Gadolinium Complex of 1,4,7,10-Tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) Conjugate of [(p-aniline benzothiazole)methyl]pyridine as a Tumor-Targeting MRI Contrast Agent

Abstract

The synthesis of a DO3A conjugate of [(p-aniline benzothiazole)methyl]pyridine ($L^2H_3$) and its gadolinium complex of the type [$Gd(L^2)(H_2O)$] ($GdL^2$) is described. The $R_1$ relaxivity ($=4.50mM^{-1}sec^{-1}$) and kinetic inertness of $GdL^2$ compares well with those of structurally analogous Dotarem$^{(R)}$ ($R_1=3.70mM^{-1}sec^{-1}$), a typical extracellular (ECF) MRI contrast agent (CA). Yet, by comparison with Dotarem$^{(R)}$, $GdL^2$ exhibits non-covalent interactions with human serum albumin (HSA) as evidenced by the ${\varepsilon}^*$ titration curve along with in vivo MR signal enhancement in both aorta and heart. Liver-specific nature of $GdL^2$ is also observed as excretion is made through gallbladder. Most notably, $GdL^2$ further demonstrates specificity toward the MDA-MB-231 breast cancer.

Introduction

Magnetic resonance imaging (MRI) is a powerful technique for noninvasive diagnosis of the human anatomy, physiology, and pathophysiology on the basis of superior spatial resolution and contrast useful in providing anatomical and functional images of the human body.1 At the clinical level, MRI techniques are mostly performed employing Gd-chelates to enhance the image contrast by increasing the water proton relaxation rate in the body.2 Despite their wide and successful applications in clinics, however, conventional Gd-based low-molecular weight contrast agents are mostly ECF agents exhibiting rapid extravasation from the vascular space. As a result, the time window for imaging is considerably reduced, thus limiting acquisition of high-resolution images.

To overcome such limitations inherent to ECF MRI CAs, there has risen the necessity for the development of new MRI CAs carrying some unique functionalities. As a representative example, one may cite the so-called ‘targeting MRI CAs’ capable of responding to particular pathologies (i.e., tumors, angiogenesis, apoptosis).3-6 It would be even more desirable to equip the targeting imaging probes with such an additional function as therapy within a single molecule, from which a new term ‘theranostic’ (a coinage of diagnostic and therapeutic) may be derived.

We have recently reported the synthesis of DO3A-(p-aniline benzothiazole) conjugate (L1H3) (Chart 1) and its Gd complex of the type [Gd(L1)(H2O)] (GdL1) and demonstrated that GdL1 is truly a single molecule theranostic agent.7 GdL1 is not only tumor-specific but also intracellular, enhancing MR images of cytosols and nuclei of tumor cells such as MCF-7, MDA-MB-231, and SK-HEP-1. Both L1H3 and GdL1 reveal antiproliferative activities as demonstrated by GI50 and TGI values obtainable from the cell counting kit- 8 (CCK-8) assays performed on these cell lines. Eχ vivo and in vivo monitoring of tumor sizes provide parallel and supportive observations for such antiproliferative activities.

Benzothiazole derivatives exhibit diverse biological properties such as anti-inflammatory, antimicrobial, and anticancer effects. As such, a great deal of research activities has been carried out in the past two decades in an effort to develop various benzothiazole derivatives with high antitumor activity.8 Their antitumor properties have been utilized in combination with molecular imaging and therapy. For instance, radioactive Gp 7 complexes (M = 99mTc, 186Re, 188Re) have been proposed as potential radiopharmaceuticals for single photon emission computed tomography (SPECT) and radioimmunotherapy (RIT).9,10 Benzothaizoles labeled with radioisotopes such as 99mTc and 11C have been proposed as potential probes for Ab-amyloid plaques in the brain.11,12 More recently, DO3A conjugates of benzothiazoles have been employed as multimodal imaging probes such as magnetic resonance imaging (MRI)/optical and MRI/SPECT.13,14

Chart 1

Motivated by such intriguing properties of benzothiazoles and by our recent observations cited above in connection with GdL1, we have decided to pursue further studies on the structure activity relationship for the theranostic application of benzothiazole derivatives. Here we wish to report the synthesis and evaluation of L2H3 and GdL2 as a potential theranostic agent.

