Pharmacokinetic Optimization of Small Molecule Janus Kinase 3 Inhibitors to Target Immune Cells

Abstract

Modulation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling is a promising method of treating autoimmune diseases, and the profound potency of clinical compounds makes this mode of action particularly attractive. Other questions that remain unanswered also include: What is the ideal selectivity between JAK1 and JAK3? Which cells are most relevant to JAK blockade? And what is the ideal tissue distribution pattern for addressing specific autoimmune conditions? We hypothesized that JAK3 selectivity is most relevant to low-dose clinical effects and interleukin-10 (IL-10) stimulation in particular, that immune cells are the most important compartment, and that distribution to inflamed tissue is the most important pharmacokinetic characteristic for in vivo disease modification. To test these hypotheses, we prepared modified derivatives of JAK3 specific inhibitors that target C909 near the ATP binding site based on FM-381, first reported in 2016; a compound class that was hitherto limited in uptake and exposure in vivo. These limits appear to be due to metabolic instability of side groups binding in the selectivity pocket. We identified derivatives with improved stability and tissue exposure. Conjugation to macrolide scaffolds with medium chain linkers was sufficient to stabilize the compounds and improve transport to organs while maintaining JAK3 affinity. These conjugates are inflammation targeted JAK3 inhibitors with long tissue half-lives and high exposure to activated immune cells.

Kinases are enzymes that catalyze the transfer of phosphate to a target substrate, usually onto a serine, threonine, or tyrosine residue. They can occur as receptors with kinase activity or as unbound, cytosolic kinases that bind to a receptor after outside stimulation. Many of the more than 500 known kinases are targets of anti-inflammatory, antiproliferative, or other drugs.¹⁻³ The Janus kinase (JAK) family, comprised of JAK1, JAK2, JAK3, and TYK2, is a group of cytosolic tyrosine kinases involved in cellular signal transduction. Extracellular signals arrive in the form of cytokines like growth factors, interleukins, and interferons. Their corresponding receptors are activated, causing JAKs to bind to them as homo- or heterodimers, after which they, in turn, phosphorylate tyrosine residues of signal transducer and activator of transcription (STAT) proteins. STATs then dimerize, translocate to the nucleus, and modulate gene transcription on a cellular level.⁴⁻⁶ JAK-STAT signaling is regulated by several groups of proteins, e.g., protein inhibitor of activated STAT (PIAS), protein tyrosine phosphatase (PTP), and suppressors of cytokine signaling (SOCS). The constitutively expressed PIAS proteins transfer small ubiquitin-like modifiers (SUMO) to STATs, which are then unable to bind to DNA.^7,8 PTPs remove phosphate groups from tyrosine residues of JAK-STAT pathway proteins.^9,10 Lastly, SOCS form protein complexes which degrade JAKs or relevant receptors by transferring ubiquitin to them or they directly block JAK-STAT signaling.¹¹

JAK3, unlike its more ubiquitously expressed isoforms, is localized only in immune cells and certain epithelial cells.¹² Given this distribution pattern, it is not surprising that mutations in the JAK3 gene are associated with disorders related to function of the immune system: Increased activity of JAK3, whether by an activating mutation or otherwise elevated signaling, contributes to a variety of autoimmune diseases and also some leukemias.¹³⁻¹⁵ However, JAK3 is coexpressed and activated alongside JAK1.¹⁶ This leads to the question of whether inhibition of either JAK1 or JAK3 or both is required to treat autoimmune diseases. Selective inhibition of one isoform could provide the correct balance of suppression of autoimmunity without influencing general immunity to pathogens.¹⁷

Answering this question using in vivo disease models requires JAK-isoform specific, stable inhibitors with comparable exposure in vivo. As of now, no JAK3 selective inhibitors have been approved.¹⁸⁻²¹ Well-known commercially available JAK inhibitors include tofacitinib, ruxolitinib, and baricitinib. Tofacitinib (1, Figure 1), a first generation clinical JAK inhibitor, was initially reported to be a JAK3-selective agent but is now known to be nonselective (JAK1 IC₅₀ = 15 nM, JAK3 55 nM, JAK2 71 nM, TYK2 472 nM).²² It is used for the treatment of rheumatoid arthritis, psoriatic arthritis, and ulcerative colitis.¹⁹⁻²¹ Ruxolitinib is a JAK1 (IC₅₀ = 3.3 nM) and JAK2 (2.8 nM)-selective inhibitor that also inhibits TYK2 (19 nM) to a lesser extent (JAK3 428 nM).²³ It has been approved for use in myelofibrosis and polycythemia vera.^24,25 Baricitinib, a selective reversible inhibitor of the JAK1 (IC₅₀ = 5.9 nM) and JAK2 (5.7 nM) isoforms (JAK3 253 nM, TYK2 14 nM), received FDA approval in 2018 and is indicated for the treatment of rheumatoid arthritis.²⁶ Upadacitinib is a JAK1 (IC₅₀ = 0.8 nM) specific inhibitor (JAK2 19 nM, JAK3 224 nM, TYK2 118 nM).^27,28 It has been approved for use in atopic dermatitis and rheumatoid arthritis, adding to the spectrum of disorders that respond to this mode of action.

Figure 1.

Structure of the pan JAK inhibitor Tofacitinib, the JAK3 selective inhibitor FM-381, and the macrolide antibiotic Azithromycin. The latter was used as a starting point for the design of carrier scaffolds to conjugate with JAK3 inhibitor warheads.

The logic of JAK optimization to date has been to focus on JAK1 specificity given that JAK2 is associated with signaling in hematopoiesis. JAK3 is implicated in relatively few signaling pathways relative to JAK1, suggesting that JAK1 would be the target with the broader anti-inflammatory relevance. JAK1 is, however, a regulator of innate immunity, whereas JAK3 is associated with signaling pathways specific to adaptive immune signaling. In particular, JAK1 inhibitors prevent signaling via the IL-10 receptor which may limit or counter-regulate their beneficial effects.²⁹ Furthermore, accumulating evidence points to an increased risk of major cardiac adverse events in patients treated with JAK inhibitors. While a definitive cause has not been identified yet, a common element of the compounds in question is the inhibition of JAK1.³⁰⁻³³

The four JAK isoforms are highly conserved, which complicates the development of specific inhibitors. JAK3, while similar to JAK1, possesses a cysteine (C909) instead of a serine near the ATP binding pocket. The reactive thiol residue can be covalently bound to electrophilic inhibitors in order to gain both potency and selectivity over the other isoforms.^34,35 The same principle has been successfully employed in designing inhibitors of other kinases that bear an accessible cysteine near the ATP binding pocket, e.g., EGFR, BTK, and KIT/PDGFR.³⁶⁻³⁸ In 2016, Forster et al. described a highly selective and potent JAK3 inhibitor, termed FM-381 (2, Figure 1) with a covalent-reversible binding mode.^39,40 This binding mode is characterized by the formation of a covalent bond to the target enzyme for as long as the inhibitor is sufficiently stabilized in the optimal position by surrounding amino acids. After denaturation of the tertiary structure, e.g., by proteasomal degradation, the inhibitor is released again in its native, unbound form.^41,42 An advantage of this binding mode is prolonged efficacy, considering that after kinase degradation, the “regenerated” inhibitors are once again available for binding. Additionally, the chance of off-target covalent binding, which bears the risk of haptenization and allergic reactions, is reduced compared to conventional covalent inhibitors.⁴³

Although well characterized in vitro, this series was not optimized in terms of in vivo properties. To test the hypothesis that JAK3 selective inhibitors could provide more nuanced disease modification effects, we set out to improve the drug-like properties of the series. We pursued two main lines of improvement: stabilizing side groups to metabolism and conjugation to immunotropic/lysosomotropic carrier structures that convey a greater volume of distribution and distribution-bias to activated immune cells.

This was done by covalently linking the discussed structures to macrolide-based carrier molecules. Macrolides, e.g., the azalide azithromycin (3, Figure 1), are known for their substantial uptake into immune cells and tissues, especially inflamed tissue. The azalides accumulate in acidic compartments, particularly in activated immune cells where they also exert nonspecific immune modulation. These effects include, for example, a decreased production of proinflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) coupled with a simultaneous increase in IL-10 levels. This makes them interesting anti-inflammatory substances even outside the context of infection, and their use in chronic inflammatory disease has been discussed.⁴⁴⁻⁵⁰

JAK3 is an inducible kinase whose expression is upregulated by inflammatory stimuli.^51,52 By conjugating inhibitors to carriers selective for tissues where we expect high concentrations of the target kinase, we set out to achieve an equal or greater clinical effect at lower molar quantities compared to the unlinked analogues, while reducing potential for off-target effects. To this end, we worked to identify potent JAK3 inhibitors that were well distributed into tissue or targeted to myeloid cells such that we could compare their effects to well-characterized substances with JAK1 binding.

Results and Discussion

JAK3 Structure–Activity Relationships

Forster et al. described a highly selective and potent JAK3 inhibitor in 2(39,40) but provided no in vivo pharmacokinetic (PK) data for 2. To better understand the series, we first assessed PK in BALB/c female mice. Although possible metabolites of 2 are reported,³⁹ we measured only the unchanged substance in plasma and in organs.

Apparent oral bioavailability (BA) was 10.4% (average of 4 studies), which is low vs tofacitinib (57% in BALB/c mice⁵³ and 29% in Sprague–Dawley rats).⁵⁴ Elimination half-life in plasma after i.v. injection was 23 min (five different studies, n = 3 each, 2 being a reference compound in all studies). The highest organ concentration of 2 was found in lung tissue 8 h after i.v. administration (Figure 2). Oral administration was associated with higher levels in ileal tissue and bile. The high levels in bile, even 8 h after administration, suggest that 2 is efficiently absorbed via the liver and eliminated into bile. The relatively long duration of retention in the bile is probably related to the fact that studies were conducted in the light period where mice are less active. Compound 2 was the lead for further structure–activity relationship (SAR) exploration and the reference for PK studies. The substance has up to 330-fold selectivity for JAK3 over JAK1 (depending on assay format and ATP concentration). A feature contributing to its potency and selectivity is the nitrile substituent present near the acrylamide. Another driver of isoform selectivity is the formation of the arginine pocket via interactions with the side chains of R911 and R953.^39,40 An important feature of JAK3 is the C909, which replaces a serine found in other isoforms.⁵⁵ The electrophilic warhead forms a covalent bond to its thiol residue when brought into proximity. We thus chose to maintain the cyanoacrylamide motif and the furyl linker moiety in order to maintain potency, while varying the side chain addressing the ATP binding pocket (a cyclohexyl group in the case of 2) and the solvent-exposed amide. While we monitored JAK3 affinity, our aim was more to detect improvements to in vivo kinetics. SAR for organ distribution within a given scaffold could also provide modeling data and help to better understand why similarly potent compounds perform differently in various disease models.

Figure 2.

Comparison of organ concentrations of 2 in mice, 8 h after administration of 12 μmol/kg p.o. or 2.4 μmol/kg i.v.; n = 3. Plotted are the mean and 95% confidence interval.

Our first series of compounds (Table 1) retained the cyclohexane residue, and modifications to the lead structure were made at the acrylamide. Alterations at this position had limited effects, leading to only negligible decreases in inhibitory potency, except for the N-(2-(dimethylamino)ethylamide 5 which was much less active (JAK3 IC₅₀ = 133 ± 9 nM). None of these substitutions improved plasma half-life and oral BA, except for 5 which had an increased half-life (31 min). This may be due to α₁-acid glycoprotein binding caused by the additional basic moiety, which would slow redistribution from protein to plasma, or due to accumulation in acidic compartments.⁵⁶ Conversely, the methyl ester-bearing 4 could not be detected in any of the plasma samples, most likely being rapidly cleaved by unspecific plasma enzymes. We expect the free acid metabolite to still possess JAK3 inhibitory activity because the acid residue is facing the solvent-exposed site of the ATP binding pocket and does not partake in critical interactions with the kinase.

Table 1. JAK3 Inhibition Values and PK Parameters of Cyclohexane Series.

^aIC₅₀ values are calculated by ELISA;⁵⁷ average ± SEM (n = 3).

^bBiological half-lives are calculated from i.v. PK studies.

^cBA = apparent oral bioavailability.

^dt_1/2 and BA could not be calculated due to a lack of quantifiable compound concentrations in PK study samples.

Organ concentrations after p.o. administration were similar compared to the lead 2, with most compounds found mainly in ileum and bile samples (see Supporting Information, Figures S7–S13, for details). As ilea were thoroughly washed before sample workup, this is most likely not just a consequence of low oral uptake, but of actual distribution and retention in gut tissues and biliary excretion (see i.v. data). Several members of this compound class were also found in renal tissues, suggesting both rapid partition from plasma to kidney and possibly elimination via urine.

For a second series of derivatives (Table 2), the cyclohexane group was exchanged for a tetrahydropyran-4-yl residue. JAK3 IC₅₀ values remained in the two-digit nanomolar range. While the direct lead analogue 10 had a 2-fold loss of potency, the same drop was not observed when comparing 11–16 to their cyclohexane analogues 4–8: With the exception of 12, where an improvement over 5 was achieved, IC₅₀ values were similar. Plasma half-lives decreased slightly, as did BA. It is possible that the tetrahydropyran ring is prone to cytochrome P450 (CYP)-mediated O-dealkylation to form aldehydes or alcohols. Alternatively, oxidation of carbons vicinal to the cyclic ether moiety seems plausible. Organ distributions mirrored the previous findings for most compounds, with lower overall concentrations. A strong preference for kidneys was found in 13 and 14, albeit with high variance between individual mice potentially due to variable renal elimination rates.

Table 2. JAK3 Inhibition Values and PK Parameters of Tetrahydropyran Series.

^aIC₅₀ values are calculated by ELISA;⁵⁷ average ± SEM (n = 3).

^bBiological half-lives are calculated from i.v. PK studies.

^cBA = apparent oral bioavailability.

^dt_1/2 and BA could not be calculated due to a lack of quantifiable compound concentrations in PK study samples.

The third series (Table 3) incorporated an N-methylpiperidine-4-yl group, which proved to be detrimental to JAK3 inhibition. Between 2 and its analogue 17, a 10-fold loss of potency was observed. The change was less drastic, but still apparent, for 18 and 19 (2- and 5-fold, respectively). Given that the tertiary amine is likely protonated at cytoplasmic pH, it appears that a cation is suboptimal at this position. This was also discussed by Flanagan et al., who mentioned loss of activity when higher pK_a values of headgroup nitrogens led to ionization.⁵⁸

Table 3. JAK3 Inhibition Values and PK Parameters of 1-Methylpiperidine Series.

^aIC₅₀ values are calculated by ELISA;⁵⁷ average ± SEM (n = 3).

^bBiological half-lives are calculated from i.v. PK studies.

^cBA = apparent oral bioavailability.

^dt_1/2 and BA could not be calculated due to a lack of quantifiable compound concentrations in PK study samples.

The compounds in this series were also eliminated or transformed rapidly in vivo: For 17 and 19, no quantifiable levels were detected in any of the plasma samples from the kinetic studies, while 18 was only detectable after i.v. application. The compounds may be particularly vulnerable to N-dealkylation of the methyl group,^59,60 which was also observed in liver microsome experiments (see below), although rapid tissue distribution may also be a factor. High concentrations of 18 were found in organs including brain after i.v. administration, (see Supporting Information, Figure S22). This is surprising given that conventional kinase inhibitors rarely cross the blood–brain barrier (BBB).^61,62 The other substance still detectable in brain samples after 8h was 19, with concentrat ions 4-fold over its JAK3 IC₅₀ after p.o. administration and about 30-fold after i.v. administration (Figure 3). When considering traditional “rules” for BBB penetration,⁶³ both compounds would seem less likely to cross into the central nervous system (CNS) compared to the other compounds, however cation transporters may explain this effect. Active transport, e.g., by organic cation transporters, into CNS or other tissues has been reported for a variety of well-established drugs that contain tertiary amines, e.g., first-generation H₁-antihistamines or several opiates.⁶⁴⁻⁶⁶ Distribution into brain tissue, coupled with a JAK3 IC₅₀ of 99 nM, would make 19 a potential tool compound for evaluating the role of JAK3 in models of neurodegenerative disease.

Figure 3.

Comparison of organ concentrations of 19 in mice, 8 h after administration of 12 μmol/kg p.o. or 2.4 μmol/kg i.v. (n = 3). Plotted is the mean and 95% confidence interval.

In a fourth series (Table 4), we replaced the cyclohexane group with a methyl cyclopropane residue. For all four compounds, activity decreased between 4- and 10-fold when compared to their analogues from previous pairings. A similar drop in potency was observed with the cyclopropyl-substituted 34. It appears that a smaller side chain does not fill the ATP binding pocket adequately, leading to reduced inhibitor binding. This is consistent with previous findings by Forster et al., where replacement of the cyclohexyl residue by a methyl group led to ca. 40-fold loss of activity.⁴⁰ Plasma half-lives in the methyl cyclopropane series were still short, with none greater than 18 min, although 20 had high AUC values (see Supporting Information, Figure S24). Oral bioavailability was improved for 20 (BA 25%, 2.5 times greater than 2) and 21 (BA 35%). While the BA for 21 was relatively high, absolute levels were low because it is a labile methyl ester.

Table 4. JAK3 Inhibition Values and PK Parameters of Methylcyclopropane Series.

^aIC₅₀ values are calculated by ELISA;⁵⁷ average ± SEM (n = 3).

^bBiological half-lives are calculated from i.v. PK studies.

^cBA = apparent oral bioavailability.

A remarkable improvement in plasma stability was found in 34 (Table 7), which had a plasma half-life of 128 min (an over 5-fold improvement over the lead compound). Based on these data, we hypothesize that the position connected to the imidazole group is particularly vulnerable to metabolic conversion, e.g., oxidation by CYP enzymes. The planar, angularly strained cyclopropane ring is more resistant to proton abstraction compared to larger carbocycles and is, therefore, metabolically stable.⁶⁷ Although the smaller residue lost potency (JAK3 IC₅₀ = 122 nM), it had a relatively high AUC vs 2, 10, and 20 (see Supporting Information, Figure S38). Like the other N,N-dimethylamides, 34 showed a preference for lung tissues after i.v. and for ileum and bile after p.o. administration.

Table 7. JAK3 Inhibition Values and PK Parameters of 33 and 34.

^aIC₅₀ values are calculated by ELISA;⁵⁷ average ± SEM (n = 3).

^bBiological half-lives are calculated from i.v. PK studies.

^cBA = apparent oral bioavailability.

As a consequence, we next set out to increase stability by quaternization of the suspected metabolic “weak point”. Series 5 (Table 5) features a phenyl residue and thus no protons at the connecting carbon. The resulting compounds showed increased half-lives when compared to their aliphatic analogues: For example, 24 has a 5-fold longer plasma half-life than 2 (133 min, Figures 4 and 5).

Table 5. JAK3 Inhibition Values and PK Parameters of Benzene Series.

^aIC₅₀ values are calculated by ELISA;⁵⁷ average ± SEM (n = 3).

^bBiological half-lives are calculated from i.v. PK studies.

^cBA = apparent oral bioavailability.

Figure 4.

Plasma concentrations of 2, 24, and 34 measured from murine plasma samples after administration of 2.4 μmol/kg i.v. (n = 3).

Figure 5.

Structural comparison of 2, 24, and 34 and their respective activities vs JAK3 and plasma half-lives in Balb/c female mice after administration of 2.4 μmol/kg i.v.

Similar increases were seen in 25 (71 min, 6-fold longer vs 6), 27 (34 min, 3-fold vs 14), and especially 26 (whose analogues 4 and 11 were not stable enough to calculate half-lives). The stability of 26, which has a half-life of 182 min, is surprising given that it contains the same methyl ester moiety that we thought responsible for the instability of 4 and related compounds. The introduction of a phenyl group also led to a noticeable loss of activity. All four members of this series have JAK3 IC₅₀ values in the three-digit nanomolar range, the lowest being 175 nM (24). The aromatic group’s rigidity compared to aliphatic analogs might be one reason for the detrimental effect on inhibitory potency. The series had poor apparent oral BA, ranging between 0.9 and 2.5%. High levels of 24 in liver tissue were observed in i.v. PK studies (see Supporting Information, Figure S28), while 26 also had high concentrations in ileum and bile samples after p.o. administration, reaching three-digit micromolar values on average. These data suggest that while uptake to these sites may have been limiting, metabolic transformation was probably the main factor reducing the observed levels.

The last series (Table 6) consisted of the nucleophilic warheads linked to macrolide carriers derived from the antibiotic drug azithromycin. Macrolides accumulate in immune cells to higher concentrations than in plasma, which can be useful for increasing distribution into inflamed tissues. Additionally, apart from their antibacterial effects, they possess certain immunomodulatory properties themselves.⁴⁴⁻⁴⁶ By virtue of their basic moieties, they become trapped in acidic compartments, driving the aforementioned distribution. It has been demonstrated that by covalently linking small molecule drugs to macrolide carriers, the drugs’ PK properties can be improved while still preserving enough of their biological activity.^47,48 Through modifications to the macrolide structure, e.g., removal of the cladinose sugar or introduction of bulky residues to the desosamine, the carriers’ antibiotic properties can be reduced, thus avoiding potential selection for bacterial macrolide resistance.⁴⁹ We used azithromycin as a model compound for the carrier due to its metabolic stability and high ratio of tissue to serum concentration.⁶⁸ Also, compared to erythromycin, the azalide is considerably more resistant to acidic conditions in the stomach and has less affinity for human ether-á-go-go-related gene (hERG) channels, the inhibition of which could result in long QT syndrome.⁶⁹⁻⁷¹

Table 6. JAK3 Inhibition Values and PK Parameters of Macrolide Conjugate Series.

