Synthesis, Spectroscopic and In-vitro Photosensitizing Efficacy of Ketobacteriochlorins Derived from Ring-B and Ring-D Reduced Chlorins via Pinacol-Pinacolone Rearrangement

Abstract

In this report we present a regioselective oxidation of a series bacteriochlorins, which on reacting with either ferric chloride (FeCl₃) or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) yielded the corresponding ring-B or ring-D reduced chlorins. The effect of the number of electron withdrawing groups present at the peripheral position, with or without a fused isocyclic ring (ring-E) did not make any significant difference in regioselective oxidation of the pyrrole rings. However, depending on the nature of substituents, the intermediate bis-dihydroxy bacteriochlorins on subjecting to Pinacol-Pinacolone reaction conditions gave various ketochlorins. The introduction of the keto- group at a particular position in the molecule possibly depends on the stability of the intermediate carbocation species. The newly synthesized bacteriochlorins show strong long-wavelength absorption and produced significant in vitro (Colon26 cells) photosensitizing ability. Among the compounds tested, the bacteriochlorins containing a keto-group at position-7 of ring-B with cleaved 5-member isocyclic ring showed the best efficacy.

Introduction

In developing effective agents for Photodynamic Therapy (PDT) the structure-activity relationship (SAR) and quantitative structure-activity relationship (QSAR) studies have been proven to be extremely useful.^1-3 The Roswell Park Cancer Institute (RPCI) group was the first to investigate the SAR and QSAR studies on a series of the alkyl ether analogs of pyropheophorbide-a (chlorophyll-a derivative) and observed a parabolic relationship between overall lipophilicity and PDT efficacy.^4,5 Among the compounds investigated, 3-(1’-hexyloxyethyl) derivative (HPPH) showed the best PDT efficacy without any significant toxicity and is currently undergoing Phase I/II clinical trials for several indications.^6-8 The SAR approach has also been found useful in developing other systems, e. g., phthalocyanines,⁹-tetra (m-hydroxyphenyl) chlorins,¹⁰ extended porphyrins (texaphyrins),¹¹ porphyceins,¹² purpurinimides^13-15 and bacteriopurpurinimides¹⁶ and other bacteriochlorin analogs.^17,18 The biological studies have indicated that besides overall lipophilicity, the position of the substituents present in the photosensitizer(s) makes a tremendous difference in long-term PDT efficacy.

Bacteriochlorins are a class of tetrapyrrolic system in which two pyrrole rings diagonal to each other are reduced.¹⁹ These chromophores exhibit long-wavelength absorption in the range of 720-800 nm depending on the nature of substituents present at the peripheral positions of the molecules. In recent years enormous interest has been generated due to the utility of bacteriochlorins in the bacterial photosynthetic reaction center²⁰ and in the treatment of cancer by photodynamic therapy (PDT). There are several bacteriochlorophyll-a analogs, which are currently under advanced human clinical trials (e.g. Tookad)²¹ or at the advanced preclinical studies (bacteriopurpurinimides) for the treatment of cancer.²² The only naturally occurring bacteriochlorin that is not involved in photosynthesis is the tolyporphyrin isolated by Prinsep et. al. from the blue-green alga Tolypothrix nodosa.²³ This compound, which enhances the cytotoxicity of adriamycin or viablastine in SK-VLB cells at doses as low as 1mg/ml, is characterized as a multidrug resistance (MDR) reversing agent. Kishi's group at Harvard University synthesized the tolyporphyrin by the extension of the Eschenmoser sulfide contraction/iminoester cyclization method with long wavelength absorption near 675 nm (ε = 22,000)²⁴. However, there are no reports regarding the in vivo photosensitizing ability of this novel compound. Lindsey and coworkers²⁵ have also reported facile syntheses of certain bacteriochlorins starting from pyrroles with variable lipophilicity following multistep synthetic methodologies. Some of these compounds exhibit interesting photophysical properties and could be potential candidates for PDT.

One of the simplest methods that has been used extensively for the conversion of porphyrins/chlorins to the corresponding vic-dihydroxy chlorins and bacteriochlorins respectively is the osmium tetroxide mediated oxidation.²⁶ These intermediate “diols” on reacting under acidic conditions produce the corresponding keto-chlorins and keto-bacteriochlorins respectively. The position of the keto- group in the resulting chlorins and bacteriochlorins depends on the stability of the intermediate carbocation, which is also influenced by the nature of the substituents (electron withdrawing or electron donating) present in the molecules (Scheme 1).

Scheme 1.

Conversion of chlorins to ketobacteriochlorins

Chang and Sotiriou²⁷ were the first to show that free-base octaethylchlorin or its metalated analog upon reaction with osmium tetroxide, can be converted into the corresponding ketobacteriochlorins in a reasonable yield. Bonnett et al.²⁸ employed this approach to prepare bacteriochlorin diols by reacting ketoethyl chlorin with osmium tetroxide. Some of these analogs showed considerable PDT efficacy in vitro. Pandey et. al. in collaboration with Smith and coworkers extended this approach to the pyropheophorbide-a, purpurin-18 and purpurinimide systems and a series of stable keto-bacteriochlorin analogs were synthesized.²⁹ Most of the resulting bacteriochlorins showed long-wavelength absorption in the range of 730-800 nm and some of them were effective both in vitro and in vivo.

So far, most of the ketobacteriochlorins [keto- group is present either at position-7 or position-8 (ring-B)] investigated for PDT efficacy are derived from ring-D reduced chlorins³⁰. The main objectives of the work presented herein were (i) to develop an efficient synthetic approach for ring-B reduced chlorins from certain selected bacteriochlorins derived from naturally occurring bacteriochlorophyll-a and then convert them into the corresponding ring-D ketobacteriochlorins (17- keto and 18 keto-), and (ii) to compare the photophysical and photosensitizing abilities of these novel structures.

Results and Discussion

For our present study, two types of bacteriochlorins 6 and 13 containing either a fused five-member isocyclic ring system or a methoxycarbonyl group present at position-13 (ring–C pyrrole) were used as a substrate and these were obtained from bacteriochlorophyll-a by following the literature procedure.³¹ We have recently reported an efficient regioselective preparation of ring-Band ring-D reduced chlorins from bacteriochlorins.³² We extended this approach for bacteriopyropheophorbide-a, which on treating with DDQ at room temperature produced mainly D-ring reduced chlorin 5, whereas on treating 6 with ferric chloride (FeCl₃) produced ring-B reduced chlorin 7 in excellent yields (Scheme 2). Analysis of the NMR data confirmed the structures of both isomers. One of the distinct differences was the resonances of the –CH₂ protons of the five member fused isocyclic ring, which showed an ABX pattern at δ 5.22 ppm in compound 5 due to reduced ring-D, whereas these protons appeared as a singlet at δ 5.44 ppm in compound 7 (ring-D oxidized).

Scheme 2.

Regioselective synthesis of ring-D and ring-B chlorins (5 and 7 respectively) from bacteriochlorin 6.

For determining the photophysical and photosensitizing abilities of various ketobacteriochlorin isomers, the keto group was regioselectively introduced either in the ring-B or ring D pyrrole ring of the bacteriochlorin system. For the synthesis of the desired analogs, the ring-D and ring-B reduced chlorins 5 and 7 were individually reacted with OsO₄ and the resulting diols 8 and 10 (both as isomeric mixtures, cis-diol up and cis-diol down with respect to the diagonal reduced ring) were isolated in 60% yield. Reaction of these diols independently with concentrated sulfuric acid under Pinacol-Pinacolone conditions produced some interesting results. For example diol 8 gave 7-ketobacteriochlorin 9 as a major and 9a as a minor product and it's exactly matched with the previously reported procedure^26c whereas diol 10 under similar reaction conditions gave mainly 18-ketobacteriochlorin 11. These results suggest that the formation of intermediate carbocation species is certainly directing the position of the keto- group, which is possibly is also being influenced by the electron withdrawing acetyl group²⁶ present at position-3 of the 7,8- and 17,18-dihydroxybacteriochlorins (Scheme 3).

Scheme 3.

Synthesis of ring-B and ring-D reduced ketobacteriochlorins from ring-B and ring-D reduced chlorins.