 

Experimental

General. All reactions were performed under an atmosphere of dinitrogen using the standard Schlenk technique. Solvents were purified and dried using standard procedures. DO3A(tBu)3 (= 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tert- butyl acetate) was prepared according to the literature method.15 All other reagents were purchased from commercial sources and used as received unless otherwise stated. Deionized water was used throughout all experiments. The 1H NMR experiments were performed on a Bruker Advance 400 Spectrometer by Center for Instrumental Analysis, Kyungpook National University (KNU). Chemical shifts were given as δ values with reference to tetramethylsilane (TMS) as an internal standard. Coupling constants are in Hz. FABmass spectra were obtained by using a JMS-700 model (Jeol, Japan) mass spectrophotometer by Korea Basic Science Institute (KBSI). Elemental analyses were conducted by using a Fisons EA 1108 model at Center for Instrumental Analysis, KNU.

Synthesis of 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-tert- butyl acetate-10-[(6-bromomethylpyridyl)-2-methyl] (1). To a solution of 2,6-bis(bromomethyl)pyridine (1.0 g, 3.9 mmol) and K2CO3 (1.2 g, 8.6 mmol) in CH3CN (50 mL) was added drop wise a solution of DO3A(tBu)3 (1.1 g, 7.8 mmol) in CH3CN (50 mL) at 0 ℃. After the addition was complete, the reaction mixture was stirred for 18 h at 45 ℃, after which solids were removed by filtration. The filtrate was evaporated under a reduced pressure. The crude compound was purified by chromatography (CH2Cl2/MeOH, 97:3) to give an ivory solid. Yield: 0.86 g (64%). 1H NMR (CDCl3) δ 7.76-7.73 (t, 1H, pyridine), 7.41-7.39 (d, 1H, pyridine), 7.26-7.24 (d, 1H, pyridine), 4.51 (s, 2H, CH2Br), 3.58-2.13 (m, 22H, CH2 in the cyclen ring), 1.51 (s, 9H, CH3), 1.42 (s, 18H, CH3). Anal. Calcd for C33H56BrN5O6·1.5HBr: C, 48.33; H, 7.07; N, 8.54. Found: C, 48.74; H, 7.05; N, 8.71. MALDI-TOF MS (m/z): calcd for C33H56BrN5O6: 698.7316 ([MH+]). Found: 698.8658 ([MH+]).

Synthesis of 10-{[6-(p-Aniline benzothiazole)methylpyridyl] methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tris-tert- butyl acetate (2). To a mixture of 1 (1.0 g, 3.9 mmol) and K2CO3 (1.0 g, 1.9 mmol) in acetone (50 mL) was added drop wise a solution of 2-(p-aniline)benzothiazole (0.8 g, 3.4 mmol) in acetone (100 mL). On completion of addition, the reaction mixture was stirred for 18 h at 45 ℃, after which any solids were removed by filtration. The filtrate was evaporated under a reduced pressure. The crude compound was purified by chromatography (CH2Cl2/MeOH, 98:2) to give a paled yellow solid. Yield: 1.4 g (58%). 1H NMR (CDCl3) δ 7.98-7.96 (d, 1H, benzothiazole), 7.84-7.79 (t, 1H, pyridine), 7.68-7.61 (d, 1H, benzothiazole), 7.46-7.40 (d, 1H, pyridine), 7.38-7.28 (m, 2H, benzothiazole), 7.18- 7.15 (d, 1H, pyridine), 6.74-6.66 (d, 2H, benzothiazole), 4.52-4.50 (d, 2H, benzothiazole), 3.19-1.78 (m, 24H, CH2 in the cyclen ring), 1.49 (m, 27H, CH3 in the cyclne ring). Anal. Calcd for C46H65N7O6S·2HBr: C, 54.92; H, 6.71; N, 9.75; S, 3.19. Found: C, 54.71; H, 6.60; N, 9.78; S, 2.50.