^aIC₅₀ values are calculated by ELISA;⁵⁷ average ± SEM (n = 3).

^bBiological half-lives are calculated from i.v. PK studies.

^cBA = apparent oral bioavailability.

Our macrolide-linked series was comprised of the previously developed cyanoacrylamide warheads linked to the desosamine moieties by a short alkyl chain. Compounds 28–30 were less potent in cell-free JAK3 inhibition assays vs their unlinked counterparts, with JAK3 IC₅₀ values in the range of 192 (28) and 751 nM (30). Despite the cyanoacrylamides facing the outside of the ATP binding pocket, (being solvent exposed) the large macrolides may still impede the inhibitors’ reversible binding mode and the displacement of ATP. However, a loss of in vitro activity does not necessarily correlate with lower potency in vivo, or even in cellular models if it increases exposure to the target. Compounds 31 and 32, whose linker is lengthened by an additional methylene group compared to the analogs 28 and 30, exhibited substantially better activity (31 IC₅₀ = 35 nM vs 28 IC₅₀ = 192 nM, 32 IC₅₀ = 109 nM vs 30 IC₅₀ = 751 nM). We propose that the slightly increased length of the alkyl chain connecting the electrophilic warhead to the carrier allows for greater flexibility and sufficiently increases the distance from the macrolide to the ATP binding pocket. This in turn improves the ability of the core structure to displace ATP and inhibit JAK3 activity. Plasma half-lives of the macrolide series range between 55 (30) and 98 (28) min, which, although considerably higher than those of the corresponding nonmacrolide analogs, are still influenced by rapid distribution into tissues and explain the high concentrations of 28–32 in organ samples taken after 8 h (Figure 6, Supporting Information, Figures S32–S36). When comparing the macrolide series to the unlinked compounds, it is apparent that the macrolide scaffold is exerting a dominant influence on the PK properties, increasing both stability and distribution to tissues. Concentrations were high in liver and kidney samples for all five compounds, implying similar distribution patterns to those of azithromycin.⁷²⁻⁷⁴ However, this partition to leukocytes and tissues was associated with an apparent oral BA of 3–6%, which indicates that while the oral route in animals gives rise to very high tissue levels, these are based on seemingly low overall uptake. One has to take into account, however, that the so-called apparent oral BA is calculated solely from the concentrations measured in blood or plasma samples, disregarding any organs or other tissues. A more suitable parameter would be the steady-state volume of distribution V_ss, which would require prolonged dosing studies to be determined. Interestingly, the N-methylpiperidine type 32 was even found in brain tissues in meaningful concentrations (7-fold over the JAK3 IC₅₀ after i.v. administration, 4-fold after p.o.). While a molecule of this size would seem unlikely to pass the BBB, it possesses the same N-methylpiperidine moiety that we consider a candidate for active transport (see above).

Figure 6.

Structure, JAK3 IC₅₀, and plasma half-life in mice of 32 (left). Comparison of organ concentrations of 32 in mice, 8 h after administration of 12 μmol/kg p.o. or 2.4 μmol/kg i.v. (n = 3). Plotted is the mean and 95% confidence interval (right).

We also prepared a single compound featuring a 4,4-difluorocyclohexyl residue, 33. We observed a 2–3-fold loss of potency compared with the cyclohexyl counterpart 2, although JAK3 IC₅₀ was still reasonably low at 28 nM. The plasma half-life of 22 min was in a similar range as 2, suggesting that metabolism likely occurs at a different position of the ring. There was a notable increase in both oral c_max (from 164 nM to 912 nM) and BA (from 10% to 46%), which we attribute to the enhanced lipophilicity of the difluorinated ring (Table 7). Like the other N,N-dimethylamides, 33 was most abundant in lung tissues after i.v. administration.

To investigate the importance of the Michael acceptor to the inhibitory potency of 2, we saturated the cyanoacrylamides’ double bond to obtain 36 (Figure 7), which caused an over 50-fold decrease in activity (IC₅₀ = 657 nM). In a closely related compound lacking the nitrile function, only a 3-fold loss of potency was reported after hydrogenation. This was seen as an indication that a covalent binding mode is not likely without additional stabilization by the nitrile group’s interaction with R911 and R953.⁴⁰ Likely the greater loss in potency compared to that compound is due to the ability of the cyanoacrylamide 2 to form a covalent bond to C909, an ability which is lost after saturation. Hydrogenation caused oral BA to increase 4-fold to 45%. This may be attributed to the cyanoacrylamides’ ability to undergo nonspecific Michael reactions with cysteine residues in the gastrointestinal contents and mucosa. Another possibility is their reduction by glutathione or similar antioxidants after uptake by gut cells.

Figure 7.

Structures, JAK3 IC₅₀ values and oral BA of tool compounds (macrolide carrier 35 and reduced lead compound 36). IC₅₀ values are calculated by ELISA⁵⁷ as average ± SEM (n = 3).

We also determined the pharmacokinetics of the unlinked macrolide carrier 35. Apparent oral BA was as low as that of 28–31. However, as with the aforementioned compounds, both i.v. and p.o. application led to high concentrations in organ tissues even after 8 h, especially when compared to the unlinked inhibitors (see Supporting Information, Figure S39, for organ concentration data). These data suggest that while macrolides maintain their properties of tissue distribution, overall uptake or extraction and detection may be hindered by large or reactive side-chains.

A general trend for many compounds, including the macrolides, was secondary plasma concentration peaks after p.o. administration at 120 or 240 min after treatment. This is in line with the high levels in bile and ileum and is consistent with hepatobiliary cycling (see Supporting Information, Figures S7–S40).

To summarize, it appears that modification of the amide residues is well-tolerated and potency is rarely affected, as they are noncritical for kinase inhibition. Instead, modifications to this site can be employed to optimize physical parameters like solubility or polarity. This has already been shown in previous reports on this compound class.⁴⁰ We have demonstrated that the amide residues can be similarly fine-tuned to adjust PK properties, especially organ distribution, for specific purposes. While modifications to the cyclohexane residue can impact distribution and uptake, it appears that good activity versus JAK3 seems to require a sufficiently large residue, ideally a six-membered ring system, to adequately fill the target pocket. A planar, rigid group (e.g., phenyl) or basic, potentially charged moieties are less tolerated than the more flexible cyclohexyl. Second, the labile residues can be modified in a manner that impedes the proton abstraction necessary for transformation, so in vivo stability is improved considerably.

Lastly, conjugation of an active kinase inhibitor warhead to a suitable carrier scaffold, e.g., a macrolide, is an elegant means of conveying the latter’s useful PK properties to the former. Using a sufficiently spaced linker, as is the case, e.g., for 31, this can be done while retaining high activity versus the target. Good potency coupled with high in vivo stability and uptake into the intended target tissues may improve overall efficacy.

Mouse Liver Microsomal Experiments

A selection of compounds was tested for in vitro stability in mouse liver microsomes (MLM) (Table 8). In contrast to the data observed in vivo, 2 demonstrated better stability than its cyclopropyl and phenyl series analogs 34 and 24: After 2 h of incubation, more than 75% of unchanged compound remained, while concentrations of 34 and 24 decreased to 54 and 44%, respectively. Unsurprisingly, 19 was the least stable derivative with <5% of the original compound measured after 2 h. The m/z values and retention times of its metabolites suggest hydrolysis of the methyl ester and N-demethylation of either the side chain or the amide. For 34, metabolites were most likely formed by demethylation, hydroxylation at different positions (as suggested by the occurrence of metabolites of the same m/z, but with different retention times) and combinations thereof. The same findings apply to 24, along with masses corresponding to loss of the phenyl side chain.

Table 8. MLM Stability of Selected Compounds.

compd	compd after 2 ha (%)	t1/2 (min)
2	79 ± 8.9	142
19	4 ± 0.4	26
34	54 ± 1.4	133
24	44 ± 5.3	103

^aDisplayed as the percentage of unchanged compound remaining vs t₀. Samples were incubated at 37° and aliquots taken at 6 different time points; n = 3.

While the calculated MLM half-lives of 24 and 34 are in line with their in vivo values, 2 appeared to have a greatly increased half-life (142 min vs 23 min in vivo). This would suggest that the rapid elimination of 2 may be more due to reactivity, tissue distribution and direct elimination rather than by liver metabolism.

Cellular Activity and Target Engagement

A selection of compounds was chosen for further in vitro characterization with bioluminescence resonance energy transfer (BRET) assays. Using fluorescent tracer K10 and HEK293T cells expressing NanoLuc JAK3, NanoBRET experiments were carried out to determine cellular JAK3 IC₅₀. In these, a luciferase-conjugated protein, in this case JAK3, is used to excite a fluorophore conjugated to a (JAK3 ligand) tracer molecule, generating a fluorescence emission based on the proximity of the bioluminescent protein and the ligand. As the ligand is displaced from the binding pocket by a competitive JAK3 inhibitor, the signal intensity declines. In addition to the assay using intact cells, a parallel assay with lysed cells was carried out to determine the compounds’ activity independently of factors like intracellular localization. The latter part plays a particular role for the macrolide conjugates, as they are expected to accumulate mainly in acidic compartments like lysosomes, which would make them less available for inhibition of the fusion protein. Along with the JAK3 inhibitor test compounds, we also included Tofacitinib (1) as a reference. The results of the assay can be found in Table 9.

Table 9. Cellular Activity and Target Engagement of JAK3 as Determined by nanoBRET.

compd	intact IC50 (nM)a,b	lysed IC50 (nM)a,c	ratio intact/lysed
1	892 ± 398	228 ± 34	3.9
2	347 ± 177	98 ± 3	3.5
6	877 ± 22	115 ± 2	7.7
17	1029 ± 399	269 ± 11	3.8
19	1361 ± 174	209 ± 11	6.5
20	n.d.d	271 ± 9	–
24	9706 ± 1533	474 ± 148	20.5
28	>45000	16490 ± 2659	–
31	19860 ± 3833	2201 ± 129	9.0
32	12564 ± 5440	765 ± 74	16.4
33	597 ± 117	131 ± 11	4.6
34	3807 ± 122	435 ± 85	8.7

^aObtained from BRET signal ratios at 11 inhibitor concentrations. IC₅₀ calculations were done using a normalized 3-parameter curve fit.

^bn = 4.

^cn = 2.

^dDisplacement could not be determined for this compound due to interference with the BRET signal.

The unconjugated compounds retained their good potency against JAK3 in the NanoBRET assay. As is to be expected, IC₅₀ values were increased by the transition to a cellular setting due to factors like cell permeability and high intracellular ATP concentrations. When ranked against one another, the compounds’ individual potency generally matched the results from the enzymatic assay: The most potent compounds are still lead compound 2 (IC₅₀ = 347 nM), the difluorinated analog 33 (IC₅₀ = 597 nM), and morpholine amide analog 6 (IC₅₀ = 877 nM). A relative improvement among the candidates was noted for the N-methylpiperidine substituted 17, whose potency was closer to the aforementioned compounds than in the enzymatic assay. The macrolide conjugates 28, 31, and 32 were notably less potent in the intact cell assay (IC₅₀ range from 12 μM to >45 μM), which is especially surprising in the case of 31, which had previously demonstrated great potency in the enzymatic assay (IC₅₀ = 35 nM). This could be possibly due to either low uptake into cells caused by hindered membrane passage, increased accumulation in acidic compartments, decreasing the compounds’ availability in the cytosol, or problems with accessing the large JAK3 NanoLuc fusion protein. In the lysed assay, their activity increased by a large proportion, which supports the aforementioned accumulation theory. A considerable improvement between assays was also observed for the phenyl residue compound 24 (9.7 μM to 0.47 μM). Overall, the data indicate good cellular target engagement for the majority of the selected compounds, complementing our previous findings on 2 and related substances.⁴⁰

Chemistry

For synthesis of the JAK inhibitors 2–36, we made slight modifications to the previously reported methods employed by Forster et al.^39,40 Our modified synthetic route as shown in Scheme 1 began by protection of position 1 of the commercially available 4-chloro-1H-pyrrolo[2,3-b]pyridine via p-tosyl chloride, followed by nitration of position 5. Next, the side chains R_1′ were added as aliphatic or aromatic amines by way of SN_Ar reaction. We modified the following hydrogenation step by using Pt–C (5%) as catalyst instead of Pd–C, allowing for reactions at rt instead of 60 °C and the replacement of the potentially hazardous EtOAc/MeOH solvent mixture with EtOAc. In place of a hydrogenation reactor, a water column was used to induce mild overpressure, leading to comparatively longer reaction times. Alternatively, reduction of the nitro group was achieved via reaction with Na₂S₂O4 in EtOH/H₂O at 70 °C with simple workup and satisfying yields.⁷⁵ The vicinal diamines were then reacted with 5-(hydroxymethyl)furan-2-carbaldehyde and oxone to obtain imidazoles. In the case of N-methylpiperidine-substituted 41c, this led to an intermediate N-oxide which was reduced by titration with TiCl₃ solution before the product was carried on to the next step. Oxidation of the furylic alcohols to aldehydes was achieved via Dess–Martin periodinane in DCM. Next, the tosyl group was removed by stirring in 1 M KOH solution in methanol. This step proved problematic for the base-labile intermediate 42d, leading to occurrence of side products and low yields. Other deprotection methods (reducing agents, carbonates, aniline, DABCO, ultrasonification with Mg) did not achieve satisfying results, either failing to generate the desired product or having even lower yields (data not shown). The finalized JAK inhibitors were then synthesized via Knoevenagel condensation with the corresponding cyanoacetamides. When employing more than 1.0 equiv of cyanoacetamide, we often observed formation of adducts of the surplus reagents to the product, as suggested by MS m/z values and TLC controls. We thus advise using equimolar amounts of starting materials. To obtain the saturated 36, we used NaBH₄ in a hydrogenation reaction at ambient temperature with short reaction times and simple workup.

Scheme 1. Synthesis of JAK Inhibitors.

Reagents and conditions: (i) NaH, p-TsCl, THF, 0 °C to rt, 92%; (ii) Me₄N·NO₃, TFAA, DCM, 0–5 °C, 72%; (iii) corresponding amine, Et₃N, iPrOH, reflux, 85–95%; (iv) Pt/C, H₂, EtOAc, rt, 78–100%; (v) Na₂S₂O₄, EtOH/H₂O, rt, 98%; (vi) 5-(hydroxymethyl)furan-2-carbaldehyde, KHSO₅, DMF/H₂O, rt, 42–85%; (vii) DMP, DCM, 0 °C to rt, 70–90%; (viii) KOH, MeOH, rt, 28–88%; (ix) corresponding cyanoacetamide, piperidine, MeOH or iPrOH, 50–70 °C, 32–77%; (x) (from 2) NaBH₄, MeOH, rt, 89%.

The macrolide-linked cyanoacetamides 35 and 47 were prepared according to Scheme 2. In a first preparative step, azithromycin was epoxidized to form 44.⁴⁴ We employed a modified procedure using excess racemic glycidol instead of epichlorohydrin and iBuOH as solvent. The formation of an epoxide and its stereochemistry were then elucidated using 2D NMR as well as X-ray crystallography (Figure 8 and Supporting Information, Figures S1–S2).

Scheme 2. Synthesis of Macrolide-Linked Cyanoacetamides 35 and 47.

Reagents and conditions: (i) glycidol, iBuOH, 75 °C, 21%; (ii) corresponding diamine, 60–105 °C, 46–66%; (iii) cyanoacetic acid, HATU, THF, rt 64–83%.

Figure 8.

Structure of 44 and a cocrystallized methanol molecule (middle) as obtained from X-ray crystallography. The modified desosamine moiety with the 2′R, 3′R epoxide is shown at the bottom. The crystallographic data can be accessed at the Cambridge Crystallographic Data Centre (www.ccdc.cam.ac.uk/data_request/cif, deposition number 2121076).

The epoxide was then opened using excess amounts of the corresponding N,N′-dimethylalkyl diamine, which acted as both reagent and solvent. Under the given conditions, both possible isomers formed, but were readily separable by chromatography. The structures for the 2′-amino substituted products were identified by 2D NMR spectroscopy (see Supporting Information, Figures S3–S6, for 45 and 46). Lastly, cyanoacetamides were formed via reaction with cyanoacetic acid, using HATU as a coupling reagent. Other cyanoacetamide reagents for the Knoevenagel condensation step were either synthesized via aminolysis of methyl- or ethyl cyanoacetate or by Schotten–Baumann reaction with cyanoacetyl chloride (see Experimental Section).

Conclusion

In this study, we explored the in vivo PK profile of lead compound 2, which had already been shown to be a highly selective and potent inhibitor of JAK3. The SAR of this compound class was further elucidated with closely related compounds, and we identified several adequately tolerated modifications that allow us to fine-tune PK properties. By variation of the cyclohexane residue, we confirmed a suspected metabolic weak point which can be circumvented by impeding the abstraction of protons or quaternization of the connecting atom, as shown with 24 and 34, respectively. While both modifications were associated with a drop in target affinity, these data suggest that it is possible to improve stability with this pharmacophore while retaining affinity for the target.

The PK studies indicated diverse tissue distribution patterns, which allows us to investigate the question of how distribution patterns impact efficacy in various in vivo disease models: For example, 28 showed high accumulation in the intestines after oral gavage, making it a potentially interesting candidate for inflammatory bowel disease, while the highly lung-selective (when applied parenterally) 2, 10, or 33 will help in elucidating the role of JAK3 in acute respiratory distress syndrome or chronic respiratory diseases.

Lastly, we reported the synthesis of macrolide-derived carriers, their conjugation to the electrophilic warheads, and the impact on activity and PK properties. The azithromycin scaffold confers a robust half-life and a tendency to accumulate into tissues, especially acidic compartments, with high concentrations in most organ samples. As shown with 31, those PK benefits can be applied to the electrophilic warhead while still retaining IC₅₀ values in the lower two-digit nanomolar range.

In conclusion, we have demonstrated the synthesis and pharmacokinetical characterization of a library of potential “in vivo probes” for JAK3 to be used in various animal models of inflammatory disease. A selection of candidates, both unconjugated compounds as well as macrolide conjugates, have already been examined by us for their anti-inflammatory potencies in vitro and in vivo and demonstrated excellent efficacy. The results of these studies will be submitted for publication in the near future. Those data suggest that it will be feasible to create targeted small molecule JAK3 inhibitors for clinical use.

Experimental Section

Experimental Animals

All animal experiments were carried out in accordance with German law. The 7–8 week old BALB/cJ female mice were purchased from Janvier Laboratories and maintained in our dedicated specific-pathogen-free animal facility. They were kept for at least 7 days after arrival for acclimatization. To reduce the number of animals per experiment, compounds were given as cassettes of three to five substances per animal, and n was chosen to be 3 per group. Body weights were approximately 20 g per mouse, with each mouse being weighed right before treatment to ensure equal doses for all animals.

Formulations for Use In Vivo

Treatment solutions were freshly prepared before the start of each study. For i.v. treatment, compounds were dissolved in DMSO and then diluted 40-fold in BALB/c female serum (four compounds, final DMSO concentration 10%) for application at 5 mL/kg to reach a dose of 2.4 μmol/kg per substance. The vehicle for p.o. treatment comprised a solution in DMSO that was diluted 40-fold in 0.5% citric acid (four compounds, final DMSO concentration 10%) with each compound being administered at a dose of 12 μmol/kg. The solutions were thoroughly homogenized via vortex mixer and ultrasonic bath.

Collection of Samples

To measure drug concentrations in plasma, mice were bled from the tail vein at eight time points after treatment. Time points differed depending on the route of application and the study (see Supporting Information). Animals were sacrificed after 8 h by CO₂ inhalation. We collected heart blood, bile, brain, ileum, liver, lungs, kidneys, and spleen. Blood was collected in heparinized tubes and centrifuged for 8 min at 8000 rpm at 4 °C. The supernatant was used to determine plasma concentrations. Both plasma and organ samples were immediately stored at −25 °C until workup for analytics.

Sample Workup for HPLC-MS

Plasma samples were diluted with ACN containing 5 nM sulfentrazone and 1 nM terbuthylazine (used as negative and positive internal standards respectively), homogenized in a FastPrep FP-120 instrument, and then centrifuged at 14,000 rpm for 7 min at 4 °C. Organ samples were treated with 1 μL proteinase K solution (0.5 mg/mL in 20 mM phosphate buffer) per mg organ weight and then worked up analogously to the plasma samples. Bile samples were diluted with water, homogenized, and then further diluted with ACN plus internal standards, followed by homogenization and centrifugation.

Sample Analysis

Compound concentrations were measured using reverse-phase HPLC with MS detection. The procedure used a mobile phase comprised of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Method: 10% B for 1 min, to 100% B in 4 min, 100% B for 2 min, to 10% B in 1 min, 10% B for 2 min, stop time 10 min, flow rate 500 μL/min, injection volume 6 μL. Using a thermostat, a constant column temperature of 45 °C was maintained, while the samples were kept at 6 °C.

Calculation of Plasma Half-Lives

Half-lives of compounds in mice were calculated using the plasma concentrations measured with the aforementioned methods. To reduce the influence of distribution processes, plasma concentrations before t = 15 min were not included. Values outside the quantification limits were disregarded for the calculation of the elimination constants and resulting half-lives.

JAK3 Inhibition Assays

To determine JAK3 IC₅₀ values, assays were a described previously (Bauer et al., 2014).⁵⁷ In short, a 96-well plates was fitted with artificial, tyrosine-rich peptides to act as phosphorylation targets. A fragment (amino acids 781–1124) of human JAK3 containing the active site was incubated with 1.4 μM ATP (twice the K_m value), leading to phosphorylation of the peptide substrate. By addition of a horseradish peroxidase-conjugated phosphotyrosine antibody followed by 3,3′,5,5′-tetra-methylbenzidine, color development proportional to bound antibody was observed. The reaction was stopped after a set time via sulfuric acid, and the optical density (OD) was determined at 450 nm. Through inclusion of potential inhibitors to the incubation step at varying concentrations, their inhibitory potencies could be determined by comparison of the resulting OD₄₅₀ values to those of control reactions.

