Multi DTPA conjugated tetrapyrollic compounds for phototherapeutic contrast agents

Abstract

Novel tetrapyrollic water soluble photosensitizing and imaging compounds and the methods of treating and imaging hyperproliferative tissue, e.g. tumors and hypervacularized tissue such as found in macular degeneration. Broadly, the compounds are tetrapyrollic photosensitizer compounds where the tetrapyrollic compound is a chlorin, bacteriochlorin, porphyrin, pyropheophorbide, purpurinimide, or bacteriopurpurinimide having 3 to 6 —CH2CONHphenylCH2CH(N(CH2COOH)2))(CH2N(CH2COOH)(CH2CH2N(CH2COOH)2)) groups or esters thereof or complexes thereof with gadolinium(III).

Description

BACKGROUND OF THE INVENTION

As described in above priority patent application Ser. No. 10/390,438, an effective tetrapyrollic photosensitizer, e.g. HPPH (a chlorophyll-a derivative) was conjugated with Gd(III)-aminophenyl-DTPA, an imaging agent. In vivo reflection spectroscopy confirmed that tumor uptake of the HPPH-aminophenylDTPA Gd (III) conjugate was higher than that of HPPH alone in the radiation-induced fibrosarcoma (RIF) tumor of C₃H mice. The subcutaneously-implanted Ward colon carcinoma in rats showed markedly increased MRI signal at twenty-four hours after intravenous injection of the conjugate. Both in vitro (RIF tumor cells) and in vivo (mice bearing RIF tumors) the conjugate produced significant efficacy. We have synthesized a molecule [two Gd (III) atoms per HPPH molecule] that also remained tumor-avid, PDT-active, and with improved MRI enhancing ability than the related mono-Gd(III) analog. Unfortunately, at the MRI dose (10 μmole/kg), these conjugates produced severe skin phototoxicity. However, replacing the hexyl- group of the pyropheophorbide-a with a PEG group, produced remarkable tumor enhancing at 8 hour postinjection, significant tumoricidal activity. The poor water-solubility problem of these conjugates was resolved by liposomal formulation.

For many years, in vivo imaging of human organs was largely dependent upon the intravenous administration of radioactive molecules for nuclear scanning or non-radioactive iodinated chemicals for radiography. However, over the last decade magnetic resonance imaging (MRI) has assumed a critical role in imaging. Unlike nuclear scanning, conventional radiography, or even computed tomography, MRI uses contrast enhancers (“contrast media”) that contain paramagnetic ions, particularly gadolinium [Gd(III)]. They are not themselves “seen” by the MRI scanner. Rather, they affect the water in body tissue so as to increase its “signal” when placed in a magnetic field. At present, three similar gadolinium(III)-derived MRI contrast agents have been approved for human clinical use in the United States, the bis-N-methylglucamine salt of Gd(III)diethylenetriaminepentaacetic acid (DTPA) (Magnavist), the bis-N-methylamide of Gd(III) DTPA (Omniscan), and the Gd(III) chelate of 20-(2-hydroxypropyl) derivative of 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-1,4,7-tetraacetic acid (Prohance). All three of these agents are carboxylate containing, water-soluble complexes. After intravenous injection they result in a transient signal increase in the vascular space and penetrate the “leaky” capillary bed of many tumors, but are rapidly excreted through the kidneys by glomerular filtration. Although several liver-specific contrast media have also been created, other organs have not been successfully targeted, and no specific tumor-avid MRI contrast agent is available to date.

Signal Intensity in MRI:

Signal intensity in MRI stems largely from the local value of the longitudinal and transverse relaxation rates of water protons, 1/T₁, and the transverse rate, 1/T₂. Signal tends to increase with increasing 1/T₁ and decrease with increasing 1/T₂. Pulse sequences that emphasize changes in 1/T₁ are referred to as “T₁-weighted,” and the opposite is true for T₂-weighted scans. Contrast agents increase both 1/T₁ and 1/T₂ to varying degrees, depending on their nature as well as the applied magnetic field. Agents like gadolinium (III) that increase 1/T₁ and 1/T₂ by roughly similar amounts are best visualized using T₁-weighted images, because the percentage change in 1/T₁ in tissue is much greater than that in 1/T₂. The longitudinal and transverse relaxivity values, r₁, and r₂, refer to the amount of increase in 1/T₁ and 1/T₂, respectively, per milimole of agent (often given as per mM of Gd). T₁ agents usually have r₂/r₁ ratios of 1-2.

Advances in MRI have strongly favored T₁ agents and thus gadolinium(III). Faster scans with higher resolution require more rapid radio-frequency pulsing and are thus generally T₁-weighted, because MR signal in each voxel becomes saturated. T₁ agents relieve this saturation by restoring a good part of the longitudinal magnetization between pulses. At the same time, a good T₁ agent would not significantly affect the bulk magnetic susceptibility of the tissue compartment in which it is localized, thus minimizing any inhomogeneities that can lead to image artifacts and/or decreased signal intensity.

The effect of these agents is to increase signal on T₁-weighted images that are negatively affected by proton density. The effect on T₂-weighed images is to decrease signal, but this effect is minimal, because most of the T₂ signal comes from the influence of proton density. Signal Intensity for the Spin Echo Imaging is expressed as: S_(TE,TR)=N_(H)[1−2e−(^TR−TE/2)/T₁=e−^TE/T₁]e−^TE/T₂

Conventional clinical MRI units produce static, cross sectional images. Newer “interventional MRI” units allow the operator to continuously image an organ while performing surgery or other manipulations.

Gd(III) is a logical choice for MRI contrast media because of its superior performance compared with other lanthanide ions. Dysprosium(III) and holmium(III) have larger magnetic moments than that of Gd(III), but the asymmetry of their electronic states leads to very rapid electron spin relaxation. The symmetric S-state of Gd(III) is a more hospitable environment for electron spins, leading to a much slower electronic relaxation rate. In the process that gives rise to relaxivity, water protons hardly feel the effects of ions like Dy(III), much like a leaf near the rapid wings of hummingbird; Gd(III) electrons, on the other hand, are more closely in tune with the proton's frequency.

A key biological factor that influences the selection of gadolinium compounds for human use is that its ligands like DTPA circulate and are excreted intact. The metal ion is “buried” in the chelation cage and will not bind to donor groups of proteins and enzymes. This in vivo stability markedly reduces the potential for toxicity from free gadolinium.

Tetrapyrrole-Based Compounds as MRI Agents:

The porphyrins and related tetrapyrrolic systems are among the most widely studied of all macrocyclic compounds. In fact, in one capacity or another these versatile molecules have influenced nearly all disciplines in chemistry. The concentration of certain porphyrins and related tetrapyrrolic or expanded porphyrin-type compounds is much higher in malignant tumors than in most normal tissues. A few years ago Sessler and coworkers discovered a new class of expanded porphyrins that is based on the Schiff base condensation between a diformyl-tripyrrane and an aromatic 1,2-diamine. This new class of expanded porphyrins has come to be known as the “texaphyrins”. Compared to the natural porphyrin system, the texaphyrins possess a larger core size and thus have the capability to form complexes with certain lanthanides, including gadolinium(III). Gd(III) texaphyrin is currently under phase I/II human clinical trials as a tumor-avid MRI contrast agent.

Some tetrapyrrole-based compounds are effective photosensitizers for cancer treatment by photodynamic therapy [PDT]. Although PDT is sometimes considered a novel, idiosyncratic therapy, it has in fact been effective in a wide variety of malignancies, including skin, lung, bladder, head and neck, breast, and esophagus. The precise mechanism(s) of PDT are unknown; however, in vitro studies suggest that singlet oxygen production is phototoxic when the photosensitizing agent encounters light. In vivo animal data suggest that tumor vasonecrosis may be the direct cause of tumor kill.

Effective PDT requires delivery of light to tumor that has absorbed a photosensitizer previously delivered by the systemic circulation after peripheral intravenous injection. Superficial visible lesions, or those that are endoscopically accessible—e.g., endobronchial or esophageal—are easily treated, but the vast majority of malignant lesions are too deep to be reached by light of the wavelength required to trigger singlet oxygen production in the current generation of photosensitizers. Although the technology to deliver therapeutic light to deep lesions via thin transmission fibers “capped” by a terminal diffuser is well-developed, a deep lesion is by definition not visible from the skin surface, and its uptake of a peripherally-injected photosensitizer is unknown; therefore, PDT of deep tumors thus far been impractical.

A relatively long-wavelength absorbing photosensitizer, the 3-(1-hexyloxy)ethyl derivative of pyropheophorbide-a 1 [HPPH], developed in our laboratory, is tumor-avid and currently in Phase VIII clinical trials at The Roswell Park Cancer Institute. We investigated this compound as a “vehicle” for delivering gadolinium complexes to tumor, with the goal of creating the first single compound that would function both as an MRI tumor-avid contrast medium and a photosensitizer for cancer therapy. [Gd(III) texaphrin is not a photosensitizer, because it does not produce singlet oxygen when exposed to light].

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A shows MRI images of tumors as compared to muscle using conjugate 3.

FIG. 1B shows MRI images of tumors as compared to muscle using conjugate 7.

FIG. 2 is a bar graph showing tumor to muscle contrast of the conjugates.

FIG. 3 is a bar graph showing tumor to fat contrast of the conjugates.

BRIEF DESCRIPTION OF THE INVENTION

The present invention deals with the synthesis of “higher pay-load’ contrast agents in which the photosensitizers are conjugated with three- and six Gd(III)DTP molecules. In contrast to conjugates with mono- and di-DTPA conjugates which were formulated in Tween 80/water and liposomal formulation, the conjugates with three- and six DTPA molecules can be formulated in phosphate buffer at 7.4 pH and show the potential for tumor imaging ability and phototoxicity. The development of a tumor-avid contrast medium for MRI would by itself represent an important step in the diagnosis of cancer, but a dual function agent presents the potential for a diagnostic body scan followed by targeted photodynamic therapy, combining two modalities into a single cost-effective “see and treat” approach.

The invention includes both the novel tetrapyrollic water soluble photosensitizing and imaging compounds and the methods of treating and imaging hyperproliferative tissue, e.g. tumors and hypervacularized tissue such as found in macular degeneration. Broadly, the compounds are tetrapyrollic photosensitizer compounds where the tetrapyrollic compound is a chlorin, bacteriochlorin, porphyrin, pyropheophorbide, purpurinimide, or bacteriopurpurinimide having 3 to 6 —CH₂CONHphenylCH₂CH(N(CH₂COOH)₂))(CH₂N(CH₂COOH)(CH₂CH₂N(CH₂COOH)₂)) groups or esters thereof or complexes thereof with gadolinium(III).

Preferably, the compound has at least one pendant —CH₂CH₂CONHC(CH₂CH₂CONHphenylCH₂CH(N(CH₂COOH)₂))(CH₂N(CH₂COOH)(CH₂CH₂N(CH₂COOH)₂)))₃ group or esters thereof or complexes thereof with gadolinium(III).

Preferred compounds of the invention have the formula: or a pharmaceutically acceptable derivative thereof, wherein:

R₁ and R₂ are each independently substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, —C(O)R_a or —COOR_a or —CH(CH₃)(OR) or —CH(CH₃)(O(CH₂)_nXR) where R_a is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, or substituted or unsubstituted cycloalkyl where R₂ may be CH═CH₂, CH(OR₂₀)CH₃, C(O)Me, C(═NR₂₁)CH₃ or CH(NHR₂₁)CH₃;

where X is an aryl or heteroaryl group;

n is an integer of 0 to 6;

R and R are independently H or lower alkyl of 1 through 8 carbon atoms;

where R₂₀ is methyl, butyl, heptyl, docecyl or 3,5-bis(trifluoromethyl)-benzyl; and

R₂₁, is 3,5,-bis(trifluoromethyl)benzyl;

R_1a and R_2a are each independently hydrogen or substituted or unsubstituted alkyl, or together form a covalent bond;

R₃ and R₄ are each independently hydrogen or substituted or unsubstituted alkyl;

R_3a and R_4a are each independently hydrogen or substituted or unsubstituted alkyl, or together form a covalent bond;

R₅ is hydrogen or substituted or unsubstituted alkyl;

R₆ and R_6a are each independently hydrogen or substituted or unsubstituted alkyl, or together form ═O;

R₇ is a covalent bond, alkylene, azaalkyl, or azaaraalkyl or ═NR₂₀ where R₂₀ is hydrogen or lower alkyl of 1 through 8 carbon atoms or —CH₂-3,5-bis(tri-fluoromethyl)benzyl or —CH₂X—R₁ or —YR¹ where Y is an aryl or heteroaryl group;

R₈ and R_8a are each independently hydrogen or substituted or unsubstituted alkyl or together form ═O;

R₉ is a pendant group containing 3 through 6

—CH₂CONHphenylCH₂CH(N(CH₂COOH)₂))—(CH₂N(CH₂COOH)(CH₂CH₂N(CH₂COOH)₂)) groups or esters thereof or complexes thereof with gadolinium(III).

R₁₀ is hydrogen, or substituted or unsubstituted alkyl and; each of R₁-R₁₀, when substituted, is substituted with one or more substituents each independently selected from Q, where Q is alkyl, haloalkyl, halo, pseudohalo, or —COOR_b where R_b is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, or OR_c where R_c is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl or CONR_dR_e where R_d and R_e are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or NR_fR_g where R_f and R_g are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or ═NR_h where R_h is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid residue;

each Q is independently unsubstituted or is substituted with one or more substituents each independently selected from Q₁, where Q₁ is alkyl, haloalkyl, halo, pseudohalo, or —COOR_b where R_b is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, or OR_c where R_c is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl or CONR_dR_e where R_d and R_e are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or NR_fR_g where R_f and R_g are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or ═NR_h where R_h is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid residue.

R₉ is preferably a —CH₂CH₂CONHC(CH₂ CH₂CONHphenylCH₂CH(N(CH₂COOH)₂))—(CH₂N(CH₂COOH)(CH₂CH₂N(CH₂COOH)₂)))₃ group or esters thereof or complexes thereof with gadolinium(III).

DETAILED DESCRIPTION OF THE INVENTION

A compound in accordance with the present invention that effectively functions both as an MRI contrast medium and a photosensitizer creates an entirely new paradigm for tumor diagnosis and therapy. After peripheral intravenous injection of this compound, a patient could be scanned with interventional MRI. The tumor site(s) could thus be defined, and, while the patient remains in the scanner, an interventional radiologist could transcutaneously insert ultra-slim needles acting as introducers for light-transmission fibers into the lesion(s). Because such fiber diameters can be small, e.g. only 400 microns, the introducer needles would produce negligible tissue damage. A light source can be coupled to the fibers, and PDT of the lesion(s) can commence, without any significant injury to other organs. Because the same molecule represents the contrast medium and the therapeutic medium, the lesion(s) can be continuously imaged during needle/fiber placement, without any ambiguity in location or “misregistration” of separate diagnostic/therapeutic images. This paradigm makes the low-toxicity and high efficacy of PDT available to virtually any location from the skull base to the floor of the pelvis.

Examples of compounds for use in accordance with the present invention were prepared and tested. Synthetic intermediates and the final products were characterized by NMR (400 MHz), mass spectrometry (EIMS & HRMS) and elemental analyses. ¹H NMR spectra were recorded on a Bruker AM-400 spectrometer. Chemical shifts are expressed in ppm. All photo physical experiments were carried out using spectroscopic grade solvents. The reactions were monitored by TLC and/or spectrophotometrically. Column chromatography was performed either over Silica Gel 60 (70-230 mesh) or neutral Alumina (Brockmann grade III, 50 mesh). UV-visible spectrums were recorded on Varian Cary 50 Bio UV-visible spectrophotometer using dichloromethane as solvent unless other wise specified.

Compound No. 1:

HPPH (100.0 mg, 0.157 mmol), amine A (97.8 mg, 0.235 mmol), EDCI (60.2 mg, 0.314 mmol) and DMAP (38.36 mg, 0.314 mmol) were taken in a dry RBF (100 ml). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over silica gel using 1-3% Methanol/Dichloromethane mixture as eluent to give product 1.Yield: 130.0 mg (80.0%).UV-vis (λmax cm⁻¹, dichloromethane): 318, 412, 506, 537, 604 & 660. ¹HNMR (400 MHz, CDCl₃): δ 9.78 (s, 1H, H-5), 9.41 (s, 1H, H-10), 8.54 (s, 1H, H-20), 5.96-5.91 (m, 2H, CH₃CHOhexyl, NH), 5.33 (d, 1H, CH-15¹, J=19.6 Hz), 5.14 (d, 1H, CH-15¹, J=19.6 Hz), 4.53 (q, 1H, H-17, J=7.2 Hz), 4.32 (m, 1H, H-18), 3.71-3.67 (m, 2H, —OCH₂-hexyl), 3.63-3.60 (m, 2H, 8-CH₂CH₃), 3.52 (s, 3H, 7-CH₃), 3.39 (s, 3H, 2-CH₃), 3.27 (s, 3H, 12-CH₃), 2.67 (m, 1H, CH-17²), 2.37 (m, 1H, H-17²), 2.26 (m, 1H, H-17¹), 2.15-2.13 (m, 9H, 3CH₂-chain & CH₃CHOhexyl), 1.97 (m, 1H, H-17¹), 1.92 (t, 6H, 3CH₂-chain, J=7.6 Hz), 1.80 (d, 3H, 18-CH₃, J=7.2 Hz), 1.73 (m, 2H, CH₂hexyl), 1.66 (t, 3H, 8-CH₂CH₃, J=7.6 Hz), 1.44 (m, 2H, CH₂hexyl), 1.30 (s, 27H, 3CO₂^tBu), 1.24 (m, 4H, 2CH₂hexyl), 0.78 (t, 3H, CH₃hexyl, J=6.8 Hz), 0.04 (brs, 1H, NH),-1.68 (brs, 1H, NH). EIMS: 1035 (MH⁺).

Compound No. 2:

Compound 1 (73.0 mg, 0.07 mmol) was stirred with 70% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. To this crude were added, amino-benzyl-DTPA-penta-tert-butyl ester (219.0 mg, 0.282 mmol), EDCI (67.0 mg, 0.352 mmol) and DMAP (43.0 mg, 0.352 mmol). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over alumina G (III) using 1-3% Methanol/Dichloromethane mixture as eluent to give product 2. Yield: 130.0 mg (58.47%).UV-vis (λmax cm⁻¹, dichloromethane): 319, 411, 506, 537, 604, 661. ¹HNMR (400 MHz, CDCl₃): δ 9.74 (splitted s, 1H, H-5), 9.36 (splitted s, 1H, H-10), 8.54 (splitted s, 1H, H-20), 8.17 (brs, 1H, NH), 7.74 (m, 1H, Ph-DTPA), 7.61-7.56 (m, 2H, Ph-DTPA), 7.43 (m, 4H, Ph-DTPA), 7.12 (m, 5H, Ph-DTPA), 6.96 (brs, 1H, NH), 5.96 (m, 1H, CH₃CHOhexyl), 5.29 (d, 1H, CH-15¹, J=19.2 Hz), 5.11 (d, 1H, CH-15¹, J=19.2 Hz), 4.47 (m, 1H, H-17), 4.26 (m, 1H, H-18), 3.64 (m, 2H, & OCH₂hexyl), 3.54 (m, 2H, 8-CH₂CH₃), 3.45-3.38 (m, 30H, 15CH₂-DTPA), 3.36 (s, 3H, 7-CH₃), 3.32 (s, 3H, 2-CH₃), 3.22 (s, 3H, 12-CH₃), 3.12 (m, 3H, CH-DTPA), 2.84-2.70 (m, 19H, 9CH₂-DTPA & CH-17²), 2.59 (m, 6H, 6CH₂-benzyl), 2.47-2.41 (m, 8H, 3CH₂-chain, CH-17² & CH-17¹), 2.19-2.15 (m, 9H, 3CH₂-chain, CH₃CH-Ohexyl), 2.06 (d, 3H, 18-CH₃, J=7.6 Hz) 2.00 (m, 1H, CH-17¹), 1.74 (m, 4H, 2CH₂-hexyl), 1.66 (t, 3H, 8-CH₂CH₃, J=7.2 Hz), 1.60 (m, 135H for 15 CO₂^tBu), 1.26 (m, 4H, 2CH₂-hexyl), 0.77 (m, 3H, CH₃-Ohexyl), 0.55 (brs, 1H, NH), −0.24 (brs, 1H, NH). HRMS: Calculated for C₁₇₂H₂₆₇N₁₇O₃₆: 3149.053, found: 3150.10 (MH⁺).

Compound No. 3:

Compound 2 (110.0 mg, 0.034 mmol) was stirred with 70% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. The crude thus obtained was dissolved in pyridine (10 ml) and put under stirring, to this stirring solution GdCl₃.6H₂O (77.9 mg, 0.21 mmol) in 1 ml of water was added slowly and resultant mixture was stirred for 16 hr. Reaction mixture was concentrated to dryness under high vacuum. Residue was washed with water (10 ml×3), acetone (10 ml×3) and finally dried under high vacuum using P₂O₅ as drying agent. Yield: 75.0 mg (77.55%). UV-vis (λmax cm⁻¹, MeOH): 620, 408, 504, 537, 604 & 660. Elemental analysis: Calculated for C₁₁₃H₁₅₁Gd₃N₁₇O₃₆: C, 48.55; H, 5.44; Gd, 16.88; N, 8.52.; O, 20.61, found: C, 48.68; H, 5.49; N, 8.57.

Compound No. 5:

Acid 4 (100.0 mg, 0.143 mmol), amine A (89.1 mg, 0.214 mmol), EDCI (54.9 mg, 0.28 mmol) and DMAP (34.9 mg, 0.28 mmol) were taken in a dry RBF (100 ml). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over silica gel using 1-2% Methanol/Dichloromethane mixture as eluent to give product 5. Yield: 134.0 mg (85.3%).UV-vis (λmax cm⁻¹, dichloromethane): 318, 411, 506, 536, 604 & 661. ¹HNMR (400 MHz, CDCl₃): δ 9.76 (splitted s, 1H, H-5), 9.38 (splitted s, 1H, H-10), 8.55 (s, 1H, H-20), 6.02 (d, 1H, CH₃CHOOTEG, J=6.4 Hz), 5.99 (brs, 1H, NH), 5.34 (d, 1H, CH-15¹, J=20.0 Hz), 5.15 (d, 1H, CH-15¹, J=20.0 Hz), 4.58 (q, 1H, H-17, J=6.8 Hz), 4.33 (m, 1H, H-18), 3.88-3.75 (m, 4H, 2CH₂—O-TEG), 3.70-3.62 (m, 6H, 3CH₂—O-TEG), 3.55 (m, 2H, 8-CH₂CH₃), 3.47-3.44 (m, 2H, CH₂—O-TEG), 3.40 (s, 3H, 7-CH₃), 3.39 (s, 3H, 2-CH₃), 3.29 (s, 3H, 12-CH₃), 3.27 (s, 3H, CH₃—O-TEG), 2.69 (m, 1H, CH-17²), 2.39 (m, 1H, CH-17²), 2.31 (m, 1H, CH-17¹), 2.14 (m, 8H, 4CH₂-chain), 2.00 (m, 1H, CH-17¹), 1.92 (m, 7H, 2CH₂-chain, CH₃CHOTEG), 1.82 (d, 3H, 18-CH₃, J=7.2 Hz), 1.68 (t, 3H, 8-CH₂CH₃, J=7.6 Hz), 1.31 (s, 27H, 3CO₂^tBu), 0.42 (brs, 1H, NH), −1.69 (brs, 1H, NH).EIMS: 1097 (MH⁺)

Compound No. 6:

Compound 5 (100.0 mg, 0.091 mmol) was stirred with 80% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. To this crude were added, amino-benzyl-DTPA-penta-tert-butyl ester (285.0 mg, 0.365 mmol), EDCI (105.0 mg, 0.54 mmol) and DMAP (66.8 mg, 0.54 mmol). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over alumina G (III) using 1-3% Methanol/Dichloromethane mixture as eluent to give product 6. Yield: 250.0 mg (85.3%). UV-vis (λmax cm⁻¹, dichloromethane): 319, 411, 506, 537, 606, 661. ¹HNMR (400 MHz, CDCl₃): δ 9.70 (splitted s, 1H, H-5), 9.44 (splitted s, 1H, H-10), 9.37 (brs, 1H, NH), 9.20 (brs, 1H, NH), 8.56 (m, 1H, NH), 8.47 (s, 1H, H-20), 7.77 (m, 1H, Ph-DTPA), 7.59 (m, 2H, Ph-DTPA), 7.44 (m, 2H, Ph-DTPA), 7.10 (m, 6H, Ph-DTPA), 6.81 (m, 1H, Ph-DTPA), 5.97 (m, 1H, CH₃CHOTEG), 5.20 (m, 2H, CH₂-15¹), 4.60 (m, 1H, H-17), 4.22 (m, 1H, H-18), 3.81-3.64 (m, 4H, 2CH₂—OTEG), 3.57-3.50 (m, 4H, 8-CH₂CH₃, CH₂—OTEG), 3.60 (m, 6H, 3CH₂OTEG), 3.38 (s, 30H, 15CH₂-DTPA), 3.35 (s, 3H, 7-CH₃), 3.23 (m, 6H, 12-CH₃, OCH₃-TEG), 3.04 (m, 3H, 3CH-DTPA), 2.70 (m, 19H, 9CH₂-DTPA, CH-17²), 2.55 (m, 7H, 3CH₂-benzyl & CH-17²), 2.32 (t, 6H, 3CH₂-chain, J=6.8 Hz), 2.23 (m, 1H, CH-17¹), 2.10 (d, 3H, CH₃CH-OTEG, J=6.4 Hz), 2.01 (m, 1H, CH-17¹), 1.77 (d, 3H, 18-CH₃, J=7.2 Hz), 1.68 (t, 3H, 8-CH₂CH₃, J=7.6 Hz), 1.59 (t, 6H, 3CH₂-chain, J=6.4 Hz), 1.44 (m, 135H, 15CO₂^tBu),-1.75 (brs, 1H, NH).

HRMS: Calculated for C₁₇₃H₂₆₉N₁₇O₃₉: 3211.077, found: 3212.20 (MH⁺).

Compound No. 7:

Compound 6 (226.0 mg, 0.07 mmol) was stirred with 80% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. The crude thus obtained was dissolved in pyridine (10 ml) and put under stirring, to this stirring solution GdCl₃.6H₂O (156.9 mg, 0.422 mmol) in 1 ml of water was added slowly and resultant mixture was stirred for 16 hr. Reaction mixture was concentrated to dryness under high vacuum. Residue was washed with water (10 ml×3), acetone (10 ml×3) and finally dried under high vacuum using P₂O₅ as drying agent. Yield: 165.0 mg (82.5%). UV-vis (λmax cm⁻¹, MeOH): 320, 408, 505, 537, 605 & 660. Elemental analysis: Calculated for C₁₁₃H₁₄₉Gd₃N₁₇O₃₉: C, 47.77; H, 5.29; Gd, 16.60; N, 8.38.; O, 21.96, found: C, 47.85; H, 5.30; N, 8.43.

Compound No. 9:

Acid 8 (82.0 mg, 0.118 mmol), amine A (73.6 mg, 0.177 mmol), EDCI (45.3 mg, 0.236 mmol) and DMAP (28.8 mg, 0.236 mmol) were taken in a dry RBF (100 ml). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over silica gel using 1-2% Methanol/Dichloromethane mixture as eluent to give product 9. Yield: 90.0 mg (69.8%). UV-vis (λmax cm⁻¹, dichloromethane): 365, 418, 509, 545 & 699. ¹HNMR (400 MHz, CDCl₃): δ 9.75 (splitted s, 1H, meso-H), 9.64 (s, 1H, meso-H), 8.55 (s, 1H, meso-H), 6.25 (m, 1H, CONH), 5.79 (q, 1H, CH₃CHObutyl, J=6.4 Hz), 5.34 (m, 1H, H-17), 4.52 (t, 2H, —NCH₂butyl, J=7.2 Hz), 4.42 (m, 1H, H-18), 3.84 (s, 3H, ring-CH₃), 3.70-3.59 (m, 4H, —OCH₂-butyl, 8-CH₂CH₃), 3.32 (s, 3H, ring-CH₃), 3.17 (s, 3H, ring-CH₃), 2.61 (m, 1H, CH-17²), 2.43 (m, 1H, H-17²), 2.27 (m, 1H, H-17¹), 2.20 (t, 6H, 3CH₂-chain, J=7.2 Hz), 2.06 (m, 3H, CH₃CHObutyl), 2.01 (t, 6H, 3CH₂-chain, J=7.6 Hz), 1.82 (m, 1H, H-17¹), 1.75 (d, 3H, 18-CH₃, J=6.0 Hz), 1.68 (t, 3H, 8-CH₂CH₃, J=7.6 Hz), 1.62 (m, 8H, 4CH₂butyl), 1.34 (s, 27H, 3CO₂^tBu), 1.10 (t, 3H, CH₃-obutyl, J=7.2 Hz), 0.87 (t, 3H, CH₃-Nbutyl, J=7.2 Hz), 0.40 (brs, 1H, NH),-0.06 (brs, 1H, NH). EIMS: 1092 (MH⁺).

Compound No. 10:

Compound 9 (80.0 mg, 0.073 mmol) was stirred with 70% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. To this crude were added, amino-benzyl-DTPA-penta-tert-butyl ester (286.0 mg, 0.366 mmol), EDCI (84.4 mg, 0.44 mmol) and DMAP (53.7 mg, 0.44 mmol). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over alumina G (III) using 1-3% Methanol/Dichloromethane mixture as eluent to give product 10. Yield: 140.0 mg (59.57%). UV-vis (λmax cm⁻¹, dichloromethane): 365, 418, 509, 546, 699. ¹HNMR (400 MHz, CDCl₃): δ 9.75 (splitted s, 1H, meso-H), 9.63 (splitted s, 1H, meso-H), 9.33 (brs, 1H, NH), 8.60 (splitted s, 1H, meso-H), 7.61 (m, 2H, Ph-DTPA), 7.58 (m, 1H, Ph-DTPA), 7.37 (m, 3H, Ph-DTPA), 7.12 (m, 5H, Ph-DTPA), 6.84 (m, 1H, Ph-DTPA), 5.76 (m, 1H, CH₃CHObutyl), 5.39 (m, 1H, H-17), 4.45 (m, 3H, H-18 & NCH₂butyl), 3.82 (s, 3H, ring-CH₃), 3.65 (m, 4H, 8-CH₂CH₃ & OCH₂butyl), 3.38 (m, 22H, 11CH₂-DTPA), 3.31 (m, 11H, 4CH₂-DTPA & ring-CH₃), 3.17 (s, 3H, ring-CH₃), 3.03 (m, 3H, CH-DTPA), 2.84-2.61 (m, 19H, 9CH₂-DTPA & CH-17²), 2.47 (m, 8H, 6CH₂-benzyl & CH-17² & CH-17¹), 2.20 (m, 6H, 3CH₂-chain), 2.04 (d, CH₃CHObutyl, J=6.8 Hz), 1.96 (m, 6H, 3CH₂-chain), 1.84 (m, 1H, CH-17¹), 1.73 (s, 3H, 17-CH₃), 1.67 (t, 3H, 8-CH₂CH₃, J=7.2 Hz), 1.60 (m, 4H, 2CH₂—Obutyl), 1.41 (m, 135H for 15 CO₂^tBu), 1.37 (m, 4H, 2CH₂—N-butyl), 1.03 (t, 3H, CH₃—Obutyl, J=6.8 Hz), 0.86 (t, 3H, CH₃—N-butyl, J=6.8 Hz),−0.09 (brs, 1H, NH). HRMS: Calculated for C₁₇₄H₂₇₀N₁₈O₃₇: 3206.104, found: 3207.250 (MH⁺).

Compound No. 11:

Compound 10 (130.0 mg, 0.04 mmol) was stirred with 70% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. The crude thus obtained was dissolved in pyridine (10 ml) and put under stirring, to this stirring solution GdCl₃.6H₂O (90.3 mg, 0.243 mmol) in 1 ml of water was added slowly and resultant mixture was stirred for 16 hr. Reaction mixture was concentrated to dryness under high vacuum. Residue was washed with water (10 ml×3), acetone (10 ml×3) and finally dried under high vacuum using P₂O₅ as drying agent. Yield: 80.0 mg (69.9%). UV-vis (λmax cm⁻¹, MeOH): 364, 415, 546 & 700.Elemental analysis: Calculated for C₁₁₄H₁₅₀Gd₃N₁₈O₃₇: C, 48.28; H, 5.33; Gd, 16.63; N, 8.89.; O, 20.87, found: C, 48.14; H, 5.40; N, 8.93.

Compound No. 12:

HPPH (100.0 mg, 0.157 mmol), Di-tert-butyl iminodiacetate (77.0 mg, 0.314 mmol), EDCI (60.2 mg, 0.314 mmol) and DMAP (38.36 mg, 0.314 mmol) were taken in a dry RBF (100 ml). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over silica gel using 1-1.5% Methanol/Dichloromethane mixture as eluent to give product 12. Yield: 120.0 mg (88.3%). UV-vis (λmax cm⁻¹, dichloromethane): 317, 411, 506, 538, 605 & 660. ¹HNMR (400 MHz, CDCl₃): δ 9.80 (s, 1H, H-5), 9.51 (s, 1H, H-10), 8.55 (s, 1H, H-20), 5.94 (q, 1H, CH₃CHOhexyl, J=6.4 Hz), 5.33 (d, 1H, CH-15¹, J=20.0 Hz), 5.17 (d, 1H, CH-15¹, J=20.0 Hz), 4.52 (q, 1H, H-17, J=7.6 Hz), 4.41 (m, 1H, H-18), 4.04 (m, 2H, CH₂chain), 3.75 (m, 2H, —OCH₂-hexyl), 3.67 (s, 3H, 7-CH₃), 3.62 (m, 2H, 8-CH₂CH₃), 3.42 (s, 3H, 2-CH₃), 3.37 (m, 2H, CH₂chain), 3.29 (s, 3H, 12-CH₃), 2.77 (m, 1H, CH-17²), 2.46 (m, 1H, CH-17²), 2.16 (m, 1H, CH-17¹), 2.13 (m, 3H, & CH₃CHOhexyl), 1.97 (m, 1H, CH-17¹), 1.84 (d, 3H, 18-CH₃, J=7.2 Hz), 1.78 (m, 2H, CH₂hexyl), 1.72 (t, 3H, 8-CH₂CH₃J=7.6 Hz), 1.49 (s, 9H, CO₂^tBu), 1.46 (m, 2H, CH₂hexyl), 1.45 (s, 9H, CO₂^tBu), 1.25 (m, 4H, 2CH₂hexyl), 0.8 (t, 3H, CH₃hexyl, J=6.8 Hz), 0.42 (brs, 1H, NH),−1.7 (brs, 1H, NH). EIMS: 865 (MH⁺).

Compound No. 13:

Compound 12 (120.0 mg, 0.139 mmol) was stirred with 70% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. To this crude were added, amine A (144.2 mg, 0.34 mmol), EDCI (106.6 mg, 0.556 mmol) and DMAP (67.8 mg, 0.556 mmol). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over silica gel using 2-6% Methanol/Dichloromethane mixture as eluent to give product 13. Yield: 190.0 mg (88.3%). UV-vis (λmax cm⁻¹, dichloromethane): 318, 411, 506, 537, 605 and 660. ¹HNMR (400 MHz, CDCl₃): δ 9.78 (s, 1H, H-5), 9.52 (s, 1H, H-10), 8.3 (s, 1H, H-20), 8.27 (brs, 1H, NH), 8.17 (brs, 1H, NH), 6.59 (brs, 1H, NH), 5.90 (m, 1H, CH₃CHOhexyl), 5.34 (d, 1H, CH-15¹, J=20.0 Hz), 5.15 (d, 1H, CH-15¹, J=20.0 Hz), 4.53 (q, 1H, H-17, J=6.0 Hz), 4.36 (m, 1H, H-18), 3.77 (m, 2H, —OCH₂-hexyl), 3.69 (m, 2H, 8-CH₂CH₃), 3.67 (s, 3H, 7-CH₃), 3.61 (m, 4H, 2CH₂chain), 3.36 (s, 3H, 2-CH₃), 3.26 (s, 3H, 12-CH₃), 2.77 (m, 1H, CH-17²), 2.66 (m, 1H, CH-17²), 2.52 (m, 1H, CH-17¹), 2.23 (m, 12H, 6CH₂-chain), 2.11 (d, 3H, CH₃CHOhexyl, J=6.4 Hz), 2.07 (m, 6H, 3CH₂-chain), 1.95 (m, 6H, 3CH₂-chain), 1.93 (m, 1H, CH-17¹), 1.81 (d, 3H, 18-CH₃, J=7.2 Hz), 1.75 (m, 2H, CH₂hexyl), 1.71 (t, 3H, 8-CH₂CH₃, J=8.0 Hz), 1.43 (m, 2H, CH₂hexyl), 1.41 (s, 27H, 3CO₂^tBu), 1.32 (s, 27H, 3CO₂^tBu), 1.24 (m, 4H, 2CH₂hexyl), 0.77 (t, 3H, CH₃hexyl, J=6.8 Hz). EIMS: 1548 (MH⁺).

Compound No. 14:

Compound 13 (100.0 mg, 0.064 mmol) was stirred with 80% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. To this crude were added, amino-benzyl-DTPA-penta-tert-butyl ester (503.5 mg, 0.64 mmol), EDCI (123.9 mg, 0.64 mmol) and DMAP (78.8 mg, 0.64 mmol). Dry N, N-dimethylformamide (15 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was concentrated under vacuum, added dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over alumina G (III) using 1-3% Methanol/Dichloromethane mixture as eluent to give product 14. Yield: 250.0 mg (70.0%). UV-vis (λmax cm⁻¹, Dichloromethane): 318, 413, 507, 539, 606 & 660. Elemental analysis: Calculated for C₃₀₉H₄₉₁N₃₁O₇₁: C, 64.25; H, 8.57; N, 7.52;O, 19.67, found: C, 64.30; H, 8.59; N, 7.56.

Compound No. 15:

Compound 14 (225.0 mg, 0.038 mmol) was stirred with 70% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. The crude thus obtained was dissolved in pyridine (10 ml) and put under stirring, to this stirring solution GdCl₃.6H₂O (173.7 mg, 0.46 mmol) in 1 ml of water was added slowly and resultant mixture was stirred for 16 hr. Reaction mixture was concentrated to dryness under high vacuum. Residue was washed with water (10 ml×3), acetone (10 ml×3) and finally dried under high vacuum using P₂O₅ as drying agent. Yield: 170.0 mg (86.7%). UV-vis (λmax cm⁻¹, MeOH): 320, 411, 507, 539, 606 & 660. Elemental analysis: Calculated for C₁₈₉H₂₅₁,Gd₆N₃₁O₇₁: C, 45.07; H, 5.02; Gd, 18.73; N, 8.62.; O, 22.55, found: C, 45.15; H, 5.10; N, 8.58.

Compound No. 16:

Acid 4 (150.0 mg, 0.214 mmol), Di-tert-butyl iminodiacetate (105.0 mg, 0.429 mmol), EDCI (82.3 mg, 0.429 mmol) and DMAP (52.0 mg, 0.429 mmol) were taken in a dry RBF (100 ml). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over silica gel using 1-1.5% Methanol/Dichloromethane mixture as eluent to give product 16. Yield: 165.0 mg (82.9%). UV-vis (λmax cm⁻¹, dichloromethane): 318, 411, 506, 536, 605 and 661. ¹HNMR (400 MHz, CDCl₃): δ 9.75 (splitted s, 1H, H-5), 9.52 (splitted s, 1H, H-10), 8.53 (s, 1H, H-20), 6.01 (m, 1H, CH₃CHOTEG), 5.31 (d, 1H, CH-15¹, J=20.0 Hz), 5.13 (d, 1H, CH-15¹, J=20.0 Hz), 4.50 (q, 1H, H-17, J=7.2 Hz), 4.36 (m, 1H, H-18), 4.02 9m, 2H, CH₂chain), 3.85 (m, 2H, CH₂—O-TEG), 3.79 (m, 2H, CH₂—O-TEG), 3.73 (m, 4H, 3CH₂—O-TEG), 3.68 (s, 3H, 7-CH₃), 3.66 (m, 2H, 8-CH₂CH₃), 3.55 (m, 2H, CH₂—O-TEG), 3.39 (s, 3H, 2-CH₃), 3.27 (s, 6H, 12-CH₃ & OCH₃-TEG), 2.75 (m, 1H, CH-17²), 2.44 (m, 1H, CH-17²), 2.41 (m, 1H, CH-17¹), 2.16 (m, 1H, CH-17¹), 2.14 (d, 3H, CH₃CHOTEG, J=6.4 Hz), 1.81 (d, 3H, 18-CH₃, J=7.6 Hz), 1.71 (t, 3H, 8-CH₂CH₃, J=7.6 Hz), 1.44 (splitted s, 9H, CO₂^tBu), 1.06 (splitted s, 9H, CO₂^tBu), 0.39 (brs, 1H, NH),-1.80 (brs, 1H, NH).EIMS: 927 (MH⁺).

Compound No. 17:

Compound 16 (140.0 mg, 0.151 mmol) was stirred with 70% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. To this crude were added, amine A (188.2 mg, 0.453 mmol), EDCI (115.9 mg, 0.604 mmol) and DMAP (73.7 mg, 0.604 mmol). Dry dichloromethane (30 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was diluted with dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over silica gel using 3-7% Methanol/Dichloromethane mixture as eluent to give product 17.Yield: 210.0 mg (86.4%). UV-vis (λmax cm⁻¹, dichloromethane): 318, 411, 506, 536, 604 & 661. ¹HNMR (400 MHz, CDCl₃): δ 9.76 (splitted s, 1H, H-5), 9.53 (s, 1H, H-10), 8.63 (s, 1H, H-20), 8.31 (splitted s, 1H, CONH), 6.56 (splitted s, 1H, CONH), 6.00 (m, 1H, CH₃CHOTEG), 5.35 (d, 1H, CH-15¹, J=20.0 Hz), 5.16 (d, 1H, CH-15¹, J=20.0 Hz), 4.53 (q, 1H, H-17, J=7.6 Hz), 4.36 (d, 1H, H-18, J=10.4 Hz), 3.85-3.80 (m, 4H, 2CH₂—O-TEG), 3.74-3.71 (m, 6H, 2CH₂chain, CH₂—O-TEG), 3.67 (s, 3H, 7-CH₃), 3.66 (m, 4H, 8-CH₂CH₃, CH₂—O-TEG), 3.53 (m, 2H, CH₂—O-TEG), 3.42-3.39 (m, 5H, CH₂—O-TEG, 2-CH₃), 3.27-3.26 (m, 6H, 12-CH₃, OCH₃TEG), 2.75 (m, 1H, CH-17²), 2.67 (m, 1H, CH-17²), 2.52 (m, 1H, CH-17¹), 2.24-2.22 (m, 13H, 6CH₂-chain, CH-17¹), 2.14 (d, 3H, CH₃CHOTEG, J=6.8 Hz), 2.09-2.04 (m, 6H, 3CH₂-chain), 1.96 (m, 6H, 3CH₂-chain), 1.81 (d, 3H, 18-CH₃, J=7.2 Hz), 1.71 (t, 3H, 8-CH₂CH₃, J=8.0 Hz), 1.41 (s, 27H, 3CO₂^tBu), 1.33 (s, 27H, 3CO₂^tBu), 0.39 (brs, 1H, NH),-1.79 (brs, 1H, NH).EIMS: 1610 (MH⁺).

Compound No. 18:

Compound 17 (100.0 mg, 0.06 mmol) was stirred with 80% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. To this crude were added, amino-benzyl-DTPA-penta-tert-butyl ester (471.0 mg, 0.60 mmol), EDCI (115.9 mg, 0.60 mmol) and DMAP (73.8 mg, 0.60 mmol). Dry N, N-dimethylformamide (15 ml) was added to it and reaction mixture was stirred at RT for 16 hr under N₂ atm. Reaction mixture was concentrated under vacuum, added dichloromethane (100 ml), washed with brine solution, organic layer separated, dried over sodium sulfate and concentrated. Crude mixture was chromatographed over alumina G (III) using 1-3% Methanol/Dichloromethane mixture as eluent to give product 18. Yield: 250.0 mg (69.0%). UV-vis (λmax cm⁻¹, Dichloromethane): 319, 412, 508, 538, 606 & 661. Elemental analysis: Calculated for C₃₀₉H₄₉₁N₃₁O₇₄: C, 63.72; H, 8.50; N, 7.46.; O, 20.33, found: C, 63.67; H, 8.57; N, 7.46.

Compound No. 19:

Compound 18 (215.0 mg, 0.0369 mmol) was stirred with 80% TFA/DCM (5.0 ml) at RT for 3 hr. Resultant mixture was concentrated and dried under high vacuum to remove trace of TFA. The crude thus obtained was dissolved in pyridine (10 ml) and put under stirring, to this stirring solution GdCl₃.6H₂O (164.6 mg, 0.44 mmol) in 1 ml of water was added slowly and resultant mixture was stirred for 16 hr. Reaction mixture was concentrated to dryness under high vacuum. Residue was washed with water (10 ml×3), acetone (10 ml×3) and finally dried under high vacuum using P₂O₅ as drying agent. Yield: 160.0 mg (85.0%). UV-vis (λmax cm⁻¹, MeOH): 320, 410, 507, 539, 606 & 661. Elemental analysis: Calculated for C₁₉₀H₂₅₃Gd₆N₃₁O₇₄: C, 44.76; H, 5.00; Gd, 18.50; N, 8.52.; O, 23.22, found: C, 44.80; H, 5.07; N, 8.51.

Relaxivity measurements for examples of the compounds as presented herein were acquired on a General Electric 4.7T/33 cm horizontal bore magnet (GE NMR instruments, Fremont, Calif.) incorporating AVANCE digital electronics (Bruker BioSpec platform with ParaVision version 3.0.2 acquisition software, Bruker Medical, Billerica, Mass.).

T1 relaxation rates (R1) were acquired for a range of contrast agent concentrations (0.02 mM to 0.10 mM) with a saturation recovery spin-echo (SE) sequence with a fixed TE=10 ms and TR times ranging from 75 to 8000 ms. Additional MR acquisition parameters are as follows: (FOV) 32×32 mm, slice thickness=1 mm, slices=3, interslice gap=2 mm, matrix=192×192, NEX=1. Signal intensities at each repetition time was obtained by taking the mean intensity within regions of interest (ROI's) using Analyze 5.0 (Biomedical Imaging Resource, Mayo Foundation, Rochester, Minn.), and R₁ and S_MAX were determined by nonlinear fitting of the equation: S_(TR)=S_MAX(1−e^−(R1*^TR))+Background Noise using Matlab's Curve Fitting Toolbox (Matlab 7.0, MathWorks Inc., Natick, Mass.). The T₁ relaxivity was then determined by obtaining the slope of concentration vs. R1 via linear regression fitting. Similarly, T₂ relaxation rates (R₂) were acquired with multi-echo, CPMG SE sequence with a fixed TR of 2500 ms and TE times ranging from 15 to 300 ms, and the number of averages=2. R₂ and S_MAX were determined as described above using the equation: S_(TE)=S_MAX(e^−(R2*^TE))+Background Noise. As before, the T2 relaxivity was then determined by obtaining the slope of concentration vs. R2 via linear regression fitting.

Results are shown in Table 1

Results:

TABLE 1
T1/T2 relaxivity of photosensitizer-Gd(III)DTPA conjugates
	T1 Relaxivity	T2 Relaxivity
Compound	(mM · s)−1	(mM · s)−1
593 (HPPH-3Gd)	3	23.9	62.81
601 (HPPH-6Gd)	15	24.72	66.54
604 (PP-Dibutyl-3Gd)	11	20.11	84.58
611 (Pyro-OTEG-3Gd)	7	13.58	40.11
612 (Pyro-OTEG-6Gd)	19	25.09	69.19

On a T1-weighted scan and at low intratumoral concentrations of the agents (<0.1 mM), shortened T1 times will dominate the effect on signal intensity. All five of the compounds exhibit much higher T1 relaxivity values (Table 1) than conventional MR contrast-enhancing agents, which have T1 relaxivities˜3 to 4 (mM·s)⁻¹. The increased relaxivities of our compounds allow for reduced doses with similar enhancement when compared to conventional compounds. These compounds were tested in vivo (next section) a injection doses of 10 μmoles/kg vs. 100 μmoles/kg prescribed for conventional MR contrast agents.

Examples of compounds of the invention were tested in vivo. Baseline MR images were acquired prior to injection of the compounds to serve as a baseline comparison. Animals were then re-scanned 8 and 24 hours after injection. Two spin-echo imaging protocols were used, all utilizing the same geometry (5-6 axial slices, 1.5 mm slice thickness, 6×6 cm FOV). The first scanning protocol was a moderately T1-weighted scan acquired with a TE/TR of 10/1200 ms. The second protocol was a heavily T1-weighted scan acquired with Chemical Shift Selective (CHESS) fat suppression with a TE/TR of 10/356 ms.

Regions of interest (ROI's) for tumor, muscle, fat, and noise were defined, and the mean intensity and standard deviation for each ROI was sampled. Tumor conspicuity was measured by determining the contrast-to-noise ratios (CNR's), which is defined as the difference in signal between two tissues divided by the standard deviation of the noise. Enhancement of tumor as compared to muscle was determined using the first scanning protocol (TE/TR=10.3/1200 ms). Due to the inherent hyperintensity of fat on T1-weighted MR scans, enhancement of the tumor as compared to fat was determined by analyzing the fat-suppressed images. Results are outlined in Tables 2 & 3, and sample images show in FIGS. 1A and 1B.

TABLE 2
Tumor to fat contrast/noise ratio of the Photosensitizer-Gd(III) conjugates
Tumor to Fat Contrast to Noise Ratio (CNR)
	CNR
	Base-		CNR Improvement
Compound	line	4 hr	8 hr	24 hr	4 hr	8 hr	24 hr
593 (HPPH-3Gd)-3	10.42	xx	15.88	19.34	xx	54%	86%
601 (HPPH-6Gd)-15	15.61	xx	23.80	22.66	xx	52%	45%
604	5.36	xx	15.78	17.84	xx	194%	233%
(PP-Dibutyl-3Gd)-11
611	6.20	xx	20.30	11.50	xx	227%	85%
(Pyro-OTEG-3Gd)-7
612	9.10	18.60	13.40	11.20	104%	47%	23%
(Pyro-OTEG-6Gd)-19

TABLE 3
Tumor to muscle contrast/noise ratio of the
photosensitizer Gd(III) conjugates
Tumor to Muscle Contrast to Noise Ratio (CNR)
	CNR	CNR Improvement
Compound	Baseline	4 hr	8 hr	24 hr	4 hr	8 hr	24 hr
593-3	8.69	xx	15.31	19.17	xx	66%	96%
601-15	6.36	xx	13.40	18.65	xx	111%	193%
604-11	9.93	xx	10.40	20.48	xx	5%	106%
611-7	9.39	20.00	21.30	14.40	113%	127%	53%
612-19	11.50	16.30	20.90	14.20	42%	82%	23%

Additionally, tumor avidity of each formulation was investigated by comparing the increase in contrast, i.e. difference in normalized signal, between tumor and normal tissues. The HPPH-Gd compounds demonstrated a much higher contrast between tumor and normal tissue than the Pyro-OTEG compounds shown in FIGS. 2 & 3. The PP-Dibutyl compound showed peak contrast at 24 hours. In the Figures, compounds coded as 593=3; 601=15; 604=11, 611=7 and 612=19. FIGS. 2 and 3 show tumor to muscle and tumor to fat contrast of the conjugates.

The relatively low increase in contrast between tumors and normal tissues with the Pyro-OTEG compounds is indicative that the increase in the CNR's is predominately a global increase of signal-to-noise in the images than a preferential accumulation of the compound within the tumor.

HPPH-formulations (593 (3) and 601 (15)) showed a continuation of increasing CNR of tumor to fat and muscle from 8 hours to 24 hours. This is indicative of a longer circulation time of these compounds as compared to the Pyro-OTEG from. Conversely, the Pyro-OTEG formulations (611 (7) & 612 (19)) also showed large increases in CNR at 4 and 8 hours, but then decreased 24 hours later. This is indicative of a much shorter circulation time, which may limit efficacy of PDT treatments if not performed within 8 hours of the administration of the agent. Furthermore, the relative increase in contrast with the Pyro-OTEG compounds [611 (7) & 612 (19)] was much lower than that of the HPPH and PP-Dibutyl compounds (593 (3), 601 (15) & 604 (11)), which is exhibited in FIGS. 2 and 3. The large increases in contrast-to-noise with these compounds is attributed more to a global increase in signal to noise (due to the presence of the agents) than tumor avidity. In “real world” application, an increase in CNR is most effective when the CNR approaches a threshold of detection, which is within the range of CNR=2 to 5 for humans. Due to the bi-functionality of these agents, serving as both PDT agents as well as MR contrast-enhancing agents, the greater tumor avidity of compounds 593 (3), 601 (15), & 604 (11) would be preferred over the Pyro-OTEG compounds so that the effect of the PDT therapy is more specific to the tumor tissue rather than host tissue. However, it is foreseeable that the more rapidly clearing Pyro-OTEG compounds would be seen as beneficial in clinical applications.

Claims

1. A tetrapyrollic photosensitizer compound said tetrapyrollic compound being a chlorin, bacteriochlorin, porphyrin, pyropheophorbide, purpurinimide, or bacteriopurpurinimide having 3 to 6 —CH2CONHphenylCH2CH(N(CH2COOH)2))(CH2N(CH2COOH)(CH2CH2N(CH2COOH)2)) groups or esters thereof or complexes thereof with gadolinium(III).

2. The compound of claim 1 having at least one pendant —CH2CH2CONHC(CH2 CH2CONHphenylCH2CH(N(CH2COOH)2))(CH2N(CH2COOH)(CH2CH2N(CH2COOH)2)))3 group or esters thereof or complexes thereof with gadolinium(III).

3. A compound of the formula:

4. The compound of claim 3 where R9 is —CH2CH2CONHC(CH2CH2CONHphenylCH2CH(N(CH2COOH)2))(CH2N(CH2COOH)(CH2CH2N(CH2COOH)2)))3 group or esters thereof or complexes thereof with gadolinium(III).

5. A compound according to claim 3 having the formula:

6. A compound according to claim 3 having the formula:

7. A compound according to claim 2 having the formula:

8. The compound of claim 3, wherein: R1 is substituted or unsubstituted alkyl; R2 is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or —C(O)Ra, where Ra is substituted or unsubstituted alkyl; R1a and R2a together form a covalent bond; R3 and R4 are each independently substituted or unsubstituted alkyl; R3a and R4a are each independently hydrogen, or together form a covalent bond; R5 is substituted or unsubstituted alkyl; R6 and R6a together form ═O; R7 is azaalkyl, or azaaralkyl; R8 and R8a together form ═O; R9 and R10 are each independently substituted or unsubstituted alkyl; each of R1-R10, when substituted, is substituted with one or more substituents each independently selected from Q, where Q is halo, pseudohalo, haloalkyl, COORb where Rb is hydrogen or alkyl, ORc where Rc is alkyl or aralkyl, NRfRg where Rf and Rg are each independently hydrogen, alkyl or aralkyl, or ═NRh where Rh is aralkyl; each Q is independently unsubstituted or is substituted with one or more substituents each independently selected from Q1, where Q1 is halo, pseudohalo, or haloalkyl.

9. The compound of claim 3, wherein: R1 is unsubstituted alkyl; R2 is substituted or unsubstituted alkyl, unsubstituted alkenyl, or —C(O)Ra, where Ra is unsubstituted alkyl; R1a and R2a together form a covalent bond; R3 and R4 are each independently unsubstituted alkyl; R3a and R4a are each independently hydrogen, or together form a covalent bond; R5 is unsubstituted alkyl; R6 and R6a together form ═O; R7 is azaalkyl, or azaaralkyl; R8 and R8a together form ═O; R10 is unsubstituted alkyl; each of R1-R10, when substituted, is substituted with one or more substituents each independently selected from Q, where Q is halo, pseudohalo, haloalkyl, COORb where Rb is hydrogen or alkyl, ORc where Rc is alkyl or aralkyl, NRfRg where Rf and Rg are each independently hydrogen, alkyl or aralkyl, or ═NRh where Rh is aralkyl; each Q is independently unsubstituted or is substituted with one or more substituents each independently selected from Q1, where Q1 is halo, pseudohalo, or haloalkyl.

10. The compound of claim 3, wherein: R1 is methyl; R1a and R2a together form a covalent bond; R3 is methyl; R4 is ethyl; R3a and R4a are each independently hydrogen, or together form a covalent bond; R5 is methyl; and R10 is methyl.

11. The compound claim 3, wherein: R2 is CH═CH2, CH(OR20)CH3, C(O)Me, C(═NR21)CH3 or CH(NHR21)CH3; where R20 is methyl, butyl, heptyl, dodecyl or 3,5-bis(trifluoromethyl)-benzyl; and R21 is 3,5-bis(trifluoromethyl)benzyl.

12. The compound of claim 3, wherein: R7 is ═NR20, where R20 is methyl, butyl, heptyl, dodecyl or 3,5-bis(trifluoromethyl)-benzyl.

13. A pharmaceutical composition, comprising a compound of claim 1 or a pharmaceutically acceptable derivative thereof in a pharmaceutically acceptable carrier.

14. A pharmaceutical composition, comprising a compound of claim 3 or a pharmaceutically acceptable derivative thereof in a pharmaceutically acceptable carrier.

15. An article of manufacture, comprising packaging material and a compound of claim 1 or a pharmaceutically acceptable derivative thereof contained within the packaging material, wherein the compound or salt thereof is effective in a photodynamic therapy treatment for ameliorating the symptoms of a hyperproliferative disorder; and the packaging material includes a label that indicates that the compound or salt thereof is used in a photodynamic therapy treatment for ameliorating the symptoms of a hyperproliferative disorder.

16. An article of manufacture, comprising packaging material and a compound of claim 3 or a pharmaceutically acceptable derivative thereof contained within the packaging material, wherein the compound or salt thereof is effective in a photodynamic therapy treatment for ameliorating the symptoms of a hyperproliferative disorder; and the packaging material includes a label that indicates that the compound or salt thereof is used in a photodynamic therapy treatment for ameliorating the symptoms of a hyperproliferative disorder.

17. A method for administering a therapy to a target, comprising: (i) administering to a subject the compound of claim 1 or a pharmaceutically acceptable derivative thereof that preferentially associates with the target, and (ii) irradiating the subject with light of a wavelength and total fluence sufficient to produce a therapeutic effect.

18. The method of claim 17, wherein the target is selected from the group consisting of: a vascular endothelial tissue, a neovasculature tissue, a neovasculature tissue present in an eye, an abnormal vascular wall of a tumor, a solid tumor, a tumor of a head, a tumor of a neck, a tumor of an eye, a tumor of a gastrointestinal tract, a tumor of a liver, a tumor of a breast, a tumor of a prostate, a tumors of a lung, a nonsolid tumor, malignant cells of one of a hematopoietic tissue and a lymphoid tissue, lesions in a vascular system, a diseased bone marrow, and diseased cells in which the disease is one of an autoimmune and an inflammatory disease.

19. A method for administering a therapy to a target, comprising: (i) administering to a subject the compound of claim 3 or a pharmaceutically acceptable derivative thereof that preferentially associates with the target, and (ii) irradiating the subject with light of a wavelength and total fluence sufficient to produce a therapeutic effect.

20. The method of claim 19, wherein the target is selected from the group consisting of: a vascular endothelial tissue, a neovasculature tissue, a neovasculature tissue present in an eye, an abnormal vascular wall of a tumor, a solid tumor, a tumor of a head, a tumor of a neck, a tumor of an eye, a tumor of a gastrointestinal tract, a tumor of a liver, a tumor of a breast, a tumor of a prostate, a tumors of a lung, a nonsolid tumor, malignant cells of one of a hematopoietic tissue and a lymphoid tissue, lesions in a vascular system, a diseased bone marrow, and diseased cells in which the disease is one of an autoimmune and an inflammatory disease.

21. The method of claim 17, further comprising the step of allowing sufficient time for any of the compound that is not preferentially associated to the target tissue to clear from non-target tissue of the subject prior to the step of irradiating.

22. A method of photodynamic therapy for treating hyperproliferative tissue in a subject, comprising: (i) administering to the subject the compound of claim 1 or a pharmaceutically acceptable derivative thereof that preferentially associates with the hyperproliferative tissue, and (ii) irradiating the subject with light of a wavelength and fluence sufficient to activate the compound, whereby the hyperproliferative tissue is destroyed or impaired.

23. A method of photodynamic therapy for treating hyperproliferative tissue in a subject, comprising: (i) administering to the subject the compound of claim 3 or a pharmaceutically acceptable derivative thereof that preferentially associates with the hyperproliferative tissue, and (ii) irradiating the subject with light of a wavelength and fluence sufficient to activate the compound, whereby the hyperproliferative tissue is destroyed or impaired.

24. A method for detecting the presence of a hyperproliferative tissue in a subject comprising: (i) administering to the subject a sufficient quantity of the compound of claim 1 or a pharmaceutically acceptable derivative thereof, said compound or pharmaceutically acceptable derivative thereof being fluorescent and that preferentially associates with the hyperproliferative tissue; and (ii) visualizing the compound within the patient by fluorescent spectroscopy.

25. A method for detecting the presence of a hyperproliferative tissue in a subject comprising: (i) administering to the subject a sufficient quantity of the compound of claim 3 or a pharmaceutically acceptable derivative thereof, said compound or pharmaceutically acceptable derivative thereof being fluorescent and that preferentially associates with the hyperproliferative tissue; and (ii) visualizing the compound within the patient by fluorescent spectroscopy.

26. A method for detecting the presence of a hyperproliferative tissue in a subject comprising: (i) administering to the subject a sufficient quantity of the compound of claim 1 or a pharmaceutically acceptable derivative thereof, said compound or pharmaceutically acceptable derivative thereof being magnetically resonant and that preferentially associates with the hyperproliferative tissue; and (ii) visualizing the compound within the patient by MRI imaging.

27. A method for detecting the presence of a hyperproliferative tissue in a subject comprising: (i) administering to the subject a sufficient quantity of the compound of claim 3 or a pharmaceutically acceptable derivative thereof, said compound or pharmaceutically acceptable derivative thereof being magnetically resonant and that preferentially associates with the hyperproliferative tissue; and (ii) visualizing the compound within the patient by MRI imaging.