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. 2022 Nov 20:10.1111/cbdd.14179. Online ahead of print. doi: 10.1111/cbdd.14179

Design, synthesis, and biological evaluation of novel ruxolitinib and baricitinib analogues for potential use against COVID‐19

Qin Lin 1, Jun Li 2, Yinping Wang 1, Jie Zang 3,✉
PMCID: PMC9878086  PMID: 36366971

Abstract

The coronavirus pandemic known as COVID‐19 caused by severe acute respiratory syndrome coronavirus 2, threatens public health worldwide. Approval of COVID‐19 vaccines and antiviral drugs have greatly reduced the severe cases and mortality rate. However, the continuous mutations of viruses are challenging the efficacies of vaccines and antiviral drugs. A drug repurposing campaign has identified two JAK1/2 inhibitors ruxolitinib and baricitinib as potential antiviral drugs. Ruxolitinib and baricitinib exert dual antiviral effect by modulation of inflammatory response via JAK1/2 and inhibition of viral entry via AAK1 and GAK. Inspired by this, in an effort to diversify chemical space, three analogues ((R)‐8, (S)‐8, and 9) of ruxolitinib and baricitinb were made using a scaffold hopping strategy. Compound 9 displayed potent and comparable potencies against AAK1, JAK1, and JAK2 compared to baricitinib. Notably, compound 9 showed better selectivity for AAK1, JAK1, and JAK2 over GAK. Besides, compound 9 displayed good druglikeness according to Lipinski's and Veber's rule. We thereby identified a potential lead compound 9, which might be used for the further development of anti‐coronaviral therapy.

Keywords: antiviral drugs, AP2‐associated protein kinase 1 (AAK1), baricitinib, coronavirus disease 2019 (COVID‐19), cyclin G‐associated kinase (GAK), Janus kinase (JAK), ruxolitinib

We developed a novel analogue of baricitinib 9, which showed comparable inhibitory activities against AAK1, JAK1, and JAK2 compared to baricitinib and better selectivity over GAK. Therefore, 9 has the potential to be used to combat COVID‐19.

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1. INTRODUCTION

The 2019 novel coronavirus (COVID‐19) pandemic has caused >371 million people infected and >5.7 million people died after 2‐years of spreading (Neamati, 2022). The development, production, and regulatory approval of various COVID‐19 vaccines have played an essential role in preventing viral spread and hospitalization (Zheng et al., 2022). Antiviral therapies including remdesivir (Rubin et al., 2020), a combination of remdesivir and baricitinib (Kalil et al., 2021), molnupiravir (Dyer, 2021), Paxlovid (a combination of nirmatrelvir and ritonavir) (Lamb, 2022), and monoclonal antibody bebtelovimab (Orders, 2022) have been either approved or granted an emergency use authorization (EUA) for the treatment of COVID‐19. However, continuous virus mutations are weakening the efficacy of vaccines and antiviral therapies. Therefore, there is still a great medical need for antiviral medicines against COVID‐19.

The replication of viruses is dependent on the host cellular machinery. A variety of host cellular proteins are involved in the progression of their life cycle, among which, cellular kinases play crucial roles (Figure 1) (Pillaiyar & Laufer, 2022). Coronavirus has been shown to enter cells through angiotensin‐converting enzyme 2 (ACE2)‐mediated endocytosis (Hoffmann et al., 2020). AP2‐associated protein kinase 1 (AAK1) and cyclin G‐associated kinase (GAK) are numb‐associated kinases, which are responsible for clathrin‐mediated viral endocytosis. Therefore, disruption of AAK1 and/or GAK is expected to block the viral entry (Figure 1) (Bekerman et al., 2017; Lu et al., 2020; Neveu et al., 2012, 2015). Cytokine storm is an aggressive inflammatory response event in which a large amount of pro‐inflammatory cytokines is rapidly released upon the virus infection. It has been shown that cytokine storm correlated directly with multi‐organ failure, unfavorable prognosis, and mortality. Controlling the cytokine storm in COVID‐19 patients is expected to improve survival rates and reduce mortality (Ragab et al., 2020). Janus kinase (JAK) is a family of intracellular, non‐receptor tyrosine kinases that phosphorylate signal transducers and activators of transcription proteins (STATs). The JAK/STAT pathway transduces cytokine‐mediated signals that are involved in cell proliferation, differentiation, and immunological responses (Vogelstein et al., 2013). An approach targeting JAK has been explored to regulate the COVID‐19 cytokine storm (Figure 1) (Chen et al., 2021; Convertino et al., 2020; Ingraham et al., 2020; Limen et al., 2022; Luo et al., 2020).

FIGURE 1.

