Discovery of novel macrocyclic MERTK/AXL dual inhibitors

Abstract

MERTK and AXL are members of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases that are aberrantly expressed and have been implicated as therapeutic targets in a wide variety of human tumors. Dual MERTK and AXL inhibition could provide anti-tumor action mediated by both direct tumor cell killing and modulation of the innate immune response in some tumors such as non-small cell lung cancer. We utilized our knowledge of MERTK inhibitors and a structure-based drug design approach to discover a novel class of macrocyclic dual MERTK/AXL inhibitors. The lead compound 43 had sub-nanomolar activity against both MERTK and AXL and good selectivity over TYRO3 and FLT3. Its target engagement and selectivity were also confirmed by NanoBRET and cell-based MERTK and AXL phosphorylation assays. Compound 43 had excellent pharmacokinetic properties (large AUC and long half-life) and mediated anti-tumor activity against lung cancer cell lines, indicating its potential as a therapeutic agent.

Graphical Abstract

INTRODUCTION

Approximately two-thirds of cancer patients in the United States, mostly those with early diagnoses, will be cured of their disease using a combination of surgery, radiation and chemotherapy. In contrast, patients with advanced and metastatic disease continue to have extremely poor prognosis, even in the era of targeted therapies. For instance, five-year survival for newly diagnosed patients with non-small cell lung cancer (NSCLC) was under 20% just a few years ago and patients with distant metastases have five-year survival rates of only 5%.¹ Clearly, new avenues of therapy and clinical trials that test combinations of therapies early in the course of a patient’s disease are needed.

MERTK and AXL are members of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases that are aberrantly expressed and have been implicated as therapeutic targets in a wide variety of human tumors.² In particular, MERTK and AXL have direct roles in tumor cell survival, chemoresistance, motility, and metastasis^3–4 and have been associated with poor prognosis. In NSCLC models, both MERTK and AXL are important for tumorigenesis and chemoresistance.⁴ In addition to their overlapping functions, MERTK and AXL regulate non-overlapping signaling events in NSCLC.⁴ Together, these data suggest that inhibition of both family members may be more effective for treatment of some cancers than targeting MERTK or AXL alone. Indeed, inhibition of MERTK sensitizes tumor cells to AXL inhibition and vice versa.⁵ Similarly, MERTK and AXL both play roles in tumor-associated macrophages and natural killer (NK) cells, where they function to promote immune suppressive phenotypes that enhance tumor growth and metastasis.^{2, 6–10} Therefore, dual MERTK and AXL inhibitors are expected to provide anti-tumor action mediated by both direct tumor cell killing and modulation of the innate immune response. To date, there are two AXL/MERTK dual inhibitors reported to be tested in in vivo systems. INCB081776 from Incyte is in Phase I clinical trial for advanced solid tumors, however, the structure and selectivity of the compound have not been publicly disclosed.¹¹ AZ14145845 is a selective in vivo tool compound published recently by AstraZeneca.¹²

In 2017, we reported a macrocyclic pyrimidine UNC2541 (1) (Figure 1A) as a potent MERTK-selective in vitro tool compound.¹³ In the optimization process, we found that the large (18-membered) and flexible macrocycle in 1 might contribute to its low cell permeability, leading to its low cellular activity (just sub-micromolar). Therefore, we decided to construct a new macrocycle, such as the one in compound 2, which is smaller and more rigid (Figure 1B) to improve cellular activity. A phenyl group was directly attached to the 4-amino group on the pyrimidine core to contribute rigidity. In addition, a 1,3-disubstitution pattern on the phenyl ring reduced the ring size of the macrocycle compared to a 1,4-disubstituion pattern that we used before.¹³ Incorporation of a double bond further rigidified the macrocycle. Furthermore, compound 2 fits our MERTK docking model and is predicted to retain three key hydrogen bonds with the hinge area of the MERTK protein (Pro672 and Met674) (Figure 1C).¹³ In this paper, we explicate how this new design led to novel macrocyclic compounds with dual activity versus MERTK and AXL.

Figure 1.

A. Structure and activity data of UNC2541 (1); B. Structure of 2; C. Docking model of 2 based on X-ray structure of 1 in complex with MERTK kinase domain (PDB ID code 5K0X)¹³.

RESULTS AND DISCUSSION

As described in Scheme 1, the proposed compound 2 was synthesized by a multistep sequence starting with commercially available methyl 2,4-dichloropyrimidine-5-carboxylate. The 4-Cl group on pyrimidine ring was replaced by aniline 3 via a S_NAr reaction to yield intermediate 4. Boc deprotection followed by an intramolecular S_NAr displacement closed the macrocycle to afford intermediate 5 in 86% yield over 2 steps. The methyl ester in 5 was hydrolyzed by NaOH and the resulting free carboxylic acid was converted to amide 6 via an amide coupling reaction. Finally, the nitro group was reduced in the presence of iron and ammonium chloride to provide the desired compound 2 in 65% yield.

Scheme 1.

Synthesis of compound 2 (General procedure A).

Gratifyingly, analog 2 was active with a 28 nM IC₅₀ in our in house microcapillary electrophoresis (MCE) MERTK assay^14–15 (Table 1). It also showed 11-fold selectivity over AXL and much higher selectivity over TYRO3 and FLT3 (111- & 64-fold respectively). When the double bond on the macrocycle was reduced, the corresponding analog 7 lost activity dramatically for MERTK (>210-fold) and became inactive for AXL and TYRO3 while FLT3 activity was not affected. On the contrary, configuration changes of double bond from trans- to cis- had only minimal effect on the activities of the 13-membered macrocycle 8 (2-fold for MERTK, AXL and FLT3 and 5-fold for TYRO3). However, for analog 9 which had a larger trans-14-membered macrocycle, the AXL activity was diminished 6-fold compared to 2 while cis-14-membered macrocycle 10 was equally potent against TAM and FLT3 compared to cis-13-membered macrocycle 8. Further increasing the size of macrocycle led to more active MERTK inhibitors 11 but with reduced selectivity for MERTK over FLT3 (15-fold) compared to 9 (66-fold). For analogs with a cis-double bond in the macrocycle, the increase of macrocycle size had a minimum effect on inhibitory activities; for example, analogs 10 and 12 were equally potent against TAM and FLT3. As reported previously, the amide group could be replaced by a 2-pyridine as a hydrogen bond donor to form an intramolecular hydrogen bond with the NH group at the C-4 position of the pyrimidine ring.¹⁶ The same strategy applied here and resulted in an equally potent MERTK inhibitor 13 compared to 2, however, with dramatically reduced selectivity for MERTK over TYRO3 and FLT3 (34- and 8-fold vs. 111- & 64-fold respectively). Therefore, the cis-13-membered macrocycle and the amide group were selected for further structure-activity relationship (SAR) studies on the phenyl ring to explore the activity and selectivity profile among the TAM family and FLT3, a frequent off-target for TAM inhibitors.

Table 1.

Size of the macrocycles.

^aValues are the mean of three or more independent assays.

In our initial design, a NH₂ group was introduced at the 4-position of the phenyl ring as a hydrogen bond donor to potentially form bond with the residues R727, N728 or D741 in the MERTK protein. Surprisingly, analog 14 (Table 2), without the 4-NH₂ group on the phenyl ring, had similar activity against the TAM family and was more potent against FLT3 (11-fold) compared to 2 (Table 1). This suggested that it was unlikely a hydrogen bond formed at this position with the MERTK protein, possibly due to the conformation change caused by rigidity of the macrocycle. Therefore, a variety of substituents such as fluoro, chloro, cyano, methyl and methoxy groups were tested at this position. The corresponding analogs 15–19 were all potent MERTK inhibitors with good selectivity for MERTK over TYRO3 (>15-fold) and FLT3 (>9-fold) (Table 2). Analog 19 was the most potent MERTK inhibitor with a 4.9 nM IC₅₀ but was less selective over FLT3 (35-fold) compared to analog 17 which had the best selectivity of MERTK over FLT3 (368-fold) but weaker potency against MERTK (4-fold compared to 19). Interestingly, moving the cyano group to the 5-position of the phenyl ring significantly reduced the activity of the corresponding analog 20 against TAM kinases (>5-fold) compared to 17. Furthermore, incorporation of one nitrogen in the phenyl ring of the macrocycle was well tolerated. The resulting analogs 21 and 22 had similar activities against TAM kinases and FLT3 compared to 14 and 8 respectively. Since the cyano group at the 4-position of the phenyl ring led to potent analog 17 with the best selectivity for MERTK over FLT3, it was retained on the phenyl ring for further SAR exploration at the amide position.

Table 2.

SAR Study on the phenyl ring of the macrocycle.

^aValues are the mean of three or more independent assays.

As shown in Table 3, replacing the amide hydrogen with a methyl group dramatically reduced the activity of analog 23 towards MERTK (22-fold) and AXL (29-fold) compared to 17, as what we previously observed for a substituted amide series.¹⁷ The weak activity of 24 against MERTK and AXL further demonstrated the importance of the H-bond donor in the amide group; bonding to Met674 (Met623) in the hinge region of MERTK (AXL). When the isopropyl group in the amide group of 17 was replaced with a 4-piperidine ring, the corresponding analog 25 had a slightly improved AXL IC₅₀ (85 nM) compared to 17 (210 nM). In addition, 4-membered 1-methylazetidine reduced MERTK (4-fold) and AXL (3-fold) activities for the corresponding analog 26, while 5-membered (S)-3-pyrrolidine led to an equally potent analog 27 compared to 17. In addition, a CH₂ linker between the amide nitrogen and 4-piperidine ring was well tolerated and yielded an equal potent analog 28 compared to 25. Surprisingly, replacement of the 4-piperidine ring in 25 with an aromatic 4-pyridine ring dramatically reduced the activity of the resulting analog 29 against TAM (>48-fold). Extending the phenyl group by introducing a N-methyl piperazine ring at its para-position recovered MERTK activity of the corresponding analog 30 compared to 25, however, it completely lost selectivity over FLT3. Adding a CH₂ linker between the phenyl and N-methyl piperazine ring led to an equally potent MERTK inhibitor 31 with some selectivity for MERTK over FLT3 (10-fold) compared to 30. On the other hand, extending the 4-piperidine group in 25 by adding a (tetrahydro-2H-pyran-4-yl)methyl group to its nitrogen led to a more potent MERTK inhibitor 32 with similar activity against AXL and better selectivity over TYRO3 (48-fold) and FLT3 (1160-fold) compared to 25. Similarly, analog 33 which contained a carbonyl group as a linker had a 4.7 nM IC₅₀ against MERTK and excellent selectivity over TYRO3 (117-fold) and FLT3 (234-fold). Furthermore, a trans-4-(morpholine-4-carbonyl) cyclohexyl group slightly decreased MERTK activity (3-fold) for analog 34 compared to 33. Surprisingly, changing the newly formed secondary amide to a primary amide increased activity for both TAMs and FLT3 (>3-fold) and yielded potent MERTK/AXL dual inhibitor 35 with 42-fold selectivity for MERTK over TYRO3 and FLT3. Ring opening of the N-methyl piperidine and di-ethylation yielded an equally potent MERTK/AXL dual inhibitor 36. To rationalize the increased potency of 35 and 36 against MERTK and AXL (as compared to TYRO3 and FLT3), we performed structural analysis of their binding modes to all four enzymes. High-confidence binding poses of 35 (Figure 2A) and 36 to MERTK were inferred from several X-ray structures of 4-amino-pyrimidine inhibitors including a macrocyclic inhibitor 1 (PDB ID: 5K0X) (see Methods for details). The resulting binding pose was readily transferred to structurally aligned AXL, TYRO3 and FLT3 with subsequent energy optimization (Figures 2B–D). The poses show perceptible variation in interaction signatures of both inhibitors to the four enzymes. As exemplified for 35 (Figures 2A, B), it is capable of “comfortably” maintaining a network of five hydrogen bonds to either MERTK or AXL. Besides four interactions inside the pocket – three with the hinge region (residues Pro672 and Met674 in MERTK) and one with a conserved lysine in the catalytic site (Lys619 in MERTK) – the protonated nitrogen in the piperidine group of 35 binds to respectively Tyr685 in MERTK and Asp639 in AXL. Both TYRO3 and FLT3 possess the same or analogous binding sites and may, in principle, form up to five hydrogen bonds with 35. However, because of a subtle variation in the overall protein folds, the conserved lysine residues in the binding pocket, as well as the solvent-exposed sites for the piperidine nitrogen, are oriented in such a way that 35 is not able to reach to both sites concurrently. Even though solvent-exposed interactions with Tyr685 and Asp639, in respectively MERTK and AXL, are less critical than the intra-pocket hydrogen bonds, their cooperative contribution might be enough to explain an approximately 10-fold difference in potencies of 35 against MERTK/AXL compared to those against TYRO3/FLT3.

Table 3.

SAR study at the amide position.

^aValues are the mean of three or more independent assays.

Figure 2.

Binding poses and major interactions of 35 with MERTK (A), AXL (B), TYRO3 (C), and FLT3 (D); poses were modeled based on X-ray MERTK structures co-crystallized with 4-amino-pyrimidine inhibitors (PDB ID: 4M3Q, 4MHA, 5K0X).

Based on SAR information presented in Table 2, fluoro, chloro, methyl and methoxy groups were all well tolerated at the R² position for MERTK/AXL potency with differing selectivity profiles. Therefore, we decided to synthesize a small, focused library of 8 compounds with a ‘mix and match’ combination of these four groups at the R² position with two R¹ groups from analogs 35 & 36 (Table 4). Indeed, this exercise turned out to be highly productive as 2 analogs (41&43) show potent inhibition of both MERTK and AXL kinase activity with IC₅₀s < 10 nM. Analog 43 (UNC8969A) also had excellent selectivity over TYRO3 and FLT3 (>60-fold compared to MERTK IC₅₀s).