For investigating the effect of the variable number of electron withdrawing groups in bacteriochlorin diols under Pinacol-Pinacolone rearrangement conditions, the isocyclic ring in methyl bacteriopheophorbide-a 12 was cleaved on reacting with sodium methoxide and the resulting bacteriochlorin 13 was obtained in excellent yield.³³ Subsequent treatment of 13 with collidine at refluxing temperature gave rhodobacteriochlorin 14 as the minor (15%) and the corresponding ring-B reduced chlorin 15 as the major product (85%), which on reacting with osmium tetraoxide/pyridine yielded 17, 18-bis-dihydroxybacteriochlorin diol 16 as an isomeric mixture (cis-hydroxy groups up or down relative to ring-B). Further treatment of 16 with concentrated sulfuric acid gave 18-keto bacteriochlorin 17 as a major product (Scheme 4). As expected, the oxidation of bacteriochlorin 13 with DDQ and FeCl₃ afforded the corresponding ring-D and ring-B reduced chlorins 18 and 22 respectively, which on reacting with osmium tetroxide produced the corresponding diols 19 and 23 in quantitative yields. Interestingly, further reaction of these diols with conc. sulfuric acid gave a mixture of 7-ketobacteriochlorin 20 and 8-ketobacteriochlorin 21 (from 19) as an isomeric mixture. Separation of these mixtures (20/21) by usual chromatographic technique was not successful. Finally, the isomeric mixture was separated by HPLC (column: Luna, eluting solvent: using ethyl acetate and hexane as the eluting solvents) delivered the pure regioisomer 8-ketoisomer 20 and regioisomer 7-ketoisomer 21 were isolated in 19% and 25% respectively. Whereas diol 23 in which the cis-hydroxyl groups were present in ring-D yielded only 18-keto bacteriochlorin 17 (in which the β-ketoester functionality present at position-15 was cleaved) as a major product. The elimination of β-ketoester functionality was not observed on treating 19 under similar reaction conditions, and these results were surprising (Scheme 5). The results presented herein confirms our previous findings³⁴, which suggests that the position and presence of the number of electron withdrawing groups present at the peripheral position of chlorin diols makes a significant impact in the stability of the intermediate carbocation during the Pinacol-Pinacolone reaction, which obviously determines the formation of the resulting keto-chlorins under acid catalyzed rearrangement. Therefore in our study to investigate the effect of more than two electron withdrawing groups under acid catalyzed conditions, methyl bacteriopheophorbide-a 12 containing a methoxycarbonyl group at position-13²-of the fused isocyclic ring was used as a substrate, which on reacting with DDQ and FeCl₃ produced exclusively the ring-D reduced 24 and ring-B reduced chlorins 25 respectively (Scheme 6). Unfortunately, the corresponding bacteriochlorin diols obtained by reacting 24 and 25 with OsO₄/pyridine as such or under acidic conditions at room temperature were not stable and produced complex mixtures, which were not characterized.

Scheme 4.

Synthesis of rhodobacteriochlorin 14 and the corresponding 18-keto-bacteriochlorin 16.

Scheme 5.

Synthesis of 7-keto and 8-keto-ring-D reduced and 18-keto-ring-B reduced bacteriochlorins.

Scheme 6.

Ring-B and ring-D reduced chlorins derived from methyl bacteriopheophorbide-a on reacting with OSO₄/H₂SO₄ gave complex mixture.

The mechanism of the formation of keochlorins under Pinacol-Pinacolone reaction is well established. However the compounds investigated in this study shows some interesting results and mechanism for the formation of these analogs is illustrated in Scheme 7. In brief, in vic-dihydroxy analog containing methyl- and ethyl group present at adjacent positions, the migration of methyl group was preferred over the ethyl group and the ethyl group migrated product was isolated in a minor quantity. Interestingly, the vic-dihydroxy analogs containing methyl and propionic ester functionality at adjacent positions on treating under similar acidic conditions gave only the methyl migrated product (18-keto-), which of course depend upon the stability of intermediate carbocation species. The previous studies from others^26c and our own laboratory³⁴ suggest that in porphyrin system the number of electron withdrawing group present at the peripheral position of the porphyrin skeleton makes a remarkable difference in the stability of intermediate cabocation(s), which dictates the formation of corresponding keto-analogs.

Scheme 7.

The proposed mechanism for the formation ring-B and ring D reduced ketobacteriochlorins obtained from their corresponding diols under Pinacol-Pinacolone reaction conditions.

The purity of ketobacteriochlorins 11, 17, 20 and 21 was ascertained by HPLC and the structures were assigned by NMR and mass spectrometry analyses. The bacteriochlorins 20 and 21, which were initially isolated as isomeric mixture, were separated into individual isomers by HPLC using Luna column, eluted with 30% Ethyl Acetate-hexane. The retention time for bacteriochlorins 11, 17, 20 and 21 was 12.20, 10.76, 21.13 and 18.47 min respectively (figure 1). A slight shoulder in HPLC chromatogram of 21 could be due to repeated use of the HPLC column for a long time. The purity of the product was also confirmed by using a reverse phase HPLC column (Symmetry C18 column, dimensions 4.6 × 150 mm), eluted with 90%CH₃OH and 10% H₂O and flow rate was adjusted to 1.0 mL/min. For details see “Supporting Material Information”.

Figure 1.

HPLC chromatogram of bacteriochlorin 11, 17, 20 and 21 (for details see the Supporting Material Information)

A detailed NMR study confirmed the structures of the proposed bacteriochlorins. As can be seen from the results summarized in Figure 2 (only partial NMR Spectra are shown) the meso-protons at position 5, 10 and 20 for bacteriochlorins 11, 17, 20 and 21 show a significant shift and which could be due to variation in electron density at these positions. Further, in 8-keto bacteriochlorin 20 the 7¹-CH₃ protons appeared as a triplet around δ 0.5 ppm whereas for the other isomer 21, these protons were observed as a multiplet at δ 0.45-0.51 ppm, which could be due to the presence of epimeric mixture. In compound 20 the 7-CH₃ was observed as a singlet at δ 1.82, while in compound 21, it exhibited two singlets around δ 1.90/1.92 ppm (epimeric mixture) for 8-CH₃. The 7-CH₂ and 8-CH₂ in isomer 20 and 21 appeared as nice quartets at δ 2.56 and δ 2.62 ppm respectively. These assignments were further confirmed by 2D NMR studies.

Figure 2.

Partial ¹H-NMR of ketobacteriochlorins 11, 17, 20 and 21 (only meso- regions are shown). For details see the “Experimental” and “Supporting Information” parts of the paper.

In order to confirm the position of dialky groups (methyl/ethyl) or methyl/propionic ester of the major regioisomers, NOESY experiment was performed on isomers 11, 17 and 21. In the case of compound 21, the resonances for the ethyl group at position C-8 were chosen as a starting point for the interpretation of the NOESY results. A peak at δ 2.62 for the CH₂CH₃ protons showed NOE correlation with the adjacent meso- proton at δ 8.97 which is C-10; the –CH₃protons of the ethyl group also showed a strong interaction with the methyl proton which is attached to the same carbon (C-8) as well as adjacent meso-10-H proton. No correlation was observed between the CH₃ protons and the acetyl protons (C-3), which further confirmed the proposed structural assignment for 21. Following a similar approach, the structures of compound 11 and 17 were also established (Figure 3).

Figure 3.

NOE correlations of ketobacteriochlorins 11, 17 and 21.

Spectroscopic Properties of Keto-Bacteriochlorins

The absorption and fluorescence characteristics of ketobacteriochlorins 9, 9a, 11, 17, 20 and 21 as epimeric mixtures were measured in dichloromethane. These bacteriochlorins exhibited the long wavelength absorptions at 725, 714, 715, 731, 737 and 732 nm and fluorescence at 736, 727, 732, 735, 765 and 741 respectively. Among all the analogs, the 18-keto-bacteriochlorin 20 exhibited the largest Stokes shift (28 nm), whereas bacteriochlorin 17 lacking the 5-member ring,but instead, containing a methoxycarbonyl- functionality at position-13, showed a much smaller shift of 4 nm. The observed shift was in the order of 20 > 11 > 9a > 9 > 21 >17 (Figure 4).

Figure 4.

Electronic absorption spectra and fluorescence emission spectra of keto-bacteriochlorins 9, 9a, 11, 17, 20 & 21 at equimolar concentration (1.2 μM in CH₂Cl₂).