Mouse Liver Microsome Experiments

Murine liver microsomes (Xenotech, CD1 male) were preincubated in an NADPH-regenerating system (5 mM glucose-6-phosphate, 1 mM NADP⁺, 5 U/mL glucose-6-phosphate dehydrogenase) with 4 mM MgCl₂ in 0.1 M tris buffer (pH 7.4) at 37 °C.

After addition of the analytes (to a final concentration 0f 100 μM), microsomes were kept at 37 °C, and aliquots were taken at several time points from 0 to 120 min (0, 10, 20, 30, 60, 120 min). Aliquots were mixed with 100 μL of 50 μM internal standard in acetonitrile, then vortexed (30 s) and centrifuged (19.800 relative centrifugal force, 15 min, 4 °C). Supernatants were then used for quantification by LC-MS using an Alliance 2695 HPLC (Waters GmbH, Eschborn) fitted with a Kinetex 2.6 u C18 100 Å column (50 × 3 mm) and an injection volume of 5 μL. Eluent A: 90% H₂O, 10% ACN, 0.1% formic acid. Eluent B: 100% ACN, 0.1% formic acid. Gradient: 10% B for 2.5 min, to 70% B in 7.5 min, 70% B for 2 min, to 10% B in 0.01 min, 10% B for 3 min. Column temperature was set to 40 °C. Detection was performed using a Micromass Quattro microtriple quadrupole mass spectrometer (Waters GmbH, Eschborn) using ESI positive mode. Spray, cone, extractor, and RF lens voltages were set to 4 kV, 30, 5, and 1 V respectively. Desolvation temperature was 350 °C, gas flow was at 650 l/h. Data were analyzed using MassLynx 4.1 software.

X-ray Crystallography

Orthorhombic crystals of 44 (CCDC number 2121076) were obtained by repeated crystallization of the product obtained as described in the synthesis section in MeOH. Diffraction data for 44 (C₃₆H₆₅NO₁₂, CH₃OH, M = 735.93) were collected at the University of Mainz on a STOE IPDS 2T, using Mo K-α radiation (λ = 0.7197 Å). The crystals belonged to space groups P2₁2₁2_1. Unit cell parameters were a = 10.2333(2) Å, b = 17.4881(5) Å, c = 22.6671(7) Å. Z = 4, R1 = 0.0459, wR2 = 0.0937. Goodness-of-fit on F² = 1.026. Data was collected at 120 K. Data visualization and image generation were performed using PyMol.⁷⁶

NanoBRET Assay

The assay was performed as described previously.⁷⁷ In brief: Full-length kinase was obtained as plasmid cloned in frame with a C-terminal NanoLuc-fusion (NV1471, Promega). Plasmid was transfected into HEK293T cells (ATCC) using FuGENE HD (Promega, E2312), and proteins were allowed to express for 20 h. Serially diluted inhibitor and NanoBRET Kinase Tracer K10 (Promega) at a concentration determined previously as the Tracer K10 K_D,app (500 nM) were pipetted into white 384-well plates (Greiner 784075) using an Echo acoustic dispenser (Labcyte). The corresponding protein-transfected cells were added and reseeded at a density of 2.5 × 10⁵ cells/mL after trypsinization and resuspending in Opti-MEM without phenol red (Life Technologies). The system was allowed to equilibrate for 2 h at 37 °C/5% CO₂ prior to BRET measurements. To measure BRET, NanoBRET NanoGlo Substrate + Extracellular NanoLuc Inhibitor (Promega, N2540) was added as stated in the manufacturer’s protocol, and filtered luminescence was measured on a PHERAstar plate reader (BMG Labtech) equipped with a luminescence filter pair (450 nm BP filter (donor) and 610 nm LP filter (acceptor)). Competitive displacement data were then graphed using GraphPad Prism 8 software using a normalized 3-parameter curve fit with the following equation: Y = 100/(1 + 10^{(X – log IC₅₀)}). For the lysed assay format, the HEK293T cells were treated the same way as described above. After the 2 h incubation time with the tracer and compound, 50 ng/mL of digitonin (CAS 11024-24-1) was added using the ECHO acoustic dispenser (Labcyte). After an incubation time of 5 min at RT, NanoBRET NanoGlo Substrate was added, and luminescence was measured on a PHERAstar plate reader.

General

Unless stated otherwise, solvents, reagents, and other materials were of commercial quality and used without further purification. Preparative column chromatography was either performed manually with glass columns and ACROS Organics 60–200 μm silica or by using an Interchim PuriFlash 5.020 automated flash chromatography system with prepacked Interchim columns containing either 15 or 50 μm silica. Gradients for flash chromatography were calculated automatically via the accompanying “TLC to Flash and Prep Chromatography” software. TLC was performed using Merck TLC Silica gel 60 F₂₅₄ plates. For detection, we used UV light at 254 nm or cerium molybdate staining solution. The purity and t_ret of intermediates and final compounds were determined on a Varian ProStar210 system coupled with a SEDEX LT-ELSD 80 LT and using a Dr. Maisch ReproSil-Pur120 C18-Aq column (75 × 3 mm, 5 μm). The mobile phase was composed of water containing 0.05% formic acid (eluent A) and methanol containing 0.05% formic acid (eluent B). Two different gradients were used depending on the analyte: 20% B for 5 min, to 100% B in 20 min, 100% B for 4 min, to 20% B in 1 min, 20% B for 5 min (method A), or: 5% B for 5 min, to 100% B in 20 min, 100% B for 4 min, to 5% B in 1 min, 5% B for 5 min (method B). The flow rate for both methods was 1.3 mL/min. Nitrogen gas was used for the nebulization and evaporation of the mobile phase. Pressure was set at 3.3 bar, and the drift tube temperature of the ELSD was 75 °C. Measurements of samples from in vivo studies were performed by reverse-phase HPLC on an Agilent series system using the 1260 HiP Degasser, the 1260 BinPump, the 1260 HiP ALS and the 1290 Thermostat, using Agilent C18 2.7 μm columns. For detection, the system was connected to an AB SCIEX API 4000 MS using a TurboSpray ion source (settings: source voltage 45 V, ion spray voltage 4.5 kV, temperature 300 °C, gas flow 5 l/min). NMR spectra were either recorded on a Bruker Avance 400 MHz, a Bruker Avance III HDX 400 MHz, or a Bruker Avance III 300 MHz. Chemical shifts are reported in ppm relative to TMS and calibrated against the residual proton peak of the respective solvent. Standard mass spectra were obtained as ESI-MS (pos. mode) from a Thermo Finnigan LCQ Deca XP system (settings: ESI voltage 3.0 kV, capillary voltage 9 V, capillary temperature 275 °C, gas flow 7 l/min).

HRMS measurements were made using a Bruker maXis 4G ESI-TOF from Daltonik at the Institute of Organic Chemistry, Eberhard-Karls-University Tuebingen, using ESI+ mode and the following settings: capillary voltage 4.5 kV, source temperature 200 °C, gas flow 6 l/min, nebulizer gas pressure 1.2 bar, end plate offset −0.5 kV, and an m/z range of 80 to 1350 m/z. All final compounds are ≥95% pure by HPLC. In case of E/Z mixtures obtained from Knoevenagel condensations, purity is calculated from the sum of both isomer peak areas.

Synthesis of 4-Chloro-1-tosyl-1H-pyrrolo[2,3-b]pyridine (37)

In a 2 L three-neck round bottomed flask, 25 g of 4-chloro-7-azaindole (163,9 mmol, 1.0 equiv) was dissolved in 700 mL of dry THF. The solution was cooled with ice/water and stirred under argon. Then, 7.6 g of NaH (60% dispersion in mineral oil, 196.6 mmol, 1.2 equiv) was added portionwise and left to stir for 15 min. Afterward, 33 g tosyl chloride (172 mmol, 1.05 equiv) was dissolved in 120 mL of dry THF and added dropwise. After complete addition, the ice bath was removed and stirring continued at ambient temperature for 2 h until TLC and MS controls indicated full consumption of 4-chloro-7-azaindole. After quenching by addition of 25 mL of saturated NH₄Cl solution, the mixture was diluted with EtOAc and transferred to a separatory funnel. Two portions of 200 mL of 1 M K₂CO₃ and one portion of 200 mL brine were used to wash the organic phase. Afterward it was dried over Na₂SO₄ and evaporated under reduced pressure. Cold MeOH was added to the obtained solid, and it was lightly ground with a glass rod. After decantation of the solvent and evaporation of residues under reduced pressure, 46 g (92%) of the title compound was obtained as a brown solid. ¹H NMR (300 MHz, CDCl₃) δ 8.20 (d, J = 5.3 Hz, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 5.3 Hz, 1H), 6.57 (d, J = 4.0 Hz, 1H), 2.23 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 147.5, 145.6, 145.2, 136.7, 135.1, 129.8, 128.1, 126.92, 122.2, 119.0, 103.3, 21.6. MS (ESI) m/z: 307.33 [M + H]⁺; HPLC t_ret = 0.6 min (as a salt) or 14.0 min (free base) (method A).

Synthesis of 4-Chloro-5-nitro-1-tosyl-1H-pyrrolo[2,3-b]pyridine (38)

44 g of (37) (143,4 mmol, 1.0 equiv) and 25.5 g of tetramethylammonium nitrate (186.4 mmol, 1.3 equiv) were dissolved in 700 mL of DCM in a 1 L three-neck round bottomed flask and stirred. Cooling to an internal temperature of 0–5 °C for the whole reaction was achieved by submersion in an ice/water bath. The flask was then saturated with argon. Over the course of 8 h, 26.3 mL of trifluoroacetic anhydride (186.4 mmol, 1.3 equiv) were added dropwise. Stirring continued overnight while allowing the mixture to reach ambient temperature. As both TLC and MS indicated substantial leftovers of educt (37), the mixture was cooled again, and an additional 10 mL of trifluoroacetic anhydride were added over 8 h. Again, stirring overnight was carried out at ambient temperature. The yellow solution was diluted with DCM and washed, in this order, with water, sat. NaHCO₃ and Na₂CO₃ solutions and brine. The resulting red organic phase was dried over Na₂SO₄ and evaporated under reduced pressure. The solid was suspended in MeOH and stored at −20 °C overnight. The suspension was filtrated, the solids washed with small amounts of cold MeOH and dried in vacuo. 36.9 g (72%) of the title compound was obtained as a beige powder. ¹H NMR (300 MHz, CDCl₃) δ 8.91 (d, J = 3.9 Hz, 1H), 8.04–7.95 (m, J = 7.8, 4.0 Hz, 2H), 7.86 (d, J = 3.9 Hz, 1H), 7.28–7.20 (m, 2H), 6.79–6.71 (m, J = 3.8 Hz, 1H), 2.32 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 147.1, 146.6, 142.2, 140.5, 134.3, 131.2, 130.1, 130.0, 128.5, 122.9, 104.5, 21.8. MS (ESI) m/z: 352.2 [M + H]⁺; HPLC t_ret = 20.9 min (method A).

General Procedure A for Nucleophilic Substitution at Position 4

In a sufficiently large round bottomed flask, 1.0 equiv of (38) was suspended in IPrOH (0.2 M) and stirred. 1.4 equiv of Et₃N and 1.25 equiv (unless noted otherwise) of amine were added, and the mixture is heated to reflux. After confirmation of full consumption of starting material by TLC and/or MS, a 2:1 mixture of H₂O and sat. NH₄Cl solution (35 mL per 1 g (38)) was added portionwise to the hot mixture, leading to precipitation. Stirring at about 80 °C oil bath temperature continued for 5 min. Afterward, the mixture was stirred for at least 30 min in an ice bath. Filtration, aqueous washing, and drying of the precipitate led to the desired products, which most of the time were usable without further purification.

N-cyclohexyl-5-nitro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-amine (39a)

Obtained from 5.03 g of 38 and 2.06 mL of cyclohexylamine following general procedure A with a reaction time of 1 h. No further purification needed after filtration and washing. Yield: 5.65 g (95%) of 39a as a red solid. ¹H NMR (300 MHz, CDCl₃) δ 9.01 (s, 1H), 7.98 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 4.0 Hz, 1H), 7.21 (d, 2H), 6.62 (d, J = 4.0 Hz, 1H), 3.97–3.77 (m, 1H), 2.31 (s, 3H), 2.10–1.96 (m, 2H), 1.86–1.69 (m, 2H), 1.50–1.12 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 148.9, 146.4, 145.9, 144.9, 134.9, 129.8, 128.6, 126.5, 123.6, 107.6, 106.7, 52.9, 33.5, 25.3, 24.4, 21.8. MS (ESI) m/z: 415.33 [M + H]⁺; HPLC t_ret = 21.7 min (method A).

5-Nitro-N-(tetrahydro-2H-pyran-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-amine (39b)

Obtained from 13.0 g of 38 and 4.78 mL of 4-aminotetrahydropyran following general procedure A with a reaction time of 2 h. No further purification needed after filtration and washing. Yield: 14.57 g (95%) of 39b as a bright yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.85 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 8.4, 2.1 Hz, 2H), 7.81–7.74 (m, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.08–6.99 (m, 1H), 4.34–4.22 (m, 1H), 3.88–3.75 (m, 2H), 3.56 (t, J = 11.2 Hz, 2H), 2.33 (s, 3H), 2.02–1.90 (m, J = 12.4 Hz, 2H), 1.74–1.57 (m, J = 19.6, 9.6 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 148.0, 146.0, 145.3, 144.1, 134.1, 130.1, 128.0, 126.4, 124.2, 107.5, 107.1, 65.0, 49.3, 32.8, 21.1. MS (ESI) m/z: 417.4 [M + H]⁺; HPLC t_ret = 17.6 min (method A).

N-(1-Methylpiperidin-4-yl)-5-nitro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-amine (39c)

Obtained from 8.35 g of 38 and 3.58 mL of 1-methylpiperidin-4-amine following general procedure A with a reaction time of 2 h. No further purification needed after filtration and washing. Yield: 8.9 g (87%) of 39c as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 9.08–8.96 (m, J = 11.6 Hz, 2H), 8.05–7.89 (m, J = 11.3, 8.4 Hz, 2H), 7.56–7.44 (m, J = 11.5, 4.1 Hz, 1H), 7.23 (t, J = 9.5 Hz, 2H), 6.69–6.58 (m, J = 10.6, 4.2 Hz, 1H), 3.94 (s, 1H), 2.77–2.62 (m, 2H), 2.36–2.17 (m, J = 19.2, 9.6 Hz, 8H), 2.11–1.98 (m, J = 6.1 Hz, 2H), 1.78–1.63 (m, 2H).¹³C NMR (75 MHz, CDCl₃) δ 148.8, 146.2, 145.9, 144.8, 134.7, 129.7, 128.5, 126.6, 123.7, 107.5, 106.5, 53.3, 50.2, 46.2, 32.5, 21.7. MS (ESI) m/z: 430.45 [M + H]⁺; HPLC t_ret = 13.1 min (method B).

N-(Cyclopropylmethyl)-5-nitro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-amine (39d)

Obtained from 8.14 g of 38 and 2.48 mL of cyclopropylmethanamine following general procedure A with a reaction time of 1 h. No further purification needed after filtration and washing. Yield: 8.35 g (93%) as yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.87 (s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 4.1 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 4.2 Hz, 1H), 3.60–3.53 (m, 2H), 2.33 (s, 3H), 1.25–1.10 (m, J = 12.3, 7.3 Hz, 1H), 0.59–0.51 (m, 2H), 0.39–0.33 (m, J = 4.8 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 148.2, 146.2, 145.5, 145.4, 134.4, 130.2, 128.2, 126.3, 123.8, 123.7, 108.1, 49.4, 40.6, 40.4, 40.1, 39.8, 39.5, 39.2, 39.0, 21.4, 10.9, 3.6. MS (ESI) m/z: 387.33 [M + H]⁺; HPLC t_ret = 20.7 min (method B).

N-(4,4-Difluorocyclohexyl)-5-nitro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-amine (39e)

Obtained from 1.95 g of 38 and 1.0 g of 4,4-Difluorocyclohexylamine (1.05 equiv) following general procedure A with a reaction time of 3 h. No further purification needed after filtration and washing. Yield: 2.18 g (87%) as yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.82 (d, J = 8.1 Hz, 1H), 8.03–7.98 (m, 2H), 7.81 (d, J = 4.2 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.14 (d, J = 4.3 Hz, 1H), 4.35–4.26 (m, J = 8.3 Hz, 1H), 2.39–2.33 (m, 5H), 2.10–1.99 (m, J = 9.4 Hz, 4H), 1.82–1.68 (m, J = 21.1, 10.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 146.0 (s), 145.2 (s), 144.3 (s), 134.1 (s), 130.3 (d, J = 10.5 Hz), 130.02 (s), 127.9 (s), 126.5 (s), 124.1 (s), 107.4 (s), 49.5 (s), 30.8 (t, J = 24.6 Hz), 28.5 (d, J = 9.5 Hz), 21.1 (s). MS (ESI) m/z: 451.33 [M + H]⁺; HPLC t_ret = 18.3 min (method A).

N-Cyclopropyl-5-nitro-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-amine (39f)

Obtained from 2.03 g of 38 and 501 μL of cyclopropylamine following general procedure A with a reaction time of 1.5 h. No further purification needed after filtration and washing. Yield: 1.83 g (85%) as yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 8.98 (s, 1H), 8.87 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 4.1 Hz, 1H), 7.21 (d, J = 8.2 Hz, 2H), 7.15 (d, J = 4.1 Hz, 1H), 3.04–2.89 (m, J = 6.6, 3.0 Hz, 1H), 2.30 (s, 3H), 0.97 (q, J = 6.7 Hz, 2H), 0.78–0.73 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 148.8, 146.9, 145.8, 145.8, 134.8, 129.8, 128.5, 126.6, 123.3, 108.9, 107.4, 27.2, 21.7, 10.1. MS (ESI) m/z: 373.40 [M + H]⁺; HPLC t_ret = 18.5 min (method A).

5-Nitro-N-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridin-4-amine (39g)

Obtained from 5.17 g of 38 and 5.5 mL of aniline (4.1 equiv) following general procedure A with a reaction time of 1 h. After filtration and washing, the crude product was suspended in a small amount of Et₂O and stored at −20 °C for 1 h, after which the red supernatant was discarded. The orange solid was dried in vacuo to yield 5.32 g (88%) of the tile compound. ¹H NMR (300 MHz, DMSO-d₆) δ 8.98 (s, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.60–7.34 (m, 9H), 2.36 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 148.3, 146.3, 145.0, 143.3, 139.1, 134.4, 130.3, 129.7, 128.2, 127.7, 126.9, 124.1, 109.4, 106.3, 99.7, 21.4. MS (ESI) m/z: 409.40 [M + H]⁺; HPLC t_ret = 20.1 min (method A).

General Procedure B for Reduction of Aromatic Nitro Groups to Amines

1.0 equiv of the nitro compound were weighed in a round bottomed flask and dissolved in EtOAc (0.075 M). The stirred mixture was thoroughly purged with argon; connection of the system to a washing flask and a subsequent water column before the gas outlet ensured a mild overpressure inside the reaction flask. A 0.1 mass equiv Pt/C (5%) was added, and the system was once again purged with argon. Afterward, the system was purged with H₂, the washing flask acting as a reservoir, and stirred at ambient temperature until TLC and/or MS indicated full conversion of educt. Consumption of H₂ could be observed semiquantitatively by measuring the height of the water column. If necessary, used up H₂ was refilled. Leftover H₂ was removed from the closed reaction flask by purge with argon and solids were filtrated over a Celite pad. The pad was washed with EtOAc and the filtrate evaporated under reduced pressure to yield the product as a solid. No further purification steps were necessary.

N4-Cyclohexyl-1-tosyl-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (40a)

Obtained from 5.65 g of (39a) following general procedure B. Yield: 5.24 g (quant.) of 40a as a purple foam. ¹H NMR (300 MHz, CDCl₃) δ 7.91 (d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.36 (d, J = 4.2 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 6.46 (d, J = 4.2 Hz, 1H), 3.66–3.53 (m, J = 11.6, 8.2 Hz, 1H), 2.25 (s, 3H), 2.08 (s, 1H), 2.00–1.94 (m, J = 7.3 Hz, 2H), 1.75–1.66 (m, 2H), 1.35–1.16 (m, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 146.1, 144.6, 140.6, 137.4, 135.7, 129.4, 127.9, 123.0, 122.3, 108.0, 104.9, 52.3, 34.3, 25.6, 24.8, 21.6. MS (ESI) m/z: 385.14 [M + H]⁺; HPLC t_ret = 14.4 min (method A).

N4-(Tetrahydro-2H-pyran-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (40b)

Obtained from 14.1 g of 39b following general procedure B. Yield: 13.08 g (quant.) of 40b as lilac foam. ¹H NMR (300 MHz, CDCl₃) δ 7.92 (d, J = 8.4 Hz, 2H), 7.76 (s, 1H), 7.39 (d, J = 4.2 Hz, 1H), 7.17–7.12 (m, J = 8.3 Hz, 3H), 6.44 (d, J = 4.2 Hz, 1H), 4.73–4.64 (m, J = 6.2 Hz, 1H), 3.95–3.89 (m, 2H), 3.44 (t, J = 16.0, 6.2 Hz, 2H), 2.26 (s, 3H), 1.92 (s, 2H), 1.53–1.44 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 146.0, 144.8, 140.1, 137.8, 135.7, 129.5, 128.0, 123.5, 122.8, 108.4, 104.5, 66.6, 49.8, 34.5, 21.7. MS (ESI) m/z: 387.4 [M + H]⁺; HPLC t_ret = 10.4 min (method B).