Table 4.

Focused library.

^aValues are the mean of three or more independent assays.

As presented in scheme 1, these macrocycles can be prepared via a linear sequence (See Experimental Section). Fortunately, each step can be scaled up without difficulty, as exemplified by the synthesis of 43 in Scheme 2. Starting with 10 g of commercially available cis-butene-1,4-diol, TBDPS protection of one hydroxyl group followed by a Mitsunobu reaction yielded intermediate 45 with 35% yield over 2 steps. Changing the protecting group from phthalimide to Boc followed by TBDPS deprotection in the presence of TBAF led to intermediate 46. The reason for changing phthalimide to a BOC protecting group was that the phthalimide group couldn’t be removed in the latter stage of the synthesis while the Boc group couldn’t be introduced at the beginning of the synthesis. A second Mitsunobu reaction of 46 with 2-methyl-5-nitrophenol attached the side chain to the phenyl ring and iron reduction of the nitro group yielded 47 in excellent yields (89% & 91% respectively). The chloride at the 4-position of methyl 2,4-dichloropyrimidine-5-carboxylate was replaced by aniline 47 via a S_NAr reaction to give intermediate 48 in high yield (93%). The macrocycle was then formed by a Boc deprotection reaction followed by an intramolecular S_NAr displacement in 70% yield over 2 steps. Unlike other ring closure reactions, this key S_NAr reaction doesn’t require a low concentration and no intermolecular S_NAr reaction was observed. To complete the synthesis, the methyl ester in macrocycle 49 was hydrolyzed by LiOH·H₂O and the carboxylic acid intermediate was converted to an amide 50 via an amide coupling reaction with methyl trans-4-aminocyclohexane-1-carboxylate. The methyl ester in amide 50 was hydrolyzed under the same basic conditions and an amide bond formation reaction with N,N’-dimethyl ethylenediamine in the presence of TBTU yielded the final compound 43. In summary, although it took 14 steps to synthesize 43 starting from commercially available cis-butene-1,4-diol, only 5 steps needed column chromatography purification and the overall yield was 4.2%.

Scheme 2.

Synthesis of compound 43 (General procedure B).

Based on MERTK and AXL activity (IC₅₀s < 15 nM) and selectivity for MERTK over TYRO3 and FLT3 (>30-fold), three compounds (35, 36 & 43) were selected for exploratory pharmacokinetic (PK) evaluation in mice via iv administration at a 3.0 mg/kg dose (Table 5). All three compounds had large AUCs (>1290 h*ng/mL), moderate clearance (24-38 mL/min/kg) and long half-lives (>5.4 h). Analogs 36 and 43 were also evaluated in mice via oral administration at 3.0 mg/kg dose. Analog 36 didn’t have any oral absorption, 43 demonstrated 4.2% oral bioavailability. Based on these data, 43 (UNC8969) was selected as the lead compound and was further evaluated in full mouse PK studies via iv (5 mg/kg) and ip (10 mg/kg) administration routes. This revealed excellent bioavailability (81%) via ip administration. Therefore, 43 can be used as an in vivo MERTK/AXL tool compound via iv or ip administration.

Table 5.

In Vivo Pharmacokinetic Parameters of selected analogues.

Compounda	administrative route	Dose (mg/kg)	T1/2 (h)	Cmax (ng/mL)	AUClast (h*ng/mL)	CL (mL/min/kg)	Bioavailability %
35 b	iv	3	5.4	6735	2119	23.6	-
36 b	iv	3	8.4	7989	1333	36.7	-
43 b	iv	3	6.3	5293	1288	38.3	-
43 b	po	3	1.9	14.4	54.2	-	4.2
43 c	iv	5	7.3	4085	1916	42.7	-
43 c	ip	10	7.0	890	3028	-	81

^aformulation: 10% NMP, 5% Solutol in normal saline (0.9% NaCl);

^bn = 2 mice per time point;

^cn = 3 mice per time point.

The selectivity of 43 against other members of the kinome was evaluated using a 32-kinase panel including MERTK, AXL and TYRO3 at 110 nM (100-fold above its MERTK IC₅₀). This 32-kinase panel provides a broad survey of kinase families by selecting representative tyrosine kinases along with a selection of serine/threonine kinases. As expected, both MERTK and AXL were fully inhibited while TYRO3 was inhibited partially (67%) at this concentration. Only one other kinase (KDR) was inhibited greater than 80% in the presence of 110 nM 43 and none of the serine/threonine kinases were appreciably inhibited (Figure 3) (details see support information).

Figure 3.

The kinase tree (generated using an online tool KinMap¹⁸). A. Full view. B. Expansion of the Tyrosine Kinase area. Size of the red dot is proportional to % inhibition.

Furthermore, 43 was tested in MERTK, AXL and TYRO3 NanoBRET assays to evaluate its cellular target engagement (Figure 4). In this assay, it bound tightly to MERTK and AXL with 20.7 and 103 nM EC₅₀ values, respectively (Figs 4A–B), and much weaker binding to TYRO3 (>40-fold) with a 4.34 uM EC₅₀ (Fig 4C). This trend is consistent with results from the MCE assay and demonstrates the selectivity of 43 for MERTK and AXL over TYRO3. Analog 43 also inhibited MERTK and AXL in cell-based assays (Figure 5). Treatment with 43 mediated dose-dependent inhibition of MERTK and AXL autophosphorylation in NSCLC cells with EC₅₀ values of 146 nM and 83 nM, respectively. Additionally, treatment with 43 inhibited colony formation in NSCLC cell cultures (Figure 6).

Figure 4.

Target engagement of 43 in NanoBRET assays. Interactions between 43 and TAM kinases were determined using a bioluminescence resonance energy transfer-based assay (NanoBRET). Cells were treated with 43 (0.5 nM – 10 μM) and binding to MERTK (A), AXL (B) and TYRO3 (C) was determined. IC₅₀ values were calculated by non-linear regression.

Figure 5.

43 inhibits MERTK and AXL with similar potency in non-small cell lung cancer cells. H4011 non-small cell lung cancer cells were cultured with the indicated concentrations of 43 or an equivalent volume of vehicle control in the presence of pervanadate to stabilize phosphorylated proteins. Target proteins were immunoprecipitated from cell lysates and total and phospho- (denoted by p) proteins were detected by immunoblot. A. Representative immunoblots; B. Relative levels of phosphorylated and total proteins were determined by densitometry. Mean values +/− standard error derived from 4 independent experiments are shown. EC₅₀ values and 95% confidence intervals of 146 nM (86-243 nM) and 83 nM (50-133 nM) were calculated by non-linear regression for MERTK and AXL, respectively.

Figure 6.

43 inhibits NSCLC colony formation. H1975 non-small cell lung cancer cells were cultured at low density for 7-8 days with the indicated concentrations of 43 or an equivalent volume of vehicle control and colonies were counted. Data were derived from two independent experiments.

CONCLUSIONS

In conclusion, we created novel macrocyclic dual MERTK/AXL inhibitors via a structure-based drug design approach. Although the synthetic sequence is linear to our lead compounds, each step can be scaled up to multigram scale and column chromatography purification was not required for most reactions. The lead compound 43 had low-nanomolar activity against both MERTK and AXL and good selectivity over TYRO3 and FLT3. Its target engagement and selectivity were further confirmed by NanoBRET and cell-based MERTK and AXL phosphorylation assays. Analog 43 also had excellent PK properties (large AUC and long half-life) and is suitable for use as an in vivo tool compound via intravenous or intraperitoneal administration routes.

EXPERIMENTAL SECTION

Synthesis of Analogues

Microwave reactions were carried out using a CEM Discover-S reactor with a vertically focused IR external temperature sensor and an Explorer 72 autosampler. The dynamic mode was used to set up the desired temperature and hold time with the following fixed parameters: PreStirring, 1 min; Pressure, 200 psi; Power, 200 W; PowerMax, off; Stirring, high. Sonication was carried out on Branson 3510 Ultrasonic Cell. Centrifugation was carried out on Eppendorf Centrifuge 5418. Flash chromatography was carried out on Teledyne ISCO Combi Flash^® R_f 200 with pre-packed silica gel disposable columns or pre-packed reverse phase C18 columns. Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F₂₅₄, 0.25 mm pre-coated TLC plates. TLC plates were visualized using UV₂₅₄ and phosphomolybdic acid with charring. All ¹H NMR spectra were obtained with a 400 or 500 or 850 MHz spectrometer using CDCl₃ (7.26 ppm), DMSO-d⁶ (2.50 ppm, quintet) or CD₃OD (3.31 ppm, quintet) as an internal reference. Signals are reported as m (multiplet), s (singlet), d (doublet), t (triplet), q (quartet), and bs (broad singlet); and coupling constants are reported in hertz (Hz). ¹³C NMR spectra were obtained with a 100 or 125 or 214 MHz spectrometer using CDCl₃ (77.2 ppm, triplet), DMSO-d⁶ (39.5 ppm, septet), or CD₃OD (49.3 ppm, septet) as the internal standard. LC/MS was performed using an analytical instrument with the UV detector set to 220 nm, 254 nm, and 280 nm, and a single quadrupole mass spectrometer using electrospray ionization (ESI) source. Samples were injected (2 μL) onto a 4.6 x 50 mm, 1.8 μM, C18 column at rt. A linear gradient from 10% to 100% B (MeOH + 0.1% acetic Acid) in 5.0 min was followed by pumping 100% B for another 2 or 4 min with A being H₂O + 0.1% acetic acid. The flow rate was 1.0 mL/min. High-resolution (positive ion) mass spectra (HRMS) were acquired using a LCMS-TOF mass spectrometer. Purity is >95% for all final compounds determined by LC-MS. Analytical HPLC was performed with prominence diode array detector (SPD-M20A). Samples were injected onto a 3.6 μm PEPTIDE XB-C18 100 Å, 150 x 4.6 mm LC column at rt. The flow rate was 1.0 mL/min. Various linear gradients were used with A being H₂O + 0.1% TFA and B being acetonitrile + 0.1% TFA. Purity is >95% for key compounds in Table 4 determined by analytical HPLC.

General Procedure A (Scheme 1):

To a solution of tert-butyl (E)-(4-hydroxybut-2-en-1-yl)carbamate (1.0 g, 5.3 mmol) in anhydrous THF (15 mL) was slowly added sodium hydride (0.43 g, 10.7 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min, then was added 3-fluoro-4-nitroaniline (0.92 g, 5.87 mmol). The reaction mixture was stirred at rt for 12 h, quenched with ice water (20 mL) and extracted with ethyl acetate (100 mL, 2×). The combined organic layers were washed with brine (30 mL), dried (Na₂SO₄) and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column (petroleum ether/ethyl acetate = 10/1 to 5/1) to afford the desired product 3 (1.3 g, 75%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 8.8 Hz, 1H), 6.23–6.17 (m, 2H), 6.00–5.91 (m, 1H), 5.84 (dtt, J = 15.6, 5.1, 1.4 Hz, 1H), 4.59 (dd, J = 5.0, 1.4 Hz, 2H), 4.35 (s, 2H), 3.80 (s, 2H), 1.45 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.44, 161.94, 161.30, 142.84, 134.84, 126.52, 123.89, 107.77, 106.20, 77.65, 69.06, 52.94, 28.19. MS (ESI) for [M+Na]⁺ (C₁₅H₂₁N₃O₅Na⁺): calcd m/z 346.15; found m/z 346.20. LC–MS: 98% purity.

A solution of 3 (1.0 g, 3.1 mmol), methyl 2,4-dichloropyrimidine-5-carboxylate (0.64 g, 3.1 mmol) and DIPEA (0.80 g, 6.2 mmol) in isopropanol (20 mL) was heated under reflux for 14 h, then cooled to rt and filtered. The solid was washed with hexane (20 mL) and dried under reduced pressure to afford the desired product 4 (1.1 g, 72 %) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.90 (s, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.45 (dd, J = 9.0, 2.2 Hz, 1H), 7.04 (s, 1H), 5.90 (dt, J = 15.7, 5.1 Hz, 1H), 5.78 (dt, J = 15.2, 5.4 Hz, 1H), 4.74 (d, J = 6.2 Hz, 2H), 3.94 (s, 3H), 3.64 – 3.56 (m, 2H), 1.36 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.46, 161.96, 161.33, 159.21, 152.27, 142.87, 134.86, 132.44, 126.55, 123.91, 113.28, 107.78, 106.23, 77.69, 69.08, 52.96, 41.05, 28.21. MS (ESI) for [M + H]⁺ (C₂₁H₂₅ClN₅O₇): calcd m/z 494.14; found m/z 494.13. LC–MS: 97% purity.

To a solution of 4 (0.85 g, 1.70 mmol) in DCM (10 mL) was added a 4.0 M HCl solution in 1,4-dioxane (5.4 mL, 21.6 mmol). The reaction mixture was stirred at rt for 1.0 h. The solvents were removed under reduced pressure. The residue was suspended in diether ether (10 mL), stirred for 20 min, and filtered. The solid was washed with diether ether (15 mL) and dried under reduced pressure to afford the desired product methyl (E)-4-((3-((4-aminobut-2-en-1-yl)oxy)-4-nitrophenyl)amino)-2-chloropyrimidine-5-carboxylate (0.64 g, 95%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.47 (s, 1H), 8.91 (s, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 2.2 Hz, 1H), 7.51 (dd, J = 8.9, 2.1 Hz, 1H), 6.17–6.07 (m, 1H), 6.02–5.92 (m, 1H), 4.80 (d, J = 4.1 Hz, 2H), 3.94 (s, 3H), 3.56 (s, 1H), 3.55–3.50 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.47, 161.94, 161.38, 159.23, 152.07, 142.96, 134.80, 129.43, 126.63, 125.86, 113.56, 107.86, 106.26, 68.49, 66.36, 52.99. MS (ESI) for [M + H]⁺ (C₁₆H₁₇ClN₅O₅): calcd m/z 394.08; found m/z 394.10. LC–MS: 96% purity.