In vitro photosensitizing efficacy

The keto-bacteriochlorins 9, 9a, 11, 17 and 20, 21 (as an isomeric mixture, and as individual isomers) were evaluated for in vitro PDT efficacy in Colon26 tumor cells. The cells were incubated with increasing concentrations of the photosensitizers (3-100 nM) for 24 h and were then exposed to variable light doses (0-2 J/cm²) at an appropriate wavelengths corresponding to the long-wavelength absorption of each compound formulated in 17% BCS in PBS. MTT assay³⁵ was performed after 48 h (for details see the Experimental Section). Among the photosensitizers evaluated, the bacteriochlorins 9, 9a containing a keto- group in ring-B, were less effective than bacteriochlorins 11, 17 containing a keto- group in ring-D. However, the photosensitizer containing a fused 5-member isocyclic ring showed lower activity than 17 bearing a –CO₂Me group at position-13. In contrast bacteriochlorins 20 and 21 (as a 43:57 mixture) containing a keto-group in ring-B showed enhanced activity than 11 and 17. To investigate the efficacy of the individual isomer, the isomeric mixture of 20 and 21 was duly separated into individual isomers by HPLC (see Figure 1 and Supporting Information) and the in vitro photosensitizing efficacy of both the isomers was investigated under similar experimental conditions except the cells were exposed to light at the appropriate long-wavelength absorptions of the photosensitizers (bacteriochlorin 20, λ_max: 737 nm and 21, λ_max: 732 nm). Under similar parameters, isomer 21 showed significantly higher efficacy than the isomer 20 (see inset figure 5C). As mentioned earlier, isomer 21 constitutes a major part of the mixture 20/21 (Scheme 5), which could explain the reason for similar PDT efficacy of the mixture as compared to isomer 21.

Figure 5.

(A) In vitro photosensitizing efficacy of bacteriochlorins 9, 9a (containing a keto- group in ring-B), 11 (containing a keto- group in ring-D), 17, and 20/21 isomeric mixture (bearing keto-groups in ring-B) at variable concentrations and light doses. The Colon 26 tumor cells were exposed to light at 1 J/cm² at 24 h post-incubation and the MTT assay was performed after 48 h. (B) In vitro dark toxicity of photosensitizers 9, 9a, 11, 17, 20/21 incubated in Colon26 tumor cells for 24 h, but did not exposed to light. (C) The inset figure shows the PDT efficacy of the separated isomers 20 and 21 at similar concentrations as the parent mixture and exposed to light (dose: 1J/cm²).

The in vitro results depicted in Scheme 6 suggest that besides the position of the keto- group (ring-B vs. ring-D), the nature of the substituents present at the periphery of bacteriochlorin system makes a significant impact in PDT efficacy. However, further study with a series of analogs is required to establish a “true” structure-activity relationship and these studies are currently in progress. Efforts are also underway to investigate a correlation between the cell-uptake, intracellular localization, STAT-3 dimerization³⁶ with in vitro/in vivo PDT efficacy and these results will be published in an appropriate journal.

Conclusion

In summary, the work discussed in this manuscript describes an efficient approach for the synthesis of ring-B and ring-D ring reduced chlorins from naturally occurring bacteriochlorophyll-a, which are otherwise difficult to synthesize. Compared to naturally occurring bacteriochlorins, the keto-bacteriochlorins obtained from the respective chlorins showed enhanced stability with significant in vitro photosensitizing efficacy. The acetyl group present at position-3 of the chlorin systems provides a unique opportunity to alter the overall lipophilicity of the molecules to investigate the effect of such modifications in in vivo clearance and PDT efficacy. An easy access to these molecules should also generate a great interest in developing new supramolecular structures and synthetic models for understanding the bacterial photosynthetic reaction centers.

Experimental Section

All reactions were carried out in heat gun-dried glassware under an atmosphere of nitrogen with magnetic stirring. Thin-layer chromatography (TLC) was done on precoated silica gel GF PE sheets (layer thickness 0.25 mm) and aluminum oxide NF PE sheets. Column chromatography was performed either over Silica Gel 60 (70-230 mesh) or neutral Alumina. In some cases preparative TLC plates were also used for the purification. Solvents were purified as follows: trace amounts of water and oxygen from THF were removed by refluxing over sodium under an inert atmosphere. Dichloromethane was dried over P₂O₅. Anhydrous DMF, triethylamine, pyridine and other common chromatographic solvents were obtained from commercial suppliers and used without further purification. NMR spectra were recorded on a 400 MHz spectrometer. All chemical shifts are reported in parts per million (δ). ¹HNMR (400 MHz) spectra were recorded at room temperature in CDCl₃ or CD₃OD solutions and referenced to residual CHCl₃ (7.26 ppm) or TMS (0.00 ppm). EI-Mass spectra were carried out on a ion-trap mass spectrometer equipped with a pneumatically assisted electrospray ionization source, operating in positive mode. UV-visible spectra were recorded on FT UV-visible spectrophotometer using dichloromethane/THF as solvent. All photophysical experiments were carried out using spectroscopic grade solvents.

HPLC Method

HPLC analysis of final products was carried out using a Waters Delta 600 System consisting of the 600 Controller, 600 Fluid Handling Unit and 2998 Photodiode Array Detector equipped with a phenomenex Luna column, 5 micron particle size, with dimensions 4.6 × 250mm. A gradient mobile phase program was used: starting at 30% ethyl acetate /70% hexane linear gradient to 70% ethyl acetate/30% hexane over 80 minutes; the flow rate was 1.0 ml/min.

Methyl 3-acetyl-17,18-dihydroxybacteriopyropheophorbide-a 10

Compound 7 (50.0 mg, 0.09 mmol) was taken in a round bottom flask(100 ml) and dissolved in 30 ml dry dichloromethane. To this, added OsO₄ (100.0 mg) and pyridine (1.0 ml) and reaction mixture was stirred vigorously at room temperature for 24 hr. Reaction was monitored by UV-vis and TLC. H₂S gas was then bubbled into the reaction mixture for 5 min. and then excess of H₂S was removed by bubbling N₂ gas for another 30 min. Water was added to reaction mixture and then extracted with dichloromethane (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. Crude thus obtained was chromatographed over silica gel using 0.5-1% CH₃OH/CH₂Cl₂ mixture as eluent to give 10 as a mixture of cis-diols (49:51). Yield: 23.0 mg (44 %). ¹HNMR (400 MHz, CDCl₃): δ 9.08 & 9.02 (s, 1H, 5-H), 8.67 & 8.64 (s, 1H, 10-H), 8.30 & 7.96 (s, 1H, 20-H), 5.24 (dd, 1H, 13CHH, J = 20 Hz)), 4.89 (dd, 1H, 13CHH), 4.30 - 4.25 (m, 1H, 8-H), 4.05 & 3.96 (m, 1H, 7-H), 3.45 & 3.43 (s, 3H, CO₂Me), 3.38 & 3.34 (s, 3H, 12-CH₃), 3.04 & 2.97 (s, 3H, 2-CH₃), 2.89 & 2.24 (s, 3H, COCH₃), 2.96-2.88 (m, 2H, 17¹-CH₂), 2.67 - 2.62 (m, 2H, 17²-CH₂), 2.41 - 2.36 (m, 2H, 8-CH₂-CH₃), 2.15 & 2.12 (s, 3H, 18-CH₃), 1.95 & 1.72 (d, 3H, 7-CH_3, J = 7.2 and 7.6 H_z), 1.11 & 1.02 (t, 3H, 8-CH₂CH₃, J = 7.2 and 7.6 Hz ), -0.09 & -0.03 (brs, 1H, NH), -1.26 & -1.23 (brs, 1H, NH) ¹³C NMR (100 MHz, CDCl₃): δ 199.0, 198.9, 196.1, 196.0, 174.2, 174.1, 170.6, 170.7, 164.8, 164.6, 164.2, 164.0, 154.8, 154.6, 146.9, 146.7, 139.6, 139.1, 137.2, 136.9, 136.6, 136.5, 136.4, 136.1, 132.3, 132.2, 130.3, 129.6, 121.7, 120.9, 109.6, 109.4, 99.2, 99.0, 97.9, 97.8, 94.6, 94.4, 84.3, 83.8, 83.75, 83.74, 55.29, 55.28, 51.8, 51.7, 48.6, 48.5, 47.9, 47.8, 33.24, 33.2, 33.1, 33.0, 30.7, 30.1, 30.0, 28.9, 28.8, 23.1, 22.9, 20.9, 20.7, 11.0, 10.8, 10.7, 10.4. EIMS (m/z): 621 (M⁺+ Na). HRMS: Calcd. For C₃₄H₃₉N₄O₆[MH]⁺ : 599.2870. Found: 599.2880.

