Experimental and Density Functional Theory Characteristics of Ibrutinib, a Bruton’s Kinase Inhibitor Approved for Leukemia Treatment

Ibrutinib, a Bruton’s tyrosine kinase that plays an essential role in the B-cell development and cancer cells, has been recently approved to treat chronic, lymphocytic, and other types of leukemia.&is study focused on investigating ibrutinib by its electronic transitions, vibrational frequencies, and electrospray mass spectra. &e experimental peaks for electronic spectrum were found at 248.0 and 281.0 nm, whereas the ]C� 0 stretching frequency was found at 1652.4 and 1639.19 cm.&ese experimental properties were compared with the corresponding theoretical calculations in which density functional theory was applied. &e optimized structure was obtained with the calculations using a hybrid function (B3LYP) and high-level basis sets [6-311G++(d,p)]. Most of the calculated vibrational frequencies showed a relatively good agreement with the experimental ones.&e electronic transitions of ibrutinib calculated using time-dependent DFTmethod were performed at two different solvation methods: PCM and SMD. &e mass spectrum of ibrutinib, its fragments, and its isotopic pattern agreed well with the expected spectra.


Introduction
e current knowledge in biology and chemistry improved our understanding of cancer treatments. For example, most cancer types express some specific dysregulated molecules called tumor-specific antigens that will play an important role in the tumor development process. Bruton's tyrosine kinase is an enzyme that is important in B-cell development [1][2][3]. Inhibition of this enzyme is found to cure various types of diseases such as B-cell lineage cancerous, including mainly different types of leukemias common in adults. Although chemotherapy kills the BTK-lineage cells, it also kills other vital cells in the body. However, the recent discovery of molecules specific to BTK enzyme inhibition energizes the kinase-inhibition field research [4][5][6][7][8][9][10][11][12][13][14][15][16]. e covalent BTK inhibitors attract great attention because it irreversibly forms a covalent bond with cysteine base (noncatalytic Cys481) located at the edge of the ATPbinding BTK which brings a transient inhibition, higher efficiency, and better specificity and duration of drug action [17]. Several studies had been conducted on finding these irreversible inhibitors, which carry based on the structural bioinformatics approach [5,6]. It is believed that kinase inhibitors dominate more than 30% of the drug-discovery industry [18,19].
Ibrutinib (or PCI-32765) is the first BTK covalent irreversible inhibitor approved by several health agencies to treat leukemia types. After extensive laboratory studies and clinical experiments, the approval was decided, providing promising treatment results with IC 50 � 0.5 nM [20][21][22][23][24][25]. Although some chemical properties of ibrutinib had been reported, most importantly, the crystal structure was solvated in different media [26]. However, many other characteristics are yet to be reported, such as structural, vibrational, and electronic properties. e purpose of this work is to investigate ibrutinib by its UV-Vis, IR, and ESI-MS characteristics. ese experimental properties were compared with the corresponding theoretical calculations using the DFT at the B3LYP level of theory and 6-311G++(d,p) basis sets.

Materials and Reagents.
Ibrutinib was purchased from BLD Pharmatech Ltd. with >98% purity (via MolPort.com). All other reagents and solvents were of spectroscopic grade and bought from Sigma Aldrich.

Experimental
Methods. Ibrutinib's UV spectrum was measured using Perkin Elmer Lamda 35 UV-Vis spectrophotometer at room temperature. Ibrutinib was dissolved and diluted with methanol to a final concentration of 1x10 −5 M. e absorption spectrum was recorded at the range of 200-800 nm with 0.5 nm slit size. FT-IR spectrum was obtained from compact Alpha FT-IR spectrometer (Bruker, Germany). e device has got multireflection ZnSe as attenuated total reflectance (ATR) and deuterated triglycine sulfate (DTGS) as detector.
e accuracy of the instrument reaches as low as 0.01 cm −1 for wavenumber and 0.1% for transmittance. Ibrutinib was mixed and ground with KBr to make a homogeneous mixture diffused by compression to get a transparent disk. Ibrutinib's mass spectra were measured using positive polarization electrospray ionization coupled with Q-TOF II Bruker high-resolution mass spectrometry. e mass spectrum of different concentrations of ibrutinib in 95% V/V methanol : formic acid solution (the formic acid solution is 5%FA : 95%H2O) was measured using ESI-MS in the range of 10 nM and 1.0 µM. e electrospray was set at a flow rate of 10 uL/min, the capillary tip is set to be at 6000 V voltage, and the temperature is set at 200 C. e parameters such as collision cell RF (400.0 VPP), the reflector (1700 V), the transfer time (28.0 μs), and prepulse storage time (15.0 μs) were found critical in obtaining the best sensitivity measurements for ibrutinib. e tandem mass spectrum (MS 2 ) was determined at a collision energy of 25 J.