A solution of methyl (E)-4-((3-((4-aminobut-2-en-1-yl)oxy)-4-nitrophenyl)amino)-2-chloropyrimidine-5-carboxylate (0.80 g, 2.0 mmol) and DIPEA (2.10 g, 2.80 mL, 20.0 mmol) in isopropanol (15 mL) was heated under reflux for 12 h, then cooled to rt, and filtered. The solid was washed with isopropanol (10 mL) and diether ether (10 mL) and dried under reduced pressure to afford the desired product 5 (0.65 g, 90%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.41 (s, 1H), 8.65 (s, 1H), 8.39–8.36 (m, 1H), 8.22–8.18 (m, 1H), 7.89 (d, J = 8.9 Hz, 1H), 6.85–6.80 (m, 1H), 6.14–6.03 (m, 1H), 5.72–5.61 (m, 1H), 5.01–4.93 (m, 2H), 3.87–3.80 (m, 5H). ¹³C NMR (214 MHz, DMSO-d₆) δ 166.81, 162.75, 160.93, 159.09, 152.08, 142.43, 138.27, 133.67, 126.33, 126.15, 111.85, 111.32, 96.08, 70.65, 51.87, 43.22. MS (ESI) for [M + H]⁺ (C₁₆H₁₆N₅O₅): calcd m/z 358.11; found m/z 358.10. LC–MS: 96% purity.

To a solution of 5 (0.82 g, 2.30 mmol) in a mixture of THF and H₂O (5:1, 10 mL) was added NaOH (0.37 g, 9.20 mmol). The reaction mixture was heated under reflux for 3.0 h. The solvents were removed under reduced pressure. The residue was dissolved in water (5.0 mL). The resulting solution was acidified with 1.0 N aqueous solution of HCl to pH ~ 3 at 0 °C and filtered. The solid was washed with water (10 mL) and diether ether (10 mL) and dried under reduced pressure to afford (E)-3⁴-nitro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (0.61 g, 78%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.06 (s, 1H), 8.89 (s, 1H), 8.71 (s, 1H), 8.61 (s, 1H), 8.16–8.13 (m, 1H), 7.91 (d, J = 8.8 Hz, 1H), 6.95–6.91 (m, 1H), 6.17–6.08 (m, 1H), 6.04–5.96 (m, 1H), 4.97 (d, J = 6.1 Hz, 2H), 4.17–4.04 (m, 2H). ¹³C NMR (214 MHz, DMSO-d₆) δ 166.96, 159.24,152.23, 151.30, 140.64, 137.82, 135.34,126.73, 126.39, 125.90, 113.27, 113.08, 98.45, 70.52, 43.33. MS (ESI) for [M + H]⁺ (C₁₅H₁₄N₅O₅): calcd m/z 344.09; found m/z 344.10. LC–MS: 96% purity.

To a solution of (E)-3⁴-nitro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (0.30 g, 0.88 mmol) and propan-2-amine (0.16 g, 2.60 mmol) in DMF (15 mL) was added 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU, 0.34 g, 1.3 mmol). The reaction mixture was stirred at 25 °C for 16 h, quenched with water, and extracted with ethyl acetate (100 mL, 2×). The combined organic layers were washed with brine (25 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column (DCM/Methanol = 20/1 to 10/1) to afford the desired product 6 (0.26 g, 76%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (s, 1H), 8.48 (s, 1H), 8.21 (d, J = 6.9 Hz, 1H), 7.48 (t, J = 7.3 Hz, 1H), 7.40–7.34 (m, 1H), 6.69 (d, J = 8.7 Hz, 1H), 6.11–5.98 (m, 1H), 5.76–5.62 (m, 1H), 5.00–4.91 (m, 2H), 4.13–4.03 (m, 1H), 3.86–3.78 (m, 2H), 1.16 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.40, 165.94, 162.33, 157.93, 152.63, 143.27, 138.09, 132.73, 126.42, 111.18, 110.14, 99.40, 70.76, 43.03, 40.78, 22.28. MS (ESI) for [M + H]⁺ (C₁₈H₂₁N₆O₄): calcd m/z 385.16; found m/z 385.20. LC–MS: 97% purity.

A suspension of 6 (50.0 mg, 0.13 mmol), iron (73.0 mg, 1.3 mmol) and ammonium chloride (14.0 mg, 0.26 mmol) in a mixture of ethanol and H₂O (5:1, 5.0 mL) was heated at 70 °C for 1.5 h. The reaction mixture was filtered through a pad of Celite. The pad of Celite was washed with ethyl acetate (10 mL). The combined organic layers were washed with brine (10 mL), dried (Na₂SO₄), and concentrated under vacuum under reduced pressure. The residue was purified by prep-HPLC to afford the title compound 2 (30 mg, 65%) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.48 (s, 1H), 7.93 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 6.86 (dd, J = 8.4, 2.2 Hz, 1H), 6.12 (dt, J = 16.0, 5.5 Hz, 1H), 5.59 (dt, J = 15.9, 6.3 Hz, 1H), 5.01 (d, J = 6.0 Hz, 2H), 4.24–4.12 (m, 1H), 3.98 (d, J = 4.8 Hz, 2H), 1.24 (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 163.75, 162.15,161.15, 158.71, 153.84, 147.12, 143.52, 135.52, 119.50, 118.20, 114.43, 112.60, 101.54, 70.32, 44.52, 42.71, 22.27. MS (ESI) for [M+H]⁺ (C₁₈H₂₃N₆O₂⁺): calcd. m/z 355.19; found m/z 355.20; LC–MS: 99% purity.

General procedure B (Scheme 2):

To a solution of (Z)-but-2-ene-1,4-diol (10.0 g, 113.5 mmol) in anhydrous THF (150 mL) was slowly added sodium hydride (5.45 g, 136.2 mmol) at 0 °C under an argon atmosphere. After stirred at 0 °C for 30 min, tert-butylchlorodiphenylsilane was added in drops. The reaction mixture was stirred at rt for 12 h, quenched with ice water (30 mL), and extracted with ethyl acetate (100 mL, 2×). The combined organic layers were washed with brine (30 mL), dried (Na₂SO₄), and concentrated under reduced pressure to provide (Z)-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-ol (25.0 g) as yellow oil, which was used in the next step without further purification. ¹H NMR (400 MHz, CDCl₃) δ 7.73–7.68 (m, 4H), 7.46–7.38 (m, 6H), 5.77–5.70 (m, 1H), 5.69–5.61 (m, 1H), 4.30–4.27 (m, 2H), 4.04–4.01 (m, 2H), 1.08 (s, 9H). MS (ESI) for [M + H]⁺ (C₂₀H₂₇O₂Si⁺): calcd m/z 327.17; found m/z 327.10. LC–MS: 97% purity.

To a solution of (Z)-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-ol (25.0 g, 76.6 mmol) and phthalimide (12.4 g, 84.2 mmol) in anhydrous THF (200 mL) were added triphenylphosphine (22.1 g, 84.2 mmol) and diisopropyl azodicarboxylate (DIAD, 17.0 g, 84.2 mmol) at 0 °C. The reaction mixture was stirred at rt for 14 h, quenched with ice water (50 mL) and extracted with ethyl acetate (100 mL, 2×). The combined organic layers were washed with brine (25 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column (hexane/ethyl acetate = 5/1 to 2/1) to afford the desired product 45 (18.2 g, 35% over 2 steps) as yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.84–7.81 (m, 2H), 7.78–7.74 (m, 4H), 7.70–7.66 (m, 6H), 7.44 (dd, J = 4.3, 1.7 Hz, 1H), 5.86–5.78 (m, 1H), 5.56–5.47 (m, 1H), 4.52 (d, J = 6.0 Hz, 2H), 4.22 (d, J = 7.0 Hz, 2H), 1.10 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 167.77, 135.62, 133.87, 133.60, 133.22, 132.17, 129.70, 127.75, 124.05, 123.19, 60.22, 35.03, 26.84, 19.17. MS (ESI) for [M + H]⁺ (C₂₈H₃₀NO₃Si⁺): calcd m/z 456.19; found m/z 456.20. LC–MS: 96% purity.

To a solution of 45 (18.0 g, 39.51 mmol) in methanol (150 mL) was added hydrazine (1.89 g, 59.27 mmol). The reaction mixture was stirred at 50 °C for 1.5 h, cooled to rt and filtered. The solid was washed with ethyl acetate (50 mL). The combined organic layers were washed with brine (20 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column (hexane/ethyl acetate = 10/1 to 2/1) to afford (Z)-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-amine (7.5 g, 58%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.71–7.64 (m, 4H), 7.46–7.34 (m, 6H), 5.70–5.57 (m, 1H), 5.53–5.44 (m, 1H), 4.28–4.20 (m, 2H), 3.15–3.08 (m, 2H), 1.04 (s, 9H). MS (ESI) for [M + H]⁺ (C₂₀H₂₈NOSi⁺): calcd m/z 326.19; found m/z 326.20. LC–MS: 96% purity.

To a solution of (Z)-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-amine (7.0 g, 21.5 mmol) and triethylamine (3.26 g, 32.3 mmol) in DCM (100 mL) was added di-tert-butyl decarbonate (Boc₂O, 4.93 g, 22.6 mmol) at rt. The reaction mixture was stirred at rt for 2.0 h, quenched with water (40 mL) and extracted with DCM (40 mL, 2×). The combined organic layers were washed with brine (25 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column (petroleum ether/ethyl acetate = 20/1 to 5/1) to afford tert-butyl (Z)-(4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-yl)carbamate (8.2 g, 90%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.70–7.67 (m, 4H), 7.45–7.37 (m, 6H), 5.75–5.66 (m, 1H), 5.50–5.39 (m, 1H), 4.29–4.24 (m, 2H), 3.62–3.54 (m, 2H), 1.43 (s, 9H), 1.06 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 155.79, 135.72, 135.68, 133.67, 131.73, 129.86, 129.72, 127.83, 127.76, 60.08, 28.52, 26.92, 19.24. MS (ESI) for [M + H]⁺ (C₂₅H₃₆NO₃Si⁺): calcd m/z 426.24; found m/z 426.20. LC–MS: 95% purity.

To a solution of tert-butyl (Z)-(4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-yl)carbamate (7.7 g, 18.1 mmol) in anhydrous THF (100 mL) was added a 1.0 M TBAF solution (19.9 ml, 19.9 mmol). The reaction mixture was stirred at rt for 1.0 h, quenched with water (20 mL) and extracted with DCM (60 mL, 2×). The combined organic layers were washed with brine (20 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column (hexane/ethyl acetate = 10/1 to 3/1) to afford the desired product 46 (3.0 g, 89%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.81–5.68 (m, 1H), 5.56–5.40 (m, 1H), 4.94 (s, 1H), 4.16 (t, J = 5.8 Hz, 2H), 3.73 (t, J = 6.7 Hz, 2H), 3.34 (s, 1H), 1.40 (s, 14H). ¹³C NMR (101 MHz, CDCl₃) δ 156.29, 131.39, 128.53, 79.84, 57.66, 37.42, 28.46. MS (ESI) for [M + H]⁺ (C₉H₁₈NO₃⁺): calcd m/z 188.12; found m/z 188.10. LC–MS: 96% purity.

To a solution of 46 (1.5 g, 8.01 mmol), triphenylphosphine (2.73 g, 10.4 mmol) and 2-methyl-5-nitrophenol (1.59 g, 10.4 mmol) in anhydrous THF (60 mL) was added DIAD (2.11, 10.4 mmol) at 0 °C. The suspension was stirred at rt for 12 h, quenched with water (20 mL) and extracted with DCM (60 mL, 2×). The combined organic layers were washed with brine (20 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The residue was purified by an ISCO silica gel column (hexane/ethyl acetate = 10/1 to 5/1) to afford tert-butyl (Z)-(4-(2-methyl-5-nitrophenoxy)but-2-en-1-yl)carbamate (2.3 g, 89%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J = 8.2, 2.2 Hz, 1H), 7.60 (d, J = 2.2 Hz, 1H), 7.23 (dd, J = 8.2, 0.9 Hz, 1H), 5.82–5.75 (m, 1H), 5.74–5.67 (m, 1H), 4.69 (d, J = 5.9 Hz, 2H), 3.86 (t, J = 6.4 Hz, 2H), 2.27 (s, 3H), 1.42 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 156.78, 155.86, 147.18, 135.35, 131.27, 130.74, 126.50, 115.99, 105.83, 64.38, 38.11, 28.48, 16.78. MS (ESI) for [M + Na]⁺ (C₁₆H₂₂N₂O₅Na⁺): calcd m/z 345.15; found m/z 345.20. LC–MS: 96% purity.