Synthesis of L2H3. Deprotection of the tert-butyl groups from 2 was performed as follows: The compound 2 (0.8 g, 1.9 mmol) was added to a solution of TFA/CH2Cl2 (1:3, 20 mL), and the mixture stirred at RT for 24 h. The solvents were removed by evaporation, the crude product dissolved in MeOH (5 mL), and diethyl ether (100 mL) added to precipitate an off-white solid. Yield: 0.6 g (96%). 1H NMR (Methanol-d4) δ 7.98-7.96 (d, 1H, benzothiazole), 7.84- 7.77(t, 1H, pyridine), 7.65-7.62 (d, 1H, benzothiazole), 7.46- 7.41 (d, 1H, pyridine), 7.38-7.28 (m, 2H, benzothiazole), 7.18-7.15 (d, 1H, pyridine), 6.74-6.66 (d, 2H, benzothiazole), 4.55-4.52 (d, 2H, benzothiazole), 3.19-1.78 (m, 24H, CH2 in the cyclen ring). Anal.Calcd for C34H41N7O6S·5H2O: C, 55.95; H, 6.49; N, 13.43; S, 4.39. Found: C, 56.40; H, 6.01; N, 13.66; S, 4.30. HR-FABMS (m/z): calcd for C34H41N7O6S: 676.2917 ([MH]+). Found: 676.2914 ([MH]+).

Synthesis of GdL2. To a solution of L2H3 (0.75 g, 0.74 mmol) in water (20 mL), was added GdCl3·6H2O (0.41 g, 0.74 mmol). The mixture was stirred at RT for 18 h during which time pH of the solution was periodically adjusted to 7.0-7.5 with NaOH (1.0 M). Water was removed by evaporation, the remaining oily residue taken up in a minimum amount of MeOH (~5 mL), and ether added drop wise for precipitation. The pale yellow solid thus formed was removed by filtration, washed with diethyl ether, and dried under vacuum. The absence of the free Gd3+ ion in the precipitate was confirmed by the xylenol orange test. The final product was obtained as a hygroscopic off-white solid. Yield: 0.4 g (67%). Anal. Calcd for Gd34H40N7O7S·9H2O: C, 40.42; H, 5.72; N, 9.71; S, 3.17. Found: C, 39.96; H, 4.37; N, 9.61; S, 2.87. HR-FABMS (m/z): calcd for Gd34H40N7O7S: 830.1845 ([MH]+). Found: 831.1926 ([MH]+).

Transmetallation Kinetics and Determination of Kinetic Constants. These experiments were performed following the procedure described in the literature.16,17 It is based on the evolution of the water proton relaxation rate (R1 P) of a buffered solution (phosphate buffer, pH 7.4) containing Gd complex (2.5 mM) and ZnCl2 (2.5 mM). The solution was made by adding a solution of 250 mmol/L solution of ZnCl2 (10 μL, 250 mM) to a buffered solution of the Gd complex (1.0 mL). After stirring the mixture vigorously, an aliquot of 300 μL was taken up for the relaxometric study. A control study, run on Gadovist®, Primovist®, Multihance®, Omniscan®, and Dotarem® with zinc acetate, has given results identical to those obtained in the presence of ZnCl2. The R1 P relaxation rate was obtained after subtraction of the diamagnetic contribution of the proton water relaxation from the observed relaxation rate R1 = (1/T1). The measurements were performed on a 3 T whole body system (Magnetom Tim Trio, siemens, Germany) at room temperature.

Relaxivity. T1 measurements were carried out using an inversion recovery method with a variable inversion time (TI) at 1.5 T (64 MHz). The magnetic resonance (MR) images were acquired at 35 different TI values ranging from 50 to 1750 msec. T1 relaxation times were obtained from the non-linear least square fit of the signal intensity measured at each TI value. For T2 measurements the CPMG (Carr- Purcell-Meiboon-Gill) pulse sequence was adapted for multiple spin-echo measurements. Thirty four images were acquired with 34 different echo time (TE) values ranging from 10 to 1900 msec. T2 relaxation times were obtained from the non-linear least squares fit of the mean pixel values for the multiple spin-echo measurements at each echo time. Relaxivities (R1 and R2) were then calculated as an inverse of relaxation time per mM. The determined relaxation times (T1 and T2) and relaxivities (R1 and R2) are finally imageprocessed to give the relaxation time map and relaxivity map respectively.

In vivo MR Imaging and Image Analysis. All animal experiments were performed in accordance with the rules of the animal research committee of Kyungpook National University. Six-week male. (Iinstiture of Cancer Resarch) ICR mice with weights of 29-31 g were for the MRI. The mice were anesthetized by 1.5% isoflurane in oxygen. Measurements were made before and after injection of GdL2, Primovist® and Multihance® via tail vein. The amount of CA per each injection was 0.1 mmol [Gd]/kg for MR images. After each measurement, the mouse was revived from anesthesia, and placed in the cage with free access to food and water. During these measurements, the animals were maintained at approximately 37 ℃ using a warm water blanket. MR images were taken with an 1.5 Tesla (T) MR unit (GE Healthcare, Milwaukee, WI, USA) equipped with a home-made small animal RF coil. The coil was of the receiver type with its inner diameter being 50 mm. The imaging parameters for SE (Spin Echo) are as follows: repetition time (TR) = 300 ms; echo time (TE) = 13 ms; 7 mm field of view (FOV); 192 × 128 matrix size; 1.2 mm slice thickness; number of acquisition (NEX) = 8. Images were obtained for 300 min after injection. The anatomical locations with enhanced contrast were identified with respect to heart, kidney and liver on post-contrast MR images. For quantitative measurement, signal intensities in specific regions of interest (ROI) measured using Advantage Window software (GE Medical, USA). The CNR (Contrast to Noise Ratio) was calculated using Eq. (1), where SNR is the signal to noise ratio.

 

Results and Discussion

Synthesis. Scheme 1 shows the preparative method leading to the formation of a new bifunctional chelate (BFC) L2H3 and its gadolinium complex GdL2. A simple reflux of an equimolar mixture of 2,6-bis(bromomethyl)pyridine and DO3A(tBu)3 in CH3CN resulted in the monoalkylation to yield 6-bromomethylpyridyl-2-methyl-DO3A(tBu)3 which was further treated with 2-(4-aminophenyl)benzothiazole (BTA) to give the DO3A(tBu)3 conjugate of pyridylmethyl- BTA. Subsequent deprotection by TFA led to the formation of L2H3. The corresponding Gd complex (GdL2) is obtained by simple complexation with an equimolar amount of gadolinium chloride in water. The complex was isolated as an off-white, hygroscopic solid by repeated precipitation with diethyl ether from the reaction mixture. All new compounds were characterized by microanalysis and various spectroscopic techniques such as 1H NMR and HR-FAB mass spectroscopy.

Scheme 1

Figure 1.Evolution of longitudinal relaxation rates R1p(t)/R1p(0) as a function of time for various MRI CAs ([Gd]0 and [ZnCl2]0 = 2.5 mM in PBS (pH 7.4) at 128 MHz and 293 K).