FIGURE 1

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Potential drug targets involved in SARS‐CoV‐2 entry and cytokine storm for treatment of COVID‐19

Drug repurposing is a frequently used strategy to explore the new use of existing drugs. As approved drugs have shown a well‐established safety profile, drug repurposing can rapidly and efficiently identify safe candidates for various diseases (Kingsmore et al., 2020; Pushpakom et al., 2019). This strategy has also been utilized to identify therapeutic molecules against COVID‐19 both in industry and academia (Richardson et al., 2020; Singh et al., 2020; Yousefi et al., 2021; Zhou et al., 2020). Baricitinib, an approved JAK1/2 inhibitor for the treatment of rheumatoid arthritis displayed antiviral activity in primary human liver cells and primary human liver spheroids (Mullard, 2018; Stebbing et al., 2020; Stebbing et al., 2021). Plenty of clinical investigations of baricitinib for usage against SARS‐CoV‐2 infection are ongoing (Ely et al., 2022; Marconi et al., 2021). Moreover, FDA has granted a EUA to the combination of Baricitinib and remdesivir in hospitalized COVID‐19 patients needing respirators (Kalil et al., 2021). Ruxolitinib is an FDA‐approved JAK1/2 inhibitor for myelofibrosis and polycythemia vera (Mascarenhas & Hoffman, 2012; Raedler, 2015). Ruxolitinib showed in vitro and in vivo activity in suppressing cytokine levels (Quintas‐Cardama et al., 2010; Walker, 2008). Besides, ruxolitinib has displayed its clinical effectiveness in COVID‐19 patients (Cao et al., 2020; Capochiani et al., 2020; La Rosee et al., 2020). Besides the regulation of cytokine levels via modulation of the JAK–STAT pathway, ruxolitinib and baricitinib also exhibit antiviral activity by targeting viral endocytosis kinases such as AKA1 and GAK (Figure 2) (Stebbing, Phelan, et al., 2020).

FIGURE 2.

FIGURE 2

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(a) Drug repurposing campaign identified ruxolitinib and baricitinb as potential antiviral compounds by inhibiting AAK1, GAK, and JAK1/2. (b) Structures of JAK1/2 inhibitors ruxolitinib and baricitinib.

Ruxolitinib and Ruxolitinib showed inherent advantages as anti‐viral drug candidates owing to their dual anti‐viral mode via anti‐inflammatory effects and viral entry inhibition. Inspired by this, we designed and synthesized two analogues ((R)‐8 and (S)‐8) of ruxolitinib and one analogue (9) of baricitinib using a scaffold hopping strategy. The three analogues were then evaluated for their inhibitory activities against AAK1, JAK1, JAK2, and GAK. Ruxolitinib analogues (R)‐8 and (S)‐8 showed a dramatic decrease in potencies against all tested kinases compared to ruxolitinib. Baricitinib analogue 9 can retain the potencies against AAK1, JAK1, and JAK2, but displayed a remarkable selectivity over GAK compared to Baricitinib. Compound 9 might serve as a potential lead for the further development of anti‐coronaviral therapy.

2. EXPERIMENTAL SECTION

2.1. Chemistry

2.1.1. General procedures

All reagents were obtained from commercial sources and were used without further purification unless otherwise stated. Reactions were monitored using thin‐layer chromatography (TLC) and/or liquid chromatography–mass spectrometry (LC–MS). TLC was performed using aluminium precoated silica gel plates and visualized using ultraviolet light. LC–MS was performed on a Bruker Daltronics instrument running a gradient of increasing MeCN/water (5%–95%) containing 0.1% formic acid, at 1 ml/min on a 50 × 20 mm C18 reverse phase (RP) column. Normal phase (NP) flash column chromatography was carried out in Teledyne ISCO CombiFlash with SepaFlash® Silica Flash Column. Preparative high‐performance liquid chromatography (HPLC) was performed on an Agilent 1100 Infinity Series equipped with a UV detector and Welch Xtimate C18 100 × 40 mm × 3 μm column with mobile phase: [water (TFA)‐ACN]. Supercritical fluid chromatography (SFC) purification was performed on DAICEL CHIRALPAK AD (250 × 30 mm, 10 μm) column with mobile phase: [0.1% NH3.H2O‐EtOH]. The final compounds are >95% pure by LC–MS (UV) unless otherwise stated. 1H NMR spectra were recorded at 400 MHz and 13C NMR spectra were recorded at 100 MHz on a Bruker Advance 400 Fourier transform spectrometer. Chemical shifts are reported in ppm and are reported with reference to the residual solvent peak. Multiplicities are reported with coupling constants.