A suspension of tert-butyl (Z)-(4-(2-methyl-5-nitrophenoxy)but-2-en-1-yl)carbamate (2.3 g, 7.13 mmol), iron (1.20 g, 21.4 mmol) and ammonium chloride (0.38 g, 7.13 mmol) in a mixture of ethanol and H₂O (5:1, 15 mL) was heated at 70 °C for 2.0 h. The reaction mixture was filtered through a pad of Celite. The pad of Celite was washed with ethyl acetate (40 mL). The combined organic layers were washed with brine (10 mL), dried (Na₂SO₄), and concentrated under vacuum under reduced pressure. The residue was purified by an ISCO silica gel column (hexane/ethyl acetate = 5/1 to 2/1) to afford the desired product 47 (1.9 g, 91%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.24 (s, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.25 (s, 1H), 6.20 (d, J = 7.9 Hz, 1H), 5.83–5.73 (m, 1H), 5.66–5.56 (m, 1H), 4.56 (d, J = 6.3 Hz, 2H), 3.86–3.79 (m, 2H), 2.08 (s, 3H), 1.43 (s, 9H). MS (ESI) for [M + H]⁺ (C₁₆H₂₅N₂O₃⁺): calcd m/z 293.18; found m/z 293.20. LC–MS: 96% purity.

A solution of 47 (1.90 g, 6.50 mmol), methyl 2,4-dichloropyrimidine-5-carboxylate (1.41 g, 6.82 mmol) and DIPEA (1.69 g, 13.0 mmol) in isopropanol (50 mL) was heated at 72 °C for 2.0 h, then cooled to rt and filtered. The solid was washed with hexane (20 mL) and dried under reduced pressure to afford the desired product 48 (2.8 g, 93%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.34 (s, 1H), 8.81 (s, 1H), 7.39 (s, 1H), 7.12 (d, J = 7.1 Hz, 1H), 7.00 (dd, J = 8.1, 2.2 Hz, 1H), 5.92–5.84 (m, 1H), 5.75–5.66 (m, 1H), 4.66 (d, J = 5.9 Hz, 2H), 3.97 (s, 3H), 3.91–3.84 (m, 2H), 2.21 (s, 3H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 166.88, 163.94, 161.12, 159.74, 156.77, 135.86, 130.84, 130.54, 124.07, 113.80, 105.94, 104.24, 80.02, 64.08, 52.79, 28.53, 16.07. MS (ESI) for [M + H]⁺ (C₂₂H₂₈ClN₄O₅⁺): calcd m/z 463.17; found m/z 463.20. LC–MS: 95% purity.

To a solution of 48 (2.0 g, 4.3 mmol) in DCM (30 mL) was added a 4.0 M solution of HCl in 1,4-dioxane (5.4 mL, 21.6 mmol). The reaction mixture was stirred at rt for 1.0 h and then concentrated under reduced pressure. The residue was suspended in diether ether (15 mL), stirred for 20 min, and filtered. The solid was washed with diether ether (10 mL) and dried under reduced pressure to afford methyl (Z)-4-((3-((4-aminobut-2-en-1-yl)oxy)-4-methylphenyl)amino)-2-chloropyrimidine-5-carboxylate (1.27 g, 81%) as a white solid. ¹H NMR (400 MHz, Methanol-d₄)) δ 10.42 (s, 1H), 8.80 (s, 1H), 7.47 (d, J = 2.2 Hz, 1H), 7.18–7.13 (m, 1H), 7.06 (dd, J = 8.1, 2.1 Hz, 1H), 6.20–6.13 (m, 1H), 5.80–5.70 (m, 1H), 4.76 (d, J = 6.7 Hz, 2H), 3.98 (s, 3H), 3.78 (d, J = 6.7 Hz, 2H), 2.22 (s, 3H). MS (ESI) for [M + H]⁺ (C₁₇H₂₀ClN₄O₃⁺): calcd m/z 363.12; found m/z 363.10. LC–MS: 95% purity.

A solution of methyl (Z)-4-((3-((4-aminobut-2-en-1-yl)oxy)-4-methylphenyl)amino)-2-chloropyrimidine-5-carboxylate (1.2 g, 3.31mmol) and DIPEA (2.14 g, 2.7 mL, 16.5 mmol) in isopropanol (20 mL) was heated at 72 °C for 1.0 h, then cooled to rt and filtered. The solid was washed with isopropanol (10 mL) and dried under reduced pressure to afford the desired product 49 (0.85 g, 79%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.02 (s, 1H), 9.52 (d, J = 2.3 Hz, 1H), 8.57 (s, 1H), 8.19–8.16 (m, 1H), 6.98 (d, J = 8.8 Hz, 1H), 6.48 (dd, J = 7.9, 2.2 Hz, 1H), 5.53 (d, J = 9.7 Hz, 1H), 5.07 (dd, J = 14.1, 9.7 Hz, 1H), 4.59 (d, J = 14.4 Hz, 1H), 4.55–4.44 (m, 1H), 3.81 (s, 3H), 3.52 (dd, J = 16.1, 4.8 Hz, 1H), 2.10 (s, 3H). MS (ESI) for [M + Na]⁺ (C₁₇H₁₉N₄O₃Na⁺): calcd m/z 349.14; found m/z 349.10. LC–MS: 96% purity.

To a solution of 49 (0.75 g, 2.30 mmol) in a mixture of THF and H₂O (5:1, 10 mL) was added LiOH·H₂O (0.48 g, 11.0 mmol). The reaction mixture was heated at 50 °C for 3.0 h. Then, the solvents were removed under reduced pressure. The residue was diluted with water (2 mL), acidified with 1.0 N aqueous HCl solution to pH ~ 3 at 0 °C, and then filtered. The solid was washed with water (10 mL) and dried under reduced pressure to afford (Z)-3⁴-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (630 mg, 88%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 9.52 (s, 1H), 8.54 (s, 1H), 8.19–8.11 (m, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.46 (dd, J = 8.0, 2.2 Hz, 1H), 5.54 (d, J = 9.4 Hz, 2H), 5.16–5.03 (m, 1H), 4.59 (d, J = 14.6 Hz, 1H), 4.55–4.45 (m, 1H), 3.54 (dd, J = 16.1, 5.9 Hz, 1H), 2.10 (s, 3H). MS (ESI) for [M + H]⁺ (C₁₆H₁₇N₄O₃⁺): calcd m/z 313.12; found m/z 313.10. LC–MS: 96% purity.

To a solution of (Z)-3⁴-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (0.63 g, 2.02 mmol), DIPEA (1.30 g, 1.67 mL, 10.1 mmol) and methyl trans-4-aminocyclohexane-1-carboxylate (0.64 g, 4.03 mmol) in DMF (15 mL) was added TBTU (0.97 g, 3.03 mmol). The reaction mixture was stirred at 25 °C for 16 h, quenched with water (20 mL) and then filtered. The solid was washed with water (15 mL) and diether ether (10 mL) and dried under reduced pressure to afford the titled compound 50 (800 mg, 87%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.15 (s, 1H), 9.55 (d, J = 2.2 Hz, 1H), 8.56 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.86–7.80 (m, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.39 (dd, J = 8.0, 2.2 Hz, 1H), 5.52 (d, J = 9.6 Hz, 2H), 5.06 (dd, J = 13.9, 9.7 Hz, 1H), 4.57 (d, J = 14.1 Hz, 1H), 4.52–4.40 (m, 1H), 3.79–3.68 (m, 1H), 3.60 (s, 3H), 3.48 (dd, J = 14.9, 6.0 Hz, 1H), 2.34–2.23 (m, 1H), 1.92 (dd, J = 27.2, 11.4 Hz, 5H), 1.50–1.29 (m, 5H). ¹³C NMR (101 MHz, DMSO-d₆) δ 175.19, 166.15, 161.63, 159.18, 157.67, 155.79, 138.55, 132.90, 129.73, 127.97, 118.02, 109.35, 106.25, 98.76, 63.65, 51.37, 47.40, 41.60, 31.16, 27.72, 15.21. MS (ESI) for [M + H]⁺ (C₂₄H₃₀N₅O₄⁺): calcd m/z 452.22; found m/z 452.20. LC–MS: 96% purity.

To a solution of 50 (0.80 g, 1.80 mmol) in a mixture of THF and H₂O (5:1, 10 mL) was added LiOH·H₂O (0.22 g, 5.3 mmol). The mixture was stirred at 25 °C for 12 h. Then, the solvents were removed under reduced pressure. The residue was diluted with water (2.0 mL), acidified with 1.0 N aqueous HCl solution to pH ~ 3 at 0 °C, and then filtered. The solid was washed with water (10 mL) and diether ether (10 mL) and dried under reduced pressure to afford (Z)-3⁴-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (710 mg, 92%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.76 (s, 1H), 9.30 (s, 1H), 8.68 (s, 1H), 8.67–8.60 (m, 2H), 7.04 (d, J = 8.7 Hz, 1H), 6.58 (dd, J = 8.0, 2.2 Hz, 1H), 5.55 (q, J = 10.4 Hz, 2H), 5.07 (dd, J = 14.4, 10.1 Hz, 1H), 4.61 (d, J = 14.4 Hz, 1H), 4.57–4.48 (m, 1H), 3.82–3.69 (m, 2H), 2.24–2.13 (m, 1H), 2.12 (s, 3H), 1.99–1.84 (m, 5H), 1.38 (q, J = 7.3 Hz, 5H). MS (ESI) for [M + H]⁺ (C₂₃H₂₈N₅O₄⁺): calcd m/z 438.20; found m/z 438.20. LC–MS: 96% purity.

To a solution of (Z)-3⁴-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (0.23 g, 0.53 mmol), N1,N1-diethylethane-1,2-diamine (0.09 g, 0.8 mmol) and DIPEA (0.34 g, 0.44 mL, 2.7 mmol) in DMF (5.0 mL) was added TBTU (0.26 g, 0.8 mmol). The reaction mixture was heated at 25 °C for 16 h, quenched with water (20 mL) and then filtered. The solid was washed with water (10 mL) and diether ether (10 mL) and purified by HPLC to afford the desired product 43 which was converted to HCl salt by the treatment with a 4.0 N solution of HCl in dioxane and dried under lyophilization as a white solid (217 mg, 76%). ¹H NMR (400 MHz,CD₃OD) δ 9.39 (d, J = 2.2 Hz, 1H), 8.42 (s, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.47 (dd, J = 8.0, 2.2 Hz, 1H), 5.69–5.55 (m, 2H), 5.05 (dd, J = 14.4, 10.3 Hz, 1H), 4.65–4.54 (m, 2H), 3.87 (tt, J = 11.4, 3.9 Hz, 1H), 3.70 (d, J = 16.0 Hz, 1H), 3.59 (t, J = 6.2 Hz, 2H), 3.35–3.28 (m, 6H), 2.31 (tt, J = 12.0, 3.5 Hz, 1H), 2.17 (s, 3H), 2.13–2.04 (m, 2H), 2.03–1.95 (m, 2H), 1.71–1.58 (m, 2H), 1.54–1.43 (m, 2H), 1.38 (t, J = 7.3 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 174.67, 166.09, 161.64, 159.19, 157.63, 155.79, 138.56, 132.90, 129.74, 127.98, 118.02, 109.38, 106.25, 98.80, 63.66, 51.68, 47.56, 46.70, 43.26, 36.82, 31.50, 28.38, 15.21, 11.90. MS (ESI) for [M + H]⁺ (C₂₉H₄₂N₇O₃⁺): calcd m/z 536.33; found m/z 536.30. LC–MS: 99% purity; mp: 261.1-263.4 °C.

(E)-3⁴-Amino-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (2)

See general procedure A.

3⁴-Amino-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphane-1⁵-carboxamide (7)

To a solution of 2 (20.0 mg, 0.056 mmol) in methanol (5.0 mL) was added palladium on carbon (20%, 4.0 mg) under hydrogen atmosphere. The resulting suspension was stirred under hydrogen atmosphere at rt for 12 h, then passed through a pad of celite pad. The celite pad was washed with methanol (10 mL). The filtrate was concentrated under reduced pressure. The residue was purified by pre-HPLC to afford the title compound 7 which was converted to HCl salt by the treatment with a 4.0 N solution of HCl in dioxane and dried under lyophilization as a white solid (10.0 mg, 0.030 mmol, 54%). ¹H NMR (400 MHz, CD₃OD) δ 8.44 (s, 1H), 8.01 (brs, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.86 (dd, J = 8.4, 1.3 Hz, 1H), 4.24–4.14 (m, 3H), 3.57 (t, J = 6.2 Hz, 2H), 2.04–1.84 (m, 4H), 1.25 (d, J = 6.6 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 164.56, 161.28, 162.43, 158.60, 134.27, 142,30, 138.11, 126.10, 113.17, 112.87, 100.14, 69.60, 43.22, 41.12. 26,31, 24.89, 22.14. MS (ESI) for [M+H]⁺ (C₁₈H₂₅N₆O₂⁺): calcd. m/z 357.20; found m/z 357.20; LC–MS: 96% purity.

(Z)-3⁴-Amino-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (8)

The title compound 8 (8.0 mg, 0.023 mmol, 13% over 6 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (Z)-(4-(5-amino-2-nitrophenoxy)but-2-en-1-yl)carbamate (1.56 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.64 (d, J = 2.3 Hz, 1H), 8.51 (s, 1H), 7.29 (d, J = 8.5 Hz, 1H), 6.82 (dd, J = 8.5, 2.3 Hz, 1H), 5.76–5.65 (m, 2H), 5.34–5.23 (m, 1H), 4.83–4.66 (m, 2H), 4.25–4.15 (m, 1H), 3.84 (dd, J = 16.3, 1.9 Hz, 1H), 1.26 (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 163.59, 163.21, 161.38, 160.29, 159.20, 158.40, 153.64, 143.23, 135.21, 128.80, 114.25, 112.85, 101.34, 70.03, 44.34, 42.51, 22.10. MS (ESI) for [M+H]⁺ (C₁₈H₂₃N₆O₂⁺): calcd. m/z 355.19; found m/z 355.19; LC–MS: 97% purity.