Computational Methods.
e structural properties of ibrutinib were determined using the density functional theory (DFT) with a hybrid function of B3LYP level of theory and a high level of basis sets with two sets of diffuse and polarization functions [6-311G++(d,p)]. e calculations were carried out using Gaussian 09 software [27] to get the most optimized geometry with the lowest energy. e final optimized structure ( Figure 1) is then confirmed to have all real vibrational states at the same theory level (no negative vibrational frequency). e time-dependent DFT (TDDFT) calculations were used to determine ibrutinib's optimized structures of the excited states in methanol utilizing two main solvation packages. Polarizable continuum model (PCM) and solvation model based on density (SMD) are widely used in literature as solvation models and will be our choice of TDDFT calculations of electronic transitions. Figure 2 shows the UV-Vis absorption spectrum of ibrutinib. e absorption mainly occurs between 200 nm and 340 nm. Two main peaks that can be recognized in the spectrum are at 248.0 nm with a molar absorptivity of 22889 L/mol.cm and at 281.0 nm with a molar absorptivity of 16004 L/mol.cm. From the relatively high molar absorptivity, one can conclude that these absorptions are allowed spectroscopically.

Experimental FT-IR Spectrum.
e FT-IR spectrum of ibrutinib is depicted in Figure 3. e spectrum shows several noticeable peaks. N-H peaks appeared at 3470.06 cm −1 and 3436.95 cm −1 . Aromatic and aliphatic C-H peaks are found between 3063.37 and 2885.62 cm −1 . Strong peaks at 1652.40 cm −1 and 1639.19 cm −1 can be assigned for the C�O stretching frequency. e series of other peaks from 1613.07 to 1520 cm −1 can be assigned for C�C and C�N stretching frequencies. Bending frequencies of HCN and HCH in addition to υCH, υCC, υOC, and υNC have their peaks between 1483 and 600 cm −1 . e strong and obvious peaks are presented in Table 1 with the help of the assignments from the theoretical calculations.

ESI-MS Spectrum.
e positive mode ESI spectrum of 1 µM ibrutinib in methanol solution is shown in Figure 4(a) with the principle peak at m/z � 441.2032. is peak corresponds to the protonated compound [ibrutinib + H] + as expected from the methanol electrospray ionization. e peak accuracy is comparable with the estimated one (441.20390) using the high mass spectrometry. e compound can be detected as low as 10 nM with [ibrutinib + H] + is the major peak having S/N ∼ 10. Figure 5 shows the tandem mass spectrum of ibrutinib in which two main peaks are found at m/z � 304.1178 and m/z � 138.1123.

Structural Optimization.
e structure of ibrutinib was optimized using a high level of DFT theory with hybrid functional B3LYP and 6-311G++(d,p) as basis sets and is shown in Figure 1. e minimum energy of the optimized molecule was computed as −1446.4520836 HF (−39359.9847812 eV). e molecule's point group was decided as C1 with a dipole moment of 4.237 Debye projected from the carbon in the center of the molecule (C8) in-plane tilted toward the oxygen (O20). e bond lengths, bond angles, and torsion angles of the optimized structure are listed in Table S1 (supplementary materials). N-H bonds were found around 1.01 Ǻ, and the C-bond lengths are between 1.08 Ǻ and 1.10 Ǻ as expected. All carbon angles involve O or N were calculated between 104.7 and 108.6. C8-C9-C14 is measured around 140.4, which is relatively large due to the ring fusing. e torsion angles C8-C9-C14-N13 and C8-C9-C14-N15 torsions are almost linear (177.7°and -3.0°), indicating that the fused moiety is planar that agrees with the aromaticity fact. e difference in the bond length between C4-H35 and C4-H34 (1.10 and 1.09 Ǻ) might be evidence of the intramolecular C2-O1···H35 hydrogen bonding.

Vibrational Frequencies.
e vibrational frequencies were calculated using the DFT method at the same level of theory and level of basis sets. e results showed that ibrutinib (57 atoms) has a C1 point group with 165 degrees of freedom that produces 165 IR modes. e main assignments of these IR frequencies are reported in Table 1.
e software Veda 4 [28] was used to extract and assign the infrared modes after scaling it with 0.97 as recommended by Andersson and Uvdal [29].

Electronic Transition.
e time-dependent DFT (TDDFT) calculations are determined for all optimized structures. PCM and SMD calculation methods are suggested to estimate the electronic transitions. ese calculations computed all molecular orbitals of each molecule, suggesting the possible and most probable electronic transitions. UV-Vis spectrum was constructed based on the energy gaps between states for each structure from these calculations. All calculations were performed using DFT coupled with B3LYP and 6-311G++(d,p) as basis sets. e final results were extracted and visualized using GaussSum [30]. Both PCM and SMD methods predicted almost identical absorption spectra with three peaks. e first peak at 310.13 nm using PCM, 309.28 using the SMD method appeared to be the major one for the PCM method with an oscillator strength of 0.566. e second peak at 399.03 nm (for PCM and 396.26 nm for SMD) is the SMD method's major peak. e last peak is found at 325.98 nm (for PCM and 326.61 nm for SMD) is considered as a minor peak with an oscillator strength of 0.03-0.04. All results are tabulated in Table 2.