2.2. Procedures for preparing compounds (S)‐8, (R)‐8, and 9

4‐Chloro‐7‐((2‐(trimethylsilyl)ethoxy)methyl)‐7H‐pyrrolo[2,3‐d]pyrimidine (1) To a solution of NaH (5.08 g, 60% in mineral oil, 126.98 mmol) in THF (100 ml) was added a solution of 4‐chloro‐7H‐pyrrolo[2,3‐d]pyrimidine (15 g, 97.68 mmol) in THF (50 ml) dropwise at 0°C for 20 min. After addition, the mixture was stirred at 0°C for 20 min, and then 2‐(chloromethoxy)ethyl‐trimethyl‐silane (21.17 g, 22.47 ml, 126.98 mmol) in THF (30 ml) was added dropwise at 0°C. The resulting mixture was stirred at 0°C for 1 h. The reaction mixture was added to water (400 ml) slowly at 0 °C, and then extracted with EtOAc/PE (200 ml, EtOAc/PE = 7:3). The organic layers were washed with brine (150 ml × 2), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (0%–15% EtOAc/PE). The titled compound (23 g, 81.03 mmol, 83%) was collected as a colourless oil. LC–MS (ESI+): m/z calcd for C12H19ClN3OSi [M + H]+: 284.10; Observed: 284.1.

6‐(Trifluoromethyl)‐1‐(triisopropylsilyl)‐1H‐indole (2) To a solution of 6‐(trifluoromethyl)‐1H‐indole (10 g, 54.01 mmol) in THF (100 ml) was added dropwise n‐BuLi (2.5 M in hexane, 28.09 ml, 70.22 mmol) at −78°C. After addition, the mixture was stirred at the same temperature for 10 min, and then TIPSCl (13.54 g, 15.02 ml, 70.22 mmol) was added dropwise at −78°C. The resulting mixture was stirred at −78°C for 20 min. The mixture was added to aqueous NH4Cl (50 ml) at −78°C and water (50 ml) and extracted with EtOAc (60 ml × 3). The combined organic layers were washed with brine (60 ml), dried over Na2SO4, filtered, and concentrated under reduced pressure. The titled compound (22 g, 64.42 mmol, crude yield: 119%) was collected as an orange oil, which was used in the next step without further purification. LC–MS (ESI+): m/z calcd for C18H27F3NSi [M + H]+: 342.19; Observed: 342.2.

3‐Bromo‐6‐(trifluoromethyl)‐1‐(triisopropylsilyl)‐1H‐indole (3) To a solution of 2 (0.5 g, 1.46 mmol) in DMF (10 ml) was added NBS (0.28 g, 1.57 mmol) at 0°C. The mixture was stirred at 0°C for 1 h. The mixture was added with water (20 ml) and extracted with EtOAc (10 ml × 3). The combined organic layers were washed with brine (10 ml), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE containing 0.1% of Et3N). The titled compound (0.34 g, 0.81 mmol, 55%) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm 7.79 (s, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.58 (s, 1H), 7.45 (dd, J = 1.0, 8.3 Hz, 1H), 1.75 (spt, J = 7.5 Hz, 3H), 1.16 (d, J = 7.5 Hz, 18H).

3‐(4,4,5,5‐Tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐6‐(trifluoromethyl)‐1‐(triisopropylsilyl)‐1H‐indole (4) To a solution of 3 (0.2 g, 0.48 mmol) in THF (15 ml) was added dropwise n‐BuLi (2.5 M in hexane, 1 ml, 2.5 mmol) at −78°C under N2 atmosphere. After addition, the mixture was stirred at the same temperature for 10 min, and then 2‐isopropoxy‐4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolane (0.18 g, 194 μl, 0.95 mmol) in THF (1 ml) was added dropwise at −78°C. The resulting mixture was stirred at −78°C for 20 min. To the reaction mixture was added aqueous NH4Cl (15 ml) at −78°C and water (15 ml). The resulting mixture was extracted with EtOAc (10 ml × 3). The combined organic layers were washed with brine (10 ml), dried over Na2SO4, filtered, and concentrated under reduced pressure. The titled compound (0.22 g, 0.47 mmol, 98%) was obtained as an orange oil, which was used in the next step without further purification. 1H NMR (400 MHz, CD3OD) δ ppm 7.89 (s, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.53 (d, J = 3.2 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 1.40–1.38 (m, 12H), 1.17 (d, J = 2.6 Hz, 18H). LC–MS (ESI+): m/z calcd for C24H37BF3NO2Si [M + H]+: 468.27; Observed 468.3.