(E)-3⁴-Amino-N-isopropyl-4-oxa-2,10-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclodecaphan-6-ene-15-carboxamide (9)

The title compound 9 (10.0 mg, 0.030 mmol, 7.5% over 6 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (E)-(5-(5-amino-2-nitrophenoxy)pent-3-en-1-yl)carbamate (1.64 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.46 (s, 1H), 7.59 (d, J = 2.1 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 6.87 (dd, J = 8.4, 2.1 Hz, 1H), 6.18 (dt, J = 15.4, 7.6 Hz, 1H), 5.66–5.57 (m, 1H), 4.85–4.80 (m, 2H), 4.25–4.15 (m, 1H), 3.49–3.45 (m, 2H), 2.37–2.30 (m, 2H), 1.25 (d, J = 6.6 Hz, 6H); MS (ESI) for [M+H]⁺ (C₁₉H₂₅N₆O₂⁺): calcd. m/z 369.20; found m/z 369.20; LC–MS: 98% purity.

(Z)-3⁴-Amino-N-isopropyl-4-oxa-2,10-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclodecaphan-6-ene-1⁵-carboxamide (10)

The title compound 10 (13.0 mg, 0.035 mmol, 8.9% over 6 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (Z)-(5-(5-amino-2-nitrophenoxy)pent-3-en-1-yl)carbamate (1.64 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.50 (s, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 6.87 (dd, J = 8.4, 2.1 Hz, 1H), 5.84–5.71 (m, 1H), 5.67–5.58 (m, 1H), 4.96 (d, J = 6.0 Hz, 2H), 4.25–4.15 (m, 1H), 3.35 (t, J = 8.0 Hz, 2H), 2.65 (brs, 2H), 1.26 (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 167.19, 165.11, 164.38, 159.28, 157.20, 153.64, 143.23, 135.21, 126.70, 114.25, 111.45, 103.14, 100.38, 69.23, 51.74, 45.31, 27.8, 21.22. MS (ESI) for [M+H]⁺ (C₁₈H₂₃N₆O₂⁺): calcd. m/z 355.19; found m/z 355.19; MS (ESI) for [M+H]⁺ (C₁₉H₂₅N₆O₂⁺): calcd. m/z 369.20; found m/z 369.20; LC-MS: 96% purity.

(E)-3⁴-Amino-N-isopropyl-4-oxa-2,11-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacycloundecaphan-6-ene-1⁵-carboxamide (11)

The title compound 11 (8.0 mg, 0.020 mmol, 11% over 6 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (E)-(6-(5-amino-2-nitrophenoxy)hex-4-en-1-yl)carbamate (1.71 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.42 (s, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 6.87 (dd, J = 8.4, 2.2 Hz, 1H), 5.86–5.76 (m, 1H), 5.71 (dt, J = 15.9, 3.9 Hz, 1H), 4.90–4.85 (m, 2H), 4.24–4.15 (m, 1H), 3.59–3.49 (m, 2H), 2.20–2.15 (m, 2H), 1.75–1.67 (m, 2H), 1.25 (d, J = 6.6 Hz, 6H); MS (ESI) for [M+H]⁺ (C₂₀H₂₇N₆O₂⁺): calcd. m/z 383.22; found m/z 383.22; LC-MS: 96% purity.

(Z)-3⁴-Amino-N-isopropyl-4-oxa-2,11-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacycloundecaphan-6-ene-1⁵-carboxamide (12)

The title compound 12 (12.0 mg, 0.031 mmol, 9.3% over 6 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (Z)-(6-(5-amino-2-nitrophenoxy)hex-4-en-1-yl)carbamate (1.71 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.46 (s, 1H), 8.35 (d, J = 2.2 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 6.85 (dd, J = 8.5, 2.2 Hz, 1H), 5.75–5.66 (m, 1H), 5.57–5.48 (m, 1H), 5.16 (dd, J = 4.4, 2.2 Hz, 2H), 4.25–4.15 (m, 1H), 3.66–3.60 (m, 2H), 2.20 (dd, J = 13.5, 7.1 Hz, 2H), 2.01–1.86 (m, 2H), 1.26 (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 167.85, 161.25,161.52, 159.21, 153.84, 148.62, 138.22, 135.52, 124.40, 120.60, 114.23, 108.20, 103.14, 69.62, 44.32, 41.61, 31.19, 25.10, 23.20. MS (ESI) for [M+H]⁺ (C₂₀H₂₇N₆O₂⁺): calcd. m/z 383.22; found m/z 383.22; LC-MS: 96% purity.

(E)-1⁵-(Pyridin-2-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-en-3⁴-amine (13)

The title compound 13 (16.0 mg, 0.046 mmol, 18% over 5 steps) (Scheme S1) was prepared according to the general procedure A (4 steps) from tert-butyl (E)-(4-(5-amino-2-nitrophenoxy)but-2-en-1-yl)carbamate (258 mg, 0.80 mmol) and 4-chloro-2-(methylthio)-5-(pyridin-2-yl)pyrimidine (190 mg, 0.80 mmol) and NaH (96 mg, 2.40 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.73 (d, J = 4.8 Hz, 1H), 8.65 (s, 1H), 8.07–7.98 (m, 3H), 7.51–7.46 (m, 1H), 7.36 (d, J = 8.4 Hz, 1H), 6.98 (dd, J = 8.4, 2.2 Hz, 1H), 6.18 (dt, J = 16.0, 5.5 Hz, 1H), 5.68–5.58 (m, 1H), 5.05 (d, J = 6.3 Hz, 2H), 4.04 (d, J = 5.0 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 159.58, 157.97, 153.35, 147.31, 141.92, 138.34, 138.23, 133.20, 126.27, 122.02, 119.90, 116.80, 113.31, 110.92, 104.77, 70.39, 43.22. MS (ESI) for [M+H]⁺ (C₁₉H₁₉N₆O⁺): calcd. m/z 347.16; found m/z 347.16; LC-MS: 96% purity.

(Z)-N-Isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (14)

The title compound 14 (16.0 mg, 0.047 mmol, 20% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (Z)-(4-(3-aminophenoxy)but-2-en-1-yl)carbamate (1.36 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 11.90 (s, 1H), 9.41 (t, J = 2.3 Hz, 1H), 8.42 (d, J = 0.8 Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 6.75 – 6.62 (m, 2H), 5.75–5.56 (m, 2H), 5.11 (dd, J = 14.3, 11.0 Hz, 1H), 4.71 (dd, J = 16.1, 10.2 Hz, 1H), 4.60–4.52 (m, 1H), 4.25–4.14 (m, 1H), 3.82–3.75 (m, 1H), 1.25 (d, J = 6.6 Hz, 6H); ¹³C NMR (214 MHz, DMSO-d₆) δ 163.71, 158.55, 157.96, 153.10, 146.31, 138.63, 130.51, 129.27, 128.61, 112.25, 111.47, 107.57, 100.76, 63.71, 53.29, 41.29, 22.03. MS (ESI) for [M+H]⁺ (C₁₈H₂₂N₅O₂⁺): calcd. m/z 340.18; found m/z 340.18; LC-MS: 96% purity.

(Z)-3⁴-Fluoro-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (15)

The title compound 15 (15.0 mg, 0.042 mmol, 13% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (Z)-(4-(5-amino-2-fluorophenoxy)but-2-en-1-yl)carbamate (1.44 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.60 (dd, J = 8.7, 2.7 Hz, 1H), 8.41 (s, 1H), 7.02 (dd, J = 11.1, 8.7 Hz, 1H), 6.62 (dt, J = 8.8, 3.2 Hz, 1H), 5.73–5.60 (m, 2H), 5.15 (dd, J = 14.2, 10.3 Hz, 1H), 4.72–4.63 (m, 2H), 4.24–4.14 (m, J = 13.3, 6.6 Hz, 1H), 3.78–3.72 (m, 1H), 1.25 (d, J = 6.6 Hz, 6H); ¹³C NMR (214 MHz, DMSO-d₆) δ 163.74, 158.23, 153.43, 147.96, 146.83, 144.68, 134.18, 131.39, 127.81, 115.71, 111.67, 110.12, 100.62, 64.35, 41.26, 39.94, 22.03. MS (ESI) for [M+H]⁺ (C₁₈H₂₁FN₅O₂⁺): calcd. m/z 358.17; found m/z 358.17; LC-MS: 97% purity.

(Z)-3⁴-Chloro-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (16)

The title compound 16 (12.0 mg, 0.030 mmol, 14% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (Z)-(4-(5-amino-2-chlorophenoxy)but-2-en-1-yl)carbamate (1.52 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.53 (d, J = 2.5 Hz, 1H), 8.44 (s, 1H), 7.28 (d, J = 8.5 Hz, 1H), 6.69 (dd, J = 8.5, 2.5 Hz, 1H), 5.76–5.60 (m, 2H), 5.17 (dd, J = 14.3, 10.3 Hz, 1H), 4.75–4.66 (m, 2H), 4.25–4.15 (m, 1H), 3.84–3.76 (m, 1H), 1.25 (d, J = 6.6 Hz, 6H); ¹³C NMR (214 MHz, DMSO-d₆) δ 164.21, 158.55, 154.34, 149.81, 146.45, 144.20,135.20, 132.17, 127.37, 122.46, 111.61, 108.53, 100.35, 64.64, 41.15, 39.98, 22.09. MS (ESI) for [M+H]⁺ (C₁₈H₂₁ClN₅O₂⁺): calcd. m/z 374.14; found m/z 374.14; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (17)

The title compound 17 (11.0 mg, 0.030 mmol, 11% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.00 g, 4.88 mmol) and tert-butyl (Z)-(4-(5-amino-2-cyanophenoxy)but-2-en-1-yl)carbamate (1.52 g, 4.88 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.75 (d, J = 1.8 Hz, 1H), 8.51 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 6.72 (dd, J = 8.4, 2.0 Hz, 1H), 5.74–5.64 (m, 1H), 5.30–5.20 (m, 1H), 4.78–4.64 (m, 2H), 4.24–4.14 (m, 1H), 3.80–3.73 (m, 1H), 1.25 (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.90, 160.10, 159.29, 154.99, 150.01, 143.20, 133.39, 131.99, 126.88, 116.49, 112.14, 107.12, 101.30, 93.69, 64.95, 51.72, 40.15, 28.46. MS (ESI) for [M+H]⁺ (C₁₉H₂₁N₆O₂⁺): calcd. m/z 365.17; found m/z 365.17; LC-MS: 96% purity.

(Z)-N-Isopropyl-3⁴-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (18)

The title compound 18 (15.0 mg, 0.04 mmol, 12% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.50 g, 7.30 mmol) and tert-butyl (Z)-(4-(5-amino-2-methylphenoxy)but-2-en-1-yl)carbamate (2.13 g, 7.30 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 11.83 (s, 1H), 9.34 (d, J = 2.2 Hz, 1H), 8.38 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.59 (dd, J = 8.0, 2.2 Hz, 1H), 5.75–5.55 (m, 2H), 5.11 (dd, J = 14.2, 10.9 Hz, 1H), 4.71 (dd, J = 16.2, 10.4 Hz, 1H), 4.66–4.60 (m, 1H), 4.25–4.13 (m, 1H), 3.92–3.73 (m, 1H), 2.18 (s, 3H), 1.25 (d, J = 6.6 Hz, 6H); ¹³C NMR (214 MHz, DMSO-d₆) δ 163.83, 158.16, 155.75, 153.40, 146.43, 136.44, 130.42, 130.03, 128.85, 120.92, 111.02, 106.99, 100.65, 63.83, 41.24, 39.97, 22.04, 15.23. MS (ESI) for [M+H]⁺ (C₁₉H₂₄N₅O₂⁺): calcd. m/z 354.19; found m/z 354.19; LC-MS: 96% purity.

(Z)-N-Isopropyl-3⁴-methoxy-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (19)

The title compound 19 (13.0 mg, 0.035 mmol, 15% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.50 g, 7.30 mmol) and tert-butyl (Z)-(4-(5-amino-2-methoxylphenoxy)but-2-en-1-yl)carbamate (2.25 g, 7.30 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 11.84 (s, 1H), 9.41 (d, J = 2.6 Hz, 1H), 8.37 (d, J = 0.9 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.68 (dd, J = 8.6, 2.6 Hz, 1H), 5.75–5.58 (m, 2H), 5.11 (dd, J = 14.2, 10.6 Hz, 1H), 4.74–4.60 (m, 2H), 4.24–4.14 (m, 1H), 3.84 (s, 3H), 3.81–3.74 (m, 1H), 1.25 (d, J = 6.6 Hz, 6H); ¹³C NMR (214 MHz, DMSO-d₆) δ 163.71, 157.48, 152.70, 146.52, 145.32, 145.12, 131.00, 130.59, 128.68, 111.92, 111.41, 108.64, 100.62, 63.86, 55.64, 41.28, 39.98, 22.02. MS (ESI) for [M+H]⁺ (C₁₉H₂₄N₅O₃⁺): calcd. m/z 370.19; found m/z 370.19; LC-MS: 97% purity.