Methyl 3-acetyl-18-keto-bacteriopyropheophorbide-a 11

Compound 10 (15.0 mg, 0.02 mmol) was taken in a round bottom flask (100 ml) and dissolved in 15 ml conc. H₂SO₄. The reaction mixture was stirred vigorously at room temperature for 30 min and the poured into ice-water. Reaction mixture was then extracted with dichloromethane (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. Crude thus obtained was chromatographed over silica gel using 1-3% CH₃OH/CH₂Cl₂ mixture as eluent to get 11. Yield: 6.0 mg (42.8 %). UV-vis (THF, λ_max (nm),(ε): 715 (9.27 × 10⁴), 650 (2.19 × 10⁴), 518 (2.19 × 10⁴), 432 (1.48 × 10⁵) and 385 (1.39 × 10⁵). ¹HNMR (400 MHz, CDCl₃): δ 9.06 (s, 1H, 5-H), 8.55 (s, 1H, 10-H), 8.51 (s, 1H, 20-H), 5.32 (s, 2H, 13-CH₂), 4.38-4.33 (m, 1H, 7-H), 4.07-4.11 (m, 1H, 8-H), 3.56 (s, 3H, CO₂Me), 3.46 (s, 3H, 12-CH₃), 3.38 (s, 3H, 2-CH₃), 3.18 (s, 3H, COCH₃), 2.86-2.80 (m, 2H, 17¹-CH₂), 2.41 - 2.35 (m, 1H, 8-CHH-CH₃), 2.17 - 2.06 (m, 3H, 17²-CH₂ & 1H of 8-CHH-CH₃), 1.89/1.88 (s, 3H, 17-CH₃), 1.83 (d, 3H, 7-CH₃ J = 6.4Hz), 1.10-1.15 (distorted triplet, 3H, 8-CH₂CH₃), 0.2 (brs, 1H, NH), -0.88 (brs, 1H, NH). ¹³C NMR (100 MHz, CDCl₃): δ 207.7, 199.0, 196.5, 173.1, 165.9, 165.6, 144.9, 141.9, 138.7, 138.2, 138.1, 134.0, 133.8, 128.0, 122.2, 115.0, 112.9, 99.3, 97.6,94.0, 55.0, 54.7, 51.4, 49.2, 47.9, 33.3, 32.4, 30.1, 28.9, 22.8, 22.4, 13.6, 11.5, 10.7; EIMS (m/z): 603 (M⁺+ Na). HRMS: Calcd. For C₃₄H₃₇N₄O₅[MH]⁺ : 581.2764 Found: 581.2770.

Methyl bacteriopheophorbide-a 12

Rhodobacter sphaeroides (containing bacteriochlorophyll a (6)) biomass (~500 gram) was suspended in 1-propanol (2 L) and stirred at room temperature in dark with constant nitrogen bubbling for 12 hours. The blue-green extract was filtered and aqueous 0.5 N HCl (150 to 200 ml) was added to the filtrate. After stirring for 25 minutes, the solution began to turn reddish. The reaction mixture was then diluted with aqueous 5% NaCl (1.5 L) and extracted with dichloromethane. The combined extracts were washed with water, dried and rotavaporated. The residue was precipitated from hexanes to give crude bacteriopheophytin-a 6 (2 g) with purity sufficient to proceed to the next step. Compound 6 was dissolved in aqueous 80% TFA (300 ml) and stirred in the dark under N₂ at 0°C for 2 hours. The solution was then diluted with ice/water (600 ml) and extracted with dichloromethane. The combined organic extracts were washed with water, treated with diazomethane and evaporated to dryness. The crude residue was precipitated from hexanes to obtain the title compound (1.20 g), m. p. 222-224°C. UV-vis (ethyl ether, λ_max nm (ε) : 358 (11.8 × 10⁴), 385 (6.76 × 10⁴), 525 (2.89 × 10⁴), 680 (1.22 × 10⁴), 749 (6.75 × 10⁴); (in CH₂Cl₂): 362 (10.8 × 10⁴), 389 (5.81 × 10⁴), 530 (2.84 × 10⁴), 683 (1.11 × 10⁴), 754 (6.27 × 10⁴). ¹H NMR δ (in CDCl₃): 8.98 (s, 1H, 5-H), 8.49 (s, 1H, 10-H), 8.41 (s, 1H, 20-H), 6.08 (s, 1H, 13²-H), 4.27 (m, 2H, 1H for 7-H, 1H for 8-H), 4.02 (m, 2H, 1H for 17-H, 1H for 18-H), 3.85 (s, 3H, 12-CH₃), 3.59 (s, 3H, 2-CH₃), 3.49 (s, 3H, 13²-COOCH₃), 3.45 (s, 3H, 17-CH₂CH₂ COOCH₃), 3.16 (s, 3H, 3-COCH₃), 2.52 (m, 2H, 17-CH₂CH₂ COOCH₃), 2.34 (m, 2H, 17-CH₂CH₂ COOCH₃), 2.25 (m, 2H, 8-CH₂CH₃), 1.80 (d, J = 7.4Hz, 3H, 7-CH₃), 1.73 (d, J = 7.9Hz, 3H, 18-CH₃), 1.12 (t, J = 7.2Hz, 3H, 8- CH₂CH₃), 0.47 (s, 1H, NH), -0.95 (s, 1H, NH). EIMS (m/z): 626 (M+1). HRMS: C₃₆H₄₁N₄O₆ [MH]⁺ : Calcd 625.3026. Found. 625.3043.

3-Acetyl-bacteriochlorin 15-glyoxilic acid trimethyl ester 13

Methyl bacteriopheophorbide-a 12 (100 mg, 016 mmol) was taken in a round bottom flask (100 ml) and dry THF (30 ml) added. 0.3 ml of NaOMe (25% in CH₃OH) was dissolved in 10 ml of dry THF and added slowly via syringe to the reaction mixture under vigorous stirring. The reaction mixture was stirred at room temperature for 4 hr and was quenched with 5% acetic acid-H₂O and extracted with dichloromethane (100 ml). Organic layer separated, washed with brine, dried over sodium sulfate and concentrated to dryness. Trace of acetic acid was removed under high vacuum. Crude product was re-dissolved in dichloromethane and treated with diazomethane. Reaction mixture was stirred for 10 min and then excess of diazomethane was removed by bubbling N₂ gas. Reaction mixture concentrated and chromatographed over silica gel using 1-3% CH₃OH/dichloromethane gradient as eluent to obtained compound as major product. Slow moving brown-red band on silica. Yield: 40.0 mg (37.2%), m.p. > 260°C (decomp.); UV-vis λ_max (in CH₂Cl₂): 782 nm (ε 5.19 × 10⁴), 748 (10.4 × 10⁴), 543 (3.12 × 10⁴), 410 (5.27 × 10⁴) and 363 (7.61 × 10⁴);.¹HNMR (400 MHz, CDCl₃): δ 9.14 (s, 1H, meso-H), 8.68 (s, 1H, meso-H), 8.54 (s, 1H, meso-H), 4.48 (m, 1H, 17-H), 4.26 (m, 1H, 8-H), 4.22 (m, 1H, 18-H), 4.10 (s, 3H, CO₂Me), 4.07 (m, 1H, 7-H), 3.91 (s, 3H, CO₂Me), 3.53 (s, 3H, CO₂Me), 3.51 (s, 3H, 12-CH₃), 3.44 (s, 3H, 2-CH₃), 3.15 (s, 3H, COCH₃), 2.31 (m, 2H, 17²-CH₂), 2.06-2.00 (m, 3H, 8-CH₂CH₃ & 17¹-CH₂), 1.83 (d, 3H, 7-CH₃, J = 7.2 Hz), 1.76 (d, 3H, 18-CH₃, J = 7.6 Hz), 1.72 (m, 1H, 17¹-CH₂), 1.06 (t, 3H, 8-CH₂CH₃, J = 7.6 Hz), -0.45 (brs, 1H, NH), -0.53 (brs, 1H, NH). EIMS (m/z): 671.3 (M+H). HRMS: Calcd. For C₃₇H₄₂N₄O₈: 670.3002. Found: 670.3030.

Rhodobacteriochlorin 14

The second band from the column. Yield: 9.0 mg (10.2%).UV-vis (CH₂Cl₂, λ_max, nm, (ε): 355.0 (1.05 × 10⁵), 521.0 (2.22 × 10⁴), 760 (8.43 × 10⁴)¹HNMR (400 MHz, CDCl₃): δ 9.58 (s, 1H, meso-H), 9.32 (s, 1H, meso-H), 8.72 (s, 1H, meso-H), 8.65 (s, 1H, meso-H), 4.43-4.33 (m, 3H, 8-H, 17-H, 18-H), 4.31 (s, 3H, CO₂Me), 4.19-4.16 (m, 1H, 7-H), 3.63 (s, 3H, CO₂Me), 3.62 (s, 3H, ring-CH₃), 3.58 (s, 3H, ring-CH₃), 3.20 (s, 3H, COCH₃), 2.67-2.56 (m, 2H, 17²-CH₂), 2.42-2.35 (m, 3H, 8-CH₂CH₃ & 17¹-CHH), 2.13-2.07 (m, 1H, 17¹-CHH), 1.83 (d, 3H, 7-CH₃, J = 4.4 Hz), 1.81 (d, 3H, 18-CH₃, J = 4.4 Hz), 1.11 (t, 3H, 8-CH₂CH₃, J = 7.6 Hz), - 1.39 (brs, 1H, NH), -1.44 (brs, 1H, NH). EIMS (m/z): 584.5 (M⁺). ¹³C NMR (100 MHz, CDCl₃): δ 198.8, 173.8, 168.5, 166.9, 166.0, 164.8, 164.6, 135.6, 135.0, 134.6, 133.9, 133.7, 132.2, 129.7, 119.7, 98.6, 98.5, 97.2, 96.3, 56.8, 54.8, 51.5, 47.6, 47.5, 33.2, 32.1, 30.9, 30.2, 29.6, 23.6, 23.5, 13.6, 13.2, 10.8. HRMS: Calcd. For C₃₄H₄₁N₄O₅[MH]⁺ : 585.3077. Found: 585.3059.

