruction and eluted with 40 µL nuclease-free water . For cDNA synthesis with the Multiscribe Reverse Transcriptase Kit  200 ng RNA were applied for clone B2 while 1000 ng RNA were applied for TR146. Quantitative real-time PCR was performed as triplicates in white 96-well plates  with 20 µL reaction volume consisting of 4 µL 1:10 in nuclease-free water diluted cDNA, 2.8 µL 3 µM primer pairs, 3.2 µL nuclease-free water, and 10 µL PowerUpThe program for qPCR was set for 20 sec at 95 °C for activation, running 40 cycles for 3 s at 95 °C, and 30 s at 60 °C, following a melting stage of 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C using the LightCycler 480 II . Data was acquired with the LightCycler480 V1.5 software .For data analysis, ΔCt values were calculated by subtracting Ct vales of the endogenous control from the respective Ct values of the analyzed target. ΔCt values of treated samples were normalized to untreated samples upon exponentiating by 2 as shown with the formula below (Equation (2)).On the day of the experiments, cell layers seeded on 6-well ThinCert were washed twice with HBSS (TR146) or basal McCoy (clone B2) on the apical and basolateral side prior to the incubation with HBSS or basal McCoy containing 10 µg/mL CRP or 1% 20 mM TRIS and 280 mM NaCl on the apical and basolateral side for 24 h. Afterwards, cell layers were washed twice with pre-cooled PBS and incubated for 5 min in PBS after the last washing step on ice. For lysis, 50 µL RIPA buffer , supplemented with complete ULTRA protease inhibitor cocktail and PhosphoSTOP minitablet  was added for 30 min on ice. Protein lysates were collected with a cell scraper, and protein concentration was determined with Pierce BCA assay kit  according to the manufacturer’s instructions.Western blotting was performed as described in detail previously ,28. In st-test, or two-way ANOVA followed by post-hoc Holm-Sidak test with α = 0.05, p < 0.05 *, p < 0.01 **, p < 0.001 ***.Results are shown as mean ± SEM unless otherwise indicated. Graphs and statistical analysis were illustrated or performed with SigmaPlot 14.0 . Statistical analysis was performed as Mann-Whitney Rank Sum test, Student’s In this study, 18 neonates  undergoing sepsis were investigated on their CRP levels in serum and saliva  resulting in a median ratio between salivary and serum concentration of 1600 . However, comparing all measured saliva to serum concentrations (n = 33) led to a highly significant correlation between clinical saliva and serum values  as shown in Estimated values of CRP in serum and saliva at time point of sepsis varied with 76.40 ± 79.79 µg/mL in serum and 93.70 ± 161.01 ng/mL in saliva .For comparison of salivary CRP concentrations from septic neonates to baseline saliva levels, samples from 22 healthy neonates .To study a possibly directed transport in more detail, 10 µg/mL CRP was applied on the basolateral (B/A) or apical side (A/B) for 24 h in the oral mucosa and the salivary gland epithelium model. Significant barrier properties of both in vitro models were confirmed by TEER values of 238.88 ± 54.92 Ω × cmapp values for the oral mucosa model resulted in a 2.58 ± 0.39-fold higher permeability of CRP from B/A upon normalization to Papp values from A/B , shown in p < 0.001; A/B Papp: 0.013 ± 0.001 × 10−6 cm/s, B/A Papp: 0.029 ± 0.003 × 10−6 cm/s). In summary, these data clearly indicated the presence of a directed transport mechanism for CRP across the in vitro models of the BSB.Apparent permeability PPrevious literature indicated the involvement of RAGE in inflammatory response upon treating endothelial progenitor cells with CRP , followeapp values described above. To examine a possible involvement and regulation of RAGE by CRP, cell samples from the transport studies were analyzed and compared to untreated cell samples at the mRNA and protein level. As a result, the mRNA regulation showed a similar tendency as the Pp = 0.56). On the contrary, applying CRP in the salivary gland model on the basolateral (B/A) side led to a significant downregulation to 0.85 ± 0.06-fold for RAGE compared to an upregulation of 2.14 ± 0.65-fold upon applying CRP on the apical (A/B) side , 1.41 ± 0.22-fold (A/B), /B) side A,B.p < 0.05) compared to control samples with 1.00 ± 0.04 (mean ± SEM)  E,F.p < 0.01, Papp: A/B 0.12 ± 0.017 × 10−6 cm/s, B/A 0.22 ± 0.030 × 10−6 cm/s), similar to above. Upon normalization to Papp values from A/B, the addition of sRAGE led to a similar apparent permeability value of CRP from A/B , but decreased the transport of CRP from B/A significantly to 1.50 ± 0.08-fold  in comparison to transport of CRP from B/A without addition of sRAGE.To investigate the role of RAGE on the transport of CRP, transport studies with 5 µg/mL soluble RAGE (sRAGE) and 10 µg/mL CRP on the basolateral or apical side were performed and compared to the permeability of CRP alone in both models for each experiment. In the oral mucosa model, transport of CRP from B/A showed a 2.22 ± 0.21-fold higher permeability compared to A/B  compared to CRP alone from A/B . In case of the transport B/A, addition of sRAGE resulted in a significantly upregulated permeability of CRP ) compared to CRP alone   .CRP is widely used as an inflammatory biomarker in serum and associated with cardiovascular diseases at levels >10 µg/mL . Recentl2 of 0.53 ± 0.23, comparable to the result of our study with a R2 value of 0.52  were concordant to the corresponding TEER values of the models and confirmed the stronger paracellular tightness of the salivary gland model.For this purpose, we applied 10 µg/mL CRP in our oral mucosa and salivary gland model, as CRP concentrations of >8.7 µg/mL are associated with infections (together with high temperature), and clinically relevant concentrations of 10–20 µg/mL have also been used for studies with in vitro models of the blood–brain barrier previously ,46. WhilSince literature data indicated a linkage between RAGE and CRP ,47, we tThis revealed that mRNA levels of RAGE are subject to regulation by CRP in an exposure side dependent manner. With regulation of RAGE after CRP treatment on the protein level, we decided to examine the possible involvement of RAGE in the transport of CRP. For this, we added soluble RAGE (sRAGE) to CRP for further transport studies.In the oral mucosa model, this resulted in a significantly decreased permeability of CRP from B/A, while addition of sRAGE in the salivary gland model led to a significantly enhanced transport from B/A . In detaCorresponding to the detected correlation of CRP in saliva and serum samples of neonates undergoing sepsis, both in vitro models of the BSB showed an elevated transport of CRP in the direction of saliva. While the favorable transport to saliva was shown in this study and the influence of RAGE on CRP transport was described, further studies regarding the underlying mechanisms would improve our current knowledge about the causal link between serum and salivary CRP and strengthen the relevance of CRP as a salivary biomarker in general. Future studies could aim at the identification of the responsible transporters, for example, by immunoprecipitation followed by mass analysis to screen for proteins binding to CRP in the cellular membrane fraction.In this study on measurement of serum and salivary CRP levels, clinical data suggest that studying individual ratios and progression is more useful than relying on the measurement of a single time-point. Additionally, in vitro data using established models of the BSB show that CRP is preferentially transported to the direction of saliva, enhancing its feasibility as a salivary marker. Moreover, RAGE seems to be involved in the transport of CRP across the BSB. Future studies could focus on elucidation of the details of the transport mechanism of CRP across the BSB.