(Z)-3⁵-Cyano-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-15-carboxamide (20)

The title compound 20 (9.0 mg, 0.025 mmol, 11% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.50 g, 7.30 mmol) and tert-butyl (Z)-(4-(3-amino-5-cyanophenoxy)but-2-en-1-yl)carbamate (2.25 g, 7.30 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.69 (t, J = 2.2 Hz, 1H), 8.50 (s, 1H), 7.11–7.04 (m, 1H), 7.02 (dd, J = 2.5, 1.2 Hz, 1H), 5.74–5.60 (m, 2H), 5.23–5.12 (m, 1H), 4.74–4.61 (m, 2H), 4.26–4.16 (m, 1H), 3.82 (dd, J = 16.2, 1.8 Hz, 1H), 1.26 (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.35, 160.54, 159.74, 155.43, 150.45, 143.65, 133.83, 132.43, 127.33, 116.93, 112.59, 107.57, 101.74, 94.14, 65.40, 52.16, 40.59, 28.90. MS (ESI) for [M+H]⁺ (C₁₉H₂₁N₆O₂⁺): calcd. m/z 365.17; found m/z 365.17; LC-MS: 96% purity.

(Z)-N-Isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(4,2)-pyridinacyclononaphan-6-ene-1⁵-carboxamide (21)

The title compound 21 (12.0 mg, 0.035 mmol, 18% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.50 g, 7.30 mmol) and tert-butyl (Z)-(4-((4-aminopyridin-2-yl)oxy)but-2-en-1-yl)carbamate (2.04 g, 7.30 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.96 (d, J = 2.1 Hz, 1H), 8.75 (s, 1H), 8.16 (d, J = 6.9 Hz, 1H), 7.17 (dd, J = 6.9, 2.1 Hz, 1H), 5.88–5.81 (m, 1H), 5.78–5.68 (m, 1H), 5.55 (dd, J = 14.0, 11.3 Hz, 1H), 5.03 (dd, J = 14.0, 2.1 Hz, 1H), 4.73 (dd, J = 16.7, 10.5 Hz, 1H), 4.29–4.17 (m, 1H), 3.91 (dd, J = 16.6, 2.3 Hz, 1H), 3.59 (s, 1H), 1.26 (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.89, 158.98, 155.14, 149.26, 144.80, 140.71, 133.90, 125.79, 125.76, 119.22, 117.32, 100.39, 65.51, 41.19, 40.15, 22.10. MS (ESI) for [M+H]⁺ (C₁₇H₂₁N₆O₂⁺): calcd. m/z 341.17; found m/z 341.17; LC-MS: 96% purity.

(Z)-3⁶-Amino-N-isopropyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(3,5)-pyridinacyclononaphan-6-ene-1⁵-carboxamide (22)

The title compound 22 (15.0 mg, 0.040 mmol, 19% over 5 steps) was prepared according to the general procedure A from methyl 2,4-dichloropyrimidine-5-carboxylate (1.50 g, 7.30 mmol) and tert-butyl (Z)-(4-((2,5-diaminopyridin-3-yl)oxy)but-2-en-1-yl)carbamate (2.15 g, 7.30 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.77 (d, J = 2.2 Hz, 1H), 8.51 (s, 1H), 7.54 (d, J = 2.2 Hz, 1H), 5.77 (t, J = 11.0 Hz, 1H), 5.64 (t, J = 11.3 Hz, 1H), 5.31 (dd, J = 13.9, 11.3 Hz, 1H), 4.92–4.85 (m, 1H), 4.66 (dd, J = 16.6, 10.5 Hz, 1H), 4.25–4.14 (m, 1H), 3.83 (dd, J = 16.5, 2.3 Hz, 1H), 1.25 (d, J = 6.6 Hz, 6H); ¹³C NMR (214 MHz, DMSO-d₆) δ 166.97, 162.12, 158.03, 152.05, 148.02, 143.88, 128.94, 128.86, 103.60, 68.68, 56.37, 44.36, 25.23. MS (ESI) for [M+H]⁺ (C₁₇H₂₂N₇O₂⁺): calcd. m/z 356.18; found m/z 356.18; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-isopropyl-N-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (23)

(General procedure C) (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (1.50 g, 4.64 mmol, 17% over 6 steps) was prepared according to the general procedure B from intermediate 46 (5.00 g, 26.7 mmol), triphenylphosphine (9.11 g, 34.8 mmol), DIAD (7.02 g, 34.8 mmol) and 2-hydroxy-4-nitrobenzonitrile (5.67 g, 34.8 mmol) as a white solid. To a solution of (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (34 mg, 0.10 mmol), propan-2-amine (11.8 mg, 0.20 mmol) and DIPEA (38.7 mg, 0.30 mmol) in DMF (15 mL) was added TBTU (114 mg, 0.30 mmol). The reaction mixture was stirred at 25 °C for 16 h, quenched with water (20 mL) and then filtered. The solid was washed with water (15 mL) and diether ether (10 mL) and dried under reduced pressure to afford the titled compound 23 (16.6 mg, 0.044 mmol, 44%) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.62 (d, J = 2.0 Hz, 1H), 8.06 (s, 1H), 7.53 (d, J = 8.5 Hz, 1H), 6.88 (dd, J = 8.5, 2.0 Hz, 1H), 5.76–5.65 (m, 2H), 5.32–5.23 (m, 1H), 4.81–4.68 (m, 2H), 4.50–4.35 (m, 1H), 3.83 (dd, J = 16.3, 2.2 Hz, 1H), 2.99 (s, 3H), 1.26 (dd, J = 6.7, 1.7 Hz, 6H); ¹³C NMR (214 MHz, DMSO-d₆) δ 167.17, 164.39, 160.08, 157.66, 156.08, 144.21, 133.14, 132.84, 126.65, 116.70, 112.15, 107.13, 106.18, 93.34, 64.91, 53.42, 41.68, 39.97, 19.60. MS (ESI) for [M+H]⁺ (C₂₀H₂₃N₆O₂⁺): calcd. m/z 379.19; found m/z 379.19; LC-MS: 96% purity.

(Z)-1⁵-(Piperazine-1-carbonyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-3⁴-carbonitrile (24)

The title compound 24 (24.0 mg, 0.061 mmol, 61% over 2 steps) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (32.0 mg, 0.10 mmol) and tert-butyl piperazine-1-carboxylate (37.2 mg, 0.20 mmol) followed by treatment with a 4.0 M HCl solution in dioxane (125 μL, 0.50 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.64 (d, J = 2.0 Hz, 1H), 8.18 (s, 1H), 7.55 (d, J = 8.5 Hz, 1H), 6.85 (dd, J = 8.4, 2.0 Hz, 1H), 5.77–5.64 (m, 2H), 5.35–5.25 (m, 1H), 4.81–4.67 (m, 2H), 4.01–3.80 (m, 5H), 3.40–3.31 (m, 4H); ¹³C NMR (214 MHz, DMSO-d₆) δ 163.60, 160.05, 157.75, 155.03, 148.14, 143.93, 133.16, 132.28, 126.83, 116.62, 112.42, 107.35, 105.04, 93.77, 64.97, 53.33, 42.16, 34.11. MS (ESI) for [M+H]⁺ (C₂₀H₂₂N₇O₂⁺): calcd. m/z 392.18; found m/z 392.18; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-(piperidin-4-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (25)

The title compound 25 ((120 mg, 0.30 mmol, 74% over 2 steps) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (129 mg, 0.40 mmol) and tert-butyl 4-aminopiperidine-1-carboxylate (120 mg, 0.6 mmol) followed by treatment with a 4.0 M HCl solution in dioxane (494 μL, 2.0 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.63 (s, 1H), 8.67 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 5.75–5.66 (m, 2H), 5.30–5.24 (m, 1H), 4.77–4.71 (m, 2H), 4.23–4.17 (m, 1H), 3.88–3.84 (m, 1H), 3.52–3.48 (m, 2H), 3.19–3.13 (m, 2H), 2.24–2.20 (m, 2H), 1.94–1.86 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.99, 160.19, 159.24, 152.13, 147.23, 143.64, 138.01, 133.40, 132.44, 126.76, 116.62, 111.71, 106.87, 100.18, 64.95, 46.56, 44.35, 42.00, 27.89. MS m/z Calcd for [M+H]⁺ (C₂₁H₂₄N₇O₂⁺): 406.2; found: 406.2; LC/MS: 96% purity.

(Z)-3⁴-Cyano-N-(1-methylazetidin-3-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (26)

The title compound 26 (16.0 mg, 0.040 mmol, 41%) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (32.0 mg, 0.10 mmol) and 1-methylazetidin-3-amine (17.2 mg, 0.20 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.69 (dd, J = 6.5, 2.0 Hz, 1H), 8.65 (d, J = 15.8 Hz, 1H), 7.54 (dd, J = 8.4, 3.6 Hz, 1H), 6.75 (ddd, J = 8.4, 3.1, 2.1 Hz, 1H), 5.77–5.63 (m, 2H), 5.26 (dd, J = 14.3, 9.5 Hz, 1H), 4.83–4.58 (m, 5H), 4.40–4.24 (m, 2H), 3.82 (dd, J = 14.5, 2.0 Hz, 1H), 3.04 (d, J = 17.3 Hz, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ 165.34, 160.18, 159.19, 143.70, 133.37, 132.51, 126.75, 116.65, 111.70, 111.61, 106.86, 99.72, 99.63, 93.13, 64.95, 60.15, 53.37, 41.48, 41.09. MS (ESI) for [M+H]⁺ (C₂₀H₂₂N₇O₂⁺): calcd. m/z 392.18; found m/z 392.18; LC-MS: 96% purity.

(S,Z)-3⁴-Cyano-N-(pyrrolidin-3-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (27)

The title compound 27 (15.0 mg, 0.037 mmol, 37% over 2 steps) was prepared according to the general procedure C from from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (96.9 mg, 0.30 mmol) and tert-butyl (S)-3-aminopyrrolidine-1-carboxylate (112 mg, 0.60 mmol) followed by treatment with a 4.0 M HCl solution in dioxane (125 μL, 5.0 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.67 (d, J = 1.9 Hz, 1H), 8.71 (d, J = 0.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 6.78 (dd, J = 8.4, 2.0 Hz, 1H), 5.76–5.64 (m, 2H), 5.31–5.21 (m, 1H), 4.81–4.67 (m, 2H), 4.66–4.58 (m, 1H), 3.83 (dd, J = 16.4, 2.2 Hz, 1H), 3.64–3.54 (m 2H), 3.45–3.36 (m, 2H), 2.41 (dt, J = 21.5, 7.6 Hz, 1H), 2.23 (td, J = 13.7, 7.1 Hz, 1H); ¹³C NMR (214 MHz, DMSO-d₆) δ 165.33, 160.14, 159.13, 156.26, 152.20, 143.45, 133.38, 132.21, 126.82, 116.55, 111.89, 106.99, 100.22, 93.44, 64.95, 53.31, 49.07, 48.89, 43.38, 29.76. MS (ESI) for [M+H]⁺ (C₂₁H₂₄N₇O₂⁺): calcd. m/z 406.20; found m/z 406.20; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-(piperidin-4-ylmethyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (28)

The title compound 28 (16.6 mg, 0.040 mmol, 40% over 2 steps) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (33 mg, 0.10 mmol) and tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (64 mg, 0.30 mmol) followed by treatment with a 4.0 M HCl solution in dioxane (75 μL, 0.30 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.68 (d, J = 2.0 Hz, 1H), 8.56 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 6.75 (dd, J = 8.4, 2.0 Hz, 1H), 5.76–5.64 (m, 2H), 5.26 (dd, J = 14.1, 10.1 Hz, 1H), 4.81–4.68 (m, 2H), 3.83 (dd, J = 14.4, 2.0 Hz, 1H), 3.46–3.38 (m, 2H), 3.33 (d, J = 6.0 Hz, 2H), 3.04–2.94 (m, 2H), 2.05–1.95 (m, 3H), 1.55–1.40 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 165.29, 160.17, 159.25, 155.79, 150.71, 143.42, 133.46, 132.16, 126.88, 116.59, 111.93, 107.01, 100.51, 93.51, 65.00, 43.74, 42.77, 40.15, 33.38, 26.22. MS (ESI) for [M+H]⁺ (C₂₂H₂₆N₇O₂⁺): calcd. m/z 420.21; found m/z 420.21; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-(pyridin-4-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (29)

The title compound 29 (16.0 mg, 0.040 mmol, 40%) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (32.0 mg, 0.10 mmol) and pyridin-4-amine (14.5 mg, 0.15 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.72 (d, J = 2.0 Hz, 1H), 8.92 (s, 1H), 8.73–8.67 (m, 2H), 8.34 (dd, J = 6.6, 5.5 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H), 6.83 (dd, J = 8.4, 2.0 Hz, 1H), 5.77–5.66 (m, 2H), 5.28 (dd, J = 14.1, 10.1 Hz, 1H), 4.81–4.69 (m, 2H), 3.84 (dd, J = 14.4, 2.0 Hz, 1H); ¹³C NMR (214 MHz, DMSO-d₆) δ 169.83, 163.36, 162.57, 156.58, 147.31, 145.14, 136.40, 136.06, 130.50, 129.77, 119.87, 118.50, 118.40, 114.77, 112.79, 111.90, 110.06, 102.50, 95.93, 68.06, 37.34. MS (ESI) for [M+H]⁺ (C₂₁H₁₈N₇O₂⁺): calcd. m/z 400.15; found m/z 400.15; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-(4-(4-methylpiperazin-1-yl)phenyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (30)