4‐(6‐(Trifluoromethyl)‐1‐(triisopropylsilyl)‐1H‐indol‐3‐yl)‐7‐((2‐(trimethylsilyl)ethoxy)methyl)‐7H‐pyrrolo[2,3‐d]pyrimidine (5) A mixture of 4 (1 g, 1.60 mmol), 1 (0.55 g, 1.94 mmol), Pd(PPh3)4 (0.056 g, 0.048 mmol), Na2CO3 (0.34 g, 3.21 mmol) in dioxane (10 ml) and H2O (2 ml) was degassed and purged with N2 for three times, and then the mixture was stirred at 100°C for 1 h under N2 atmosphere. The reaction mixture was added with water (10 ml) and extracted with EtOAc (5 ml × 3). The combined organic layers were washed with brine (5 ml), dried over Na2SO4, filtered, and concentrated under reduced pressure. The titled compound (1.6 g, 2.72 mmol, crude yield: 170%) was obtained as a yellow oil, which was used in the next step without further purification. LC–MS (ESI+): m/z calcd for C30H43F3N4OSi2 [M + H]+: 589.30; Observed 589.3.

4‐(6‐(Trifluoromethyl)‐1H‐indol‐3‐yl)‐7‐((2‐(trimethylsilyl)ethoxy)methyl)‐7H‐pyrrolo[2,3‐d]pyrimidine (6) To a solution of 5 (9.5 g, 6.61 mmol, 41% purity) in THF (90 ml) was added TBAF (1 M in THF, 14 ml, 14 mmol). The mixture was stirred at 25°C for 30 min. The reaction mixture was quenched with water (200 ml) and extracted with EtOAc (50 ml × 3). The combined organic layers were washed with brine (150 ml × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (30%–50% EtOAc/PE). The titled compound (2.4 g, 5.36 mmol, 81%) was obtained as a yellow oil. LC–MS (ESI+): m/z calcd for C21H23F3N4OSi [M + H]+: 433.17. Observed: 433.2.

3‐Cyclopentyl‐3‐(6‐(trifluoromethyl)‐3‐(7‐((2‐(trimethylsilyl)ethoxy)methyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐yl)‐1H‐indol‐1‐yl)propanenitrile (7a) To a solution of 6 (0.2 g, 0.46 mmol) in MeCN (4 ml) was added DBU (0.14 g, 139 μl, 0.92 mmol) and (E)‐3‐cyclopentylprop‐2‐enenitrile (0.17 g, 1.39 mmol). The mixture was stirred at 25°C for 18 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (1%–100% EtOAc/PE containing 10% MeOH). The titled compound (0.062 g, 0.11 mmol, 24%) was obtained as a yellow oil. LC–MS (ESI+): m/z calcd for C29H34F3N5OSi [M + H]+: 554.26; Observed: 554.2.

2‐(1‐(Ethylsulfonyl)‐3‐(6‐(trifluoromethyl)‐3‐(7‐((2‐(trimethylsilyl)ethoxy)methyl)‐7H‐pyrrolo[2,3‐d]pyrimidin‐4‐yl)‐1H‐indol‐1‐yl)azetidin‐3‐yl)acetonitrile (7b) To a solution of 6 (0.26 g, 0.60 mmol) in MeCN (5 ml) was added DBU (0.23 g, 227 μl, 1.50 mmol) and 2‐(1‐ethylsulfonylazetidin‐3‐ylidene)acetonitrile (0.22 g, 1.20 mmol). The mixture was stirred at 25°C for 18 h. The mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (0%–50% EtOAc/PE). The titled compound was obtained (0.4 g, 0.59 mmol, 98%) as a yellow oil. LC–MS (ESI+): m/z calcd for C28H33F3N6O3SSi [M + H]+ 619.21; Observed: 619.2.

(R)‐3‐(3‐(7H‐Pyrrolo[2,3‐d]pyrimidin‐4‐yl)‐6‐(trifluoromethyl)‐1H‐indol‐1‐yl)‐3‐cyclopentylpropanenitrile (R)‐8 and (S)‐3‐(3‐(7H‐Pyrrolo[2,3‐d]pyrimidin‐4‐yl)‐6‐(trifluoromethyl)‐1H‐indol‐1‐yl)‐3‐cyclopentylpropanenitrile (S)‐8 To a solution of 7a (0.12 g, 0.22 mmol) in MeOH (4 ml) was added TFA (4.00 ml, 54.02 mmol). The mixture was stirred at 25°C for 1 h. The mixture was concentrated under reduced pressure, then to the dried residue was added DCM (4 ml) and ammonium hydroxide solution (2.73 g, 33% NH3 in H2O, 3 ml, 25.71 mmol), which was stirred at 25°C for 30 min. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep‐HPLC (26%–56% buffer B/buffer A). The titled racemic mixture (0.084 g, 0.20 mmol, 91%) was obtained as a white solid. 1H NMR (400 MHz, DMSO‐d 6) δ ppm 12.13 (br s, 1H), 8.96 (d, J = 8.3 Hz, 1H), 8.82 (s, 1H), 8.74 (s, 1H), 8.29 (s, 1H), 7.62 (d, J = 3.6 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.13 (d, J = 3.6 Hz, 1H), 5.17–5.04 (m, 1H), 3.61 (dd, J = 9.6, 17.3 Hz, 1H), 3.31–3.24 (m, 1H), 2.78–2.63 (m, 1H), 1.97–1.81 (m, 1H), 1.79–1.66 (m, 1H), 1.63–1.38 (m, 4H), 1.27–1.20 (m, 1H), 1.08–0.94 (m, 1H). 13C NMR (100 MHz, DMSO‐d 6) δ ppm 153.10, 152.34, 151.31, 137.01, 128.89, 127.11, 126.86, 124.40, 124.27, 123.82, 123.51, 119.00, 117.61, 115.25, 113.45, 108.71, 100.99, 57.14, 45.18, 29.95, 25.48, 24.84, 23.02. SFC analysis showed two peaks: (AD_ETOH_DEA_5_40_4Ml_4MIN_5CM): Peak1 RT = 1.077 min, Peak2 RT = 1.164 min. The white solid was further separated by SFC to give (R)‐8 (0.045 g, 0.11 mmol, 50%, SFC RT = 1.077 min) as a white solid. LC–MS (ESI+): m/z calcd for C23H21F3N5 [M + H]+: 424.17; Observed: 424.1. And to give (S)‐8 (0.035 g, 0.083 mmol, 38%, SFC RT = 1.164 min) as white solid. LC–MS (ESI+): m/z calcd for C23H21F3N5 [M + H]+: 424.17; Observed: 424.1.

2‐(3‐(3‐(7H‐Pyrrolo[2,3‐d]pyrimidin‐4‐yl)‐6‐(trifluoromethyl)‐1H‐indol‐1‐yl)‐1‐(ethylsulfonyl)azetidin‐3‐yl)acetonitrile (9) To a solution of 7b (0.38 g, 0.61 mmol) in DCM (10 ml) was added TFA (1.81 ml, 24.44 mmol). The mixture was stirred at 25°C for 1 h. The mixture was concentrated under reduced pressure. The crude was dissolved in MeOH (4 ml) and ammonium hydroxide solution (8.23 g, 33% NH3 in H2O, 9.05 ml, 77.53 mmol) was added. The resulting solution was stirred at 25°C for 1 h. The mixture was filtered, and washed with MeOH (5 ml) and MeCN (5 ml) to give a crude product. The crude product was purified by prep‐HPLC. The titled compound was obtained (0.18 g, 0.37 mmol, 60%) as a white solid. 1H NMR (400 MHz, DMSO‐d 6 ) δ ppm 12.15 (br s, 1H), 8.97 (d, J = 8.5 Hz, 1H), 8.84 (s, 1H), 8.52 (s, 1H), 7.74 (s, 1H), 7.61 (br d, J = 9.8 Hz, 2H), 7.14 (br s, 1H), 4.78 (br d, J = 8.8 Hz, 2H), 4.56 (br d, J = 8.8 Hz, 2H), 3.69 (s, 2H), 3.25 (q, J = 7.1 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO‐d 6) δ ppm 152.68, 152.37, 151.30, 133.84, 132.30, 130.25, 126.98, 126.78, 124.71, 124.19, 124.07, 123.87, 123.56, 118.38, 118.34, 117.46, 115.00, 113.84, 109.12, 109.09, 101.13, 58.97, 53.60, 43.14, 27.31, 7.75. LC–MS (ESI+): m/z calcd for C22H19F3N6O2S [M + H]+: 489.13; Observed: 489.1.

2.3. Kinase inhibition assay

The ADP‐Glo™ Kinase Assay measures ADP formed from a kinase reaction. The ADP is converted into ATP, which is used to generate light in a luciferase reaction. The luminescence generated correlates with kinase activity. In brief, compounds in various concentrations are incubated with kinases (AAK1, JAK1, JAK2, and GAK) for 10 min, followed by the incubation with ATP/substrate mix for 60–180 min. ADP‐Glo™ Reagent is added to stop the kinase reaction (40 min). Kinase Detection Reagent is added to convert ADP to ATP and introduce luciferase and luciferin to detect ATP (40 min).

2.4. Molecular docking studies

Molecular docking of compound 9 with AAK1, JAK1, JAK2, and GAK was performed in the Glide module of Schrödinger (version 13.0.137). The protein structures of AAK1 (PDB code: 7LVI), JAK1 (PDB code: 6RSD), JAK2 (PDB code: 6WTO), and GAK (PDB code: 5Y7Z) were obtained from the Protein Data Bank database. All proteins were initially prepared in PyMOL (version 2.5.2) and exported as monomers. In the phase of protein preparation, all proteins underwent the process of aligning bond orders, adding hydrogens, and removing water. Minimizing restraint was carried out in the force field of OPLS3. The docking site for each protein was defined as centroid of co‐crystallized ligand. All possible molecular states were generated at the target pH 7.0 ± 2.0 in the force field of OPLS3 in the LigPrep module. Ligand docking and scoring were performed using Glide with default settings and with the SP (standard precision) scoring function. PyMOL was applied for analyses and visualization of docking poses.

3. RESULTS AND DISCUSSION

3.1. Rational design of novel ruxolitinib and baricitinib analogues

The co‐crystal structure of ruxolitinib with JAK2 revealed that ruxolitinib was held deep inside the ATP site, anchored through H‐bonding interactions between the pyrrolopyrimidine moiety and backbone of Glu930 and Leu932 of the hinge region. Moreover, van der Waals (vdW) hydrophobic interactions were formed between ruxolitinib and P‐loop as well as the DFG motif (Figure 3a). Baricitinib adopts a similar binding pose to ruxolitinib (Figure 3b). The common core shared by ruxolitinib and baricitinib has a high overlap in the binding site, while the 1‐(ethylsulfonyl)azetidine group interacts with the P‐loop site in a similar way as cycloheptyl group of ruxolitinib (Figure 3c).

FIGURE 3.

FIGURE 3

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X‐ray crystal structure of JAK2 in complex with JAK2 inhibitors. (a) X‐ray crystal structure of JAK2 in complex with ruxolitinib (green carbon atoms, PDB code 6WTN). (b) X‐ray crystal structure of JAK2 in complex with baricitinib (salmon carbon atoms, PDB code 6WTO) (c) superposition of binding pose of ruxolitinib (green carbon atoms) and baricitinib (salmon carbon atoms) with JAK2. Residues in JAK2 hinge area forming a hydrogen bond with JAK2 inhibitors are shown as stickers with cyan carbon atoms. Hydrogen bonds are shown as yellow dotted lines.

As the N2 of the central pyrazole in ruxolitinib and baricitinb is not involved in any specific interactions and the N2 to C3 region of central pyrazole protrudes out of the active site. The N2 of pyrazole was removed, and a phenyl ring was fused to the original N2 to C3 region forming indole to replace the original pyrazole. A trifluoromethyl group was introduced at C6 of the indole ring to modulate physicochemical properties to improve metabolic stability and enhance membrane permeation. One baricitinb analogue (9) and both R and S‐enantiomers of ruxolitinib analogues ((R)‐8 and (S)‐8) were prepared to explore the influence of chirality on the kinase inhibitory activities and selectivity (Figure 4).

FIGURE 4.

FIGURE 4

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Design of novel ruxolitinib and baricitinib analogues using scaffold hopping strategy

3.2. Chemistry

The synthesis of ruxotinib analogues ((R)‐8 and (S)‐8) started from commercially available 6‐(trifluoromethyl)‐1H‐indole, of which the indole NH group was protected with TIPS in the presence of TIPSCl and n‐BuLi at −78°C to afford intermediate 2. Bromination of 2 with NBS at C3 yielded intermediate 3, which was converted into boronate intermediate 4 in the presence of n‐BuLi and 2‐isopropoxy‐4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolane at −78°C. Trimethylsilylethoxymethyl (SEM) protected intermediate 1, which was prepared from commercially available 4‐chloro‐7H‐pyrrolo[2,3‐d]pyrimidine, coupled with boronate intermediate 4 using Pd(PPh3)4 as catalyst afforded intermediate 5. Deprotection of TIPS group from 5 with TBAF afforded common intermediate 6. Michael addition of 6 with (E)‐3‐cyclopentylprop‐2‐enenitrile in the presence of DBU afforded intermediate 7a, which was converted into desired racemic product 8 by TFA‐mediated SEM deprotection. Chiral separation of 8 by chiral SFC afforded R‐enantiomer (R)‐8 and S‐enantiomer (S)‐8. Following the same procedure of Michael addition and SEM deprotection, common intermediate 6 can be converted into desired product 9 (Scheme 1).

SCHEME 1.

SCHEME 1

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Synthesis of ruxolitinib analogues ((R)‐8 and (S)‐8) and baricitinib analogue (9) a

a Reagents and Conditions: (a) NaH, THF, 0 °C, 20 min, then 2‐(chloromethoxy)ethyl‐trimethyl‐silane, 1 h, 81%; (b) triisopropylsilyl chloride, n‐BuLi, THF, −78°C, 30 min, 119% (crude); (c) NBS, DMF, 0°C, 1 h, 55%; (d) 2‐isopropoxy‐4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolane, n‐BuLi, THF, −78°C, 30 min, 98%; (e) Pd(PPh3)4, Na2CO3, dioxane, water, 100°C, 1 h, 170% (crude); (f) TBAF, THF, 28°C, 30 min, 81%; (g) (E)‐3‐cyclopentylprop‐2‐enenitrile for 7a, or 2‐(1‐ethylsulfonylazetidin‐3‐ylidene)acetonitrile for 7b, DBU, MeCN, 25°C, 18 h, 24% for 7a, 98% for 7b; (h) TFA, DCM, 25°C, 1 h, then DCM, NH3.H2O, 25°C, 1 h, 50% for (R)‐8, 38% for (S)‐8 and 60% for 9.

3.3. In vitro kinase inhibition

ADP‐Glo™, (Zegzouti et al., 2009) a bioluminescent‐based enzyme assay, was used to determine the inhibitory activity of target compounds towards AAK1, JAK1, JAK2, and GAK. The results are shown in Table 1. Replacement of pyrazole ring in ruxolitinib with trifluoromethyl substituted indole ring gave R‐enantiomer (R)‐8, which shared the same stereochemistry as ruxolitinib. To our surprise, R‐enantiomer (R)‐8 showed slightly decreased (3–6‐fold) potency for AAK1 and GAK but dramatically decreased (59–88‐fold) potency for JAK1/2 compared to ruxolitinib. Similar to (R)‐8, the S‐enantiomer (S)‐8 was 87–144‐fold less potent than ruxolitinib for JAK1/2. Compared to ruxolitinib, S‐enantiomer (S)‐8 showed comparable potency for GAK, but it displayed more than 22‐fold lower potency for AAK1. To conclude, the substitution of pyrazole ring in ruxolitinib with trifluoromethyl substituted indole ring was not tolerated towards JAK1/2 regardless of R or S configuration. However, it was tolerated for R‐enantiomer (R)‐8 towards AAK1 and GAK and for S‐enantiomer (S)‐8 towards GAK. Replacing the pyrazole ring in baricitinib with trifluoromethyl substituted indole ring gave 9 with comparable potency for AAK1, JAK1, and JAK2. This indicates trifluoromethyl substituted indole ring may operate as a viable replacement for the pyrazole ring in baricitinib for the inhibition of AAK1, JAK1, and JAK2. However, 9 showed much weaker inhibition of GAK (IC50 > 10 μM) than baricitinib (IC50 = 1.35 μM), which suggests the replacement of pyrazole ring with trifluoromethyl substituted indole ring in baricitinib might cause unfavorable interactions towards GAK. Compared to baricitinib, 9 showed improved selectivity of AAK1, JAK1, and JAK2 over GAK. However, the superior selectivity of 9 is unlikely to be beneficial for the anti‐coronavirus effect as inhibition of GAK is advantageous and can lead to the blockage of viral entry as mentioned above. On the other hand, the improved selectivity of 9 might be useful for other indications such as autoimmune diseases, in which inhibition of GAK is not required. The increased selectivity can reduce the risk of off‐target caused toxicity.

TABLE 1.

Biochemical activity of baricitinib, ruxolitinib, and their analogues ((R)‐8, (S)‐8, and 9) a

Cpd. Structures IC50 (nM)
AAK1 JAK1 JAK2 GAK
(R)‐8 graphic file with name CBDD-9999-0-g009.jpg 2698 39.30 38.63 5003
(S)‐8 graphic file with name CBDD-9999-0-g004.jpg >10,000 96.31 38.49 2295
Ruxolitinib graphic file with name CBDD-9999-0-g005.jpg 457 0.67 0.44 1730
9 graphic file with name CBDD-9999-0-g011.jpg 161 2.49 0.95 >10,000
Baricitinib graphic file with name CBDD-9999-0-g012.jpg 56 0.60 0.43 1352
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a

Assays were performed in duplicate and IC50 values were presented as mean values. The SEM values are <20% of the means.

3.4. Molecular docking and analysis of binding interaction of compound 9

To gain deeper insights into the molecular basis of the inhibitory activity of the best analogue 9. We committed molecular docking study of compound 9 with AAK1, JAK1, JAK2, and GSK. Figure 5a shows the binding mode of 9 in the ATP pocket of AAK1. Compound 9 forms dual hydrogen bonds between its pyrrolopyrimidine moiety and the backbone of Cys129 of the hinge region. 1‐(ethylsulfonyl)azetidine group interacts with the P‐loop site and the cyanide group formed a hydrogen bond with Asp194. The trifluoromethyl‐substituted indole ring sits in the middle of the ATP site. Figure 5b shows the binding mode of 9 in the ATP pocket of JAK1. Dual hydrogen bonds are formed between pyrrolopyrimidine moiety and the backbone of Glu957 and Leu959 in the hinge region. Moreover, a hydrogen bond is formed between Glu883 and one of sulfonamide oxygen in compound 9. The newly fused trifluoromethyl phenyl is projected towards the solvent region. Figure 5c shows the binding mode of 9 in the ATP pocket of JAK2. Compound 9 is bound to JAK2 in a similar way to baricitinib (Figure 3b). The only difference lies in that the newly fused trifluoromethyl phenyl protrudes out of the active site out the active site. The good binding mode between 9 and AAK1, JAK1, and JAK2 reasonably explained the comparable inhibitory activities of 9 against AAK1, JAK1, and JAK2 compared to baricitinib. Figure 5d shows the binding mode of 9 in the ATP pocket of GAK. Only one hydrogen bond was formed between 9 and the hinge region (Cys126). Moreover, the introduction of a fused trifluoromethyl phenyl group causes a steric clash with Lys69 and Cys190 of GAK. This might explain the dramatic decrease of inhibitory activity of 9 against GAK compared to baricitinib.

FIGURE 5.

FIGURE 5

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Molecular docking study of 9 with AAK1, JAK1, JAK2, and GAK. (a) Binding mode of 9 in the active site of AAK1. (b) Binding mode of 9 in the active site of JAK1. (c) Binding mode of 9 in the active site of JAK2. (d) Binding mode of 9 in the active site of GAK. Compound 9 is shown as a stick with magenta carbon atoms. Residues forming interaction with 9 are shown as stickers with cyan carbon atoms. Hydrogen bonds are shown as yellow dotted lines.

3.5. Prediction of druglikeness

Lipinski's rule of five, formulated by Christopher A. Lipinski, is a rule of thumb to determine if a chemical compound has desired physicochemical properties that would make it a likely orally active drug in humans. In general, the criteria includes no more than 5 hydrogen bond donors (HBD), no more than 10 hydrogen bond acceptors (HBA), no more than 500 daltons of molecular weight (MW), and no more than 5 of octanol–water partition coefficient (log P) (Lipinski et al., 2001). Veber's Rule, introduced by Daniel F. Veber, emphasized only two criteria for predicting orally active drugs, which includes no more than 10 rotatable bonds and no greater than 140 Å2 of polar surface area (Veber et al., 2002). The physiochemical properties of ruxolitinib, baricitinib, and their analogues in respect of Lipinski's and Veber's rules (Table 2) were carefully checked to evaluate their druglikeness. Ruxolitinib and baricitinib, as approved drugs, as well as compound 9 comply with both Lipinski's and Veber's rules. However, the cLogP value (5.25) of (R)‐8 and (S)‐8 violate Lipinski's rule about LogP, which should be no more than 5.

TABLE 2.

Physiochemical properties of baricitinib, ruxolitinib, and their analogues ((R)‐8, (S)‐8, and 9)

Cpd. cLogP a MW a HBD a HBA a Rotatable bonds tPSA a Number of violations of Lipinski's or Veber's rule
(R)‐8 5.25 423.44 1 3 5 70.29 1
(S)‐8 5.25 423.44 1 3 5 70.29 1
Ruxolitinib 2.48 306.37 1 4 4 83.18 0
9 2.58 488.49 1 5 5 107.67 0
Baricitinib −0.19 371.42 1 6 4 120.56 0
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a

Obtained from Marvinsketch 21.14.

4. CONCLUSION

In the present work, based on the co‐crystal structures of JAK2 in complex with ruxolitinib and baricitinib, three analogues of ruxolitinib and baricitinib were rationally designed using a scaffold hopping strategy. Ruxolitinib analogues (R)‐8 and (S)‐8 showed a dramatic decrease in potencies towards JAK1 and JAK2 and a mild decrease in potencies towards AAK1 and GAK. To our delight, baricitinib analogue 9 showed potent and comparable activities as baricitinib against AAK1, JAK1, and JAK2. Moreover, 9 showed better selectivity of AAK1, JAK1, and JAK2 over GAK. In summary, the merit of our research is the identification of a novel baricitinib analogue 9, which showed potent activity against AAK1, JAK1, and JAK2 as well as good selectivity over GAK. Moreover, compound 9 complies Lipinski's and Veber's rules. These findings warrant further investigation of 9 in more advanced preclinical studies to fully understand its potential as an effective antiviral therapeutic.

CONFLICT OF INTEREST

The authors declare no competing financial interest.

Lin, Q. , Li, J. , Wang, Y. , & Zang, J. (2022). Design, synthesis, and biological evaluation of novel ruxolitinib and baricitinib analogues for potential use against COVID‐19. Chemical Biology & Drug Design, 00, 1–12. 10.1111/cbdd.14179

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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