Discussion
e main functional groups that can be easily distinguished by IR are the N-H and C�O bonds. e υNH 2 stretching frequencies appeared experimentally at 3470.05 and 3436.95 cm −1 , while the calculations revealed them at 3600.64 and 3478.42 cm −1 . e main reason for a significant shift in the IR in organic compounds is probably due to the hydrogen bonding [31]. In ibrutinib, there might be either intramolecular forces (56H of NH 2 interacting with 13N of pyrimidine) or intermolecular forces of a possible dimerization.
is may encounter the difference in the υNH 2 stretching frequency between the theory and the experimental.
Also, υ(O�C) stretching frequencies appeared experimentally at 1652.4 and 1639.19 cm −1 , while the DFT estimated them at 1653.85 and 1601.47 cm −1 . e two υ(O�C) stretching frequencies differ theoretically at 1.45 and 37.7 cm −1 from the experimental. is change is considered   within the agreement between the theory and the experiment encountering the bulkiness of the molecule. Kara et al. concluded that a 20 cm −1 difference between B3LYP and experimental for C�O for a relatively medium-sized molecule is a good agreement [32]. Atac et al. suggested that the C�O difference between DFT and experimental can go up to 35 cm −1 differences [33]. At the same time, Do et al. found the difference between the theory and the experimental goes up to 58 cm −1 for a large molecule like C 60 fullerenes (even though the molecule is highly symmetric and has no functional group other than C�C) [34].
Other functional groups such as C�C and C�N were laid in 1400-1600 cm −1 for both experimental and theoretical calculations. e υCH, υCC, υNC, υOC, βHCH, and βHCN are interfering in the range of 1483-600 cm −1 . In general, most of the peaks from the DFT calculations with the high   level of theory and basis sets agreed relatively well with the experimental counterpart peaks, as shown in Table 1 and Figure 3.
On the other hand, the electronic spectrum of ibrutinib showed two peaks at 248.0 and 281.0 nm. In contrast, the calculations revealed two significant peaks at around 310 nm and 399 nm (see Figure 2). ese transitions are n-π * transitions which are very sensitive to the environment, such as solvent and forces between the functional groups especially with ibrutinib that has many functional groups. e peak at 310.13 (for PCM) nm 309.28 (for SMD) was assigned as HOMO to LUMO +2 electronic transition for both methods. e second peak at 399.03 nm (for PCM and 396.26 nm for SMD) was assigned as HOMO to LUMO with 98% contribution. e last peak found at 325.98 nm (for PCM and 326.61 nm for SMD) is considered a minor peak for the HOMO to LUMO +1 transition (see Table 2). Both methods seem to fail to predict the correct electronic spectra based on the experimental results shown in Figure 2. Although there is a significant difference between the    [35]. On the other hand, the difference in the electronic spectrum, using the best method (B3LYP) for medium-sized molecules (1-naphthol), is estimated as 26 nm (0.29 eV) [36]. One can expect that the existence of many functional groups and the bulkiness of the molecule could result in a greater difference between the theory and experiment, giving the fact that the main transition which is n-π * transition is very sensitive to the environment, such as solvent and forces between the functional groups. e precision of the peak [ibrutinib + H] + in mass spectrometry reached better precision than 2 ppm, which is expected from Bruker QTOF II mass spectrometry. e isotopic pattern of 1 μM ibrutinib is shown in Figure 4(b) with a resolution of 95000 and S/N is larger than 10000. e isotope intensities and values agreed relatively well with the estimated ones obtained from ChemDraw ® software. e tandem mass spectrum of ibrutinib ( Figure 5) shows two main fragments at m/z � 304.1178 and m/z � 138.1123 formed from the breaking of the N-C bond between the pyrazole and the piperidine, which agreed with the previous studies of LC-MS of ibrutinib [37][38][39].

Conclusion
is study is the first to investigate the UV-Vis and infrared spectra for ibrutinib. High resolution with isotopic patterned mass spectrum is also reported with a direct infusion of ESI-MS from methanol solution. DFT with a high level of theory, B3LYP, and basis sets, 6-311G++(d,p), is used to determine the optimized geometry of ibrutinib and compare the electronic transitions and vibrational frequencies.

Data Availability
Data are available on request by contacting the author aeyesmaeel@kau.edu.sa or pchem.ismail@gmail.com.

Conflicts of Interest e author declares no conflicts of interest.
Acknowledgments is project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant number G-384-662-1439. e author, therefore, acknowledges with thanks DSR for technical and financial support. Table S1: bond lengths, bond angles, and torsion angles of ibrutinib determined for the optimized molecule by density functional theory study. (Supplementary Materials)