N4-(1-Methylpiperidin-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (40c)

Obtained from 8.85 g of 39c following general procedure B. Yield: 8.07 g (98%) of 40c as a pink to lilac foam. ¹H NMR (300 MHz, CDCl₃) δ 7.93 (t, J = 7.6 Hz, 2H), 7.73 (s, 1H), 7.38 (d, J = 4.1 Hz, 1H), 7.13 (d, J = 8.5 Hz, 2H), 6.44 (d, J = 4.2 Hz, 1H), 4.66 (d, J = 8.3 Hz, 1H), 3.69–3.45 (m, 1H), 2.88 (s, 1H), 2.79–2.67 (m, J = 11.6 Hz, 2H), 2.28–2.20 (m, J = 7.6 Hz, 6H), 2.10 (t, J = 10.5 Hz, 2H), 2.03–1.90 (m, J = 10.0 Hz, 3H), 1.58–1.43 (m, 2H). ¹³C NMR NMR (75 MHz, CDCl₃) δ 146.0, 144.7, 140.4, 137.6, 135.7, 129.5, 127.9, 123.3, 122.7, 108.2, 104.7, 54.2, 50.0, 46.2, 33.4, 21.6. MS (ESI) m/z: 400.5 [M + H]⁺; HPLC t_ret = 8.4 min (method B).

N4-(Cyclopropylmethyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (40d)

Obtained from 8.35 g of 39d following general procedure B. Yield: 7.56 g (98%) as dark yellow foam. ¹H NMR (300 MHz, DMSO-d₆) δ 7.88 (d, J = 8.3 Hz, 2H), 7.57 (s, 1H), 7.42 (d, J = 4.2 Hz, 1H), 7.33 (d, J = 8.1 Hz, 2H), 6.81 (d, J = 4.2 Hz, 1H), 5.47 (t, J = 5.6 Hz, 1H), 4.32 (s, 2H), 3.39–3.27 (m, 2H), 2.29 (s, 3H), 1.11–1.01 (m, 1H), 0.51–0.42 (m, 2H), 0.32–0.18 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 144.7, 143.6, 137.2, 135.0, 132.7, 129.5, 127.3, 125.9, 122.3, 107.8, 106.0, 48.9, 21.0, 11.3, 3.4. MS (ESI) m/z: 357.27 [M + H]⁺; HPLC t_ret = 11.7 min (method A).

N4-(4,4-Difluorocyclohexyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (40e)

Obtained from 3.43 g of 39e following general procedure B. Due to impurities, most likely from the educt, purification by flash chromatography (cyclohexane/acetone, automatic gradient) was performed after the filtration step. Yield: 2.51 g (78%) as greyish foam. ¹H NMR (300 MHz, CDCl₃) δ 7.94 (d, J = 8.3 Hz, 2H), 7.45–7.39 (m, J = 4.2 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 6.50 (d, J = 4.2 Hz, 1H), 4.63 (d, J = 8.6 Hz, 1H), 3.90–3.74 (m, J = 8.5 Hz, 1H), 2.29 (s, 3H), 2.07–1.98 (m, J = 9.5 Hz, 4H), 1.90–1.78 (m, 4H), 1.62–1.50 (m, J = 20.4, 10.3 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 172.6, 146.4, 144.6, 139.3, 135.5, 135.2, 129.2, 127.6, 127.4, 123.0, 107.6, 104.3, 49.9, 31.5, 28.8, 20.8. MS (ESI) m/z: 421.33 [M + H]⁺; HPLC t_ret = 17.7 min (method A).

N4-Cyclopropyl-1-tosyl-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (40f)

Obtained from 1.65 g of 39f following general procedure B. Yield: 1.51 g (100%) as pale pink solid. ¹H NMR (300 MHz, CDCl₃) δ 7.90 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 11.5 Hz, 1H), 7.34 (d, J = 4.1 Hz, 1H), 7.12 (d, J = 8.1 Hz, 2H), 6.93 (d, J = 4.1 Hz, 1H), 5.02 (s, 1H), 2.84–2.68 (m, J = 4.0, 2.5 Hz, 2H), 2.23 (s, 3H), 1.21–1.15 (m, J = 8.3, 5.1 Hz, 1H), 0.83–0.71 (m, 2H), 0.61–0.52 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 146.0, 144.7, 141.6, 137.0, 135.7, 129.5, 127.9, 122.8, 122.3, 109.1, 105.9, 26.6, 21.6, 9.7. MS (ESI) m/z: 343.3 [M + H]⁺; HPLC t_ret = 10.5 min (method A).

Alternative Procedure C for Reduction of Nitro Groups

In a round bottomed flask, 1.0 equiv of the nitro compound was dissolved in EtOH (0.1 M) and stirred at ambient temperature. A solution of 6.0 equiv of Na₂S₂O₄ in H₂O (0.8 M) was added portionwise. Stirring continued at 70 °C until TLC and/or MS indicated full consumption of starting material, usually overnight. The EtOH was mostly evaporated under reduced pressure and the resulting aqueous mixture poured into a separatory funnel containing DCM. After washing twice with DCM, the combined organic phases were dried over Na₂SO₄ and evaporated in vacuo to yield the product as a solid. If needed, further purification was carried out by manual column chromatography or flash chromatography.

N4-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (40g)

Obtained from 5.32 g of 39g following procedure C with stirring overnight. No further purification. Yield: 4.8 g (98%) of the title compound as a solid. ¹H NMR (300 MHz, CDCl₃) δ 7.92 (d, J = 7.0 Hz, 2H), 7.86 (s, 1H), 7.29 (d, J = 4.1 Hz, 1H), 7.20–7.11 (m, 3H), 6.93 (t, J = 7.6 Hz, 1H), 6.80 (d, J = 7.6 Hz, 2H), 5.98 (s, 1H), 5.89 (d, J = 4.1 Hz, 1H), 2.26 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 144.9, 144.3, 141.7, 136.5, 135.7, 132.3, 129.6, 129.3, 129.1, 127.9, 124.3, 122.4, 119.4, 114.2, 104.5, 21.7. MS (ESI) m/z: 379.33 [M + H]⁺; HPLC t_ret = 15.2 min (method A).

General Procedure D for Imidazole Ring Closure

In a round bottomed flask, the vicinal diamine obtained in the previous step was dissolved in DMF (0.2 M). 1.2 equiv of 5-(hydroxymethyl)furan-2-carbaldehyde was added at ambient temperature, and the mixture was stirred for 15 min. Subsequently, 3% (v/v) water and 0.7–1.0 equiv of KHSO₅ were added. At times, further addition of KHSO₅ was necessary during the course of the reaction to drive conversion to product, which was controlled by MS. To the reaction mixture, 0.2 M K₂CO₃ solution (2 equiv) was added, resulting in brownish precipitate. The solids were filtered, dried under reduced pressure, and purified either by manual column chromatography or flash chromatography.

(5-(1-Cyclohexyl-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)methanol (41a)

Obtained from 4.97 g of 40a following general procedure. Flash chromatography with DCM/MeOH (automatic gradient). Yield: 5.21 g (82%) as a light brown powder. ¹H NMR (300 MHz, CDCl₃) δ 8.88 (s, 1H), 8.10 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 4.0 Hz, 1H), 7.33–7.16 (m, J = 8.5 Hz, 2H), 6.91 (d, J = 4.0 Hz, 1H), 6.87 (d, J = 3.4 Hz, 1H), 6.41 (d, J = 3.3 Hz, 1H), 4.71 (s, 2H), 2.32 (s, 3H), 2.21–2.09 (m, J = 0.9 Hz, 2H), 1.98–1.67 (m, J = 17.0 Hz, 5H), 1.61–1.23 (m, 5H). ¹³C NMR (75 MHz, CDCl₃) δ 157.1, 145.3, 144.5, 143.3, 142.8, 138.1, 136.8, 135.2, 133.1, 129.6, 128.2, 124.8, 114.5, 109.5, 107.7, 104.6, 57.1, 30.7, 26.9, 25.6, 24.8, 21.6. MS (ESI) m/z: 491.4 [m + H]⁺; HPLC t_ret = 19.6 min (method A).

(5-(1-(Tetrahydro-2H-pyran-4-yl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)methanol (41b)

Obtained from 14.0 g of 40b using general procedure D. Manual column chromatography with DCM → DCM/EtOAc 1:2. Yield: 7.56 g (42%) of 41b as yellow to white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.79 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 4.0 Hz, 1H), 7.18 (d, J = 8.9 Hz, 2H), 6.98 (d, J = 4.1 Hz, 1H), 6.87 (d, J = 3.4 Hz, 1H), 6.36 (d, J = 3.4 Hz, 1H), 5.13–4.93 (m, 1H), 4.64 (s, 2H), 4.14–3.99 (m, 3H), 3.43 (t, J = 11.4 Hz, 2H), 2.26 (s, 3H), 1.96 (s, 1H), 1.80–1.66 (m, J = 13.0, 4.3 Hz, 2H), 1.22–1.10 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 157.1, 145.4, 144.2, 143.6, 142.9, 138.3, 137.1, 135.3, 132.9, 129.7, 128.3, 125.1, 114.7, 109.8, 107.8, 104.9, 67.3, 57.3, 53.3, 30.5, 21.7. MS (ESI) m/z: 493.3 [M + H]⁺; HPLC t_ret = 16.6 min (method A).

(5-(1-(1-Methylpiperidin-4-yl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)methanol (41c)

Obtained from 6 g of 40c following general procedure D. MS spectra taken from reaction control samples showed an m/z of 522, suggesting N-oxidation of the methylpiperidine due to KHSO₅. Before workup, the mixture was treated with 15 mL of a solution of TiCl₃ (12%) in HCl. After 2 h of stirring at ambient temperature, MS showed no remainder of the 522 m/z peak. The mixture was diluted with CHCl₃, and 60 mL of 15% KOH was added. After transfer into a separatory funnel, the aqueous phase was extracted several times with ChCl₃. The combined organics were concentrated under reduced pressure, washed once with sparing amounts of water, and dried over Na₂SO₄. They were then evaporated in vacuo, and the crude product was purified using flash chromatography with EtOAc/MeOH (automatic gradient). Yield: 3.21 g (42%) of 41c as a beige solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.77 (s, 1H), 8.06 (d, J = 4.1 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 4.1 Hz, 1H), 7.11 (d, J = 3.4 Hz, 1H), 6.59 (d, J = 3.4 Hz, 1H), 4.94–4.81 (m, 1H), 4.54 (s, 2H), 2.99 (d, J = 11.4 Hz, 2H), 2.54–2.39 (m, 3H), 2.29 (s, 3H), 2.28 (s, 3H), 2.16–2.06 (m, 2H), 1.91 (d, J = 8.6 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.0, 145.5, 144.3, 142.6, 142.0, 137.4, 137.0, 134.7, 132.5, 129.9, 127.7, 125.5, 114.6, 109.0, 107.5, 105.3, 55.8, 54.4, 53.7, 45.9, 29.4, 21.0. MS (ESI) m/z: 506.38 [M + H]⁺; HPLC t_ret = 12.9 min (method B).

(5-(1-(Cyclopropylmethyl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)methanol (41d)

Obtained from 7.56 g of 40d following general procedure D. No further purification. Yield: 8.38 g (85%) as brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (s, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 4.1 Hz, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.23 (dd, J = 3.7, 2.7 Hz, 2H), 6.58 (d, J = 3.4 Hz, 1H), 5.47 (s, 1H), 4.67 (d, J = 7.0 Hz, 2H), 4.54 (s, 2H), 2.29 (s, 3H), 1.33–1.15 (m, 1H), 0.44–0.34 (m, J = 7.4, 3.4, 2.0 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 157.7, 145.4, 143.8, 143.4, 142.2, 136.7, 136.2, 134.7, 134.0, 129.8, 127.6, 125.5, 113.9, 109.1, 107.0, 102.3, 55.7, 49.2, 20.9, 11.4, 3.1. MS (ESI) m/z: 463.33 [M + H]⁺; HPLC t_ret = 21.8 min (method A).

(5-(1-(4,4-Difluorocyclohexyl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)methanol (41e)

Obtained from 2.4 g of 40e following general procedure D. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 2.05 g (68%) as brown foam. ¹H NMR (400 MHz, DMSO-d₆) δ 8.77 (s, 1H), 8.10 (d, J = 4.1 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 3.4 Hz, 1H), 7.06 (bs, 1H), 6.59 (d, J = 3.4 Hz, 1H), 5.18–5.04 (m, 1H), 4.55 (s, 2H), 2.52–2.49 (m, 1H), 2.46–2.39 (m, J = 11.2 Hz, 1H), 2.32 (s, 3H), 2.25–2.19 (m, J = 4.6 Hz, 3H), 2.10–2.04 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 158.0, 145.5, 144.2, 142.5, 142.1, 137.3, 136.7, 134.6, 132.6, 129.8, 127.7, 125.6, 123.6, 114.6, 108.9, 107.2, 103.8, 55.7, 53.5, 31.6, 26.2, 21.0. MS (ESI) m/z: 527.47 [M + H]⁺; HPLC t_ret = 22.9 min (method A).

(5-(1-Cyclopropyl-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)methanol (41f)

Obtained from 1.45 g of 40f following general procedure D. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 1.11 g (54%) as brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 4.0 Hz, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 3.4 Hz, 1H), 7.15 (d, J = 4.0 Hz, 1H), 6.53 (d, J = 3.4 Hz, 1H), 5.41 (t, J = 5.9 Hz, 1H), 4.54 (d, J = 5.7 Hz, 2H), 3.84–3.77 (m, 1H), 2.32 (s, 3H), 1.43–1.39 (m, J = 6.7 Hz, 2H), 0.93–0.89 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 157.8, 145.3, 145.0, 142.8, 142.2, 136.6, 135.7, 135.0, 134.7, 129.6, 127.5, 124.9, 114.5, 108.7, 107.6, 103.7, 55.8, 26.9, 21.0, 9.9. MS (ESI) m/z: 449.47 [M + H]⁺; HPLC t_ret = 16.7 min (method A).

(5-(1-Phenyl-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)methanol (41g)

Obtained from 4.35 g of 40g following general procedure D. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 3.38 g (61%) as brown solid. ¹H NMR (300 MHz, CDCl₃) δ 8.82 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.63–7.52 (m, 3H), 7.45 (d, J = 4.0 Hz, 1H), 7.41–7.33 (m, 2H), 7.21–7.13 (m, 2H), 6.14 (d, J = 3.4 Hz, 1H), 5.89 (d, J = 3.5 Hz, 1H), 5.72 (d, J = 4.5 Hz, 1H), 4.53 (s, 2H), 3.22 (s, 1H), 2.21 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 157.0, 145.3, 144.1, 143.5, 143.1, 137.9, 136.3, 136.2, 135.4, 135.3, 130.6, 130.3, 129.7, 128.2, 128.1, 125.1, 113.7, 109.2, 107.6, 101.3, 57.3, 21.7. MS (ESI) m/z: 485.40 [M + H]⁺; HPLC t_ret = 16.6 min (method A).

General Procedure E for Dess–Martin Oxidation of Furylic Alcohols

The products obtained in the previous step were dissolved in DCM (0.2 M) and cooled in an ice bath. 1.2 equiv of Dess–Martin Periodinane was added and the ice bath removed, allowing the stirred mixture to slowly reach ambient temperature. Reaction progress was monitored by TLC and/or MS. After complete conversion, saturated NaHCO₃ solution was added. Phases were separated in a separatory funnel. After extraction of the aqueous phase with four to five portions of DCM, the combined organics were dried over Na₂SO₄, and DCM was removed in vacuo. Further purification was carried out either by manual column chromatography or flash chromatography.

5-(1-cyclohexyl-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (42a)

Obtained from 5.2 g of 41a following general procedure E with a reaction time of 3 h. Flash chromatography with cyclohexane/acetone (automatic gradient). Yield: 3.73 g (72%) of 42a as a brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.74 (s, 1H), 8.78 (s, 1H), 8.03 (d, J = 8.2 Hz, 2H), 7.96 (d, J = 4.0 Hz, 1H), 7.68 (t, J = 6.5 Hz, 1H), 7.39–7.32 (m, J = 8.7 Hz, 3H), 7.11 (d, J = 4.0 Hz, 1H), 5.01–4.88 (m, J = 12.0 Hz, 1H), 2.31 (s, 3H), 2.24–2.18 (m, J = 11.4 Hz, 2H), 2.00–1.90 (m, 4H), 1.79–1.73 (m, J = 7.8 Hz, 1H), 1.58–1.44 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.0, 152.7, 148.3, 145.2, 142.3, 142.2, 137.7, 136.7, 134.6, 133.1, 129.6, 127.7, 125.1, 123.1, 115.3, 107.2, 104.5, 57.0, 30.3, 25.1, 24.2, 21.0. MS (ESI) m/z: 489.09 [M + H]; HPLC t_ret = 20.5 min (method A).

5-(1-(Tetrahydro-2H-pyran-4-yl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (42b)

Obtained from 2.02 g of 41b following general procedure E with stirring overnight. After workup, the product was carried on to the next step without further purification. Yield: 1.9 g (90%) of crude 42b as an orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.85 (s, 1H), 8.11 (d, J = 4.1 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 3.8 Hz, 1H), 7.47 (d, J = 3.8 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 4.1 Hz, 1H), 5.31–5.20 (m, 1H), 4.11 (dd, J = 11.4, 4.6 Hz, 2H), 3.61–3.53 (m, J = 11.2 Hz, 2H), 2.47–2.39 (m, J = 11.6, 7.3 Hz, 2H), 2.31 (s, 3H), 1.98 (dd, J = 12.6, 4.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.8, 152.9, 145.6, 142.7, 138.0, 136.9, 134.4, 131.1, 130.3, 129.9, 127.8, 126.3, 125.8, 120.4, 115.7, 107.4, 105.0, 66.3, 53.2, 30.2, 21.0. MS (ESI) m/z: 491.4 [M + H]⁺; HPLC t_ret = 17.2 min (method A).

5-(1-(1-Methylpiperidin-4-yl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (42c)

Obtained from 1.34 g of 41c following general procedure E with stirring overnight. Manual column chromatography (EtOAc → EtOAc/MeOH 9:1). Yield: 935 mg (70%) as yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ 9.76–9.74 (m, 1H), 8.83 (s, 1H), 8.11 (d, J = 4.1 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 3.8 Hz, 1H), 7.45 (d, J = 3.8 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 4.0 Hz, 1H), 5.05–4.94 (m, 1H), 3.05 (d, J = 7.2 Hz, 2H), 2.50 (dd, J = 3.6, 1.8 Hz, 4H), 2.33 (s, 6H), 2.04–1.94 (m, J = 10.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.9, 152.9, 148.1, 145.7, 142.8, 142.2, 138.0, 137.0, 134.6, 132.8, 130.0, 127.8, 125.8, 123.3, 115.7, 107.5, 105.4, 69.8, 54.3, 45.7, 29.3, 21.1. MS (ESI) m/z: 504.6 [M + H]⁺; HPLC t_ret = 15.0 min (method A).

5-(1-(Cyclopropylmethyl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (42d)

Obtained from 5.18 g of 41d following general procedure E with stirring overnight. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 4.33 g (80%) as yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 9.71 (s, 1H), 8.89 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 4.0 Hz, 1H), 7.39 (s, 2H), 7.25 (d, J = 8.1 Hz, 2H), 6.84 (d, J = 4.0 Hz, 1H), 4.68 (d, J = 6.9 Hz, 2H), 2.14 (s, 3H), 1.43–1.31 (m, 1H), 0.59–0.40 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 177.0, 152.9, 149.9, 145.3, 143.3, 142.2, 138.5, 136.9, 135.4, 134.8, 129.7, 128.3, 125.5, 122.43, 114.7, 107.4, 101.4, 50.5, 30.9, 21.6, 11.7, 3.8. MS (ESI) m/z: 461.33 [M + H]⁺; HPLC t_ret = 22.7 min (method A).

5-(1-(4,4-Difluorocyclohexyl)-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (42e)

Obtained from 2.04 g of 41e following general procedure E with stirring overnight. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 1.84 g (90%) as brown solid. ¹H NMR NMR (400 MHz, DMSO-d₆) δ 9.77 (s, 1H), 8.84 (s, 1H), 8.13 (d, J = 4.1 Hz, 1H), 8.03 (d, 2H), 7.75 (d, J = 3.8 Hz, 1H), 7.46 (d, J = 3.8 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.07 (s, 1H), 5.22–5.12 (m, J = 14.3, 10.0 Hz, 1H), 2.47–2.36 (m, J = 11.0 Hz, 2H), 2.32 (s, 3H), 2.29–2.10 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 179.1, 153.1, 148.0, 145.8, 143.0, 142.4, 138.2, 137.0, 134.7, 130.1, 127.9, 126.1, 122.8, 115.9, 107.5, 104.1, 54.0, 32.1, 31.8, 30.8, 26.5, 21.2. MS (ESI) m/z: 525.40 [M + H]⁺; HPLC t_ret = 20.0 min (method A).

5-(1-Cyclopropyl-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (42f)

Obtained from 1.02 g of 41f following general procedure E with overnight stirring. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 800 mg (79%) as pale brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.78 (s, 1H), 8.05–8.02 (m, 2H), 7.99 (d, J = 4.0 Hz, 1H), 7.77 (d, J = 3.8 Hz, 1H), 7.58 (d, J = 3.8 Hz, 1H), 7.42 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 4.0 Hz, 1H), 3.96–3.89 (m, 1H), 2.33 (s, 3H), 1.46–1.40 (m, 2H), 0.99–0.94 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.9, 152.6, 148.0, 145.6, 144.0, 142.5, 140.5, 137.6, 135.9, 135.5, 134.7, 130.0, 127.7, 125.5, 115.7, 107.8, 104.2, 30.7, 21.1, 9.9. MS (ESI) m/z: 447.33 [M + H]⁺; HPLC t_ret = 17.1 min (method A).

5-(1-Phenyl-6-tosyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (42g)

Obtained from 1.75 g of 41g following general procedure E with a reaction time of 3 h. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 1.35 g (78%) as brown solid. ¹H NMR (300 MHz, CDCl₃) δ 9.56 (s, 1H), 8.94 (s, 1H), 8.00 (d, 2H), 7.62 (m, J = 9.0, 3.8, 2.1 Hz, 3H), 7.51 (d, J = 4.0 Hz, 1H), 7.44–7.39 (m, 2H), 7.22–7.17 (m, 2H), 7.08 (d, J = 3.8 Hz, 1H), 6.41 (d, J = 3.8 Hz, 1H), 5.78 (d, J = 4.0 Hz, 1H), 2.28 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 177.9, 152.8, 147.7, 145.0, 143.3, 142.1, 138.2, 136.0, 135.6, 135.1, 135.0, 130.4, 130.0, 129.3, 127.9, 127.5, 125.0, 118.9, 113.9, 107.2, 100.9, 21.3. MS (ESI) m/z: 483.40 [M + H]⁺; HPLC t_ret = 8.5 min (method A).

General Procedure F for Tosyl Cleavage

The aldehyde from the previous step was dissolved or suspended in a 1 M solution of KOH in MeOH. The mixture was stirred at ambient temperature. After indication of complete consumption of educt by TLC and/or MS, saturated NH₄Cl solution was added to quench. The mixture was transferred to a separatory funnel, and EtOAc and water were added until clear, separate phases appeared. The organic phase was washed two to three times with water and subsequently brine, dried over Na₂SO₄, and reduced in vacuo. Purification of this residue, if necessary, was carried out either by manual column chromatography or flash chromatography.

5-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (43a)

Obtained from 3.63 g of 42a following general procedure F with a reaction time of 45 min. Flash chromatography with cyclohexane/acetone (automatic gradient). Yield: 988 mg (40%) of 43a as an orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.12 (s, 1H), 9.74 (s, 1H), 8.70 (s, 1H), 7.76 (d, J = 3.7 Hz, 1H), 7.58–7.55 (m, 1H), 7.38 (d, J = 3.7 Hz, 1H), 6.86–6.80 (m, J = 3.3, 1.8 Hz, 1H), 4.95 (dt, J = 16.5, 8.4, 4.2 Hz, 1H), 2.42–2.27 (m, 2H), 2.03–1.84 (m, 4H), 1.80–1.65 (m, J = 22.2 Hz, 1H), 1.59–1.42 (m, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 178.4, 152.5, 149.1, 144.7, 140.4, 136.3, 134.8, 133.0, 124.1, 123.7, 114.6, 104.0, 99.8, 56.6, 30.2, 25.2, 24.3. MS (ESI) m/z: 335.42 [M + H]; HPLC t_ret = 14.9 min (method A).

5-(1-(Tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (43b)

Obtained from 1.75 g of 42b following general procedure F with a reaction time of 1 h. No further purification after workup with water/EtOAc. Yield: 1.04 g (83%) as an orange powder. ¹H NMR (300 MHz, DMSO-d₆) δ 11.92 (s, 1H), 9.57 (s, 1H), 8.51 (s, 1H), 7.57 (d, J = 3.8 Hz, 1H), 7.41 (d, J = 2.6 Hz, 1H), 7.23 (d, J = 3.7 Hz, 1H), 6.66 (d, J = 1.7 Hz, 1H), 5.12–4.95 (m, 1H), 4.05–3.90 (m, 2H), 3.88–3.79 (m, 1H), 2.53–2.37 (m, 1H), 2.37–2.29 (m, 1H), 1.85–1.72 (m, J = 9.1 Hz, 2H), 1.04–0.95 (m, 1H). ¹³C NMR (75 MHz, DMSO-d₆) δ 178.8, 152.7, 148.9, 144.8, 140.6, 136.5, 135.0, 132.8, 124.5, 123.6, 115.0, 104.3, 100.4, 66.5, 53.0, 30.2. MS (ESI) m/z: 337.4 [M + H]⁺; HPLC t_ret = 12.6 min (method A).

5-(1-(1-Methylpiperidin-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (43c)

Obtained from 890 mg of 42c following general procedure F with a reaction time of 1 h. No further purification after workup with water/EtOAc. Yield: 395 mg (64%) as yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ 12.07 (s, 1H), 9.74 (s, 1H), 8.68 (s, 1H), 7.76 (d, J = 3.7 Hz, 1H), 7.63–7.55 (m, 1H), 7.40 (d, J = 3.7 Hz, 1H), 6.99 (s, 1H), 5.04–4.92 (m, 1H), 3.03 (d, J = 10.9 Hz, 2H), 2.75–2.64 (m, 2H), 2.58–2.47 (m, 4H), 2.31–2.28 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.4, 152.4, 148.8, 144.5, 140.4, 136.1, 134.8, 132.5, 124.0, 123.4, 114.6, 104.2, 100.4, 54.5, 53.7, 45.9, 29.2. MS (ESI) m/z: 350.64 [M + H]⁺; HPLC t_ret = 12.7 min (method A).

5-(1-(Cyclopropylmethyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (43d)

Obtained from 3.3 g of 42d following general procedure F. Since TLC and MS indicated slow conversion of product to side products, the reaction was quenched after 20 min. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 618 mg (28%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 1H), 9.69 (s, 1H), 8.62 (s, 1H), 7.57 (d, J = 3.8 Hz, 1H), 7.39–7.35 (m, 2H), 6.75–6.71 (m, J = 3.3, 1.8 Hz, 1H), 4.71 (d, J = 7.0 Hz, 2H), 1.50–1.39 (m, 1H), 0.51–0.43 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 177.1, 152.1, 149.7, 144.8, 139.8, 135.7, 134.2, 134.2, 123.7, 123.0, 113.3, 103.6, 96.4, 49.6, 11.3, 3.1. MS (ESI) m/z: 307.33 [M + H]⁺; HPLC t_ret = 15.1 min (method A).

5-(1-(4,4-Difluorocyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (43e)

Obtained from 300 mg of 42e following general procedure F with a reaction time of 2 h. No further purification after workup with water/EtOAc. Yield: 187 mg (88%) as solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H), 9.77 (s, 1H), 8.70 (s, 1H), 7.75 (d, J = 3.7 Hz, 1H), 7.64–7.57 (m, 2H), 7.42 (d, J = 3.8 Hz, 1H), 5.23–5.12 (m, 1H), 2.31–2.24 (m, J = 13.3 Hz, 4H), 2.18–2.09 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.8, 152.7, 152.3, 148.6, 144.7, 140.8, 136.3, 136.1, 134.9, 132.7, 124.5, 122.9, 114.9, 104.2, 99.2, 53.5, 32.0, 31.8, 31.5, 26.3, 26.2. MS (ESI) m/z: 371.46 [M + H]⁺; HPLC t_ret = 19.7 min (method A).

5-(1-Cyclopropyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (43f)

Obtained from 700 mg of 42f following general procedure F with a reaction time of 1.5 h. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 133 mg (30%) as orange solid. ¹H NMR (300 MHz, DMSO-d₆) δ 11.91 (s, 1H), 9.74 (s, 1H), 8.61 (s, 1H), 7.70 (d, J = 4.7, 3.8 Hz, 1H), 7.53–7.41 (m, J = 5.9, 3.9 Hz, 2H), 6.91–6.83 (m, 1H), 3.90 (dt, J = 7.3, 3.8 Hz, 1H), 1.51–1.36 (m, 2H), 1.04–0.94 (m, J = 7.5 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 178.1, 152.3, 148.9, 144.9, 141.7, 135.9, 135.2, 133.7, 123.7, 123.3, 114.3, 104.4, 98.6, 30.5, 9.9. MS (ESI) m/z: 293.46 [M + H]⁺; HPLC t_ret = 11.8 min (method A).

5-(1-Phenyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-carbaldehyde (43g)

Obtained from 1.1 g of 42g following general procedure F with a reaction time of 4 h. No further purification after workup with water/EtOAc. Yield: 310 mg (41%) as yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.05–12.02 (m, 1H), 9.63 (s, 1H), 8.80 (s, 1H), 7.78–7.75 (m, J = 6.1, 2.2 Hz, 4H), 7.57 (d, J = 3.8 Hz, 1H), 7.37–7.34 (m, 2H), 6.45 (d, J = 3.8 Hz, 1H), 5.66–5.63 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.3, 152.1, 148.4, 145.3, 140.3, 136.1, 135.9, 135.1, 133.9, 130.3, 130.1, 128.0, 124.3, 123.5, 113.3, 103.9, 96.2. MS (ESI) m/z: 329.53 [M + H]⁺; HPLC t_ret = 15.2 min (method A).

General Procedure G for Knoevenagel Condensation

Products obtained from the tosyl deprotection step were dissolved in MeOH or IPrOH (0.2 M) and 0.1 equiv of piperidine. The corresponding cyanoacetamide (1.0 to 1.1 equiv) was added, and the stirred solution was heated to 50–70 °C, either in an oil bath or a shaking incubator, or left at ambient temperature. Reaction times ranged between 1 and 3 h, and complete conversion was determined by TLC and MS. Products were either isolated by filtration after storage at −20 °C or by flash chromatography. Typically, mixtures of E/Z isomers were obtained, leading to complex NMR spectra.

(E/Z)-2-Cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N,N-dimethylacrylamide (2)

Obtained from 620 mg of 43a and 229 mg of 2-cyano-N,N-dimethylacetamide (1.1 equiv) in 10 mL of MeOH following general procedure G at 60 °C oil-bath temperature. TLC and MS showed complete conversion of educts after 30 min. The mixture was stored at −20 °C overnight, and the resulting precipitate was collected by filtration and washed sparingly with cold MeOH. No further purification was necessary. Yield: 460 mg (58%) of 2 as yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.08 (s, 1H), 8.69 (s, 1H), 7.78 (s, 1H), 7.52 (d, J = 28.3 Hz, 2H), 7.39 (s, 1H), 6.83 (s, 1H), 5.03–4.86 (m, 1H), 3.35 (s, 6H), 2.38–2.32 (m, 2H), 2.15–1.98 (m, J = 17.8 Hz, 2H), 1.96–1.87 (m, 2H), 1.83–1.69 (m, 1H), 1.63–1.43 (m, J = 37.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.7, 149.5, 148.4, 144.6, 140.5, 136.3, 135.3, 135.0, 132.8, 124.0, 121.7, 116.3, 116.2, 104.1, 100.3, 100.2, 56.1, 30.0, 30.0, 24.9, 24.2. ESI-HRMS: [M + H]⁺ calculated for C₂₄H₂₄N₆O₂: 429.20335, found 429.20374; HPLC t_ret = 21.1 min (method A).

(E/Z)-2-Cyano-N,N-dimethyl-3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (10)

Obtained from 680 mg of 43b and 249 mg of 2-cyano-N,N-dimethylacetamide (1.1 equiv) in 10 mL of MeOH following general procedure G at 60 °C oil bath temperature. TLC and MS indicated full conversion of educts after 2 h. The mixture was stored at −20 °C overnight and the resulting precipitate collected by filtration and washed sparingly with cold MeOH. No further purification was necessary. Yield: 350 mg (40%) as an orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.95 (s, 1H), 8.60–8.47 (m, J = 5.4 Hz, 1H), 7.62 (s, 1H), 7.48–7.39 (m, 1H), 7.29 (dd, J = 9.2, 3.7 Hz, 2H), 6.75–6.61 (m, J = 20.2 Hz, 1H), 5.27 (q, 1H), 3.98–3.88 (m, 2H), 3.57–3.43 (m, J = 11.4 Hz, 2H), 3.26 (s, 3H), 3.06–2.95 (m, 1H), 2.88–2.76 (m, J = 11.0 Hz, 2H), 2.32 (s, 1H), 1.98–1.64 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.6, 149.4, 148.5, 144.4, 140.2, 136.1, 135.2, 135.0, 132.4, 124.0, 122.2, 116.1, 115.9, 104.1, 101.4, 100.5, 66.0, 51.8, 30.3, 29.4. ESI-HRMS: [M + H]⁺ calculated for C₂₃H₂₂N₆O₃: 431.18262, found 431.18322; [M + H]⁺; HPLC t_ret = 19.1 min (method A).

Methyl (E/Z)-N-(2-Cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acryloyl)-N-methylglycinate (4)

Obtained from 700 mg of 43a and 425 mg of 49 (1.2 equiv) in 11 mL of MeOH following general procedure G at 60 °C oil bath temperature. TLC and MS indicated consumption of educts after 2 h. The solvents were evaporated under reduced pressure, and the crude product was purified by column chromatography (EtOAc → EtOAc/MeOH 9:1). Yield: 470 mg (46%) as red solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.08 (s, 1H), 8.67 (d, J = 8.2 Hz, 1H), 7.87–7.73 (m, 1H), 7.58–7.53 (m, J = 6.0, 3.0 Hz, 1H), 7.39 (d, J = 3.6 Hz, 1H), 7.32 (dd, J = 26.1, 3.8 Hz, 1H), 6.88–6.80 (m, J = 9.0, 3.2, 1.8 Hz, 1H), 5.01–4.87 (m, 1H), 3.69 (s, 3H), 3.23 (s, 3H), 3.09 (s, 1H), 1.98 (s, 1H), 1.92–1.89 (m, 2H), 1.80–1.71 (m, 2H), 1.65–1.41 (m, 4H), 1.25–1.06 (m, J = 9.7, 6.5, 3.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.8, 163.5, 162.2, 149.4, 148.8, 144.5, 140.4, 136.2, 134.9, 132.7, 124.1, 119.8, 116.4, 115.7, 115.5, 104.1, 100.3, 69.7, 56.0, 51.8, 36.5, 29.9, 24.8, 24.2. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₆N₆O₄: 487.20883, found 487.20970; HPLC t_ret = 19.8 and 20.7 min (E/Z mixture, method A).

Methyl (E/Z)-N-(2-Cyano-3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acryloyl)-N-methylglycinate (11)

Obtained from 980 mg of 43b and 595 mg of 49 (1.2 equiv) in 14 mL of MeOH following general procedure G at 60 °C oil bath temperature. Full consumption of starting material was observed after 3 h. The mixture was stored at −20 °C overnight, and the resulting precipitate was filtrated and washed sparingly with cold MeOH. No further purification was necessary. Yield: 796 mg (56%) as an orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.08 (s, 1H), 8.69 (s, 1H), 7.88–7.70 (m, J = 24.6, 13.4 Hz, 1H), 7.61–7.56 (m, J = 2.7 Hz, 1H), 7.53–7.43 (m, J = 13.7 Hz, 1H), 7.43–7.32 (m, 1H), 6.92–6.81 (m, 1H), 5.46–5.05 (m, 1H), 4.36–4.19 (m, 1H), 4.17–4.03 (m, J = 16.2, 11.4, 4.7 Hz, 2H), 3.73–3.62 (m, 3H), 3.59–3.55 (m, J = 6.4 Hz, 3H), 3.28–3.23 (m, 1H), 3.09 (t, J = 28.2 Hz, 1H), 2.74–2.58 (m, 2H), 2.54–2.49 (m, 1H), 2.06–1.86 (m, J = 15.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.8, 163.8, 152.8, 149.7, 144.7, 140.5, 136.4, 135.3, 132.8, 124.4, 116.6, 115.9, 114.9, 104.5, 100.9, 100.4, 66.6, 66.3, 52.2, 48.7, 36.8, 30.2, 29.74. ESI-HRMS: [M + Na]⁺ calculated for C₂₅H₂₄N₆O₅: 511.17004, found 511.17050; HPLC t_ret = 16.7 and 18.5 min (E/Z mixture, method A).

(E/Z)-2-Cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N-(2-(dimethylamino)ethyl)-N-methylacrylamide (5)

Obtained from 1.0 g of 43a and 506 mg of 50 (1.0 equiv) in 15 mL of MeOH following general procedure G at 60 °C oil bath temperature. Full consumption of educts was observed after 1 h. Manual column chromatography (EtOAc → EtOAc/MeOH 6:1), during which part of product was lost due to glassware malfunction. Yield: 225 mg (15%) as an orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.10 (s, 1H), 8.68 (s, 1H), 7.89 (d, J = 7.9, 0.9 Hz, 2H), 7.58 (d, J = 7.7, 1.7 Hz, 2H), 7.17–7.08 (m, 1H), 4.98–4.88 (m, 1H), 3.46 (t, J = 6.6 Hz, 2H), 3.33 (t, J = 6.7 Hz, 2H), 2.92 (s, 3H), 2.64–2.56 (m, J = 12.0, 5.4 Hz, 2H), 2.34 (s, 6H), 2.13–2.03 (m, 2H), 1.24–1.12 (m, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 163.4, 149.6, 144.7, 144.1, 140.7, 139.9, 136.4, 135.1, 132.9, 131.0, 129.3, 127.9, 124.3, 116.5, 104.3, 100.4, 93.7, 56.3, 55.0, 45.6, 44.3, 35.3, 30.1, 25.0. ESI-HRMS: [M + H]⁺ calculated for C₂₇H₃₁N₇O₂: 486.26120, found 486.26138; HPLC t_ret = 15.5 and 16.2 min (E/Z mixture, method A).

(E/Z)-2-Cyano-N-(2-(dimethylamino)ethyl)-N-methyl-3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (12)

Obtained from 105 mg of 43b and 52.5 mg of 50 (1.0 equiv) in 1.5 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1 h. The mixture was stored at −20 °C overnight, and the precipitate was collected by filtration and washed sparingly with cold MeOH. No further purification was necessary. Yield: 70 mg (46%) as an orange to red solid. ¹H NMR (400 MHz, CDCl₃) δ 12.25 (s, 1H), 8.83 (s, 1H), 7.67 (s, 1H), 7.53–7.45 (m, 1H), 7.38 (d, J = 3.7 Hz, 1H), 7.23 (d, J = 3.7 Hz, 1H), 7.10–7.05 (m, J = 2.2 Hz, 1H), 5.53–5.40 (m, 1H), 4.22 (dd, J = 11.5, 4.7 Hz, 2H), 3.74 (t, J = 11.6 Hz, 2H), 3.62 (t, J = 6.2 Hz, 2H), 3.20 (s, 3H), 2.89 (qd, J = 12.5, 5.1 Hz, 2H), 2.58 (t, J = 6.6 Hz, 2H), 2.28 (s, 6H), 2.02 (dd, J = 12.6, 4.6 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 149.7, 148.5, 144.6, 140.4, 136.3, 135.2, 132.6, 124.3, 122.2, 116.4, 116.2, 104.4, 102.0, 100.7, 66.2, 56.1, 52.1, 45.3, 44.9, 35.5, 29.7. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₉N₇O₃: 488.24046, found 488.24070; HPLC t_ret = 14.2 and 15.4 min (E/Z mixture, method A).

(E/Z)-2-Cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N-(2-(((2S,3R,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)(methyl)amino)ethyl)-N-methylacrylamide (28)

Obtained from 100 mg of 43a and 257 mg of 35 (1.0 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1 h. Manual column chromatography (EtOAc → EtOAc/MeOH 4:1). Yield: 255 mg (73%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.08 (s, 1H), 8.67 (s, 1H), 7.70 (s, 1H), 7.57–7.52 (m, J = 9.1, 6.3 Hz, 2H), 7.46 (s, 1H), 7.38 (d, J = 3.6 Hz, 1H), 4.96–4.82 (m, J = 20.8 Hz, 4H), 4.77–4.72 (m, 1H), 4.59 (s, 1H), 4.34–4.25 (m, 3H), 4.11–4.03 (m, 2H), 4.01–3.90 (m, J = 3.4 Hz, 2H), 3.78 (s, 1H), 3.62 (d, J = 7.0 Hz, 1H), 3.50 (s, 3H), 3.42 (d, J = 7.5 Hz, 2H), 3.23 (t, J = 4.7 Hz, 3H), 3.06–3.03 (m, 1H), 2.94–2.89 (m, 2H), 2.69 (s, 1H), 2.41–2.30 (m, 6H), 2.26–2.17 (m, 6H), 2.11 (s, 1H), 2.04–2.00 (m, J = 11.4 Hz, 2H), 1.94–1.87 (m, 5H), 1.79–1.72 (m, 4H), 1.60–1.45 (m, 9H), 1.22 (s, 3H), 1.17–1.16 (m, 3H), 1.14 (s, 1H), 1.11–1.10 (m, J = 3.2 Hz, 2H), 1.05 (d, J = 5.0 Hz, 4H), 1.01–0.99 (m, 3H), 0.95–0.93 (m, 2H), 0.87–0.82 (m, 6H), 0.79–0.75 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 177.3, 149.6, 148.4, 144.7, 140.6, 136.3, 135.1, 132.9, 124.2, 121.7, 116.4, 104.2, 100.4, 99.6, 94.7, 77.4, 77.1, 76.2, 73.7, 73.6, 72.8, 72.3, 69.8, 68.6, 67.1, 65.7, 64.9, 56.2, 55.8, 48.8, 48.7, 45.8, 45.0, 41.7, 41.0, 38.3, 35.8, 34.6, 30.7, 30.3, 30.1, 29.6, 29.0, 25.0, 24.3, 22.0, 21.7, 21.4, 21.0, 18.4, 17.5, 14.8, 14.7, 10.9, 10.9, 9.12, 8.7, 6.8. ESI-HRMS: [M + H]⁺ calculated for C₆₂H₉₄N₈O₁₄: 1175.69623, found 1175.69397; HPLC t_ret = 21.4 and 22.1 min (E/Z mixture, method B).

(E/Z)-2-Cyano-N-(2-(((2S,3R,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)(methyl)amino)ethyl)-N-methyl-3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (29)

Obtained from 98 mg of 43b and 220 mg of 35 (0.9 equiv) in 1.5 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1 h. Purification by storage at −20 °C over a weekend and subsequent filtration of precipitate and washing with cold MeOH. Yield: 144 mg (42%) as orange platelets. ¹H NMR (400 MHz, DMSO-d₆) δ 12.07 (s, 1H), 8.69 (s, 1H), 7.71 (s, 1H), 7.58 (dd, 1H), 7.48–7.44 (m, 2H), 6.91–6.87 (m, 1H), 5.47–5.36 (m, J = 11.5, 5.8 Hz, 1H), 4.90 (s, 1H), 4.85 (d, J = 3.0 Hz, 1H), 4.73 (d, J = 8.2 Hz, 1H), 4.63 (s, 1H), 4.32–4.24 (m, J = 8.3 Hz, 3H), 4.13–4.05 (m, 4H), 3.99–3.90 (m, 2H), 3.82–3.78 (m, 1H), 3.68–3.64 (m, J = 8.8, 4.3 Hz, 3H), 3.22 (s, 6H), 3.04 (d, J = 6.5 Hz, 1H), 2.94–2.86 (m, J = 15.7, 6.7 Hz, 2H), 2.71–2.61 (m, J = 11.9, 7.4 Hz, 5H), 2.49–2.47 (m, 2H), 2.40 (s, 1H), 2.36–2.31 (m, 1H), 2.27–2.15 (m, 5H), 2.00 (dd, J = 7.5 Hz, 2H), 1.92–1.80 (m, 3H), 1.79–1.62 (m, 3H), 1.53–1.43 (m, J = 15.5 Hz, 4H), 1.20 (s, 3H), 1.18–1.14 (m, J = 5.8 Hz, 4H), 1.12–1.09 (m, 4H), 1.07–1.03 (m, J = 5.6 Hz, 5H), 1.00–0.98 (m, 3H), 0.95–0.91 (m, J = 6.3 Hz, 4H), 0.86–0.75 (m, J = 19.0, 9.0 Hz, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 177.3, 149.7, 148.6, 144.6, 140.4, 136.3, 135.2, 132.7, 124.3, 122.3, 116.4, 104.4, 102.1, 100.8, 99.6, 94.7, 83.6, 77.4, 77.1, 76.3, 74.9, 73.7, 72.7, 72.4, 69.3, 68.6, 66.3, 64.9, 61.6, 52.1, 48.7, 48.6, 44.9, 41.7, 41.0, 36.6, 35.7, 34.6, 29.7, 27.6, 26.0, 22.1, 21.7, 21.0, 20.9, 18.4, 17.6, 14.8, 10.9, 8.7, 6.7. ESI-HRMS: [M + H]⁺ calculated for C₆₁H₉₂N₈O₁₅: 1177.67549, found 1177.67436; HPLC t_ret = 17.7 min (method A).

(E/Z)-2-Cyano-N,N-dimethyl-3-(5-(1-(1-methylpiperidin-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (17)

Obtained from 83 mg of 43c and 29 mg 2-cyano-N,N-dimethylacetamide (1.1 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 70 mg (66%) as an orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.05 (s, 1H), 8.68 (s, 1H), 7.76 (s, 1H), 7.60–7.53 (m, J = 2.9 Hz, 1H), 7.48 (d, J = 3.7 Hz, 1H), 7.41 (d, J = 3.7 Hz, 1H), 7.08–7.02 (m, J = 3.1, 1.9 Hz, 1H), 5.12–4.96 (m, 1H), 3.10–2.90 (m, 6H), 2.76–2.60 (m, J = 24.7, 12.4, 4.1 Hz, 3H), 2.28 (s, 3H), 2.24–2.12 (m, J = 10.8 Hz, 3H), 2.00 (d, J = 7.8 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.8, 149.6, 148.4, 144.6, 140.6, 136.3, 135.3, 135.2, 132.7, 124.2, 122.1, 116.3, 116.0, 104.5, 101.9, 101.0, 54.5, 53.3, 46.2, 34.4, 29.1. ESI-HRMS: [M + H]⁺ calculated for C₂₄H₂₅N₇O₂: 444.21425, found 444.21472; HPLC t_ret = 14.7 min (method A).

(E/Z)-2-Cyano-N-(2-(((2S,3R,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)(methyl)amino)ethyl)-N-methyl-3-(5-(1-(1-methylpiperidin-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (30)

Obtained from 83 mg of 43c and 204 mg of 35 (1.0 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 2.5 h. Purification by manual column chromatography (EtOAc → EtOAc(MeOH 6:1). Yield: 183 mg (65%) as an orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.07 (s, 1H), 8.68 (s, 1H), 7.70 (s, 1H), 7.58–7.55 (m, 1H), 7.45 (d, J = 3.8 Hz, 1H), 7.42 (d, J = 3.7 Hz, 1H), 7.09–7.06 (m, 1H), 5.00 (d, J = 3.8 Hz, 3H), 4.91–4.90 (m, 1H), 4.88–4.84 (m, 1H), 4.79–4.71 (m, 1H), 4.63–4.55 (m, J = 17.7 Hz, 1H), 4.10–4.02 (m, 2H), 3.99–3.93 (m, 1H), 3.76–3.70 (m, J = 7.7, 3.8 Hz, 3H), 3.50 (s, 3H), 3.25–3.21 (m, J = 5.2 Hz, 4H), 3.19–3.12 (m, 7H), 3.05–2.99 (m, 3H), 2.95–2.87 (m, J = 12.5, 8.8, 3.6 Hz, 7H), 2.73–2.65 (m, 4H), 2.36–2.29 (m, 4H), 2.08–1.97 (m, J = 7.0 Hz, 3H), 1.88–1.81 (m, J = 13.6, 7.2, 3.5 Hz, 6H), 1.61–1.50 (m, 7H), 1.23 (s, 4H), 1.19 (s, 1H), 1.17 (s, 2H), 1.14 (s, 1H), 1.12–1.09 (m, 3H), 1.05 (d, J = 5.4 Hz, 4H), 1.01–0.98 (m, J = 7.3 Hz, 2H), 0.96–0.90 (m, J = 6.7 Hz, 2H), 0.88–0.83 (m, 4H), 0.81–0.75 (m, J = 14.8, 7.4 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 177.4, 149.8, 148.4, 144.7, 136.3, 135.2, 132.7, 124.4, 122.2, 116.6, 104.5, 102.3, 101.2, 99.7, 94.7, 82.6, 77.5, 77.1, 75.9, 73.8, 72.9, 72.4, 69.9, 68.7, 67.1, 65.0, 62.7, 55.9, 54.3, 48.8, 45.6, 45.1, 41.7, 41.2, 40.7, 36.1, 34.6, 33.5, 32.2, 30.5, 29.7, 29.1, 28.9, 25.4, 22.0, 21.8, 21.0, 20.9, 18.5, 17.6, 14.9, 14.8, 11.0, 9.16, 8.7, 7.4. ESI-HRMS: [M + H]⁺ calculated for C₆₂H₉₅N₉O₁₄: 1190.70713, found 1190.70519; HPLC t_ret = 15.6 and 16.0 min (E/Z mixture, method A).

(E/Z)-2-Cyano-N-(2-(dimethylamino)ethyl)-N-methyl-3-(5-(1-(1-methylpiperidin-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (18)

Obtained from 78 mg of 43c and 38 mg 50 (1.0 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 35 mg (32%) as red solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.17–11.98 (m, J = 18.4 Hz, 1H), 8.81–8.60 (m, 1H), 7.72–6.98 (m, 5H), 5.33–5.01 (m, J = 55.6, 17.6 Hz, 1H), 4.28–3.85 (m, 6H), 3.23–2.09 (m, 18H). ¹³C NMR (101 MHz, DMSO-d₆) δ 163.6, 150.1, 149.4, 145.0, 141.3, 137.2, 136.8, 135.9, 133.8, 123.4, 121.0, 116.3, 116.0, 105.3, 102.8, 102.3, 56.5, 55.2, 54.4, 46.6, 45.7, 29.8, 29.7, 29.3. ESI-HRMS: [M + H]⁺ calculated for C₂₇H₃₂N₈O₂: 501.27210, found 501.27228; HPLC t_ret = 9,6 and 10,0 min (E/Z mixture, method B)

Methyl (E/Z)-N-(2-Cyano-3-(5-(1-(1-methylpiperidin-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acryloyl)-N-methylglycinate (19)

Obtained from 225 mg of 43c and 110 mg of 49 in 2.3 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 2 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 177 mg (55%) as red solid. ¹H NMR (400 MHz, CDCl₃) δ 8.71 (s, 1H), 7.80–7.72 (m, 1H), 7.45 (d, J = 3.3 Hz, 1H), 7.42 (s, 1H), 7.38–7.35 (m, 1H), 7.29–7.23 (m, 1H), 5.12–4.99 (m, 1H), 4.23–4.12 (m, 2H), 3.88 (s, 3H), 3.79 (s, 3H), 3.36–3.30 (m, 2H), 3.18–3.11 (m, 2H), 2.90–2.83 (m, J = 9.5 Hz, 2H), 2.43 (s, 3H), 2.12–2.05 (m, J = 12.3 Hz, 2H).¹³C NMR (101 MHz, CDCl₃) δ 169.0, 160.2, 155.5, 141.2, 139.9, 135.3, 132.4, 128.7, 126.7, 126.0, 124.7, 115.1, 113.4, 108.0, 106.4, 96.3, 92.5, 46.0, 43.3, 41.3, 36.9, 29.3, 20.7, 20.5. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₇N₇O₄: 502.21973, found 502.22012; HPLC t_ret = 14.4 min (method A).

(E/Z)-2-(Morpholine-4-carbonyl)-3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylonitrile (13)

Obtained from 105 mg of 43b and 49 mg of N-cyanoacetylmorpholine (1.0 equiv) in 1 mL of MeOH following general procedure G at ambient temperature overnight. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 109 mg (74%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.12 (s, 1H), 8.70 (s, 1H), 7.80 (s, 1H), 7.60 (t, J = 3.0 Hz, 1H), 7.49 (dd, J = 12.8, 3.8 Hz, 2H), 6.90–6.88 (m, J = 3.3, 1.9 Hz, 1H), 5.48–5.38 (m, 1H), 3.71–3.58 (m, J = 20.0, 9.8 Hz, 12H), 2.70–2.63 (m, 2H), 2.04–1.97 (m, J = 12.6, 4.9 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.1, 149.6, 148.8, 144.6, 140.4, 136.4, 135.7, 135.2, 132.6, 124.3, 122.8, 116.4, 116.2, 104.4, 100.9, 100.8, 66.2, 65.9, 52.1, 46.6, 29.7. ESI-HRMS: [M + H]⁺ calculated for C₂₅H₂₄N₆O₄: 473.19318, found 473.19354; HPLC t_ret = 19.7 min (method A).

(E/Z)-2-(4-Methylpiperazine-1-carbonyl)-3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylonitrile (14)

Obtained from 84 mg of 43b and 44 mg of 52 (1.05 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 7 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 70 mg (58%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.23 (s, 1H), 8.82 (s, 1H), 7.89 (s, 1H), 7.72 (s, 1H), 7.60 (dd, J = 11.2, 3.6 Hz, 2H), 7.01 (s, 1H), 5.61–5.49 (m, 1H), 4.27–4.18 (m, 2H), 3.76–3.69 (m, 4H), 2.86–2.74 (m, 2H), 2.63 (s, 6H), 2.34 (s, 3H), 2.15–2.11 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.0, 149.7, 148.7, 144.6, 140.4, 136.3, 135.5, 135.2, 132.7, 124.3, 122.6, 116.4, 116.2, 104.4, 101.3, 100.8, 66.2, 54.2, 52.11, 45.4, 45.2, 29.7. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₇N₇O₃: 486.22481, found 486.22558; HPLC t_ret = 16.4 min (method A).

(E/Z)-3-(5-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-2-(morpholine-4-carbonyl)acrylonitrile (6)

Obtained from 94 mg of 43a and 43 of mg N-cyanoacetylmorpholine (1.0 equiv) in 1 mL of MeOH following general procedure G at 65 °C oil bath temperature with a reaction time of 3 h. Purification by storage at −20 °C over a weekend and subsequent filtration of precipitate and washing with cold MeOH. Yield: 60 mg (46%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.10 (s, 1H), 8.69 (s, 1H), 7.79 (s, 1H), 7.57 (s, 1H), 7.51 (d, J = 3.5 Hz, 1H), 7.41 (d, J = 3.5 Hz, 1H), 6.84 (s, 1H), 5.04–4.85 (m, J = 12.0 Hz, 1H), 3.69–3.57 (m, J = 7.8 Hz, 8H), 2.42–2.30 (m, J = 10.6 Hz, 2H), 2.07–1.98 (m, J = 10.5 Hz, 2H), 1.98–1.85 (m, J = 11.3 Hz, 2H), 1.82–1.47 (m, J = 44.4, 37.2, 11.2 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.1, 149.6, 148.6, 144.7, 140.6, 136.4, 135.8, 135.2, 132.8, 124.2, 122.3, 116.5, 116.0, 104.2, 101.1, 100.5, 65.9, 56.0, 30.0, 24.9, 24.3. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₆N₆O₃: 471.21392, found 471.21436; HPLC t_ret = 21.2 min (method A).

(E/Z)-2-Cyano-3-(5-(1-(4,4-difluorocyclohexyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N,N-dimethylacrylamide (33)

Obtained from 140 mg of 43e and 45 mg of 2-cyano-N,N-dimethylacetamide (1.05 equiv) in 1.5 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 2 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 75 mg (42%) as orange solid. ¹H NMR (400 MHz, CDCl₃) δ 11.97 (s, 1H), 8.70 (s, 1H), 7.52 (s, 1H), 7.28 (s, 1H), 7.27–7.22 (m, J = 3.8 Hz, 1H), 7.06–7.04 (m, J = 2.3 Hz, 1H), 6.80 (s, 1H), 5.29–5.16 (m, J = 12.4, 6.1 Hz, 1H), 3.10–2.99 (m, 2H), 2.92–2.81 (m, J = 17.4 Hz, 6H), 2.77–2.69 (m, J = 12.4, 5.7 Hz, 2H), 2.21–2.09 (m, J = 15.9 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 163.1, 149.6, 149.6, 145.0, 140.5, 136.8, 136.5, 135.6, 133.2, 123.5, 122.1, 116.3, 115.7, 104.7, 101.8, 100.2, 100.2, 53.4, 31.8, 29.3, 26.2. ESI-HRMS: [M + H]⁺ calculated for C₂₄H₂₂F₂N₆O₂: 465.18451, found 465.18520; HPLC t_ret = 15.4 and 17.4 min (E/Z mixture, method A).

(E/Z)-2-(Piperidine-1-carbonyl)-3-(5-(1-(tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylonitrile (15)

Obtained from 100 mg of 43b and 45 mg of 3-oxo-3-(piperidin-1-yl)propanenitrile (1.0 equiv) in 1 mL of MeOH following general procedure G at 70 °C oil bath temperature with a reaction time of 2 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 67 mg (48%) as bright yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 11.82 (s, 1H), 8.80 (s, 1H), 7.58 (s, 1H), 7.39 (d, J = 41.0 Hz, 2H), 7.20 (s, 1H), 7.03 (s, 1H), 5.49–5.35 (m, 1H), 4.17 (d, J = 7.7 Hz, 2H), 3.69 (t, J = 11.9 Hz, 2H), 3.57 (s, 4H), 2.92–2.79 (m, J = 8.5 Hz, 2H), 1.98 (d, J = 9.5 Hz, 2H), 1.69–1.57 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 162.4, 150.2, 149.6, 144.9, 141.2, 136.6, 135.9, 133.9, 123.8, 121.8, 116.6, 116.1, 105.5, 103.1, 102.2, 67.2, 52.8, 30.2, 25.8, 24.4. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₆N₆O₃: 471.21392, found 471.21473; HPLC t_ret = 20.1 min (method A).

(E/Z)-3-(5-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-2-(piperidine-1-carbonyl)acrylonitrile (7)

Obtained from 105 mg of 43a and 48 mg of 3-oxo-3-(piperidin-1-yl)propanenitrile (1.0 equiv) in 1 mL if MeOH following general procedure G at 65 °C oil bath temperature with a reaction time of 2 h. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 55 mg (37%) as orange solid. ¹H NMR (400 MHz, CDCl₃) δ 12.00 (s, 1H), 8.81 (s, 1H), 7.63 (s, 1H), 7.45–7.36 (m, 2H), 7.23 (s, 1H), 6.83 (s, 1H), 4.98–4.86 (m, J = 12.1 Hz, 1H), 3.56 (s, 4H), 2.46–2.39 (m, J = 12.0 Hz, 2H), 2.06–1.92 (m, 4H), 1.68–1.39 (m, 10H). ¹³C NMR (101 MHz, CDCl₃) δ 162.4, 150.2, 149.2, 145.0, 141.6, 137.1, 136.7, 135.8, 134.2, 123.5, 120.4, 116.3, 116.0, 105.3, 103.4, 101.3, 57.2, 53.1, 30.6, 26.1, 25.8, 24.9, 24.4. ESI-HRMS: [M + H]⁺ calculated for C₂₇H₂₈N₆O₂: 469.23465, found 469.23521; HPLC t_ret = 21.9 min (method A).

(E/Z)-3-(5-(1-(Tetrahydro-2H-pyran-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-2-(thiomorpholine-4-carbonyl)acrylonitrile (16)

Obtained from 67 mg of 43b and 34 mg of 53 (1.0 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1.5 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 33 mg (34%) as orange solid. ¹H NMR (400 MHz, CDCl₃) δ 11.81 (s, 1H), 8.89 (s, 1H), 7.70 (s, 1H), 7.48 (d, J = 35.0 Hz, 2H), 7.30 (d, J = 10.6 Hz, 1H), 7.12 (s, 1H), 5.50 (s, 1H), 4.26 (d, J = 7.3 Hz, 2H), 4.02–3.92 (m, 4H), 3.83–3.72 (m, J = 11.1 Hz, 2H), 3.01–2.87 (m, J = 8.4 Hz, 2H), 2.77 (s, 3H), 2.36 (s, 1H), 2.06 (d, J = 8.4 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 163.1, 150.2, 150.0, 145.2, 140.9, 137.4, 137.1, 136.1, 133.9, 123.6, 122.5, 116.7, 116.1, 105.4, 102.3, 102.0, 67.2, 52.9, 30.2, 27.5. ESI-HRMS: [M + H]⁺ calculated for C₂₅H₂₄N₆O₃S: 489.17034, found 489.17085; HPLC t_ret = 15.1 and 16.0 min (E/Z mixture, method A).

(E/Z)-2-Cyano-3-(5-(1-(cyclopropylmethyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N,N-dimethylacrylamide (20)

Obtained from 83 mg of 43d and 30 mg of 2-cyano-N,N-dimethylacetamide (1.0 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1.5 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 78 mg (72%) as orange solid. ¹H NMR (400 MHz, CDCl₃) δ 11.62 (s, 1H), 8.77 (s, 1H), 7.67 (s, 1H), 7.42 (d, J = 3.6 Hz, 2H), 7.25 (d, J = 3.4 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H), 4.85 (d, J = 6.6 Hz, 2H), 3.34–2.92 (m, J = 51.1 Hz, 6H), 1.37–1.26 (m, J = 4.9 Hz, 1H), 0.55–0.25 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 163.6, 150.1, 149.6, 144.8, 141.3, 137.1, 135.9, 135.8, 135.2, 124.1, 122.2, 116.3, 115.6, 105.0, 102.2, 97.6, 50.2, 39.3, 37.0, 12.1, 3.7. ESI-HRMS: [M + H]⁺ calculated for C₂₂H₂₀N₆O₂: 401.17205, found 401.17250; HPLC t_ret = 13.9 min (method A).

(E/Z)-3-(5-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-2-(thiomorpholine-4-carbonyl)acrylonitrile (8)

Obtained from 120 mg of 43a and 61 mg of 53 (1.0 equiv) in 1.5 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1.5 h. Purification by storage at −20 °C over a weekend and subsequent filtration of precipitate and washing with cold MeOH. Yield: 80 mg (46%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.99 (s, 1H), 8.63 (s, 1H), 7.69 (s, 1H), 7.43 (d, J = 3.5 Hz, 2H), 7.29 (d, J = 3.7 Hz, 1H), 6.81–6.74 (m, J = 11.4 Hz, 1H), 4.99–4.85 (m, J = 11.9 Hz, 1H), 3.87–3.70 (m, J = 14.8 Hz, 4H), 2.66 (s, 3H), 2.46 (s, 1H), 2.40–2.30 (m, 2H), 2.02–1.88 (m, 4H), 1.75 (d, J = 10.7 Hz, 1H), 1.63–1.43 (m, J = 31.3, 17.0 Hz, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 162.2, 149.5, 148.5, 144.1, 140.6, 135.7, 135.7, 135.0, 133.0, 131.5, 123.8, 121.7, 116.1, 115.6, 104.3, 101.4, 100.4, 56.0, 48.8, 29.9, 26.6, 24.9, 24.2. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₆N₆O₂S: 487.19107, found 487.19119; HPLC t_ret = 17.3 and 19.5 min (E/Z mixture, method A).

(E/Z)-3-(5-(1-Cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-2-(4-hydroxypiperidine-1-carbonyl)acrylonitrile (9)

Obtained from 149 mg of 43a and 75 mg of 51 (1.0 equiv) in 1.5 mL of MeOH following general procedure G at 55 °C oil bath temperature with a reaction time of 1 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. The filtrate was concentrated in vacuo, subjected to flash chromatography (EtOAc/MeOH, automatic gradient), and then added to the collected solid. Total yield: 149 mg (69%) as red solid. ¹H NMR (300 MHz, DMSO-d₆) δ 12.10 (s, 1H), 8.77–8.66 (m, J = 5.8 Hz, 1H), 7.77 (s, 1H), 7.58 (s, 1H), 7.51 (d, J = 3.7 Hz, 1H), 7.41 (d, J = 3.7 Hz, 1H), 6.86 (s, 1H), 5.05–4.72 (m, 2H), 3.99–3.72 (m, J = 35.5, 21.2 Hz, 3H), 2.53 (s, 1H), 2.44–2.33 (m, J = 10.3 Hz, 2H), 2.12–2.02 (m, 2H), 1.99–1.76 (m, J = 41.2, 10.1 Hz, 5H), 1.68–1.41 (m, J = 30.9, 16.9 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ 161.8, 149.6, 148.4, 144.7, 140.6, 136.4, 135.2, 135.1, 132.8, 124.2, 121.9, 116.4, 116.0, 104.2, 101.9, 100.4, 65.1, 56.1, 33.9, 30.0, 25.0, 24.3. ESI-HRMS: [M + H]⁺ calculated for C₂₇H₂₈N₆O₃: 485.22957, found 485.22985; HPLC t_ret = 17.0 min (method A).

(E/Z)-2-Cyano-3-(5-(1-cyclopropyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N,N-dimethylacrylamide (34)

Obtained from 110 mg of 43f and 42 mg of 2-cyano-N,N-dimethylacetamide (1.0 equiv) in 1 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 1.5 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 75 mg (52%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.98 (s, 1H), 8.64 (s, 1H), 7.78 (s, 1H), 7.52–7.49 (m, 3H), 6.94–6.88 (m, 1H), 4.06–3.94 (m, 1H), 3.18–2.97 (m, 6H), 1.46–1.38 (m, 2H), 0.96–0.91 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.9, 149.4, 148.3, 144.9, 141.9, 135.9, 135.3, 135.2, 133.9, 124.1, 122.0, 116.1, 115.2, 104.5, 101.7, 98.7, 37.4, 27.4, 9.7. ESI-HRMS: [M + H]⁺ calculated for C₂₁H₁₈N₆O₂: 387.15640, found 387.15668; HPLC t_ret = 14.0 min (method A).

(E/Z)-2-Cyano-N,N-dimethyl-3-(5-(1-phenyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (24)

Obtained from 220 mg of 43g and 85 mg of 2-cyano-N,N-dimethylacetamide (1.0 equiv) in 2 mL of MeOH following general procedure G in a screw-cap tube at 60 °C incubator temperature with a reaction time of 1 h. Purification by storage at −20 °C overnight and subsequent filtration of precipitate and washing with cold MeOH. Yield: 124 mg (44%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.79–8.72 (m, 1H), 7.82–7.63 (m, 6H), 7.57–7.48 (m, 1H), 7.35–7.27 (m, 2H), 5.65–5.55 (m, J = 16.1, 3.2 Hz, 1H), 3.34 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 162.6, 149.0, 147.2, 145.3, 140.4, 135.9, 135.2, 135.0, 134.0, 130.3, 130.1, 128.0, 124.3, 120.2, 115.7, 114.6, 113.6, 103.8, 102.7, 96.2, 37.4. ESI-HRMS: [M + H]⁺ calculated for C₂₄H₁₈N₆O₂: 423.15640, found 423.15676; HPLC t_ret = 16.5 min (method A).

(E/Z)-2-(Morpholine-4-carbonyl)-3-(5-(1-phenyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylonitrile (25)

Obtained from 35 mg of 43g and 16 mg of N-cyanoacetylmorpholine (1.0 equiv) in 2 mL of EtOH following general procedure G in a screw-cap tube at 60 °C incubator temperature with a reaction time of 3 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 35 mg (70%) as yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.98 (s, 1H), 8.74 (s, 1H), 7.82–7.60 (m, 6H), 7.55 (d, J = 9.5 Hz, 1H), 7.36–7.23 (m, 2H), 5.59 (d, J = 17.8, 1.8 Hz, 1H), 3.75–3.39 (m, 8H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.8, 149.0, 147.3, 145.3, 140.3, 136.2, 135.9, 135.4, 135.0, 134.0, 130.3, 130.1, 128.0, 124.3, 120.5, 114.6, 113.6, 103.8, 102.0, 96.2, 65.9, 46.5. ESI-HRMS: [M + Na]⁺ calculated for C₂₆H₂₀N₆O₃: 487.14891, found 487.14961; HPLC t_ret = 16.8 min (method A).

Methyl (E/Z)-N-(2-Cyano-3-(5-(1-phenyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acryloyl)-N-methylglycinate (26)

Obtained from 41 mg of 43g and 21 mg of 49 in 0.5 mL of MeOH following general procedure G in a screw-cap tube at 60 °C incubator temperature overnight. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 28 mg (47%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.04 (s, 2H), 8.83 (s, 1H), 7.81–7.75 (m, 7H), 7.38–7.36 (m, 1H), 6.50 (d, J = 3.8 Hz, 1H), 5.69–5.66 (m, J = 4.0, 2.2 Hz, 1H), 4.37–4.24 (m, 1H), 3.76 (s, 3H), 3.28 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.2, 163.4, 152.3, 148.9, 145.3, 140.3, 136.2, 135.9, 135.4, 134.0, 130.1, 130.0, 128.0, 125.3, 115.4, 114.6, 112.0, 110.2, 103.8, 97.0, 96.2, 52.7, 52.0, 29.0. ESI-HRMS: [M + H]⁺ calculated for C₂₆H₂₀N₆O₄: 481.16188, found 481.16229; HPLC t_ret = 15.7 and 16.7 min (E/Z/ mixture, method A).

(E/Z)-2-(4-Methylpiperazine-1-carbonyl)-3-(5-(1-phenyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylonitrile (27)

Obtained from 40 mg of 43g and 21 mg of 52 in 1 mL of EtOH following general procedure G in a screw-cap tube at 60 °C incubator temperature overnight. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 40 mg (70%) as orange platelets. ¹H NMR (300 MHz, MeOD) δ 8.98 (s, 1H), 7.99–7.94 (m, J = 5.0, 2.2 Hz, 2H), 7.93 (s, 1H), 7.85–7.80 (m, 2H), 7.77 (s, 1H), 7.63 (d, J = 3.9 Hz, 1H), 7.46 (d, J = 3.4 Hz, 1H), 6.76 (d, J = 3.9 Hz, 1H), 6.02 (d, J = 3.4 Hz, 1H), 3.96–3.89 (m, 4H), 2.79–2.73 (m, 4H), 2.59 (s, 3H). ¹³C NMR (75 MHz, MeOD) δ 163.3, 150.5, 148.0, 145.8, 142.1, 137.9, 137.0, 136.5, 136.2, 134.7, 131.2, 130.9, 128.6, 124.9, 120.3, 116.3, 115.9, 105.5, 103.3, 97.7, 55.0, 46.0. ESI-HRMS: [M + H]⁺ calculated for C₂₇H₂₃N₇O₂: 478.19860, found 478.19891; HPLC t_ret = 11.0 min (method A).

(E/Z)-3-(5-(1-(Cyclopropylmethyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-2-(4-methylpiperazine-1-carbonyl)acrylonitrile (22)

Obtained from 80 mg of 43d and 40 mg of 52 in 3 mL of MeOH following general procedure G at 60 °C oil bath temperature with a reaction time of 4 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 91 mg (77%) as red solid. ¹H NMR (300 MHz, DMSO-d₆) δ 11.81 (s, 1H), 8.43 (s, 1H), 7.55 (s, 1H), 7.37–7.26 (m, 3H), 6.73–6.65 (m, J = 1.5 Hz, 1H), 4.67 (d, J = 6.9 Hz, 2H), 3.47–3.21 (m, 4H), 2.31–2.14 (m, 4H), 1.99 (s, 3H), 0.93–0.89 (m, 1H), 0.25–0.06 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 162.0, 149.2, 149.2, 144.9, 140.1, 135.8, 135.3, 134.4, 134.4, 124.3, 122.9, 116.4, 115.2, 103.8, 100.8, 97.0, 54.2, 49.1, 45.5, 45.5, 12.0, 3.1. ESI-HRMS: [M + H]⁺ calculated for C₂₅H₂₅N₇O₂: 456.21425, found 456.21429; HPLC t_ret = 17.5 and 18.6 min (E/Z mixture, method B).

Methyl (E/Z)-N-(2-Cyano-3-(5-(1-(cyclopropylmethyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acryloyl)-N-methylglycinate (21)

Obtained from 97 mg of 43d and 60 mg of 49 (1.1 equiv) in 1 mL of MeOH following general procedure G in a screw-cap tube at 60 °C incubator temperature with a reaction time of 2 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 112 mg (77%) as orange solid. ¹H NMR (300 MHz, DMSO-d₆) δ 12.03 (s, 1H), 8.66 (s, 1H), 7.86 (s, 1H), 7.60–7.48 (m, J = 9.1, 6.3 Hz, 3H), 6.95–6.89 (m, 1H), 4.88 (d, J = 12.6 Hz, 2H), 4.12 (d, J = 7.4 Hz, 2H), 3.70 (s, 3H), 3.28 (s, 3H), 1.03 (d, J = 6.1 Hz, 1H), 0.47–0.33 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 169.2, 163.7, 149.5, 149.1, 144.9, 144.8, 140.0, 135.8, 134.5, 124.3, 116.0, 115.8, 115.3, 103.8, 100.1, 97.2, 97.0, 52.7, 51.8, 49.2, 25.5, 12.0, 3.1. ESI-HRMS: [M + H]⁺ calculated for C₂₄H₂₂N₆O₄: 459.17753, found 459.17752; HPLC t_ret = 15.8 min (method A).

(E/Z)-3-(5-(1-(Cyclopropylmethyl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-2-(piperidine-1-carbonyl)acrylonitrile (23)

Obtained from 107 mg of 43d and 48 mg of 3-oxo-3-(piperidin-1-yl)propanenitrile (1.0 equiv) in 1 mL of MeOH following general procedure G in a screw-cap tube at 60 °C incubator temperature with a reaction time of 1.5 h. Flash chromatography (cyclohexane/IPrOH, automatic gradient). Yield: 100 mg (71%) as orange solid. ¹H NMR (300 MHz, CDCl₃) δ 11.39 (s, 1H), 8.78 (s, 1H), 7.59 (s, 1H), 7.44–7.35 (m, 2H), 7.23 (d, J = 3.8 Hz, 1H), 6.71 (d, J = 2.5 Hz, 1H), 4.85 (d, J = 6.8 Hz, 2H), 3.65–3.53 (m, J = 18.4 Hz, 4H), 1.64 (s, 6H), 0.86–0.73 (m, J = 15.2, 8.2 Hz, 1H), 0.49–0.32 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 163.0, 150.5, 150.1, 145.7, 141.6, 137.0, 137.0, 136.0, 135.7, 124.2, 122.3, 116.9, 115.8, 105.2, 103.0, 98.0, 50.6, 26.3, 25.9, 24.9, 12.5, 4.1. ESI-HRMS: [M + H]⁺ calculated for C₂₅H₂₄N₆O₂: 441.20335, found 441.20352; HPLC t_ret = 15.9 and 18.8 min (E/Z mixture, method A).

(E/Z)-2-Cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N-(3-(((2S,3R,4S,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)(methyl)amino)propyl)-N-methylacrylamide (31)

Obtained from 73 mg of 47 and 211 mg of 43a in 1.5 mL of MeOH following general procedure G in a screw-cap tube at 60 °C incubator temperature with a reaction time of 2 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 129 mg (50%) as orange solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.08 (s, 1H), 8.69–8.67 (m, J = 2.1 Hz, 1H), 7.75 (s, 1H), 7.57–7.53 (m, 2H), 7.47 (d, J = 3.7 Hz, 1H), 7.39 (d, J = 3.8 Hz, 1H), 5.01–4.85 (m, 4H), 4.80–4.73 (m, 1H), 4.60–4.53 (m, 1H), 4.42–4.17 (m, 3H), 4.11–4.02 (m, J = 14.9, 6.5 Hz, 2H), 3.96–3.60 (m, J = 74.2, 37.1, 24.6 Hz, 3H), 3.50 (s, 1H), 3.43–3.39 (m, 2H), 3.38 (s, 1H), 3.36 (s, 1H), 3.34 (d, J = 1.4 Hz, 1H), 3.23 (t, 3H), 3.17 (s, 2H), 3.05–2.99 (m, 2H), 2.93–2.88 (m, 2H), 2.68 (s, 1H), 2.48–2.45 (m, 2H), 2.40–2.29 (m, 5H), 2.26–2.20 (m, J = 10.8 Hz, 2H), 2.06–1.99 (m, 3H), 1.96–1.86 (m, 6H), 1.78–1.69 (m, J = 10.5 Hz, 5H), 1.61–1.45 (m, J = 30.7, 17.5 Hz, 9H), 1.22–1.20 (m, 2H), 1.17 (s, 1H), 1.15 (s, 1H), 1.10 (s, 3H), 1.08 (s, 2H), 1.06 (s, 2H), 1.04–0.98 (m, 7H), 0.87–0.79 (m, 6H), 0.78–0.73 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 178.5, 177.2, 149.6, 148.5, 144.7, 140.5, 136.3, 135.1, 132.8, 124.1, 121.8, 116.3, 115.9, 114.7, 104.1, 100.3, 99.61, 94.5, 77.4, 76.9, 73.5, 72.3, 65.5, 64.9, 56.1, 54.0, 48.6, 48.6, 45.7, 44.9, 41.6, 38.2, 36.9, 34.45, 30.3, 30.0, 29.0, 27.4, 25.3, 25.0, 24.3, 21.9, 21.7, 20.9, 20.8, 18.3, 17.5, 17.4, 15.1, 14.8, 10.9, 8.5. ESI-HRMS: [M + H]⁺ calculated for C₆₃H₉₆N₈O₁₄: 1189.71188, found 1189.71202; HPLC t_ret = 14.9 and 15.1 min (E/Z mixture, method A).

(E/Z)-2-Cyano-N-(3-(((2S,3S,4R,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-3-yl)(methyl)amino)propyl)-N-methyl-3-(5-(1-(1-methylpiperidin-4-yl)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)acrylamide (32)

Obtained from 132 of 47 and 53 mg of 43c in 3 mL of MeOH following general procedure G at 60 °C oil bath temperature for 6 h. Flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 145 mg (80%) as an orange to yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 11.51 (s, 1H), 8.79 (s, 1H), 7.72 (s, 1H), 7.46–7.44 (m, 1H), 7.41–7.36 (m, 1H), 7.32 (d, J = 3.7 Hz, 1H), 7.22–7.18 (m, 1H), 5.05–4.98 (m, 2H), 4.73–4.69 (m, 1H), 4.47–4.40 (m, J = 7.2 Hz, 1H), 4.27–4.22 (m, 1H), 4.11–3.98 (m, J = 18.3, 13.3, 6.6 Hz, 2H), 3.72–3.65 (m, J = 7.0 Hz, 4H), 3.64–3.61 (m, 1H), 3.53–3.46 (m, 3H), 3.32–3.27 (m, J = 9.0 Hz, 4H), 3.24–3.19 (m, 2H), 3.11–3.07 (m, J = 10.0 Hz, 2H), 3.04–2.98 (m, 3H), 2.92–2.85 (m, J = 8.0 Hz, 2H), 2.74–2.67 (m, 2H), 2.48–2.43 (m, 2H), 2.38 (s, 3H), 2.30–2.27 (m, J = 9.8 Hz, 6H), 2.04–1.94 (m, J = 16.0, 9.5 Hz, 5H), 1.86 (dd, J = 12.2, 7.7 Hz, 2H), 1.67–1.61 (m, 1H), 1.54–1.41 (m, 3H), 1.32–1.29 (m, J = 6.5 Hz, 3H), 1.28–1.24 (m, 4H), 1.22–1.20 (m, J = 1.9 Hz, 3H), 1.19–1.18 (m, 4H), 1.18–1.17 (m, 2H), 1.16–1.14 (m, J = 6.4 Hz, 4H), 1.08–1.05 (m, 6H), 1.03–0.99 (m, J = 4.3 Hz, 3H), 0.90–0.80 (m, J = 7.4 Hz, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 178.3, 163.3, 161.9, 150.1, 149.6, 144.9, 141.3, 137.4, 136.8, 135.9, 133.9, 123.7, 121.3, 116.4, 116.0, 105.4, 103.1, 102.3, 95.1, 84.0, 78.5, 78.2, 74.5, 74.2, 73.8, 73.1, 71.0, 70.0, 68.7, 65.6, 65.5, 62.5, 58.1, 55.2, 54.3, 51.0, 49.5, 46.6, 45.9, 45.3, 42.7, 41.9, 37.0, 36.5, 35.0, 30.4, 30.1, 29.8, 29.7, 27.5, 26.8, 25.5, 22.1, 21.7, 21.4, 18.4, 16.6, 15.1, 11.3, 9.4, 8.8, 7.7. ESI-HRMS: [M + H]⁺ calculated for C₆₃H₉₇N₉O₁₄: 1204.72278, found 1204.72277; HPLC t_ret = 17.0 and 19.0 min (E/Z mixture, method B).

Epoxidation of Azithromycin: (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-Ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-11-(((1R,2S,4R,6R)-4-methyl-3,7-dioxabicyclo[4.1.0]heptan-2-yl)oxy)-1-oxa-6-azacyclopentadecan-15-one (44)

15.55 g of (3) (20,8 mmol, 1.0 equiv) was dissolved in 105 mL of iBuOH and the stirred solution heated to 75 °C. 45 mL of glycidol (685 mmol, 33 equiv) was added dropwise as a 1:1 mixture in iBuOH over 1 h. Stirring continued for 3 h, at which point MS indicated no further conversion of azithromycin. The solution was concentrated to almost dryness under reduced pressure, and 300 mL of H₂O was added. The suspension was left to stir at ambient temperature overnight, and the solids were filtered. Recrystallization in MeOH yielded 3,07 g (21%) of (44) as fine, colorless to white platelets. ¹H NMR (400 MHz, CDCl₃) δ 5.08 (d, J = 4.8 Hz, 1H), 4.90 (s, 1H), 4.68 (d, J = 9.5 Hz, 1H), 4.18 (d, J = 4.4 Hz, 1H), 4.03 (dq, J = 12.4, 6.1 Hz, 1H), 3.67–3.61 (m, 2H), 3.50–3.44 (m, J = 15.8 Hz, 2H), 3.42–3.38 (m, 1H), 3.37 (s, 3H), 3.33–3.30 (m, J = 4.7 Hz, 1H), 3.19 (d, J = 3.0 Hz, 1H), 3.00 (t, J = 10.2 Hz, 1H), 2.95–2.90 (m, 1H), 2.81–2.66 (m, 2H), 2.54 (d, J = 10.7 Hz, 1H), 2.37 (s, 1H), 2.34–2.30 (m, J = 5.2 Hz, 3H), 2.20 (d, J = 11.0 Hz, 1H), 2.04–1.95 (m, J = 14.1 Hz, 3H), 1.93–1.84 (m, J = 14.7, 7.4 Hz, 1H), 1.74–1.61 (m, J = 29.9, 14.7 Hz, 2H), 1.56 (dd, J = 15.2, 4.7 Hz, 1H), 1.51–1.41 (m, 1H), 1.35–1.27 (m, J = 11.5, 5.1 Hz, 7H), 1.24–1.18 (m, 7H), 1.14 (d, J = 6.1 Hz, 3H), 1.11–1.06 (m, J = 8.9 Hz, 6H), 0.98 (d, J = 7.2 Hz, 3H), 0.94–0.86 (m, J = 15.9, 7.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 178.2, 97.8, 94.7, 85.9, 77.8, 76.8, 76.5, 73.9, 73.5, 73.0, 72.5, 69.8, 65.2, 62.2, 61.6, 53.8, 51.5, 49.0, 44.9, 41.6, 41.4, 36.0, 34.4, 32.3, 27.0, 26.4, 21.6, 21.3, 21.0, 20.6, 18.0, 16.0, 14.5, 11.0, 8.9, 6.9. MS (ESI) m/z: 704,60 [M + H]⁺; HPLC t_ret= 14.1 min (method A).

(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-Ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,6R)-3-hydroxy-6-methyl-4-(methyl(2-(methylamino)ethyl)amino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one (45)

6.0 g of (44) (8.52 mmol, 1.0 equiv) was dissolved in 10 mL of N,N′-dimethylethane-1,2-diamine in a 25 mL round bottomed flask and stirred at 60 °C. After 2 days, MS indicated full consumption of starting material. The crude product was taken up in silica gel and subjected to column chromatography (EtOAc → EtOAc/MeOH 2:1), yielding 4.47 g (66%) of the title compound as a solid. Another 1.25 g of product with identical m/z but different t_ret and NMR spectra were also isolated, likely to be from opening of the epoxide at the 2′ position instead of 3′. ¹H NMR (400 MHz, DMSO-d₆) δ 7.51 (s, 1H), 4.90 (s, 1H), 4.86 (d, J = 4.6 Hz, 1H), 4.75 (dd, J = 10.1, 2.4 Hz, 1H), 4.31–4.28 (m, J = 2.3 Hz, 1H), 4.12–4.04 (m, 2H), 4.02 (d, J = 7.1 Hz, 1H), 3.86–3.83 (m, 3H), 3.53 (d, 2H), 3.43 (s, 1H), 3.25 (s, 3H), 3.16 (s, 1H), 3.07–2.99 (m, 1H), 2.84–2.76 (m, J = 13.1, 5.9 Hz, 1H), 2.74–2.69 (m, J = 5.7 Hz, 2H), 2.66 (d, J = 6.9 Hz, 2H), 2.47 (s, 3H), 2.42 (s, 3H), 2.36 (d, J = 10.4 Hz, 1H), 2.27 (s, 1H), 2.21 (s, 3H), 2.11–2.06 (m, J = 6.9, 4.7 Hz, 1H), 1.98 (s, 1H), 1.93–1.74 (m, 3H), 1.66 (d, J = 14.2 Hz, 1H), 1.54–1.45 (m, 3H), 1.44–1.25 (m, 3H), 1.21 (s, 3H), 1.17 (s, 2H), 1.15 (d, J = 1.3 Hz, 1H), 1.11 (s, 3H), 1.09–1.05 (m, 5H), 1.00 (s, 3H), 0.96–0.92 (m, J = 6.9, 3.2 Hz, 4H), 0.88–0.82 (m, 6H), 0.81–0.76 (m, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 177.11, 99.7, 94.6, 83.4, 77.4, 77.0, 76.3, 74.9, 73.6, 72.6, 72.3, 69.0, 68.7, 65.6, 64.8, 63.2, 61.5, 59.6, 53.9, 48.6, 47.6, 45.7, 44.9, 41.6, 36.8, 35.6, 34.0, 27.6, 26.0, 22.0, 21.6, 20.9, 20.6, 18.3, 17.6, 14.8, 14.0, 10.8, 8.7, 6.6. MS (ESI) m/z: 859,73 [M + H]⁺; HPLC t_ret = 13,7 min (method A)

2-Cyano-N-(2-(((2S,3R,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)(methyl)amino)ethyl)-N-methylacetamide (35)

566 mg of cyanoacetic acid (6.66 mmol, 1.1 equiv) and 2.3 g of HATU (6.06 mmol, 1.0 equiv) were suspended in 35 mL of dry THF and 1 mL of Et₃N (7.27 mmol, 1.2 equiv) and stirred at ambient temperature for 30 min. 4.8 g of (45) (6.06 mmol, 1.0 equiv) was added in one portion, and the mixture was left to stir for 2 days, after which TLC and MS confirmed full conversion of starting material. The reaction mixture was diluted with EtOAc, transferred to a separatory funnel, and extracted three times with 5% citric acid solution. The combined aqueous phases were basified by portionwise addition of K₂CO₃ and extracted with three portions of EtOAc. The combined organics were evaporated under reduced pressure, and the crude product was subjected to column chromatography (EtOAc → EtOAc/MeOH 8:1), yielding 3.35 g (64%) of the title compound as an off-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.56 (s, 1H), 4.90 (s, 1H), 4.85 (s, 1H), 4.74 (d, J = 9.7 Hz, 1H), 4.65 (dd, J = 16.7, 2.4 Hz, 1H), 4.41–4.27 (m, 3H), 4.13–4.02 (m, 2H), 3.98–3.91 (m, 1H), 3.78–3.63 (m, 2H), 3.58–3.47 (m, J = 15.0, 11.1 Hz, 2H), 3.23 (s, 4H), 3.12–3.00 (m, J = 16.8, 9.9 Hz, 1H), 2.97–2.87 (m, 2H), 2.83 (d, J = 2.7 Hz, 1H), 2.69 (s, 6H), 2.66–2.61 (m, 1H), 2.52–2.44 (m, 3H), 2.37 (d, J = 9.8 Hz, 1H), 2.29–2.17 (m, 5H), 2.08 (s, 1H), 1.98 (s, 1H), 1.94–1.81 (m, 2H), 1.79–1.26 (m, 7H), 1.21 (d, J = 2.2 Hz, 2H), 1.16 (d, J = 6.4 Hz, 3H), 1.14–1.13 (m, 1H), 1.11 (s, 2H), 1.09–1.04 (m, 6H), 1.01 (s, 3H), 0.97–0.92 (m, J = 4.8 Hz, 4H), 0.87–0.82 (m, 4H), 0.78 (t, J = 7.4 Hz, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 177.2, 162.8, 116.1, 99.6, 94.6, 82.9, 77.4, 77.1, 76.3, 74.9, 73.6, 72.7, 72.3, 69.8, 68.7, 65.5, 64.8, 61.6, 59.8, 54.2, 53.7, 48.7, 45.0, 41.7, 41.4, 35.6, 34.6, 30.6, 27.6, 26.1, 24.9, 24.5, 22.1, 21.6, 21.0, 20.7, 18.4, 17.6, 14.8, 14.1, 10.9, 8.8, 6.7. ESI-HRMS: [M + H]⁺ calculated for C₄₃H₇₈N₄O₁₃: 859.56382, found 859.56332; HPLC t_ret = 14.7 min (method A)

Methyl Methylglycinate (48)

In an ice-cooled round bottomed flask, 25 g (281 mmol, 1.0 equiv) of sarcosine was dissolved in 350 mL of MeOH. 21.4 mL of SOCl₂ (295 mmol, 1.05 equiv) was added dropwise. After complete addition, the mixture was stirred for 30 min at 0 °C. At this point, the ice bath was removed, and the mixture was heated to reflux for 3 h. The reflux condenser was removed, and the mixture was concentrated under the hood. To the crude product, a sparing amount of MeOH and a larger amount of Et₂O were added. After vigorous stirring for about 10 min, the product was left to crystallize overnight. The solids were filtrated and dried in vacuo. Yield: 33.5 g (86%) of the title compound. ¹H NMR (300 MHz, MeOD) δ 4.88 (d, J = 8.5 Hz, 2H), 4.09 (s, 1H), 3.92 (s, 3H), 2.86 (s, 3H). ¹³C NMR (75 MHz, MeOD) δ 168.1, 53.4, 48.7, 33.6. MS (ESI) m/z: 104.16 [M + H]⁺; HPLC t_ret = 0.5 min (method B).

Methyl N-(2-Cyanoacetyl)-N-methylglycinate (49)

In a round bottomed flask, 8.25 g (97 mmol, 1.0 equiv) of cyanoacetic acid were dissolved in 250 mL of DCM. The flask was fitted with a septum connected to a gas bubbler, purged with argon, and put on an ice bath. Three mL of DMF was added by syringe, followed by 8.7 mL of oxalyl chloride (101.9 mmol, 1.05 equiv), after which gas development was observed. The mixture was stirred on ice for 40 min. The ice bath was then removed, and 10 g of 48 (97 mmol, 1.0 equiv) was added. Following that, 27 mL of Et₃N (194 mmol, 2.0 equiv) was slowly added, and the mixture was left to stir overnight. After addition of water and sat. NaHCO₃ (2:1), the two phases were transferred to a separatory funnel and extracted two times with DCM. The combined organics were dried over Na₂SO₄ and evaporated under reduced pressure, and the crude product was purified by flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 12.5 g (75%) of the title compound as a brownish oil. ¹¹H NMR (300 MHz, DMSO-d₆) δ 4.10 (d, J = 2.6 Hz, 4H), 3.66 (s, 3H), 3.00 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 169.3, 163.9, 115.8, 51.8, 49.2, 36.3, 24.7. MS (ESI) m/z: 171.13 [M + H]⁺; HPLC t_ret = 1.3 min (method B).

2-Cyano-N-(2-(dimethylamino)ethyl)-N-methylacetamide (50)

180 mg of methyl-2-cyanoacetate (1.82 mmol, 1.0 equiv) and 236 μL of N,N,N′-trimethylethane-1,2-diamine were stirred overnight at ambient temperature. The brown mixture was taken up in Et₂O and water and transferred to a separatory funnel, and the aqueous phase was washed two times with Et₂O. The organic phase was discarded, and the aqueous phase was dried in vacuo, yielding 245 mg (80%) of the tile compound as a dark oil with no further purification steps taken. ¹H-NMRn (400 MHz, DMSO-d₆) δ 3.99 (s, 2H), 3.40 (t, J = 6.7 Hz, 2H), 2.91 (s, 3H), 2.55–2.37 (m, 2H), 2.21 (s, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ 162.7, 116.12, 56.1, 45.5, 45.3, 35.4, 25.0. MS (ESI) m/z: 170.2 [M + H]⁺; HPLC t_ret = 7,8 min (method B).

3-(4-Hydroxypiperidin-1-yl)-3-oxopropanenitrile (51)

In a round bottomed flask, 1.58 g of 4-hydroxypiperidine (15.68 mmol, 1.0 equiv), 1.67 mL of ethyl cyanoacetate (15.68 mmol, 1.0 equiv), and 11 mg of sodium ethoxide (0.157 mmol, 0.01 equiv) were dissolved in 6 mL of EtOH and stirred over a weekend at ambient temperature, after which TLC and MS indicated consumption of starting material. Flash chromatography (cyclohexane/acetone, automatic gradient). Yield: 800 mg (30%) of the title compound as red oil. ¹H NMR (300 MHz, DMSO-d₆) δ 4.74 (d, J = 4.0 Hz, 1H), 3.99 (s, 2H), 3.90–3.77 (m, 1H), 3.76–3.64 (m, 1H), 3.57–3.45 (m, J = 12.8, 4.5 Hz, 1H), 3.20–2.97 (m, J = 16.4, 11.2, 3.3 Hz, 2H), 1.81–1.61 (m, 2H), 1.49–1.19 (m, J = 17.2, 8.9, 3.9 Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆) δ 161.1, 116.3, 65.6, 43.4, 34.0, 24.8. MS (ESI) m/z: 169.20 [M + H]⁺; HPLC t_ret = 1.2 min (method B).

3-(4-Methylpiperazin-1-yl)-3-oxopropanenitrile (52)

In an ice cooled round bottomed flask, 2.21 mL of N-methylpiperazine (20.0 mmol, 1.0 equiv) and 1.78 mL of methyl cyanoacetate (20.0 mmol, 1.0 equiv) were stirred overnight and allowed to reach ambient temperature. Volatile residues were then evaporated under reduced pressure, and the crude solid was suspended in Et₂O and triturated with a glass rod. After decanting with Et₂O three times, the solid was collected by filtration and dried in vacuo. Yield: 2.36 g (71%) of the title compound as brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ 4.01 (s, 2H), 3.44 (t, 2H), 3.32 (t, 2H), 2.31 (t, 2H), 2.25 (t, 2H), 2.18 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.3, 116.0, 54.3, 53.9, 45.4, 45.2, 41.5, 24.6. MS (ESI) m/z: 168.27 [M + H]⁺; HPLC t_ret = 0.5 min (method B).

3-Oxo-3-thiomorpholinopropanenitrile (53)

In a round bottomed flask, 2.0 mL of thiomorpholine (19.4 mmol, 1.0 equiv), 2.07 mL of ethyl cyanoacetate (19.4 mmol, 1.0 equiv), and 13 mg of sodium ethoxide (0.194 mmol, 0.01 equiv) were dissolved in 6 mL of EtOH and stirred at 65 °C overnight. Volatiles were removed under reduced pressure, and the residue was subjected to flash chromatography (EtOAc/MeOH, automatic gradient). HPLC still showed impurities, but the product was successfully used in further reactions without additional purification. Yield: 1.84 g (56%) of the title compound as beige solid. ¹H NMR (400 MHz, DMSO-d₆) δ 4.03 (s, 2H), 3.76–3.68 (m, 2H), 3.62–3.55 (m, 2H), 2.68–2.60 (m, 2H), 2.60–2.53 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.5, 115.9, 48.1, 44.3, 24.9. MS (ESI) m/z: 168.93 [M – H]⁻; HPLC t_ret = 13.1 min (method B).

2-Cyano-3-(5-(1-cyclohexyl-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-2-yl)furan-2-yl)-N,N-dimethylpropanamide (36)

In a screw-cap tube, 100 mg of 2 (0.23 mmol, 1.0 equiv) and 10 mg of NaBH₄ (0.26 mmol, 1.1 equiv) were dissolved in 1 mL of MeOH and gently shaken at ambient temperature. TLC and MS indicated full conversion of educt after 30 min. H₂O was added dropwise to convert excess NaHB₄, and the organic solvent was evaporated under reduced pressure. The mixture was diluted with sat. Na₂CO₃ and DCM and transferred to a separatory funnel. After extraction with DCM (three times), the combined organics were dried over Na₂SO₄ and evaporated in vacuo to yield 89 mg (89%) of the title compound as yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 11.93 (s, 1H), 8.94–8.83 (m, 1H), 7.49 (s, 1H), 6.87 (d, J = 2.9 Hz, 2H), 6.51 (d, J = 3.0 Hz, 1H), 4.83 (t, J = 12.1 Hz, 1H), 4.10 (dd, J = 8.0, 6.9 Hz, 1H), 3.45 (qd, J = 15.3, 7.6 Hz, 2H), 3.12 (s, 3H), 3.02 (s, 3H), 2.58–2.44 (m, J = 9.5 Hz, 2H), 2.12–1.97 (m, J = 13.0 Hz, 4H), 1.89 (s, 1H), 1.61–1.47 (m, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.2, 152.2, 145.1, 144.6, 143.1, 136.7, 135.5, 133.8, 123.3, 116.3, 113.8, 110.7, 105.2, 100.9, 57.1, 37.6, 36.6, 34.2, 30.9, 28.7, 25.90, 25.04. ESI-HRMS: [M + H]⁺ calculated for C₂₄H₂₆N₆O₂: 431.21900, found 431.21942; HPLC t_ret = 16.4 min (method A).

(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-Ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-11-(((2S,3R,6R)-3-hydroxy-6-methyl-4-(methyl(3-(methylamino)propyl)amino)tetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one (46)

In a screw-cap tube, 519 mg of 44 (0.738 mmol, 1.0 equiv) was suspended in excess N1,N3-dimethylpropane-1,3-diamine (about 0.5 mL) and left in a shaking incubator at 105 °C overnight, after which MS indicated full conversion of educt. The mixture was transferred to a round bottomed flask, concentrated under reduced pressure, and subjected to flash chromatography (acetone/MeOH, automatic gradient). Yield: 270 mg (46%) as colorless crystals ¹H NMR (600 MHz, DMSO-d₆) δ 4.89 (d, J = 2.5 Hz, 1H), 4.86 (d, J = 4.8 Hz, 1H), 4.75 (dd, J = 10.2, 2.7 Hz, 1H), 4.32–4.29 (m, J = 2.1 Hz, 1H), 4.09 (dq, J = 12.4, 6.1 Hz, 1H), 3.96–3.91 (m, J = 17.0, 7.6, 4.5 Hz, 1H), 3.77 (d, J = 2.4 Hz, 1H), 3.51 (d, J = 7.7 Hz, 1H), 3.43 (s, 2H), 3.25 (s, 3H), 2.89 (d, J = 9.4 Hz, 1H), 2.84–2.75 (m, 1H), 2.68–2.64 (m, 2H), 2.61 (dt, J = 13.0, 6.6 Hz, 1H), 2.47–2.44 (m, J = 7.1 Hz, 2H), 2.43 (s, 3H), 2.38–2.34 (m, 1H), 2.27–2.22 (m, J = 11.1 Hz, 4H), 2.20 (s, 3H), 2.15–2.07 (m, 2H), 1.91–1.82 (m, 2H), 1.77 (ddd, J = 14.1, 7.5, 2.6 Hz, 1H), 1.72–1.68 (m, 1H), 1.52–1.46 (m, 4H), 1.45–1.41 (m, J = 13.6 Hz, 2H), 1.38–1.34 (m, 1H), 1.27 (dd, J = 14.2, 7.7 Hz, 1H), 1.21 (s, 3H), 1.16 (d, J = 6.2 Hz, 3H), 1.11 (s, 3H), 1.07 (d, J = 7.5 Hz, 3H), 1.05 (d, J = 6.1 Hz, 3H), 1.00 (s, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 7.4 Hz, 3H), 0.83 (d, J = 6.9 Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H). 13C-NMR (75 MHz, DMSO-d₆) δ 177.1, 99.7, 94.5, 83.3, 77.4, 77.0, 76.3, 75.2, 73.6, 72.6, 72.3, 68.9, 68.8, 65.5, 64.9, 64.8, 63.0, 61.6, 58.5, 54.9, 49.9, 48.6, 44.9, 42.2, 41.8, 40.2, 36.9, 36.3, 35.6, 34.6, 27.9, 27.6, 26.0, 22.0, 21.8, 20.9, 18.4, 17.7, 15.1, 14.8, 10.9, 8.0, 6.6. MS (ESI) m/z: 806.73 [M + H]⁺; HPLC t_ret = 8.9 min (method B).

2-Cyano-N-(3-(((2S,3R,6R)-2-(((2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl)oxy)-3-hydroxy-6-methyltetrahydro-2H-pyran-4-yl)(methyl)amino)propyl)-N-methylacetamide (47)

In a round-bottomed flask, 44 mg of cyanoacetic acid, (0.519 mmol, 1.1 equiv), 197 mg of HATU (0.519 mmol, 1.1 equiv), and 72 μL of Et₃N (0.519 mmol, 1.1 equiv) were stirred in 5 mL of dry THF at ambient temperature for 1 h. At this point, 380 mg of 46 (0.471 mmol, 1.0 equiv) was added, and the mixture was stirred until TLC and MS indicated full consumption of educts. The mixture was diluted with sat. NaHCO₃ and DCM and transferred to a separatory funnel. The aqueous phase was extracted four times with DCM, and the combined organics were dried over Na₂SO₄ and evaporated under reduced pressure. The crude product was then purified by flash chromatography (EtOAc/MeOH, automatic gradient). Yield: 340 mg (83%) as off-color solid. ¹H NMR (400 MHz, CDCl₃) δ 5.09–5.00 (m, 1H), 4.68 (d, J = 7.3 Hz, 1H), 4.41 (dd, J = 16.5, 7.2 Hz, 1H), 4.24 (dd, J = 13.8, 3.4 Hz, 1H), 4.06 (dt, J = 9.3, 5.8 Hz, 1H), 3.97–3.76 (m, 1H), 3.69–3.61 (m, J = 6.9 Hz, 2H), 3.50 (s, 1H), 3.46–3.38 (m, J = 12.9, 6.3 Hz, 2H), 3.32 (d, J = 3.7 Hz, 3H), 3.29–3.24 (m, J = 9.2 Hz, 1H), 3.07–3.00 (m, 3H), 2.92 (s, 2H), 2.82–2.72 (m, J = 8.3 Hz, 3H), 2.59–2.51 (m, 2H), 2.48 (t, J = 6.1 Hz, 1H), 2.35 (bs, 3H), 2.33–2.30 (m, J = 5.0 Hz, 1H), 2.30–2.27 (m, J = 5.7 Hz, 2H), 2.24 (s, 2H), 2.01 (d, J = 18.7 Hz, 2H), 1.88 (ddd, J = 14.2, 7.6, 2.1 Hz, 1H), 1.82–1.72 (m, J = 16.3, 10.2 Hz, 3H), 1.69–1.62 (m, 1H), 1.57 (dd, J = 15.1, 4.8 Hz, 1H), 1.53–1.45 (m, 1H), 1.42 (s, 6H), 1.32–1.29 (m, J = 6.6 Hz, 7H), 1.24 (s, 3H), 1.22–1.19 (m, J = 7.7, 4.3 Hz, 7H), 1.11 (d, J = 5.2 Hz, 2H), 1.08 (s, 3H), 1.02 (dd, J = 12.2, 7.5 Hz, 3H), 0.93–0.87 (m, J = 14.7, 7.3 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 177.3, 164.6, 151.9, 134.4, 129.3, 124.9, 121.1, 116.2, 94.5, 77.4, 76.9, 73.5, 72.4, 65.6, 65.0, 54.1, 53.5, 48.7, 47.3, 45.8, 44.9, 41.6, 37.0, 36.1, 35.3, 34.5, 33.2, 30.4, 29.9, 25.0, 24.5, 21.7, 20.9, 20.7, 18.4, 17.4, 14.8, 10.9, 8.7, 8.5, 7.3. MS (ESI) m/z: 873.80 [M + H]⁺; HPLC t_ret = 11.9 min (method B).

Glossary

Abbreviations Used

ACN

acetonitrile

BA

bioavailability

BTK

Bruton’s tyrosine kinase

BRET

bioluminescence resonance energy transfer

ELSD

evaporative light scattering detector

Et₂O

diethyl ether

Et₃N

triethylamine

EtOAc

ethyl acetate

EtOH

ethanol

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

iBuOH

isobutanol

IFN

interferon

IL-10

interleukin 10

IPrOH

isopropanol

JAK

janus kinase

MeOH

methanol

PDGFR

platelet derived growth factor receptor

SEM

standard error of mean

STAT

signal transducer and activator of transcription

TYK2

tyrosine kinase 2

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00054.

• In vivo PK data of final compounds, 2D NMR spectra, X-ray crystallographic diffraction data, HPLC traces of final compounds and 35 SMILES codes of final compounds (PDF)

Author Contributions

J.L. was responsible for the design, synthesis, and characterization of the featured test compounds as well as the planning and coordination of in vitro and in vivo studies. C.F. contributed to the planning and coordination of in vivo studies. Quantification of samples generated from PK studies was performed by L.R., A.S., and J. G., and M.K. was responsible for the MLM stability experiments. D.S. carried out the X-ray crystallography of 44. NanoBRET experiments were performed by L.B., and M.B., M.F., and S.L. were responsible for general guidance and supervision of the project. Additionally, M.F. was responsible for the initial design, development, and characterization of 2.

The authors declare the following competing financial interest(s): J.L., J.G., L.R., A.S., J.G., C.P. and M.B. are employees of Synovo GmbH, a pharmaceutical company that has an interest in the development of this class of compounds.

Special Issue

Published as part of the ACS Pharmacology & Translational Science virtual special issue “New Drug Modalities in Medicinal Chemistry, Pharmacology, and Translational Science”.