Cinchona alkaloids is widened by 6′-Amino-cinchonine and 6′-Amino-cinchonidine, novel compounds which incorporate a primary amino function in the quinolinic ring system. These key intermediates open the field for a range of fruitful chemistry. Here is described a short and direct pathway for the synthesis of triazole containing derivatives of the above-mentioned substances using the [3 + 2] Huisgen cycloaddition. For this purpose, the amines were first converted into the corresponding azides. Based on this, non-substituted and silyl-protected triazoles were synthesized as examples. Furthermore, didehydrated derivatives of quincorine and quincoridine were used as addition partners, resulting in compounds that carry the quinuclidine ring of the cinchona alkaloids at both ends. Some of these compounds were examined radiographically to investigate the position of the quinuclidine ring to the triazole. The solid-state structures of compounds 10, 11 and 28 were determined by X-ray diffraction analyses.The substance class of the well-known  The protocol could also be successfully applied to amines 2 to 6 with similarly good yields .Azides 22 and didehydrocincorine 23 . Two trends can be observed. The yields in the cinchonidine series  were better than in the cinchonine series . Although the difference is not strikingly large, it cannot be ignored. Furthermore, in three pairs, the yields of the hydrogenated compounds were higher than the yields of the corresponding vinyl species.The synthesis of compounds 10 and 11  can be generated by desilylation of the compounds 14, 16, 18, 20. Overall, all reactions proceeded with good purity and good to very good yields. The aim of future work will be to investigate the potential of these compounds in, for example, catalysis or medicinal chemistry. In terms of structural chemistry, the intramolecular cyclisation of azide alkynes 5 and 6 is a challenge for every synthetic chemist.The aim of this work was to investigate the synthetic potential of 6′-Amino-cinchonine 1, 6′-Amino-cinchonidine g azides . Since tAll reagents were purchased from commercial sources  and used without further purification. The solvents used were of analytical grade. The 1H and 13C NMR spectra were recorded at room temperature on a Bruker Avance 300, Bruker Avance 400 or Bruker Avance 600, operating at 300 MHz, 400 MHz or 600 MHz for 1H and 75 MHz, 100 MHz or 150 MHz for 13C. Chemical shifts (δ) were reported as relative to the tetramethylsilane peak set at 0.00 ppm. In the case of multiplets, the signals were reported as intervals. The signals were abbreviated as s, singlet; d, doublet; t, triplet; q, quadruplet; and m, multiplet. Mass spectra were recorded on a Finnigan MAT 8400-MSS and Finnigan MAT 4515. High resolution mass spectra were recorded on a Finnigan MAT 95 XP. The reactions were monitored by TLC and performed on silica gel plates 40 × 80 mm Polygram Sil G/UV254 (Macherey-Nagel). Visualization on TLC was achieved by UV light. Column chromatography was performed with Merck silica gel 60 (70–200 mesh).Cinchona core followed the rules of Paul Rabe, with the side chain numbered based on the IUPAC rules.The numbering of the 2 (1.2 eq) in H2O (0.33 g/mL) was added to a mixture of Cinchona amine (1.0 eq) and 15% HCl (0.1 g amine/mL) cooled at −5 °C. After the addition was complete, the reaction mixture was stirred at this temperature for 60 min. A solution of NaN3 (1.7 eq) in H2O (0.2 g/mL) was added dropwise to the reaction mixture at 0 °C. After the addition was finished, the reaction mixture was maintained at 0 °C for 2 h and then stirred at room temperature for 12 h. The product was precipitated by addition of NaOH (20%) until pH 10. Upon filtration, the solid was washed with distilled water and dried. Crystallization from DCM presented the Cinchona azides as yellow solids in good yields (70–90%)General procedure: a dropwise a solution of NaNO(S)-(6-Azidoquinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (8): the title compound was synthesized from 1  to afford 8  as a yellow-orange solid. 1H-NMR  δ 8.79 , 8.08 , 7.82 , 7.74 , 7.52 , 6.17 , 5.66–5.52 , 5.17–5.08 , 3.50–3.43 , 3.35–3.32 , 2.96–2.74 , 2.37–2.29 , 2.23–2.16 , 1.93–1.68 , 1.66–1.51 , and 1.31–1.18 ; 13C-NMR  δ 151.27 , 150.32 , 146.64 ; 141.73 ; 140.31 , 132.14 , 128.03 , 123.87 , 120.87 , 115.15 , 112.90 , 72.49 , 61.27 , 50.82 , 50.19 , 41.29 , 29.65 , 27.20 , and 22.25 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 173 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.60; IR: see spectrum in the supporting information; HR-MS  was calculated for C19H21N5O1 + H+: 336.18189 Da, found 336.18185 Da.