Kinetic Inertness. The kinetic inertness of GdL2 is represented by the evolution of the normalized paramagnetic longitudinal relaxation rates, R1p(t)/R1p(0) as a function of time (Figure 1). Here, the relative value of R1p at any time t is a good estimator of the extent of transmetallation of gadolinium by zinc: GdL + Zn2+ → Gd3+ + [ZnL]−1. Of various endogenous ions, Zn(II) has been noted to displace more gadolinium ions due to its higher concentration in the blood than any other ions such as Cu(II), Ca(II), K(I).16 Quite expectedly, GdL2 exhibits high kinetic stability comparable with those of Gadovist® and Dotarem® employing the same type of macrocyclic chelate DO3A and DOTA (= 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetrakisacetic acid), respectively. Virtually no significant change in R1 relaxivity is observed either with DO3A or DOTA for as long as 3 days. In contrast, however, other MRI CAs such as Multihance®, Primovist®, and Omniscan®, all employing the acyclic DTPA (= diethylenetriaminepentaacetic acid) analogues show significant drops in R1 for the same period of time. Thus, it can be seen that the macrocyclic effect is obviously operative in the case of kinetic stability as well as thermodynamic one.

Relaxivity. The R1 relaxivity in water of GdL2 compares well enough with Dotarem®, Multihance®, and Primovist® (Table 1). In general, relaxivities drop when measured in PBS (pH 7.4) due to the formation of solid gadolinium phosphate which precipitates out of the solution. For the reason unknown to us, such a drop was more dramatic with GdL2 (to exhibit R1 as low as 1.0 mM−1s−1) than with Dotarem®, Multihance®, and Primovist®.

Table 1.aVaues in parentheses are those measured in PBS.

Figure 2.The plot of ε* values against [HSA]/[Gd] at 37 ℃ in phosphate-buffered saline (pH 7.4) for a 0.1 mmolar solution of Gd(DO3A-Py-BTA) and 0-22.5% w/v HSA at 64 MHz.

Interaction with HSA. The evidence for the non-covalent lipophilic interactions between GdL2 and HSA can be adequately provided by the so-called ε* titration (Figure 2). The curve represents the extent of relaxation enhancement as a function of the HSA concentration. A non-linear increase in the ε* value with an increase in the HSA concentration typically indicates the presence of protein binding.18

In vivo MRI. In vivo coronal images of mice obtained by tail vein injection with GdL2 reveal strong signal enhancement in liver, heart, and abdominal aorta (Figure 3). Gallbladder is also enhanced 40 minutes after injection indicating excretion is partially made through bile via hepatocyte in addition to the ordinary renal excretion. The contrast-to-noise ratio (CNR) curve provides a supporting evidence for the bile excretion (Figure 4).

Figure 3.In vivo MR T1 weighted spin echo (SE) coronal images of mice obtained by tail vein injected with GdL2 (0.1 mmol/kg): H, heart; L, liver; B, bladder, G, gallbladder; A, abdominal aorta; K, kidney (64 MHz).

Figure 4.CNR profiles obtained by GdL2 for liver, gallbladder, and kidney.

Figure 5.CNR profiles and in vivo MR axial images of mice bearing MDA-MB-231 tumor obtained with GdL2 and Dotarem® (0.1 mmol [Gd]/kg).

Tumor Targeting. GdL2 shows a strong affinity toward the MDA-MB-231 tumor cell as observed from the in vivo CNR profiles along with MR images (Figure 5). The maximum intensity endures for as long as 2 h in contrast to Dotarem®, most of which excretes out of the body within 2 h.

Judging from our previous observations that GdL1 possesses antiproliferative properties against a series of tumors, structurally related GdL2 is considered to reveal similar behaviors as a single molecule theranostic agent. In this regard, it is to be highly recommended to pursue further a study on the structure-activity relationship (SAR) with GdL2, which is the subject of our future communication.

 

Conclusions

In conclusion, we have prepared DO3A conjugate of pyridylbenzothaizole (L2H3) and its gadolinium complex of the type [Gd(L2)(H2O)] (GdL2). The R1 relaxivity and kinetic stability of GdL2 compare well with clinically available ECF MRI CAs such as Dotarem® and Gadovist®. In vivo MR images of mice with GdL2 show strong signal enhancement in gallbladder and aorta as well as liver, heart, and kidney. Further, GdL2 shows a strong affinity toward the MDA-MB- 231 tumor cell.