Ring-B reduced chlorin 15

3-Acetyl-bacteriochlorin 15-glyoxilic acid trimethyl ester 13 (100.0 mg, 0.15 mmol) was refluxed in collidine (15 ml) for 30 min. Progress of the reaction was monitored by UV-vis and TLC. After completion, reaction mixture was concentrated to dryness using high vacuum and purified on alumina (G-III) column using dichloromethane-hexane mixture as eluent. Yield: 50.0 mg (57.6%). UV-vis (CH₂Cl₂, λ_max, nm, (ε): 410.0 (8.16 × 10⁴), 509.0 (7.50 × 10³), 683.1 (4.16 × 10⁴) ¹HNMR (400 MHz, CDCl₃): δ 10.50 (s, 1H, meso-H), 9.71 (s, 1H, meso-H), 9.60 (s, 1H, meso-H), 8.95 (s, 1H, meso-H), 4.60 (m, 1H, 8-H), 4.40 (s, 3H, CO₂Me), 4.36 (m, 1H, 7-H), 4.12 (t, 2H, 17¹-CH₂, J = 7.6 Hz), 3.77 (s, 3H, ring-CH₃), 3.75 (s, 3H, ring-CH₃), 3.70 (s, 3H, CO₂Me), 3.35 (s, 3H, ring-CH₃), 3.26 (s, 3H, COCH₃), 3.18 (t, 2H, 17²-CH₂, J = 7.6 Hz), 2.51-2.46 (m, 1H, 8-CHHCH₃), 2.20-2.15 (m, 1H, 8-CHHCH₃), 1.92 (d, 3H, 7-CH₃, J = 7.6 Hz), 1.13 (t, 3H, 8-CH₂CH₃, J = 7.6 Hz), -1.79 (brs, 2H, NH). ¹³C NMR (100 MHz, CDCl₃): δ 198.8, 173.5, 171.5, 168.9, 166.5, 152.7, 151.9, 140.8, 138.7, 138.1, 137.9, 137.3, 137.2, 133.5, 132.1, 130.1, 124.3, 101.9, 100.9, 96.7, 94.9, 57.4, 51.6, 47.8, 37.0, 33.3, 30.2, 29.6, 23.8, 21.9, 14., 13.2, 11.5, 10.8. EIMS (m/z): 583.8 (M⁺+1). HRMS: Calcd. For C₃₄H₃₉N₄O₅[MH]⁺ : 583.2920. Found: 583.2938.

3-Acetyl-17,18-bis hydroxyl-ring-B reduced chlorin 16

Acetyl-chlorin-dimethyl ester 15 (50.0 mg, 0.08 mmol) was taken in a round bottom flask (100 ml) and dissolved in 30 ml dry dichloromethane. To this were added OsO₄ (100.0 mg) and pyridine (1.0 ml) and reaction mixture was stirred vigorously at room temperature for 24 hr. Reaction was monitored UV-vis and TLC. H₂S gas was then bubbled into the reaction mixture for 5 min. and then excess of H₂S was removed by bubbling N₂ gas for 30 min. Added water to reaction mixture and extracted with dichloromethane (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. The crude reaction product thus obtained was chromatographed over silica gel using 0.5-1% CH₃OH/CH₂Cl₂ mixture as eluent to give product as a mixture of cis-diols (44:56). Yield: 30.0 mg (57%). UV-vis λ_max (in CH₂Cl₂) (ε): 358.1 nm (7.88 ×10⁴), 388.1 nm (7.35 ×10⁴), 524 nm (2.49 ×10⁴), 751.9 nm (7.88 ×10⁴) ¹HNMR (400 MHz, CDCl₃): δ 9.72 & 9.69 (s, 1H, meso-H), 9.34 & 9.33 (s, 1H, meso-H), 8.83 & 8.79 (s, 1H, meso-H), 8.71 & 8.64 (s, 1H, meso-H), 4.43-4.36 (m, 1H, 8-H), 4.27 & 4.20 (m, 1H, 7-H), 4.12 & 4.10 (s, 3H, CO₂Me), 3.67 & 3.61 (s, 3H, CO₂Me), 3.49 & 3.36 (s, 3H, ring-CH₃), 3.31 (s, 3H, ring-CH₃), 2.90 & 2.87 (s, 3H, COCH₃), 2.85 (m, 2H, 17¹-CH₂), 2.76 & 2.66 (m, 2H, 17²-CH₂), 2.41 & 2.31 (m, 1H, 8-CHH-CH₃), 2.11 & 2.00 (m, 1H, 8-CHH-CH₃), 2.06 & 1.87 (s, 3H, 18-CH₃), 1.91 & 1.78 (d, 3H, 7-CH₃, J = 7.6 Hz), 1.15 & 1.08 (t, 3H, 8-CH₂CH₃, J = 7.6 Hz), -1.30 & -1.37 (brs, 1H, NH), -1.42 & -1.44 (brs, 1H, NH). ¹³C NMR (100 MHz, CDCl₃): δ 198.7, 198.6, 177.4, 177.3, 175.0, 170, 169.2, 169.1, 166.9, 166.3, 165.0, 163.9, 158.5, 158.4, 154.4, 154.2, 153.0, 143.0, 137.1, 137.0, 135.7,135.6, 135.5, 135.4, 134.9, 134.8, 133.3, 133.4, 120.0,119.9, 99.4, 99.2, 98.5, 98.3, 95.0, 96.1, 94.8 , 94.2, 86.0, 86.4, 84.1, 84.0, 56.9, 57.1, 56.6, 56.5, 52.0, 51.9, 48.1, 47.8, 33.2, 33.1, 30.3, 30.2, 29.9, 30.0, 29.3, 29.2, 25.0, 25.1, 23.5, 23.3, 13.67, 13.64, 13.36, 13.14, 10.69, 10.64; EIMS (m/z): 617 (M⁺+1). HRMS: Calcd. For C₃₄H₄₀N₄O₇[MH]⁺ : 617.2975. Found: 617.2980.

3-Acetyl-18-ketobacteriochlorin 17

Bacteriochlorin diol 16 (25.0 mg, 0.04 mmol) was taken in a round bottom flask (100 ml) and dissolved in 15 ml conc. H₂SO₄. The reaction mixture was stirred vigorously at room temperature for 30 min and then poured into ice-water. Reaction mixture was then extracted with dichloromethane (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. Crude thus obtained was chromatographed over silica gel using 1-3 % CH₃OH/CH₂Cl₂ mixture as eluent to get the product; Yield: 15.0 mg (61.9%). UV-vis (CH₂Cl₂, λ_max, nm, (ε): 403.1(7.68×10⁴), 425.0 (9.10×10⁴), 507.0 (1.17×10⁴), 539.0 (2.72 ×10³), 725.00 (8.70 ×10⁴). ¹HNMR (400 MHz, CDCl₃): δ 10.30 (s, 1H, 5-H), 9.44 (s, 1H, 10-H), 8.88 (s, 1H, 20-H), 8.89 (s, 1H, 15-H), 4.48-4.51 (m, 1H, 7-H), 4.37 (s, 3H, 17-CO₂Me), 4.24-4.28 (m, 1H, 8-H), 3.69 (s, 3H, 13-CO₂Me), 3.68 (s, 3H, 2-CH₃), 3.32 (s, 3H, 12-CH₃), 3.24 (s, 3H, COCH₃), 2.99-2.95 (m, 2H, 17¹-CH₂), 2.49 - 2.39 (m, 2H, 8-CHH-CH₃ & 1H of 17²-CH₂), 2.20 - 2.10 (m, 2H, 8-CHH-CH₃ & 17²-CH₂ ), 1.95/1.97 (singlets, 3H, 17-CH₃), 1.86 (d, 3H, 7-CH₃, J = 7.2 Hz), 1.09-1.15 (m, 3H, 8-CH₂CH₃,), - 1.50 (brs, 1H, NH), -1.6 (brs, 1H, NH). ¹³C NMR (100 MHz, CDCl₃): δ 208.6, 198.7, 173.1, 172.0, 166.5, 166.3, 162.8, 145.2, 136.8, 136.4, 135.1, 132.2, 131.5, 127.9, 123.4, 114.6, 98.6, 97.6, 94.6, 56.7, 53.4, 53.0, 52.2, 51.3, 48.3, 33.3, 30.4, 29.7, 28.8, 23.4, 23.0, 13.9, 12.9, 10.8; EIMS (m/z): 621.3 (M+Na). HRMS: C₃₄H₃₉N₄O₆[MH]⁺, Calcd. 599.2870 Obsd. 599.2882.

3-Aetyl-chlorin-15-glyoxilic acid trimethyl ester 18

3-Acetyl-bacteriochlorin 15-glyoxilic acid trimethyl ester 13 (45 mg, 0.06 mmol) was dissolved in dichloromethane (15 ml). To this mixture was added slowly a CH₂Cl₂ solution of DDQ (30 mg, 0.13 mmol). The resulting mixture was stirred at room temperature for 10 min and washed with water three times. The organic layer was separated and dried over anhydrous Na₂SO₄ and solvent was removed under vacuum. The residue obtained was purified with preparative plates using 2% acetone/dichloromethane.Yield: 35.0 mg (78.6 %). UV-vis (CH₂Cl₂, λ_max, nm, (ε)): 410.9 (8.48 ×10⁴), 504.9 (7.24 ×10³), 546 (8.77 ×10³), 692 (2.79 ×10⁴) ¹H NMR δ (in CDCl₃): 9.94 (s, 1H, 5H), 9.68 (s, 1H, 10H), 8.70 (s, 1H, 20H), 4.66 (dd, 1H, 18 H, J = 6.8 Hz), 4.35 (m, 1H, 17H, J = 7.2 Hz), 4.15 (s, 3H, CO₂Me), 3.90 (s, 3H, CO₂Me), 3.72 (m, 3H, 8 CH₂ +17¹-CH₂), 3.66 (s, 3H, CO₂Me), 3.53 (s, 3H, 12 CH₃), 3.73 (s, 3H, 2 CH₃), 3.25 (s, 3H, 7-CH₃), 3.20 (s, 3H, COCH₃), 2.05- 2.17 (m, 3H, 17²-CH₂ +17¹-CH₂), 1.68 (t, 3H, 8CH₃, J = 7.6 Hz), 1.81 (d, 3H, 18 CH₃, J = 8 Hz), 1.68 (t, 3H, 8CH₃, J = 7.6 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 198.5, 186.4, 173.2, 171.4, 167.4, 166.4, 163.5, 155.4, 150.1, 145.3, 140.6, 139.9, 137.4, 136.7, 135.6, 135.1, 135.0, 131.4, 121.1, 106.6, 105.4, 104.3, 94.7, 53.3, 52.8, 52.1, 51.5, 49.2, 33.2, 31.4, 30.9, 23.2, 19.4, 17.4, 13.4, 13.0, 11.1. EIMS (m/z): 691.1 (M⁺+ Na).HRMS: Calcd. For C₃₇H₄₁N₄O₈[MH]⁺: 669.2924. Found: 669.2916.

3-Acetyl-7,8-dihydroxybacteriochlorin-15-glyoxilic acid trimethyl ester 19

Compound 18 (30.0 mg, 0.04 mmol) was taken in a round bottom flask (100 ml) and dissolved in 30 ml dry dichloromethane (DCM). To this were added OsO₄ (100 mg) and pyridine (1.0 ml) and reaction mixture was stirred vigorously at room temperature for 24 hr. Reaction was monitored UV-vis and TLC. H₂S gas was then bubbled into the reaction mixture for 5 min and then excess of H₂S was removed by bubbling N₂ gas for 30 min. Added water to reaction mixture and extracted with DCM (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. Crude thus obtained was purified with silica gel preparative plates using 5% CH₃OH/CH₂Cl₂ to give 19 as a mixture of cis-diols (38:62). Yield: 27.0 mg (64%). UV-vis (CH₂Cl₂, λ_max, nm, (ε): 761 (7.1 ×10⁴), 529 (3.47 ×10⁴), 386 (9.9 ×10⁴), 358 (1.3 × 10⁵). ¹H NMR δ (in CDCl₃): 9.31 & 9.28 (s, 1H, 5H), 8.95 & 8.93 (s, 1H, 10H), 8.55 & 8.50 (s, 1H, 20H), 4.37-4.39 (m, 1H, 18 H), 4.25-4.26 (m, 1H, 17H), 4.09 & 4.07 (s, 3H, CO₂Me), 3.93 & 3.90 (s, 3H, CO₂Me), 3.72-3.60 (m, 3H, 8 CH₂+17¹-CH₂), 3.56 & 3.53 (s, 3H, CO₂Me), 3.45 & 3.43(s, 3H, 12 CH₃), 3.38 & 3.34 (s, 3H, 2 CH₃), 2.97 & 2.84 (s, 3H, 7 CH₃), 2.26- 2.41 (m, 3H, 17²-CH₂ +17¹-CH₂), 2.10 & 2.22(s, 3H, COCH₃), 1.67 & 1.80 (d, 3H, 18-CH₃, J = 7.6 Hz), 0.68 & 0.86 (t, 3H, 8-CH_3, J = 7.2 and 7.6 Hz), -0.41 & -0.44 (brs, 1H, NH), -0.49 & -0.53 (brs, 1H, NH); ¹³C NMR (100 MHz, CDCl₃): δ 198.2, 198.1, 186.4, 186.3, 173.3, 173.1, 169.7, 168.8, 166.6, 166.5, 166.1, 166.0, 165.8, 165.7, 164.8, 164.3, 163.2, 163.3, 161.5, 160.4, 136.0, 136.1, 135.9, 135.7, 134.3, 133.2, 132.7, 132.4, 132.0, 131.9, 131.5, 131.2, 120.7, 120.1, 108.8, 108.7, 100.3, 100.2, 98.6, 97.1, 96.5, 86.3, 85.8, 84.9, 82.7, 82.4, 53.3, 52.8, 52.1, 52.0, 51.9, 51.7, 51.6, 51.5, 49.1, 49.0, 32.9, 32.8, 31.3, 31.2, 31.0, 30.9, 22.7, 22.6, 20.7, 20.0, 13.8, 13.7, 13.3, 13.2, 12.8, 12.7, 8.3, 8.2; EIMS (m/z): 703 (M+H). HRMS: Calcd. For C₃₇H₄₃N₄O₁₀[MH]⁺ : 703.2979. Found: 703.3000.

3-Acetyl-8-keto- and 3-Acetyl-7-ketobacteriochlorin-15-glyoxilic acid trimethyl ester 20 and 21

Compound 19 (20.0 mg, 0.04 mmol) was taken in a round bottom flask (100 ml) and dissolved in 15 ml conc. H₂SO₄. The reaction mixture was stirred vigorously at room temperature for 30 min and the poured into ice-water. Reaction mixture was then extracted with dichloromethane (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. Crude thus obtained was chromatographed using preparative plates using 50% Ethyl acetate/hexane to give an isomeric mixture of 20 & 21 (43:57). This isomeric mixture was then separated by HPLC using the conditions described above in the experimental sections to yield pure isomer of 20 and 21.

3-Acetyl-8-ketobacteriochlorin-15-glyoxilic acid trimethyl ester (20)

Yield: 3.8mg (19%).UV-vis (CH₂Cl₂, λ_max, nm, (ε): 392.1 (1.24 ×10⁵), 508.0 (1.0 ×10⁴), 546.0 (2.0 ×10³), 737.0 (5.1 ×10⁴). ¹HNMR (400 MHz, CDCl₃): δ 9.45 (s, 1H, 5-H), 9.10 (s, 1H, 10-H), 8.51 (s, 1H, 20-H), 4.43-4.45 (m, 1H, 18-H), 4.21- 4.27 (m, 1H, 17-H), 4.11 (s, 3H, CO₂Me), 3.91(s, 3H, CO₂Me ), 3.56, 3.53 and 3.51 (each s,, 3H, 12-CH₃, 2-CH₃ & CO₂Me), 3.18 (s, 3H, COCH₃), 2.56 (q, 2H, 7-CH₂, J = 8 Hz), 2.36-2.37 (m, 1H, 17¹-CH₂), 2.04- 2.09 (m, 3H, 2H of 17²-CH₂ and 1H of 17¹-CH₂), 1.82 (s, 3H, 7-CH₃), 1.74 (d, 3H, 18-CH₃, J = 7.6 Hz), 0.50 (t, 3H, 7-CH₂CH_3, J = 8 Hz), 0.19 (brs, 1H, NH), 0.13 (brs, 1H, NH). ¹³C NMR (100 MHz, CDCl₃): δ 209.2, 198.0, 185.8, 174.1, 173.2, 168.4, 166.4, 166.1, 163.25, 143.1, 140.39,137.8, 137.5, 137.4, 134.7, 133.8, 129.3, 127.8, 101.3, 98.4, 96.03, 55.1, 53.3, 52.0, 51.6, 49.9, 33.1, 31.5, 31.4, 31.2, 31.04, 30.9, 29.6, 22.6, 22.5, 13.4, 12.9, 12.8, 8.8; EIMS (m/z): 707.3 (M⁺+ Na). HRMS: Calcd. For C₃₇H₄₁N₄O₉[MH]⁺ : Calcd: 685.2874 Found: 685.2890.

3-Acetyl-7-ketobacteriochlorin-15-glyoxilic acid trimethyl ester (21)

Yield: 4.5mg (25%). UV-vis (CH₂Cl₂, λ_max, nm, (ε): 385 (1.19 ×10⁵), 428.0 (1.29 ×10⁵), 511.0 (1.96 ×10⁴), 544.0 (1.08 ×10⁴), 732.0 (9.1 ×10⁴). ¹HNMR (400 MHz, CDCl₃): δ 9.83 (s, 1H, 5-H), 8.97 (s, 1H, 10-H), 8.81 (s, 1H, 20-H), 4.49-4.52 (m, 1H, 18-H), 4.35- 4.37 (m, 1H, 17-H), 4.15 (s, 3H, CO₂Me), 3.97/3.96 (s, 3H, CO₂Me ), 3.58 & 3.54 (s, 9H, 12 CH₃, 2-CH₃ & CO₂Me), 3.29 (s, 3H, COCH₃), 2.62 (q, 2H, 8-CH₂CH₃, J = 7.8 Hz), 2.36-2.37 (m, 1H, 17¹ -CH₂), 2.05- 2.09 (m, 3H, 2H of 17²-CH₂ and 1H of 17¹-CH₂), 1.90/1.92 (s, 3H, 8-CH₃), 1.78-1.80(m, 3H, 18-CH₃), 0.45-0.51 (m, 3H, 8-CH₂CH₃), -1.07 (brs, 1H, NH), -1.01 (brs, 1H, NH). ¹³C NMR (100 MHz, CDCl₃): δ 208.7, 198.1, 198.1, 186.4, 173.0, 169.2, 166.7, 166.3, 163.0, 162.95,162.93 147.7, 139.4, 136.7, 136.6, 135.6, 135.2, 134.6, 134.2 , 133.3, 133.2, 98.9, 98.8, 97.3, 54.6, 53.2, 52.4, 51.6, 48.8, 33.3, 31.8, 31.7, 31.3, 30.5, 29.7, 23.2, 23.0, 22.9, 13.1, 13.0, 8.8, 8.7; EIMS (m/z): 707.3 (M⁺+ Na). HRMS: Calcd. For C₃₇H₄₁N₄O₉[MH]⁺ : Calcd: 685.2874 Found: 685.2852.

3-Acetyl-15-glyoxilic acid-ring-B reduced chlorin trimethyl ester 22

Acetyl-bacteriochlorin 15-glyoxilic acid trimethyl ester 13 (45 mg, 0.06 mmol) was dissolved in dichloromethane (20 ml). To this mixture was added slowly a nitromethane solution of FeCl₃.6H₂O (72 mg, 0.267 mmol). The resulting mixture was stirred at room temperature for 30 min, quenched by addition of 5ml methanol, and washed with water three times. The organic layer was separated and dried over anhydrous Na₂SO₄ and solvent was removed under vacuum. The residue obtained was purified with preparative plates using 2% acetone/dichloromethane Yield: 39.0 mg (87.2%). UV-vis (CH₂Cl₂, λ_max, nm, (ε): 410.9 (8.09 ×10⁴), 512.1 (1.00 ×10⁴), 679 (3.28 ×10⁴). ¹H NMR δ (in CDCl₃): 9.85 (s, 1H, 5H), 9.56 (s, 1H, 10H), 8.90 (s, 1H, 20H), 4.53 (q, 1H, 7-H, J = 24 Hz), 4.35 (m, 1H, 8H), 4.15 (s, 3H, CO₂Me), 3.90 (s, 3H, CO₂Me), 3.80 (s, 3H, CO₂Me), 3.63 (s, 3H, 12 CH₃), 3.52 (s, 3H, 2 CH₃), 3.32 (s, 3H, 18 CH₃), 3.26 (s, 3H, COCH₃), 2.58-2.64 (m, 2H, 17¹-CH₂), 2.43- 2.45 (m, 1H, 8-CHH), 2.10-2.14 (m, 1H, 8-CHH), 1.89 (d, 3H, 7CH₃, J = 6.8 Hz), 1.08 (t, 3H, 8- CH₂CH₃, J = 7.6 Hz), -1.2 (brs, 1H, NH), -1.60 (brs, 1H, NH). ¹³C NMR (100 MHz, CDCl₃): δ 198.4, 189.3, 173.0, 172.0, 169.9, 167.0, 161.8, 149.6, 149.4, 141.8, 138.5, 138.2, 137.8, 137.7, 137.4, 131.0, 130.8, 129.9, 127.5, 113.0, 102.8, 97.8, 96.0, 58.0, 54.0, 53.5, 51.6, 47.1, 35.0, 33.2, 30.0, 23.6, 23.4, 14.0, 12.6, 11.8, 10.8. EIMS (m/z): 691.1 (M⁺+ Na). HRMS: Calcd. For C₃₇H₄₁N₄O₈[MH]⁺: 669.2924. Found: 669.2912.

3-Acetyl-17,18-dihydroxy-15-glyoxilicacid-bacteriochlorin trimethyl ester 23

Compound 22 (40.0 mg, 0.06 mmol) was taken in a round bottom flask (100 ml) and dissolved in 30 ml dry DCM. To this were added OsO₄ (75.0 mg) and pyridine (1.0 ml) and reaction mixture was stirred vigorously at RT for 24 hr. Reaction was monitored by UV-vis and TLC. H₂S gas was then bubbled into the reaction mixture for 5 min. and then excess of H₂S was removed by bubbling N₂ gas for another 30 min. Water was added to reaction mixture and extracted with dichloromethane (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. Crude thus obtained was purified with silica gel preparative plates using 5% MeOH/CH₂Cl₂. Yield: 23 mg (55%). ¹H NMR δ (in CDCl₃): 9.45 & 9.43 (s, 1H, 5H), 8.88 & 8.86 (s, 1H, 10H), 8.75 & 8.73 (s, 1H, 20H), 4.38-4.40 (m, 1H, 8 H), 4.28-4.29 (m, 1H, 7H), 4.16 & 4.15 (s, 3H, CO₂Me), 3.63 & 3.67 (s, 3H, CO₂Me), 3.51 & 3.50 (s, 3H, CO₂Me), 3.43 & 3.42 (s, 3H, 12 CH₃ ), 3.14 & 3.13(s, 3H, 2 CH₃), 3.10 & 3.09 (s, 3H, 18 CH₃), 2.52 (s, 3H, 3COCH₃), 2.31- 2.24 (m, 2H, 17-CH₂), 2.19- 2.24 (m, 2H, 8-CH₂), 2.04- 2.08 (m, 2H, 17-CH₂), 1.73 & 1.89 (d, 3H, 7 CH_3, J = 7.2 and 7.6 Hz), 1.08 (t, 3H, 8CH₃, J = 5.6 Hz), -0.354 & -0.415 (brs, 1H, NH), -0.52 & -0.60 (brs, 1H, NH), ¹³C NMR (100 MHz, CDCl₃): δ 198.3, 198.4, 175.2 (2C ?), 174.4, 174.3, 171.0, 170.9, 170.1, 170.0, 167.2, 167.1, 166.2, 166.1, 160.2, 159.8, 159.5, 159.4, 158.2, 157.8, 153.0, 143.0, 137.4, 137.2, 135.6, 135.8, 134.1, 134.2, 133.6, 133.5, 131.6, 131.4, 131.3,129.8, 115.2, 115.0, 104.7, 104.3, 103.8, 103.9, 95.9, 96.0, 92.7, 92.8, 83.0, 83.2, 57.1, 57.0, 55.0, 52.6, 52.1, 52.0, 51.5, 51.5, 46.8, 47.4, 33.3, 33.5, 28.9, 29.5, 23.4, 23.3, 22.7,22.6, 19.2,19.3, 14.0, 14.1, 13.6, 13.5, 11.9, 11.8, 10.8, 10.7. EIMS (m/z): 725 (M⁺ + Na). HRMS: Calcd. For C₃₇H₄₁N₄O₁₀[M-H]⁺: 701.2823. Found: 701.2855.

3-Acetyl-15-glyoxilic acid-8-ketobacteriochlorin trimethyl ester 17

Compound 23 (15.0 mg, 0.02 mmol) was taken in a round bottom flask (100 ml) and dissolved in 15 ml conc. H₂SO₄. The reaction mixture was stirred vigorously at room temperature for 30 min and then poured into ice-water. Reaction mixture was then extracted with dichloromethane (100 ml), organic layer separated, washed with water, dried over sodium sulfate and concentrated. The residue obtained was purified with silica gel preparative plates using 2% acetone/dichloromethane to give 23 as a mixture of cis-diols (47:53). Yield: 5.5 mg (43%). UV-vis (THF, λ_max, nm, (ε): 403.1(7.68×10⁴), 425.0 (9.10×10⁴), 507.0 (1.17×10⁴), 539.0 (2.72 ×10³), 725.00 (8.70 ×10⁴), ¹HNMR (400 MHz, CDCl₃ δ 10.30 (s, 1H, meso-H), 9.44 (s, 1H, meso-H), 8.88 (s, 1H, meso-H), 4.48-4.51 (m, 1H, 8-H), 4.37 (s, 3H, CO₂Me), 4.24-4.28 (m, 1H, 7-H), 3.69 (s, 3H, CO₂Me), 3.68 (s, 3H, 12CH₃), 3.32 (s, 3H, 2 - CH₃), 3.24 (s, 3H, COCH₃), 2.97-2.91 (m, 2H, 17¹-CH₂), 2.49 - 2.39 (m, 2H, 8-CHH-CH₃ & 1H of 17²-CH₂), 2.09 - 2.15 (m, 2H, 8-CHH-CH₃ & 17²-CH₂ ), 1.96 (d, 3H, 17-CH₃, J = 7.2 Hz), 1.86 (d, 3H, 7-CH₃, J = 7.2 Hz), 1.09-1.15 (m, 3H, 8-CH₂CH₃), 1.50 (brs, 1H, NH), -1.6 (brs, 1H, NH). EIMS (m/z): 621.3 (M+Na).

Ring-D reduced methyl pheophorbide-a 24

Methyl bacteriopheophorbide 12 (20 mg, 0.032 mmol) was dissolved in dichloromethane (5 ml) under Ar atm. To this mixture, a solution of DDQ (5.9 mg, 0.026 mmol) in dry CH₂Cl₂ was added slowly. The resulting mixture was stirred at room temperature for 5 min and the entire reaction mixture was washed with water three times. The organic layer was separated and dried over anhydrous Na₂SO₄ and solvent was removed under vacuum. The residue obtained was purified with preparative plates using 3% acetone/ DCM affording 17 mg of the methyl pheophorbide-a. Yield: 17.0 mg (85%). UV-vis (CH₂Cl₂, λ_max, nm, (ε): 691 (3.45 ×10⁴). ¹H NMR δ (in CDCl₃): 9.95 (s, 1H, 5-H), 9.61 (s, 1H, 10-H), 8.77 (s, 1H, 20-H), 6.31 (s, 1H, 13²-H), 4.53 (m, 1H for 18-H), 4.25 (m, 1H for 17-H), 3.89 (s, 3H, 12-CH₃), 3.72 (s, 3H, 2-CH₃ ), 3.63 (s, 3H, 13²-COOCH₃), 3.57 (s, 3H, 17-CH₂CH₂COOCH₃), 3.27 (s, 6H, 7-CH₃ and 3-COCH₃), 2.62 (m, 2H, 17-CH₂CH₂COOCH₃), 2.28 (m, 4H, 2H of 17-CH₂CH₂COOCH₃, 2H of 8-CH₂CH₃), 1.83 (d, J = 7.6Hz, 3H, 18-CH₃), 1.70 (t, J = 7.2Hz, 3H, 8- CH₂CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 199.2, 189.5, 173.2, 169.4, 162.0, 153.2, 151.9, 148.8, 145.0, 139.1, 137.6, 135.9, 135.6, 134.4, 130.4, 129.8, 105.7, 104.0, 100.7, 94.2, 64.8, 52.9, 51.6, 51.4, 49.8, 33.4, 31.0, 29.8, 23.2, 19.4, 17.3, 13.8, 13.4, 12.2, 11.4,11.2; EIMS (m/z): 623 (M+1). HRMS: C₃₆H₃₉N₄O₆[MH]⁺ , Calcd 623.2870. Found: 623.2864.

Ring-B reduced methyl pheophorbide-a 25

Methyl bacteriopheophorbide 12 (20 mg, 0.032 mmol) was dissolved in dichloromethane (20 ml). To this mixture, a solution of FeCl₃.6H₂O (24 mg, 0.089 mmol) in nitromethane was added slowly. The resulting mixture was stirred at room temperature for 30 min, quenched by addition of 5 ml methanol, and washed with water three times. The organic layer was separated and dried over anhydrous Na₂SO₄ and solvent was removed under vacuum. The residue obtained was purified with silica gel preparative plates using 2% acetone/dichloromethane.Yield: 16.0 mg (80%). UV-vis (CH₂Cl₂, λ_max, nm, (ε): 682 (3.65 ×10⁴). ¹H NMR δ (in CDCl₃): 9.51 (s, 1H, 5-H), 9.35 (s, 1H, 10-H), 8.77 (s, 1H, 20-H), 6.67 (s, 1H, 13²-H), 4.53 (m, 1H for 7-H), 4.30 (m, 1H for 8-H), 3.91 (m, 2H, 17-CH₂CH₂COOCH₃), 3.81 (s, 3H, 12-CH₃), 3.72 (m, 6H, 3H of 18-CH₃ and 3H of 2-CH₃), 3.60 (s, 3H, 13²-COOCH₃), 3.29 (s, 3H, 17-CH₂CH₂COOCH₃), 3.23 (s, 3H, 3-COCH₃), 2.90 (m, 2H of 8-CH₂CH₃), 2.08 (m, 2H, 17-CH₂CH₂COOCH₃), 1.88 (d, J = 7.2Hz, 3H, 18-CH₃), 1.15 (t, J = 7.2Hz, 3H, 8-CH₂CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 203.3, 189, 172.9, 169.7, 166.4, 159.2, 155.4, 141.3, 140.2, 138.4, 133.3, 132.2, 127.9, 114.6, 113.3, 114.6, 100.1, 96.9, 96.2, 96.1, 66.2, 55.8, 53.1, 51.7, 48.3, 35.6, 33.3, 30.1, 29.2, 23.3, 22.1, 13.8, 11.9, 11.4, 10.8, 10.7; EIMS (m/z): 623 (M+1). HRMS: C₃₆H₃₉N₄O₆[MH]⁺ , Calcd 623.2870. Found: 623.2828.

In vitro photosensitizing efficacy

The photosensitizing activity of the compounds were determined as described before³⁰. The tumor cell lines used are Colon 26 (Mouse colon tumor). The Colon-26 tumor cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin. Tumor cells were maintained in an atmosphere of 5% CO₂, 95% air and 100% humidity at 37°C. For determining the PDT efficacy of the compounds, the cells were plated in 96 well plates at a cell density of 3000 cell/well in complete media. After 16 hrs of incubation at 37°C, the photosensitizers were added at variable concentrations and incubated at 37 °C for 24 hr in the dark. Prior to light treatment, the cells were replaced with drug-free complete media. Cells were then illuminated with light from an argon-pumped dye laser set at 725-741 nm respectively for each of the drugs as per their absorption wavelength in 17% bovine calf serum (BCS) measured before, at a dose rate of 1.6 mW/cm² for 0-2 J/cm². After PDT the cells were incubated for a further 48 h at 37 °C in the dark. Following the 48h incubation, 10 μL of 5.0 mg/mL solution of 3-[4,5-dimethylthiazol-2-yl]-2-5-diphenyltetrazoliumbromide (MTT) in PBS (Sigma, St. Louis, MO) was added to each well. After 4 h incubation at 37°C, the MTT and the media were removed, and 100 μL of DMSO was added to solubilize the formazan crystals. The 96- well plate was read on a microtiter plate reader (BioTek Instruments, Inc., ELx800 Absorbance Microplate Reader) at an absorbance of 570 nm. The results were plotted as a percent survival of the corresponding dark (drug no light) control for each compound tested. Each data point represents the mean from two separate experiments, with 6 replicate wells and the error bars are the standard deviation.