(R)-(6-Azidoquinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (9): the title compound was synthesized from 2  to afford 9  as a yellow-orange solid. 1H-NMR  δ 8.76 , 8.05 , 7.82 , 7.71 , 7.52 , 5.78 , 5.53–5.49 , 5.02–4.94 , 3.61–3.51 , 3.12–2.94 , 2.72–2.49 , 2.48–2.18 , 1.93–1.81 , 1.80–1.78 , and 1.72–1.50 ; 13C-NMR  δ 151.22 , 150.27 , 146.62 ; 142.78 ; 140.24 , 132.15 , 127.95 , 123.25 , 120.93 , 114.90 , 112.99 , 72.40 , 61.53 , 57.59 , 43.82 , 40.95 , 29.18 , 28.25 , and 22.61 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 144 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.62; IR: see spectrum in the supporting information; HR-MS  was calculated for C19H21N5O1 + H+: 336.18189 Da, found 336.18183 Da.(S)-(6-Azidoquinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (10): the title compound was synthesized from 3  to afford 10  as a yellowish solid. 1H-NMR  δ 8.78 , 8.04 , 7.86 , 7.54 , 7.48 , 5.20–5.16 , 2.96–2.88 , 2.68–2.62 , 2.52–2.44 , 2.00–2.76 , 1.65–1.62 ; 1.53–1.30 , and 0.86 ; 13C-NMR  δ 150.30 , 149.50 , 145.62 , 136.85 , 131.70 , 126.94 , 121.56 , 119.76 , 112.15 , 70.73 , 61.14 , 49.89 , 49.11 , 37.08 , 27.11 , 26.01 , 25.03 , 24.01 , and 11.90 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 191 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.50; IR: see spectrum in the supporting information; HR-MS  calculated for C19H23N5O1 + H+: 338.19754 Da, found 338.19756 Da.(R)-(6-Azidoquinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (11): the title compound was synthesized from 4  to afford 8  as a yellow solid. 1H-NMR  δ 8.78 , 8.05 , 7.91 , 7.55 , 7.51 , 5.18–5.13 , 3.21–2.98 , 2.87–2.77 , 2.45–2.31 , 2.18–2.06 , 1.71–1.50 , 1.40–1.15 , and 0.86 ; 13C-NMR  δ 149.87 , 149.50 , 145.75 ; 136.92 , 131.79 , 126.87 , 121.45 , 119.97 , 112.73 , 71.44 ,60.59 , 57.53 , 41.64 , 37.14 , 28.18 , 27.20 , 25.14 , 24.35 , and 12.04 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 163 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.54; IR: see spectrum in the supporting information; HR-MS  calculated for C19H23N5O1 + H+: 338.19754 Da, found 338.19757 Da.(S)-(6-Azidoquinolin-4-yl)-5-ethynylquinuclidin-2-yl)methanol (12): the title compound was synthesized from 5  to afford 12  as a yellow solid. 1H-NMR  δ 8.73 , 8.07 , 7.68 , 7.53 , 7.36 , 5.58–5.48 , 3.28–3.20 , 3.18–3.09 , 3.03–2.94 , 2.84–2.74 , 2.68–2.55 , 2.51–2.44 , 2.28–2.16 , 2.15 , 2.07–1.93 , and 1.60–1.41 ; 13C-NMR  δ 149.50 , 147.93 , 146.11 , 138.16 , 132.22 , 126.81 , 121.60 , 119.47 , 111.73 , 87.31 , 72.01 , 69.31 , 60.25 , 50.28 , 49.41 , 28.03 , 27.86 , 25.11 , and 23.51 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 112 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.55; IR: see spectrum in the supporting information; HR-MS  calculated for C19H19N5O1 + H+: 334.16624 Da, found 334.16647 Da.(R)-(6-Azidoquinolin-4-yl)-5-ethynylquinuclidin-2-yl)methanol (13): the title compound was synthesized from 6  to afford 13  as a yellow solid. 1H-NMR  δ 8.74 , 8.06 , 7.95 , 7.51 , 7.21 , 5.61–5.55 , 3.42–3–3.34 , 3.24–3.16 , 2.98–2.88 , 2.75–2.61 , 2.61–2.48 , 2.44–2.37 , 2.16 , 2.06–1.94 , 1.90–1.89 , and 1.81–1.59 ; 13C-NMR  δ 149.65 , 148.02 , 146.31 , 138.27 , 131.97 , 126.86 , 121.53 , 119.67 , 111.68 , 87.26 , 71.87 , 69.21 , 60.62 , 56.31 , 49.42 , 28.17 , 27.96 , 24.94 , and 23.47 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 96 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.57; IR: see spectrum in the supporting information; HR-MS  calculated for C19H19N5O1 + H+: 334.16624 Da, found 334.16633 Da.Cinchona azide (1 eq.) and trimethylsilyl acetylene (1.5 eq.) were suspended in an H2O: MeOH (1:1) mixture to deliver a substrate concentration of 0.1 M Cu2SO4  and sodium ascorbate . The reaction mixture was stirred at room temperature until complete conversion of the azide (12–24 h). After that, the reaction was quenched by adding an aqueous solution of ammonium chloride and ammonium hydroxide (pH 10/12). The mixture was extracted with DCM (three times), and the combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated in vacuo. The residues obtained were purified by flash column chromatography using MTBE: MeOH as a gradient elution to afford the corresponding TMS-triazole derivatives.General procedure: in a round-bottom flask the corresponding (S)-(6-(4-(Trimethylsilyl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (14): the title compound was synthesized from 8  to afford 14  as a colourless wax. 1H-NMR  δ 8.77 , 8.49 , 8.10–8.06 , 7.87 , 7.35 , 6.32 , 6.06 , 5.24–5.18 , 4.22–3.96 , 3.34–3.08 , 3.05–2.88 , 2.58–2.42 , 2.37–2.21 , 1.96–1.87 , 1.85–1.71 , 1.69–1.54 , 1.15–1.00 , and 0.93–0.79  0.46 3); 13C-NMR  δ 150.13 , 147.41 , 146.93 , 146.62 ; 137.55 ; 134.68 , 131.88 , 128.35 , 124.52 , 121.47 , 119.27 , 116.78 , 110.91 , 67.67 , 60.37 , 49.61 , 48.71 , 38.28 , 27.67 , 24.31 , 19.00 , and -1.06 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 147 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.19; IR: see spectrum in the supporting information; HR-MS  calculated for C24H31N5O1Si1 + H+: 434.23706 Da, found 434.23733 Da.(R)-(6-(4-(Trimethylsilyl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (16): the title compound was synthesized from 9  to afford 16  as a colourless wax. 1H-NMR  δ 8.61 , 8.34 , 8.28 , 7.97 , 7.90 , 7.52 , 5.87–5.75 , 5.69 , 5.00–4.85 , 3.77–3.57 , 3.17–3.03 , 2.78–2.62 , 2.41–2.28 , 1.92–1.79 , 1.62–1.48 , 0.92–0.81  0.41 3); 13C-NMR  δ 150.44 , 149.23 , 147.56 , 146.96 ; 140.20 ; 134.34 , 131.78 , 127.79 , 125.50 , 121.54 , 119.57 , 115.03 , 112.93 , 69.83 , 60.52 , 56.12 , 43.07 , 39.15 , 29.63 , 27.52 , 21.12 , and -1.14 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 113 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.15; IR: see spectrum in the supporting information; HR-MS  calculated for C24H31N5O1Si1 + H+: 434.23706 Da, found 434.23742 Da.(S)-(6-(4-(Trimethylsilyl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (18): the title compound was synthesized from 10  to afford 18  as a colourless honey-type liquid. 1H-NMR  δ 8.77 , 8.47 , 8.13–8.09 , 7.81 , 7.34 , 6.38 , 4.19–3.92 , 3.28–3.04 , 3.08–2.91 , 2.52–2.37 , 2.31–2.17 , 1.92–1.85 , 1.81–1.70 , 1.61–1.49 , 1.29–1.15 , 0.95–0.83  0.79 , and 0.42 3); 13C-NMR  δ 150.20 , 148.27 , 147.43 , 146.70 ; 134.39 , 131.74 , 128.03 , 124.98 , 121.33 , 119.33 , 111.88 , 68.77 , 60.33 , 50.42 , 49.67 , 36.38 , 26.88 , 25.58 , 24.69 , 19.48 , 11.75 , and -1.15 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 166 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.11; IR: see spectrum in the supporting information; HR-MS  calculated for C24H33N5O1Si1 + H+: 436.25271 Da, found 436.25296 Da.(R)-(6-(4-(Trimethylsilyl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (20): the title compound was synthesized from 11  to afford 20  as a honey-type liquid. 1H-NMR  δ 8.63 , 8.35 , 8.24 , 8.00 , 7.95 , 7.51 , 5.84–5.73 , 5.69 3.73–3.57 , 3.16–3.02 , 2.77–2.63 H-6), 2.47–2.33 , 1.93–1.77 , 1.57–1.40 , 1.29–1.15 , 0.78 , and 0.43 3); 13C-NMR  δ 150.50 , 149.18 , 147.58 , 147.04 ; 134.45 , 131.89 , 127.83 , 125.49 , 121.64 , 119.53 , 112.84 , 69.96 , 60.36 , 57.97 , 43.23 , 36.95 , 27.39 , 27.37 , 25.18 , 20,76 , 11.88 , and -1.11 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 136 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.16; IR: see spectrum in the supporting information; HR-MS  calculated for C24H33N5O1Si1 + H+: 436.25271 Da, found 436.25305 Da.Cinchona TMS-triazole (1.00 eq.) was suspended in a mixture tBu-OH/H2O (1:1 ratio) and K2CO3 (2.0 equiv) was added. The reaction mixture was rigorously stirred for 48 h. Upon completion of the reaction, EtOAc was added and the organic layer was separated, washed with water, brine, dried over Na2SO4, filtered and concentrated in vacuo. The residues obtained were purified by flash column chromatography using MTBE: MeOH as a gradient elution to afford the corresponding triazole products.General procedure: a purified (S)-quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (15): the title compound was synthesized from 14  to afford 15  as a white solid. 1H-NMR  δ 8.68 , 8.34 , 8.23 , 8.04 , 7.93 , 7.77 , 7.58 , 6.04 , 5.69–5.66 , 5.08–5.01 , 3.33–3.25 , 3.06–3.00 , 2.88–2.78 , 2.72–2.64 , 2.26–2.19 , 2.12–2.03 , 1.80–1.75 , 1.56–1.45 , and 1.28–1.20 ; 13C-NMR  δ 150.71 , 150.24 , 147.13 ; 140.30 ; 134.54 , 134.22 , 131.86 , 125.78 , 122.07 , 121.41 , 119.60 , 114.73 , 113.70 , 71.11 , 60.25 , 49.73 , 49.11 , 39.78 , 28.01 , 26.16 , and 21.42 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 117 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.56; IR: see spectrum in the supporting information; HR-MS  calculated for C21H23N5O1 + H+: 362.19754 Da, found 362.19785 Da.(R)-quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (17): the title compound was synthesized from 16  to afford 17  as a white solid. 1H-NMR  δ 8.59 , 8.49 , 8.37 , 8.00 , 7.95 , 7.74 , 7.59 , 5.77–5.68 , 5.70 , 4.96–4.85 , 3.67–3.53 , 3.12–2.98 , 2.73–2.58 , 2.35–2.25 , 1.92–1.76 , and 1.60–1.48 ; 13C-NMR  δ 150.19 , 150.09 , 146.62 ; 140.77 ; 134.14 , 134.12 , 131.31 , 125.31 , 122.07 , 121.14 , 119.27 , 114.41 , 112.73 , 70.03 , 60.22 , 56.06 , 42.70 , 39.06 , 27.29 , 26.73 , and 20.92 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 92 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.63; IR: see spectrum in the supporting information; HR-MS  calculated for C21H23N5O1 + H+: 362.19754 Da, found 362.19796 Da.(S)-quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (19): the title compound was synthesized from 18  to afford 19  as an off-white solid. 1H-NMR  δ 8.54 , 8.26 , 8.35 , 8.98 , 7.65 , 7.98 , 7.94 , 5.87–5.77 , 3.31–3.20 , 3.11–3.08 , 2.85–2.73 , 2.69–2.58 , 2.21–2.15 , 2.32–2.23 , 1.85–1.80 , 1.51–1.40 , 1.22–1.14 , and 0.81 ; 13C-NMR  δ 13C-NMR  δ 150.81 , 149.80 , 147.27 , 134.62 , 134.30 , 132.07 , 125.75 , 122.12 , 121.40 , 119.60 , 113.56 , 70.93 , 60.38 , 50.74 , 49.87 , 37.12 , 26.76 , 26.02 , 25.08 , 21.09 , and 11.92 ; H,H-Cosy, HSQC and HMBC were made. Melting point: Substance decompose at T = 143 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.45; IR: see spectrum in the supporting information; HR-MS  calculated for C21H25N5O1 + H+: 364.21319 Da, found 364.21347 Da.(R)-quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (21): the title compound was synthesized from 20  to afford 21  as an off-white solid. 1H-NMR  δ 8.68 , 8.37, 8.29 , 8.05 , 7.96 , 7.80 , 7.57 , 5.69–5.59 , 3.47–3.41 , 3.11–2.96 , 2.65–2.55 , 2.35–2.29 , 1.79–1.71 , 1.60–1.41 , 1.34–1.18 , and 0.79 ; 13C-NMR  δ 150.79 , 150.07 , 147.25 ; 134.60 , 134.36 , 132.01 , 125.80 , 122.27 , 121.49 , 119.65 , 113.51 , 70.85 , 60.39 , 58.18 , 43.03 , 37.20 , 27.88 , 27.52 , 25.27 , 21.70 , and 11.96 ; H,H-Cosy, HSQC and HMBC were made. Melting point: the substance decomposed at T = 106 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.58; IR: see spectrum in the supporting information; HR-MS  calculated for C21H25N5O1 + H+: 364.21319 Da, found 364.21358 Da.2(1.2 eq) in H2O (0.33 g/mL) was added to a mixture of Cinchona amine (1.0 eq) and 15% HCl (0.1g amine/mL) cooled at −5 °C. After the completion of the addition, the reaction mixture was stirred at this temperature for 60 min. A solution of NaN3 (1.7 eq) in H2O (0.2 g/mL) was added dropwise to the reaction mixture at 0 °C. After the addition was finished, the reaction mixture was maintained at 0 °C for 2 h and then stirred at room temperature for 12 h. The product was precipitated by the addition of NaOH (20%) until pH 10. Upon filtration, the solid was washed with distilled water and dried. Crystallization from DCM presented the Cinchona azides as yellow solids in good yields (70–90%).General procedure: a dropwise a solution of NaNO(S)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (24): the title compound was synthesized from 8  to afford 24  as a reddish solid. 1H-NMR  δ 8.87–8.72 (1 H), 8.41–8.28 (2 H), 8.39–7.54 (2 H), 7.69–7.58 (1 H), 6.27–6.56 (1 H), 5.73–5.65 (1 H), 5.21–5.11 (2 H), 3.75–3.29 (4 H), 3.11–2.75 (6 H), 2.89–2.75 (2 H), 2.46–2.33 (1 H), 2.11–2.01 (2 H), 1.78–1.35 (7 H), 1.29–1.15 (1 H), and 0.89–0.76 (1 H); 13C-NMR  δ 151.20 (Cq), 150.54 (CH), 150.34 (Cq), 146.96 (Cq); 140.45 (CH), 134.55 (CH), 131.79 (CH), 125.49 (Cq), 121.19 (CH), 119.36 (CH), 119.22 (CH), 114.73 (CH2), 112.50 (CH), 70.67 (CH), 62.64 (CH2), 59.94 (CH), 57.30 (CH), 49.83 (CH2), 49.30 (CH2), 48.89 (CH2), 49.77 (CH2), 39.98 (CH), 33.36 (CH), 28.15 (CH), 27.55 (CH2), 26.35 (CH), 26.16 (CH2), 23.71 (CH2), and 20.08 (CH2); Melting point: the substance decomposed at T = 126 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.21; IR: see spectrum in the supporting information; HR-MS  calculated for C29H36N6O2 + H+: 501.29725 Da, found 501.29739 Da.(S)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (25): the title compound was synthesized from 8  to afford 25  as a pink solid. 1H-NMR  δ 8.71–8.64 (1 H), 8.37–8.25 (2 H), 8.00–7.88 (2 H), 7.62–7.56 (1 H), 6.16–6.04 (1 H), 5.82–5.70 (1 H), 5.13–5.01 (2 H), 3.64–3.19 (4 H), 3.16–2.82 (6 H), 2.80–2.69 (2 H), 2.32–2.21 (1 H), 2.19–2.08 (2 H), 1.82–1.43 (7 H), 1.25–1.13 (1 H), and 0.94–0.83 (1 H); 13C-NMR  δ 151.73 (Cq), 150.36 (CH), 150.24 (Cq), 146.82 (Cq); 140.17 (CH), 134.35 (CH), 131.42 (CH), 125.60 (Cq), 121.08 (CH), 119.35 (CH), 119.12 (CH), 114.71 (CH2), 112.86 (CH), 70.3 (CH), 63.33 (CH2), 59.59 (CH), 57.28 (CH), 54.75 (CH2), 49.61 (CH2), 49.20 (CH2), 40.44 (CH2), 39.75 (CH), 33.02 (CH), 27.96 (CH), 27.20 (CH2), 27.06 (CH), 25.97 (CH2), 24.89 (CH2), and 20.92 (CH2). Melting point: the substance decomposed at T = 119 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.20; IR: see spectrum in the supporting information; HR-MS  calculated for C29H36N6O2 + H+: 501.29725 Da, found 501.29742 Da.(R)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (26): the title compound was synthesized from 9  to afford 26  as a light red solid.1 H-NMR  δ 8.61–8.54 (1 H), 8.67–8.54 (2 H), 8.13–7.87 (2 H), 7.68–7.46 (1 H), 6.96–6.64 (1 H), 5.34–5.23 (1 H), 5.17–5.03 (2 H), 3.78–3.35 (4 H), 3.26–2.92 (6 H), 2.63–2.46 (2 H), 2.39–2.26 (1 H), 2.12–2.01 (2 H), 1.76–1.32 (7 H), 1.19–1.07 (1 H), and 0.97–0.85 (1 H); 13C-NMR  δ 152.08 (Cq), 150.67 (CH), 148.43 (Cq), 147.67 (Cq); 141.21 (CH), 133.96 (CH), 132.54 (CH), 126.31 (Cq), 122.45 (CH), 120.08 (CH), 119.23 (CH), 115.76 (CH2), 111.47 (CH), 69.93 (CH), 62.48 (CH2), 61.13 (CH2), 60.67 (CH), 57.79 (CH2), 56.36 (CH2), 54.76 (CH2), 42.98 (CH2), 40.54 (CH), 34.65 (CH), 27.87 (CH), 26.03 (CH), 25.53 (CH2), 24.52 (CH2), 21.73 (CH), and 19.26 (CH2). Melting point: the substance decomposed at T = 110 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.16; IR: see spectrum in the supporting information; HR-MS  calculated for C29H36N6O2 + H+: 501.29725 Da, found 501.29747 Da.(R)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-vinylquinuclidin-2-yl)methanol (27): the title compound was synthesized from 9  to afford 27  as a pink solid. 1H-NMR  δ 8.71–8.64 (1 H), 8.37–8.25 (2 H), 8.00–7.88 (2 H), 7.62–7.56 (1 H), 6.16–6.04 (1 H), 5.82–5.70 (1 H), 5.13–5.01 (2 H), 3.64–3.19 (4 H), 3.16–2.82 (6 H), 2.80–2.69 (2 H), 2.32–2.21 (1 H), 2.19–2.08 (2 H), 1.82–1.43 (7 H), 1.25–1.13 (1 H), and 0.94–0.83 (1 H); 13C-NMR  δ 151.99 (Cq), 150.65 (CH), 147.30 (Cq), 147.26 (Cq); 140.48 (CH), 133.16 (CH), 132.17 (CH), 125.47 (Cq), 121.58 (CH), 119.79 (CH), 119.33 (CH), 115.07 (CH2), 112.09 (CH), 69.33 (CH), 63.43 (CH2), 60.54 (CH2), 60.34 (CH), 57.43 (CH2), 55.89 (CH2), 54.16 (CH2), 43.25 (CH2), 40.19 (CH), 33.11 (CH), 27.49 (CH), 26.96 (CH), 26.79 (CH2), 24.71 (CH2), 21.00 (CH), and 19.88 (CH2). Melting point: the substance decomposed at T = 103 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.12; IR: see spectrum in the supporting information; HR-MS  calculated for C29H36N6O2 + H+: 501.29725 Da, found 501.29753 Da.(S)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (28): the title compound was synthesized from 10  to afford 28  as an off-white solid. 1H-NMR  δ 8.75–8.54 (1 H), 8.28–8.14 (2 H), 8.01–7.82 (2 H), 7.59–7.54 (1 H), 5.73–5.61 (1 H), 3.79–3.27 (4 H), 3.21–2.88 (6 H), 2.81–2.61 (2 H), 2.41–2.32 (1 H), 2.26–2.18 (2 H), 1.89–1.31 (9 H), 1.21–1.12 (1 H), and 0.99–0.81 (4 H); 13C-NMR  δ 152.71 (Cq), 150.98 (CH), 150.37 (Cq), 147.63 (Cq), 135.01 (CH), 132.23 (CH), 126.29 (Cq), 121.78 (CH), 119.73 (CH), 118.61 (CH), 113.93 (CH), 71.35 (CH), 62.67 (CH2), 60.59 (CH), 58.79 (CH), 55.23 (CH2), 50.69 (CH2), 49.13 (CH2), 41.65 (CH2), 40.72 (CH), 33.19 (CH), 28.17 (CH), 27.64 (CH2), 27.13 (CH), 26.27 (CH2), 25.93 (CH2), 24.63 (CH2), 21.47 (CH2), and 12.65 (CH3). Melting point: the substance decomposed at T = 145 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.19; IR: see spectrum in the supporting information; HR-MS  calculated for C29H38N6O2 + H+: 503.31290 Da, found 503.31304 Da.(S)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (29): the title compound was synthesized from 10  to afford 29  as an off-white solid; 1H-NMR  δ 9.03–8.73 (1 H), 8.41–8.29 (2 H), 8.13–7.91 (2 H), 7.68–7.59 (1 H), 6.23–5.81 (1 H), 3.69–3.23 (4 H), 3.17–2.81 (6 H), 2.75–2.34 (2 H), 2.32–2.23 (1 H), 2.16–2.01 (2 H), 1.87–1.34 (9 H), 1.27–1.14 (1 H), and 0.91–0.87 (4 H); 13C-NMR  δ 151.54 (Cq), 151.12 (CH), 150.27 (Cq), 146.12(Cq), 135.69 (CH), 130.19 (CH), 125.94 (Cq), 121.98 (CH), 120.15 (CH), 119.72 (CH), 113.26 (CH), 71.3 (CH), 63.99 (CH2), 60.57 (CH), 57.52 (CH), 55.75 (CH2), 49.69 (CH2), 49.13 (CH2), 41.64 (CH2), 40.73 (CH), 33.92 (CH), 27.56 (CH), 27.21 (CH2), 27.16 (CH), 25.47 (CH2), 25.22 (CH2), 24.83 (CH2), 21.62 (CH2), and 12.35 (CH3). Melting point: the substance decomposed at T = 138 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.21; IR: see spectrum in the supporting information; HR-MS  calculated for C29H38N6O2 + H+: 503.31290 Da, found 503.31298 Da.(R)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (30): the title compound was synthesized from 11  to afford 30  as an off-white solid. 1H-NMR  δ 8.62–8.49 (1 H), 8.38–8.27 (2 H), 7.99–7.81 (2 H), 7.71–7.60 (1 H), 5.89–5.65 (1 H), 5.17–4.89 (2 H), 3.73–3.29 (4 H), 3.21–2.94 (6 H), 2.81–2.73 (2 H), 2.54–2.36 (1 H), 2.28–2.17 (2 H), 1.93–1.49 (9 H), 1.29–1.14 (1 H), and 0.97–0.73 (4 H); 13C-NMR  δ 152.21 (Cq), 151.25 (CH), 147.91 (Cq), 146.66 (Cq); 133.73 (CH), 131.17 (CH), 125.97 (Cq), 122.68 (CH), 120.19 (CH), 119.13 (CH), 112.34 (CH), 69.78 (CH), 64.41 (CH2), 61.64 (CH2), 60.52 (CH), 58.67 (CH2), 56.37 (CH2), 54.73 (CH2), 44.87 (CH2), 40.36 (CH), 33.57 (CH), 28.19 (CH), 27.91 (CH), 26.71 (CH2), 25.52 (CH2), 24.11 (CH2), 21.68 (CH), 20.18 (CH2), and 12.37 (CH3). Melting point: the substance decomposed at T = 132 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.14; IR: see spectrum in the supporting information; HR-MS  calculated for C29H38N6O2 + H+: 503.31290 Da, found 503.31312 Da.(R)--6-(Hydroxymethyl)quinuclidin-3-yl)-1H-1,2,3-triazol-1-yl)quinolin-4-yl)-5-ethylquinuclidin-2-yl)methanol (31): the title compound was synthesized from 11  to afford 31  as an off-white solid. 1H-NMR  δ 8.89–8.71 (1 H), 8.39–8.31 (2 H), 8.23–8.12 (2 H), 7.73–7.66 (1 H), 5.79–5.65 (1 H), 5.56–5.23 (2 H), 3.81–3.39 (4 H), 3.25–2.93 (6 H), 2.85–2.71 (2 H), 2.54–2.35 (1 H), 2.23–2.13 (2 H), 1.97–1.51 (9 H), 1.33–1.21 (1 H), and 0.89–0.68 (4 H); 13C-NMR  δ 152.00 (Cq), 150.69 (CH), 150.35 (Cq), 147.34 (Cq); 135.10 (CH), 132.16 (CH), 125.65 (Cq), 121.59 (CH), 119.93 (CH), 119.42 (CH), 112.46 (CH), 69.27 (CH), 63.47 (CH2), 60.59 (CH2), 60.33 (CH), 57.67 (CH2), 57.43 (CH2), 54.13 (CH2), 40.22 (CH2), 36.83 (CH), 33.10 (CH), 27.40 (CH), 27.00 (CH), 26.89 (CH2), 25.16 (CH2), 24.79 (CH2), 20.99 (CH), 20.10 (CH2), and 11.83 (CH3). Melting point: the substance decomposed at T = 121 °C; TLC (TBME: MeOH: ammonia 25%/100:10:1): rf-value = 0.14; IR: see spectrum in the supporting information; HR-MS  calculated for C29H38N6O2 + H+: 503.31290 Da, found 503.31306 Da.α radiation. The reflections were indexed, integrated, and appropriate absorption corrections were applied as implemented in the CrysAlisPro software package [10 and 11, whilst for 28 geometrical restraints and constraints for the displacement parameters were employed. For 28, the structure contains a partly occupied HCl and two partly occupied units of water. The absolute structure parameter suggests numerically a very small contribution of an inversion twined component. As the only heavy atom (Cl1) is only partly occupied, a twin refinement was considered not reliable and was not conducted. During refinement and analysis of the crystallographic data, the programs OLEX2 and Diamond were used [The single crystals were mounted on a Hampton loop using perfluoroether oil and placed in the cold nitrogen gas stream on the diffractometer . The dat package . The str package ,32. Carbere used ,34. Unle