The title compound 30 (15.0 mg, 0.028 mmol, 30%) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (32 mg, 0.10 mmol) and 4-(4-methylpiperazin-1-yl)aniline (53 mg, 0.30 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.65 (d, J = 2.0 Hz, 1H), 8.71 (s, 1H), 7.63–7.53 (m, 3H), 7.09–7.05 (m, 2H), 6.83 (dd, J = 8.4, 2.0 Hz, 1H), 5.72 (m, 2H), 5.28 (dd, J = 14.1, 10.3 Hz, 1H), 4.82–4.72 (m, 2H), 3.86 (dd, J = 14.0, 9.3 Hz, 3H), 3.62 (d, J = 12.7 Hz, 2H), 3.30–3.26 (m, 2H), 3.10–3.01 (m, 2H), 2.98 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 166.13, 164.07, 160.21, 159.29, 146.36, 143.84, 135.12, 133.37, 131.20, 132.66, 130.92, 128.79, 126.77, 122.39, 116.71, 116.18, 111.83, 106.92, 100.87, 93.09, 64.97, 52.20, 45.85, 46.71, 42.03. MS (ESI) for [M+H]⁺ (C₂₇H₂₉N₈O₂⁺): calcd. m/z 497.24; found m/z 497.24; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (31)

The title compound 31 (15.0 mg, 0.029 mmol, 30%) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (32.0 mg, 0.10 mmol) and 4-((4-methylpiperazin-1-yl)methyl)aniline (30.8 mg, 0.15 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.66 (d, J = 2.0 Hz, 1H), 8.77 (s, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.55 (t, J = 8.9 Hz, 2H), 6.83 (dd, J = 8.4, 2.0 Hz, 1H), 5.80–5.65 (m, 2H), 5.28 (dd, J = 14.2, 10.2 Hz, 1H), 4.83–4.72 (m, 2H), 4.15 (s, 2H), 3.92–3.83 (m, 1H), 3.31 (s, 8H), 2.95 (s, 3H); ¹³C NMR (214 MHz, DMSO-d₆) δ 167.83, 163.31, 162.37, 160.80, 157.38, 146.92, 142.62, 136.47, 135.71, 135.02, 133.68, 129.89, 127.96, 124.13, 123.61, 119.81, 114.99, 110.07, 103.85, 96.29, 68.08, 56.53, 52.68, 52.34, 50.77, 45.17. MS (ESI) for [M+H]⁺ (C₂₈H₃₁N₈O₂⁺): calcd. m/z 511.26; found m/z 511.26; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-(1-((tetrahydro-2H-pyran-4-yl)methyl)piperidin-4-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (32)

The title compound 32 (17.0 mg, 0.030 mmol, 34%) was prepared according to the general procedure C from (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (32.0 mg, 0.10 mmol) and 1-((tetrahydro-2H-pyran-4-yl)methyl)piperidin-4-amine (39.6 mg, 0.20 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.65 (d, J = 2.0 Hz, 1H), 8.88 (s, 1H), 8.66 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 6.81–6.73 (m, 1H), 5.75–5.65 (m, 2H), 5.26 (dd, J = 14.1, 10.1 Hz, 1H), 4.80–4.67 (m, 2H), 4.23–4.13 (m, 1H), 3.97 (dd, J = 11.5, 2.7 Hz, 2H), 3.84 (dd, J = 16.4, 1.9 Hz, 1H), 3.71 (d, J = 12.8 Hz, 1H), 3.60–3.40 (m, 3H), 3.20–3.10 (m, 2H), 3.06 (d, J = 7.0 Hz, 2H), 2.32–1.97 (m, 5H), 1.77–1.70 (m, 2H), 1.45–1.32 (m, 2H); ¹³C NMR (214 MHz, DMSO-d₆) δ 165.27, 160.22, 159.27, 157.29, 143.78, 133.38, 132.59, 126.72, 116.69, 111.63, 106.82, 100.24, 100.08, 93.07, 66.33, 64.97, 61.38, 53.37, 51.48, 44.87, 30.78, 30.59, 29.82. MS (ESI) for [M+H]⁺ (C₂₇H₃₄N₇O₃⁺): calcd. m/z 504.27; found m/z 504.27; LC-MS: 96% purity.

(Z)-3⁴-cyano-N-(1-(tetrahydro-2H-pyran-4-carbonyl)piperidin-4-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-15-carboxamide (33)

The title compound 33 (20.0 mg, 0.040 mmol, 39%) was prepared according to the general procedure C from (Z)-3⁴-cyano-N-(piperidin-4-yl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (32.0 mg, 0.10 mmol) and 1-((tetrahydro-2H-pyran-4-yl)methyl)piperidin-4-amine (26.0 mg, 0.20 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.67 (d, J = 1.8 Hz, 1H), 8.52 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 6.78 (dd, J = 8.4, 2.0 Hz, 1H), 5.78–5.63 (m, 2H), 5.26 (dd, J = 14.1, 10.0 Hz, 1H), 4.81–4.67 (m, 2H), 4.55 (d, J = 13.2 Hz, 1H), 4.20–4.10 (m, 2H), 3.96 (dd, J = 11.9, 3.0 Hz, 2H), 3.82 (dd, J = 16.2, 2.1 Hz, 1H), 3.57–3.44 (m, 2H), 3.28–3.22 (m, 1H), 3.05–2.93 (m, 1H), 2.87–2.74 (m, 1H), 2.13–1.95 (m, 2H), 1.85–1.70 (m, 2H), 1.67–1.39 (m, 4H); ¹³C NMR (214 MHz, DMSO-d₆) δ 172.18, 165.53, 160.29, 159.35, 144.51, 133.43, 133.28, 126.47, 116.88, 111.18, 106.43, 99.67, 92.18, 66.29, 64.91, 46.52, 43.73, 36.26, 32.31, 31.16, 29.09. MS (ESI) for [M+H]⁺ (C₂₇H₃₂N₇O₄⁺): calcd. m/z 518.25; found m/z 518.25; LC-MS: 96% purity.

(Z)-3⁴-Cyano-N-(trans-4-(morpholine-4-carbonyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (34)

trans-4-((Z)-3⁴-Cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (0.45 g, 1.0 mmol, 65% over 2 steps) was prepared according to the general procedure B from the intermediate (Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxylic acid (0.50 g, 1.55 mmol) and methyl trans-4-aminocyclohexane-1-carboxylate (0.48 mg, 3.1 mmol) as a white solid. The title compound 34 (16.6 mg, 0.044 mmol, 44%) was prepared according to general procedure C from trans-4-((Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (34 mg, 0.10 mmol) and morpholine (17 mg, 0.20 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD+DMSO-D₆) δ 9.71 (s, 1H), 8.52 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 5.72–5.68 (m, 2H), 5.29–5.22 (m, 1H), 3.93–3.92 (m, 2H), 3.86–3.80 (m, 2H), 3.72–3.60 (m, 4H), 3.27–3.26 (m, 2H), 2.71–2.70 (m, 2H), 2.06–2.04 (m, 2H), 1.87–1.84 (m, 2H), 1.64–1.58 (m, 2H), 1.62–1.47 (m, 2H); ¹³C NMR (214 MHz, DMSO-d₆) δ 173.58, 165.35, 160.25, 159.33, 158.28, 158.11, 144.46, 133.36, 133.26, 126.49, 116.85, 111.26, 106.43, 99.87, 92.22, 64.87, 51.54, 51.10, 48.12, 43.36, 38.01, 31.12, 28.21. MS m/z Calcd for [M+H]⁺ (C₂₇H₃₂N₇O₄⁺): 518.25; found: 518.30; LC/MS: 96% purity.

(Z)-3⁴-Cyano-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (35)

The title compound 35 (25.0 mg, 0.050 mmol, 60%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (35.8 mg, 0.080 mmol) and 1-methylpiperidin-4-amine (18.2 mg, 0.16 mmol) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 9.62 (s, 1H), 8.60 (s, 1H), 7.55 (d, J = 8.3 Hz, 1H), 6.80 (dd, J = 8.3, 2.0 Hz, 1H), 5.77–5.65 (m, 2H), 5.26 (dd, J = 14.2, 9.8 Hz, 1H), 4.81–4.67 (m, 2H), 3.96–3.80 (m, 3H), 3.55 (d, J = 12.5 Hz, 2H), 3.12 (t, J = 11.7 Hz, 2H), 2.87 (s, 3H), 2.17–2.00 (m, 5H), 1.90 (d, J = 13.3 Hz, 2H), 1.84–1.71 (m, 2H), 1.67–1.56 (m, 2H), 1.52–1.39 (m, 2H). ¹³C NMR (101 MHz, CD₃OD) δ 178.18, 165.06, 161.98, 160.93, 153.93, 145.91, 143.86, 134.69, 131.51, 129.13, 117.02, 113.61, 109.19, 103.80, 97.14, 66.31, 54.82, 51.31, 49.99, 45.19, 43.79, 41.43, 32.43, 30.39, 29.56. MS m/z Calcd for [M+H]⁺ (C₂₉H₃₇N₈O₃⁺): 545.30; found: 545.30; LC/MS: 98% purity.

(Z)-3⁴-Cyano-N-(trans-4-((2-(diethylamino)ethyl)carbamoyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (36)

The title compound 36 (25.0 mg, 0.050 mmol, 60%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-cyano-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (35.8 mg, 0.080 mmol) and N1,N1-diethylethane-1,2-diamine (18.6 mg, 0.16 mmol) as a white solid. ¹H NMR (850 MHz, CD₃OD) δ 9.84 (d, J = 2.1 Hz, 1H), 8.51 (s, 1H), 7.44 (d, J = 8.3 Hz, 1H), 6.61 (dd, J = 8.4, 2.0 Hz, 1H), 5.70–5.61 (m, 2H), 5.22 (dd, J = 14.2, 10.6 Hz, 1H), 4.76–4.70 (m, 1H), 4.60 (dd, J = 16.5, 10.0 Hz, 1H), 3.88–3.82 (m, 1H), 3.72–3.66 (m, 1H), 3.54 (t, J = 6.2 Hz, 2H), 3.30–3.26 (m, 8H), 2.25 (tt, J = 12.2, 3.6 Hz, 1H), 2.10–2.04 (m, 2H), 1.97–1.92 (m, 2H), 1.66–1.58 (m, 2H), 1.46–1.39 (m, 2H), 1.34 (t, J = 7.3 Hz, 6H). ¹³C NMR (214 MHz, CD₃OD) δ 180.36, 167.43, 162.21, 161.09, 145.86, 134.33, 134.02, 128.04, 117.70, 112.25, 108.10, 102.03, 94.53, 66.17, 53.32, 49.55, 49.39, 45.08, 40.99, 36.14, 32.56, 29.56, 9.31. MS m/z Calcd for [M+H]⁺ (C₂₉H₃₉N₈O₃⁺): 547.31; found: 547.31; LC/MS: 98% purity.

(Z)-3⁴-Fluoro-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (37)

trans-4-((Z)-3⁴-Fluoro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (300 mg, 0.68 mmol, 8.5% over 8 steps) was prepared according to the general procedure B from the intermediate 46 (5.00 g, 26.7 mmol) and 2-fluoro-5-nitrophenol (5.46 g, 34.8 mmol) as a white solid. The title compound 37 (20.0 mg, 0.037 mmol, 70%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-fluoro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (34 mg, 0.10 mmol) and 1-methylpiperidin-4-amine (22.8 mg, 0.20 mmol) as a white solid. ¹H NMR (850 MHz, DMSO-d₆) δ 11.84 (s, 1H), 9.51 (d, J = 8.6 Hz, 1H), 8.79 (s, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.14 (dd, J = 11.2, 8.6 Hz, 1H), 6.73–6.70 (m, 1H), 5.63–5.56 (m, 2H), 5.18–5.13 (m, 1H), 4.67 (d, J = 13.3 Hz, 1H), 4.59–4.53 (m, 1H), 3.79–3.69 (m, 3H), 3.35 (d, J = 10.5 Hz, 2H), 3.26–3.20 (m, 1H), 3.03–2.96 (m, 2H), 2.68 (s, 3H), 2.11–2.05 (m, 1H), 1.92–1.86 (m, 4H), 1.78–1.68 (m, 4H), 1.46–1.40 (m, 2H), 1.36 (m, 2H). ¹³C NMR (214 MHz, DMSO-d₆) δ 174.72, 174.40, 163.89, 158.27, 147.95, 146.81, 144.72 (C-F), 144.67 (C-F), 134.30, 131.53, 127.82, 115.80 (C-F), 115.71 (C-F), 111.66, 110.10, 100.53, 64.36, 52.44, 48.80, 48.02, 43.28, 42.98, 42.28, 31.19, 28.75, 28.18. MS m/z Calcd for [M+H]⁺ (C₂₈H₃₇FN₇O₃⁺): 538.29; found: 538.30; LC/MS: 98% purity.

(Z)-3⁴-Chloro-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (38)

trans-4-((Z)-3⁴-Chloro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (200 mg, 0.44 mmol, 13% over 8 steps) was prepared according to the general procedure B from the intermediate 46 (5.00 g, 26.7 mmol) and 2-chloro-5-nitrophenol (5.93 g, 34.8 mmol) as a white solid. The title compound 38 (18.0 mg, 0.030 mmol, 75%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-chloro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (25.0 mg, 0.050 mmol) and 1-methylpiperidin-4-amine (17.1 mg, 0.15 mmol) as a white solid. ¹H NMR (850 MHz, DMSO-d₆) δ 9.55 (s, 1H), 8.75 (s, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 8.6 Hz, 1H), 5.59 (d, J = 9.5 Hz, 2H), 5.21–5.12 (m, 1H), 4.71 (d, J = 16.2 Hz, 1H), 4.58–4.53 (m, 1H), 3.78–3.69 (m, 4H), 3.36 (d, J = 12.7 Hz, 2H), 3.03–2.97 (m, 2H), 2.69 (s, 3H), 2.10–2.05 (m, 1H), 1.95–1.86 (m, 4H), 1.76 (d, J = 13.5 Hz, 2H), 1.72–1.65 (m, 2H), 1.47–1.40 (m, 2H), 1.39–1.31 (m, 2H). ¹³C NMR (214 MHz, DMSO-d₆) δ 174.69, 174.39, 164.21, 158.68, 153.00, 137.85, 131.66, 129.40, 127.67, 122.48, 114.20, 112.33, 108.49, 100.44, 64.62, 52.49, 48.89, 47.95, 43.24, 43.01, 42.31, 31.23, 28.80, 28.19. MS m/z Calcd for [M+H]⁺ (C₂₈H₃₇ClN₇O₃⁺): 554.26; found: 554.30; LC/MS: 98% purity.

(Z)-3⁴-Methyl-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (39)

The title compound 39 (20.0 mg, 0.036 mmol, 72%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (22.0 mg, 0.050 mmol) and 1-methylpiperidin-4-amine (17.1 mg, 0.15 mmol) as a white solid. ¹H NMR (850 MHz, DMSO-d₆) δ 9.31 (s, 1H), 8.72 (s, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 5.62–5.53 (m, 2H), 5.08 (dd, J = 14.3, 10.8 Hz, 1H), 4.62 (dd, J = 14.2, 2.4 Hz, 1H), 4.56 (ddd, J = 15.0, 10.2, 4.2 Hz, 1H), 3.77–3.69 (m, 4H), 3.39–3.35 (m, 2H), 3.04–2.97 (m, 2H), 2.69 (s, 3H), 2.13 (s, 3H), 1.95–1.87 (m, 4H), 1.79–1.75 (m, 2H), 1.68 (qd, J = 13.6, 4.0 Hz, 2H), 1.47–1.40 (m, 2H), 1.38–1.32 (m, 2H). ¹³C NMR (214 MHz, DMSO-d₆) δ 174.50, 174.36, 163.01, 161.67,158.18, 155.76, 135.60, 130.06, 126.13, 118.20, 115.21, 108.31, 101.70, 94.60, 63.83, 52.50, 48.91, 47.99, 43.23, 43.00, 42.32, 31.19, 28.80, 28.16, 15.26. MS m/z Calcd for [M+H]⁺ (C₂₉H₄₀N₇O₃⁺): 534.31; found: 534.30; LC/MS: 98% purity.

(Z)-3⁴-Methoxy-N-(trans-4-((1-methylpiperidin-4-yl)carbamoyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (40)

trans-4-((Z)-3⁴-Methoxyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (200 mg, 0.44 mmol, 8.3% over 8 steps) was prepared according to the general procedure B from the intermediate 46 (5.00 g, 26.7 mmol) and 2-chloro-5-nitrophenol (5.93 g, 34.8 mmol) as a white solid. The title compound 40 (18.0 mg, 0.030 mmol, 65%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-methoxy-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (25.0 mg, 0.050 mmol) and 1-methylpiperidin-4-amine (17.1 mg, 0.15 mmol) as a white solid. ¹H NMR (850 MHz, DMSO-d₆) δ 11.85 (s, 1H), 9.33 (d, J = 2.6 Hz, 1H), 8.76 (s, 1H), 7.96 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 6.72–6.68 (m, 1H), 5.61–5.53 (m, 2H), 5.05 (dd, J = 14.3, 10.1 Hz, 1H), 4.60 (d, J = 16.3 Hz, 1H), 4.55 (ddd, J = 16.3, 9.5, 4.2 Hz, 1H), 3.80–3.76 (m, 2H), 3.75 (s, 3H), 3.74–3.70 (m, 2H), 3.36 (d, J = 10.5 Hz, 2H), 3.26–3.21 (m, 1H), 3.03–2.96 (m, 2H), 2.70 (s, 3H), 2.08 (tt, J = 11.9, 3.5 Hz, 1H), 1.92–1.86 (m, 4H), 1.78–1.74 (m, 2H), 1.46–1.39 (m, 2H), 1.39–1.33 (m, 2H). ¹³C NMR (214 MHz, DMSO-d₆) δ 174.69, 174.38, 163.75, 157.47, 146.51, 145.17, 130.99, 130.59, 128.72, 125.10, 111.93, 111.50, 108.66, 100.64, 63.87, 55.64, 52.42, 48.09, 43.27, 42.96, 42.27, 31.15, 28.73, 28.16, 26.39. MS m/z Calcd for [M+H]⁺ (C₂₉H₄₀N₇O₃⁺): 550.31; found: 550.30; LC/MS: 98% purity.

(Z)-N-(trans-4-((2-(Diethylamino)ethyl)carbamoyl)cyclohexyl)-3⁴-fluoro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (41)

The title compound 41 (22.0 mg, 0.040 mmol, 66%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-fluoro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (27.0 mg, 0.060 mmol) and N1,N1-diethylethane-1,2-diamine (14.0 mg, 0.12 mmol) as a white solid. ¹H NMR (850 MHz, DMSO-d₆) δ 11.84 (s, 1H), 10.34 (s, 1H), 9.51 (d, J = 11.7 Hz, 1H), 8.80 (s, 1H), 8.79 (s, 1H), 8.70 (s, 1H), 8.29 (t, J = 5.7 Hz, 1H), 7.14 (dd, J = 11.2, 8.6 Hz, 1H), 6.71 (dt, J = 8.8, 3.0 Hz, 1H), 5.63–5.56 (m, 2H), 5.15 (dd, J = 14.1, 10.1 Hz, 1H), 4.67 (d, J = 13.6 Hz, 1H), 4.59–4.53 (m, 1H), 3.80–3.70 (m, 3H), 3.42 (q, J = 6.2 Hz, 2H), 3.15–3.11 (m, 4H), 3.08 (q, J = 6.4 Hz, 2H), 2.12 (tt, J = 11.9, 3.6 Hz, 1H), 1.92–1.87 (m, 2H), 1.83 (d, J = 13.6 Hz, 1H), 1.48–1.41 (m, 2H), 1.40–1.34 (m, 2H), 1.22 (t, J = 7.2 Hz, 6H). ¹³C NMR (214 MHz, DMSO-d₆) δ 175.34, 163.92, 158.27, 147.94, 146.80, 144.72, 144.66, 134.31, 131.52, 127.81, 115.80, 115.71, 111.64, 110.10, 100.52, 64.35, 49.84, 48.03, 46.54, 43.11, 39.96, 33.51, 31.14, 28.04, 8.41. MS m/z Calcd for [M+H]⁺ (C₂₈H₃₉FN₇O₃⁺): 540.30; found: 540.30; LC/MS: 99% purity.

(Z)-3⁴-Chloro-N-(trans-4-((2-(diethylamino)ethyl)carbamoyl)cyclohexyl)-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (42)

The title compound 42 (24.0 mg, 0.040 mmol, 69%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-chloro-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (28.6 mg, 0.060 mmol) and N1,N1-diethylethane-1,2-diamine (14.0 mg, 0.12 mmol) as a white solid. ¹H NMR (850 MHz, DMSO-d₆) δ 11.87 (s, 1H), 10.25 (s, 1H), 9.53 (s, 1H), 8.80 (s, 1H), 8.27 (t, J = 5.7 Hz, 1H), 7.31 (d, J = 8.6 Hz, 1H), 6.72 (dd, J = 8.5, 2.5 Hz, 1H), 5.59 (d, J = 9.9 Hz, 2H), 5.17 (dd, J = 14.2, 10.1 Hz, 1H), 4.71 (d, J = 13.6 Hz, 1H), 4.61–4.46 (m, 1H), 3.79–3.71 (m, 2H), 3.42 (q, J = 6.3 Hz, 2H), 3.16–3.10 (m, 4H), 3.08 (q, J = 6.4 Hz, 2H), 2.12 (tt, J = 12.1, 3.6 Hz, 1H), 1.93–1.87 (m, 2H), 1.83 (d, J = 13.2 Hz, 2H), 1.47–1.41 (m, 2H), 1.40–1.34 (m, 2H), 1.22 (t, J = 7.3 Hz, 6H). ¹³C NMR (214 MHz, DMSO-d₆) δ 175.37, 164.04, 158.61, 152.99, 137.66, 131.43, 129.41, 127.72, 114.82, 112.43, 108.56, 100.56, 64.63, 49.87, 48.01, 46.57, 43.11, 33.53, 31.14, 28.03, 8.41. MS m/z Calcd for [M+H]⁺ (C₂₈H₃₉ClN₇O₃⁺): 556.27; found: 556.30; LC/MS: 98% purity.

(Z)-N-(trans-4-((2-(Diethylamino)ethyl)carbamoyl)cyclohexyl)-3⁴-methyl-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (43)

See general procedure B.

(Z)-N-(trans-4-((2-(Diethylamino)ethyl)carbamoyl)cyclohexyl)-3⁴-methoxy-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamide (44)

The title compound 44 (25.0 mg, 0.045 mmol, 73%) was prepared according to the general procedure C from trans-4-((Z)-3⁴-methoxy-4-oxa-2,9-diaza-1(4,2)-pyrimidina-3(1,3)-benzenacyclononaphan-6-ene-1⁵-carboxamido)cyclohexane-1-carboxylic acid (28.0 mg, 0.060 mmol) and N1,N1-diethylethane-1,2-diamine (14.0 mg, 0.12 mmol) as a white solid. ¹H NMR (850 MHz, DMSO-d₆) δ 11.84 (s, 1H), 9.33 (s, 1H), 8.78 (d, J = 7.4 Hz, 1H), 8.31–8.26 (m, 1H), 6.90 (d, J = 8.7 Hz, 1H), 6.69 (dd, J = 8.7, 2.5 Hz, 1H), 5.61 – 5.53 (m, 2H), 5.05 (dd, J = 14.3, 10.0 Hz, 1H), 4.60 (d, J = 14.3 Hz, 1H), 4.55 (ddd, J = 16.2, 9.3, 4.2 Hz, 1H), 3.79–3.75 (m, 2H), 3.75 (s, 3H), 3.42 (q, J = 6.2 Hz, 2H), 3.16–3.10 (m, 4H), 3.08 (q, J = 6.0 Hz, 2H), 2.13 (tt, J = 12.0, 3.5 Hz, 1H), 1.92–1.87 (m, 2H), 1.84 (d, J = 13.9 Hz, 2H), 1.48–1.41 (m, 2H), 1.41–1.35 (m, 2H), 1.22 (t, J = 7.2 Hz, 6H). ¹³C NMR (214 MHz, DMSO-d₆) δ 175.33, 173.20, 163.82, 157.50, 146.51, 145.12, 131.05, 130.64, 128.69, 124.20, 111.94, 111.43, 108.64, 100.59, 63.86, 55.65, 49.85, 48.06, 46.54, 43.10, 40.43, 33.50, 31.11, 28.02, 8.40. MS m/z Calcd for [M+H]⁺ (C₂₉H₄₂N₇O₃⁺): 552.32; found: 552.30; LC/MS: 98% purity.

3D Structures of Ligand Protein Complexes.

Several X-ray structures of MERTK kinase domain in complex with 4-amino-pyrimidine inhibitors (putatively binding similarly to 35 and 36) including a macrocyclic inhibitor UNC2541 (PDB ID: 4M3Q, 4MHA, 5K0X) were used as structural templates to infer the binding poses of 35 and 36 to MERTK. We assumed that 35 and 36 bind to AXL (5U6B), FLT3 (4XUF) and TYRO3 (AF-Q06418-F1) similarly to how they bind MERTK. Protein Preparation Wizard available through Maestro (release 2020-2; Schrödinger, LLC) was used to prepare the complexes for the energy minimization using the OPLS force field (DOI: 10.1021/ja9621760). In addition to the default settings, missing side chains and missing loops were added using Prime. To avoid unnatural interatomic clashes, restrained minimization with heavy atoms convergence at RMSD 0.3 Å was performed.

Cell Based Kinase Inhibition Assays

H4011 NSCLC cells were cultured in DMEM medium in the absence of serum for 2 hours and then treated with 43 or an equivalent volume of DMSO vehicle for an additional 2 hours. Cultures were then stimulated with 20% fetal bovine serum in the presence of 120 μM freshly prepared pervanadate for 4 min. Cell lysates were prepared in 50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% glycerol, and 1% Triton X-100, supplemented with protease inhibitors (Roche Molecular Biochemicals, #11836153001). MERTK and AXL proteins were immunoprecipitated with anti-MERTK (R&D Systems, #MAB8912) or anti-AXL (R&D Systems, #AF154) antibody and Protein G agarose beads (InVitrogen). Phospho-proteins were detected by immunoblot using an anti-phospho-MERTK antibody (Phopshosolutions, Inc) directed against a phospho-peptide that is shared by all three TAM kinases, then membranes were stripped, and total proteins were detected using anti-MERTK (Abcam, #ab52968) or anti-AXL (R&D Systems, #AF154) antibodies. Relative phosphorylated and total protein levels were determined by densitometry using Image J software and IC₅₀ values were calculated by non-linear regression.

Clonogenic Assay

H1975 NSCLC cells were cultured in 12-well dishes (1200 cells/well) in DMEM medium with 20% fetal bovine serum with 43 or an equivalent volume of DMSO vehicle for 7-8 days. Colonies were stained with 0.2% w/v crystal violet in 25% methanol and counted using a GelCount automated colony counter (Oxford Optronix).

ABBREVIATIONS USED

Boc₂O

di-tert-butyl decarbonate

t-BuOH

tert-butanol

HATU

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

DCM

dichloromethane

DIAD

diisopropyl azodicarboxylate

DIPEA

N,N-Diisopropylethylamine

MCE

microcapillary electrophoresis

MeCN

acetonitrile

NSCLC

non-small cell lung cancer

i-PrOH

isopropyl alcohol

SAR

structure-activity relationship

TBDPSCl

tert-butylchlorodiphenylsilane

TBTU

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate