Synthesis, isolation and characterization
The target water-soluble FP-adducts and their precursor porphyrins were synthesized through a multi-step synthetic pathway as outlined in Figure S1. The initial synthetic step involved a one-pot three-component reaction involving methyl-4-formylbenzoate (3.2 eq), 4-hydroxybenzaldehyde (1.8 eq) and pyrrole (4 eq) resulting in the formation of the A4, A3B, cis-A2B2, trans-A2B2, AB3 and B4 porphyrins 5,10,15,20-tetra-(4-methoxycarbonylphenyl)porphyrin (PBe4), 5,10,15-tri-(4-methoxycarbonylphenyl)-20-(4-hydroxyphenyl) porphyrin (PBe3OH), 5,10-di-(4-methoxycarbonylphenyl)-15,20-di-(4-hydroxyphenyl) porphyrin (c-PBe2(OH)2), 5,15-di-(4-methoxycarbonylphenyl)-10,20-di-(4-hydroxyphenyl) porphyrin (t-PBe2(OH)2) and 5-(4-methoxycarbonylphenyl)-10,15,20-tri-(4-hydroxyphenyl) porphyrin (PBe(OH)3) and 5,10,15,20-tetra-(4-methoxycarbonylphenyl)porphyrin (TCPP) respectively. The stoichiometry was chosen to obtain a higher yield of the A3B porphyrin. While TLC indicated the formation of all six porphyrins, isolation and purification of only PBe4, PBe3OH, c-PBe2(OH)2, t-PBe2(OH)2 and PBe(OH)3 was possible with appreciable yields. The compounds were isolated through gravity percolation column chromatography, silica gel 60–200 mesh was used as the stationary phase, while the mobile phase employed was either DCM or differing percentages (1% − 5%) of MeOH in DCM. Wherever required, column chromatography was run multiple times to obtain pure compounds. TCPP, with four highly polar meso-carboxyphenyl groups, did not elute out in a significant amount. It was synthesized later through base hydrolysis of PBe4. The yield of the macrocycles PBe4, PBe3OH, c-PBe2(OH)2, t-PBe2(OH)2 and PBe(OH)3 were calculated to be 8%, 8%, 6%, 2% and 7% respectively. The isolated porphyrins were characterized by 1HNMR, 13CNMR, MALDI-TOF, UV-Vis, Emission and Fluorescence Lifetime spectroscopic techniques, whichever is applicable. The 1HNMR and MALDI-TOF data are fundamental for differentiation between the isolated porphyrins. The effect of the porphyrin ring diamagnetic anisotropy results in a large spread of 1HNMR resonances with the inner NH resonances being shifted to around − 2.6 ppm, whereas the meso-substituted protons are shifted further downfield with, the deshielding being higher for meso-substituents with electron withdrawing groups, the β-pyrrolic protons at the periphery of the macrocyclic ring are also downshifted.51,52 The observed β-pyrrole splitting patterns (Table-S1) for the synthesized compounds are consistent with AB3/AB3, cis-A2B2, trans-ABAB systems as reported in literature.4,5,47,53,54 As can be seen in the 1HNMR spectral data (Figures S4, S6-S8, SI), the pyrrolic protons are split into doublet/singlet/doublet, doublet/singlet/singlet/doublet and doublet/doublet corresponding to the differing meso-substitution patterns. The porphyrins consistently show a negative peak at the far right, beyond the signal for TMS; the specific peak can be attributed to the highly shielded inner NH protons. Apart from that, the -OCH3 peak is consistently seen around 4.1 ppm in the recorded spectral data. The aromatic phenyl ring protons resonated between 8.4 and 7.4 ppm. The multiplicity, proton integration and spin-spin coupling constant for each compound is summarized in Table S1. The 1HNMR spectral data and the mass values (Figure S13-S16, SI) obtained through MALDI-TOF spectroscopy are indicative of the purity of the synthesized porphyrin macrocycles.
The UV-Vis spectra of PBe3OH, c-PBe2(OH)2, t-PBe2(OH)2 and PBe(OH)3, recorded in DMF conform to the D2h micro symmetry in accordance with the Four-Orbital Model suggested by Gouterman.55 The spectra of the porphyrin macrocycles consistently show an intense absorption near 420 nm (Soret Band) and much less intense Q-band absorption between 500 and 700 nm (Table S2). Interestingly, with an increase in the number of more polar meso-hydroxyphenyl moieties, a slight red shift in Soret band absorption is observed (Figure S2A). The Q-bands show subtle differences indicating variations in the skeletal structure of the compounds; however, no specific trend can be attributed to the structural differences. The emission spectra of the porphyrins PBe4, c-PBe2(OH)2, and t-PBe2(OH)2 and recorded in DMF exhibit twin emission peaks (Table S2, Figure S2B) corresponding to the Q(0,0) and Q(0,1) emission band, expected for a free base system. In addition to the twin emissions, PBe3OH and PBe(OH)3, however, exhibit a low-intensity emission band centred at 645 and 616 nm respectively, this could be attributed to a Q (1,0) transition. The solutions were excited at the absorption maxima of their respective Soret bands.
The compounds PBe4, PBe3OH, c-PBe2(OH)2, t-PBe2(OH)2, PBe(OH)3 were hydrolysed (Figure S1) to obtain tetra-(4-carboxyphenyl)porphyrin (TCPP), 5,10,15-tri-(4-carboxyphenyl)-20-(4-hydroxyphenyl) porphyrin (PB3OH), 5,10-di-(4- carboxyphenyl)-15,20-di-(4-hydroxyphenyl) porphyrin (c-PB2(OH)2), 5,15-di-(4- carboxyphenyl)-10,20-di-(4-hydroxyphenyl) porphyrin (t-PB2(OH)2), and 5-(4- carboxyphenyl)-10,15,20-tri-(4-hydroxyphenyl) porphyrin (PB(OH)3), bearing combinations of meso-(4-hydroxyphenyl) and meso-(4-carboxyphenyl) moieties (Fig. 1). In brief, the weighed-out amount of the ester precursors was treated with crushed NaOH in DMF, for 30 minutes, followed by the addition of water. The pH of the reaction mixture was adjusted to 4.5 when the desired product precipitated out. The compounds were recovered by centrifugation. TCPP, PB3OH, c-PB2(OH)2, t-PB2(OH)2 and PB(OH)3 were obtained in 78%, 85%, 98%, 78%, and 82% yield, respectively.
The detailed analysis of 1HNMR spectral plots (Figures S9-S12, SI) of PB3OH, c-PB2(OH)2, t-PB2(OH)2 and PB(OH)3 is shown in Table S3. The data conforms to the splitting pattern expected of a cis-A2B2, trans-A2B2 and AB3 system.4,5,47,53,54 The β-pyrrole protons resonate as a multiplet centred at 8.88 ppm for c-PB2(OH)2, for t-PB2(OH)2 and PB(OH)3 however, the protons are split into two doublets (centered at 8.81 and 8.91 ppm) and a doublet-singlet-doublet (at 8.79 (J = 2.4 Hz,)-8.82 (s)-9.95 (J = 6.1 Hz) ppm) respectively. The aromatic phenyl protons of the compounds resonated between 7.0 to 8.5 ppm as outlined in Table S3.
The peak for the labile protons of -COOH and -OH groups could not be detected for compounds c-PB2(OH)2 and PB(OH)3, possibly as a result of deuterium exchange. All three compounds c-PB2(OH)2, t-PB2(OH)2, and PB(OH)3 show the inner pyrrolic proton resonances at −2.87, −2.91 and −2.88 ppm respectively. MALDI-TOF data lent further support in favour of the molecular structure of the compounds, the theoretical values of mass conform well with the experimental values (Figures S17-S20, SI). The UV-Vis and emission spectral data for c-PB2(OH)2, t-PB2(OH)2, PB(OH)3, PB3OH and TCPP, corresponding extinction coefficient and Stokes shifts are included in detail in Table S4. The UV-Vis spectral plot (Figure S3 A) of the compounds exhibits the intense Soret band and Q-bands expected of the porphyrinic system.55 The Soret band maxima of t-PB2(OH)2 is red shifted to 426 nm as compared to that of the other compounds. Not-so-significant redshifts in the Soret band maxima are also seen in PB3OH, c-PB2(OH))2, and PB(OH)3 as compared to that of TCPP The Q-band absorptions show progressive redshifts as the number of meso-hydroxyphenyl substituents increases in the porphyrin macrocycle, the red shift however does not follow any regular pattern. The Qx(0,0) band of c-PB2(OH)2, and PB(OH)3 shows strong redshift with absorption values of 690 and 684 nm respectively. It may be because the electron-donating capability of the hydroxyphenyl moieties boosts the aromatic ring current of the porphyrin macrocycle thereby bringing about a change in the (π, π*) transitions.
The emission spectra (Figure S3B and Table S4) of PB3OH and TCPP exhibit twin emission peaks corresponding to Q(0,0) and Q(0,1) transitions. However, for c-PB2(OH))2, t-PB2(OH))2 and PB(OH)3, triple emission peaks were observed at 612, 652, 710 nm, 612, 653, 708 nm and 614, 657, 710 nm corresponding to Q(1,0), Q(0,0) and Q(0,1) bands.56
The fluorescence quantum yields (\({\varPhi }_{f}\)) of the target hydrophilic compounds (Table 1) were determined at the same concentration (10 µM) using TCPP in ethanol (\({\varPhi }_{f}=0.044)\) as the standard. The following equation was used to determine the \({\varPhi }_{f}\) values:
$${\varPhi }_{f}=\frac{I{{(1-10}^{-A})}_{std}{\eta }^{2}}{{I}_{std}{(1-10}^{-A}){\eta }_{std}^{2}}$$
where \({\varPhi }_{f}\), I, A, and η are the fluorescence quantum yield, integral area of fluorescence, absorbance in λexc, and refractive index of the selected solvents (H2O = 1.333 and Ethanol = 1.3614). The subscript “std” refers to the standard molecule.
Table 1
Fluorescence Quantum yield of c-PB2(OH)2, t-PB2(OH)2, PB(OH)3, PB3OH and TCPP recorded in water at a concentration of 10 µM.
|
Fluorescence Quantum Yield
|
Compound
|
\({\varPhi }_{f}\)
|
PB3OH
|
0.044
|
c-PB2(OH)2
|
0.025
|
t-PB2(OH)2
|
0.003
|
PB(OH)3
|
0.001
|
TCPP
|
0.059
|
The compound TCPP exhibited the highest \({\varPhi }_{f}\) value, and as the number of carboxyphenyl groups within the porphyrin moiety decreased, the \({\varPhi }_{f}\) values also showed a progressive decrease.
Anti-HIV studies
In this study, the carboxyphenyl porphyrins tested against HIV-1 virus were TCPP, THPP, PB3OH, c-PB2(OH)2, t-PB2(OH)2 , and PB(OH)3. The reference controls were THPP (tetra-hydroxyphenyl porphyrin) and TCPP (tetra-carboxyphenyl porphyrin). Enfuvirtide (T20), an FDA-approved fusion and entry inhibitor, was used as the positive control.57 The anti-viral studies for these novel compounds were conducted under non-photodynamic (non-PDT) and photodynamic (PDT) conditions.
For the photodynamic therapy, the cells were incubated with serial dilutions of the test compounds before being irradiated for 45 minutes in an irradiation box. The irradiation box had the dimensions of length 20", width 6", and height 8", and it was equipped with two Philips Essential Master PL-L 36W/865/4P linear, compact fluorescent lamps. The surface of a 96-well microplate was deemed to be 15 cm from the light source. 250 Jcm-2 of light was determined to be present at the surface.
a). Carboxyphenyl porphyrins were not toxic to the cells under non-PDT and PDT conditions
The effect of the carboxyphenyl porphyrins on the cell viability was tested on HEK293T and TZM-bl cell lines under non-PDT and PDT conditions by employing the Cell-Titre blue Assay as described in the methods section. The compounds were tested at the concentration ranging from 0.1 µM to 50 µM. We observed that none of the porphyrins were cytotoxic under non-PDT conditions with a CC50 value greater than 30 µM. PB3OH, TCPP, and THPP had a CC50 value lower than 5 µM in the PDT conditions (Table S5). For the HIV-1 subtype C K3016 virus, the EC50 value of these porphyrins was found to be more than 5 µM. They were toxic to the cells at higher concentrations. The compounds c-PB2(OH)2, t-PB2(OH)2, and PB(OH)3 were non-toxic to the cells even at the higher concentrations (CC50 >30 µM). Following this assay, the non-toxic concentrations of the porphyrins (up to 5 µM for non-PDT and 500 nM for PDT conditions) were used to perform the anti-viral studies.
b). Carboxyphenyl porphyrins reduced HIV-1 virus infectivity under non-PDT conditions
The antiviral activity of the carboxyphenyl porphyrins was assessed by determining their effect on HIV-1 gene expression and virus release. HEK-293T cells were transfected with plasmid DNA containing either the HIV-1 subtype B virus (NL4-3) or the HIV-1 subtype C virus (K3016). The cells were then treated with the test compounds at a concentration ranging from 500 nM to 5 µM and incubated at 37 °C for 24 h. HIV-1 p24 ELISA was used to determine the amount of virus released in the supernatant. Both the cells and virus lysates were immunoblotted with the total HIV IgG antibody. The virus release efficiency (%VRE) in the presence of test compounds was compared to the negative control, 5% DMSO in water (-). We observed that incubating cells with the test compounds did not affect viral gene expression and the subsequent virus release from the cells for both subtypes compared to the control (Figure S22). A TZM-bl cell-based single-cycle infectivity assay was used to assess the infectivity of the viruses produced in the presence or absence of porphyrins.58,59 The expression of a luciferase reporter gene under the control of the HIV-1 LTR promoter is used in this assay, which is regarded as a sensitive and quantitative marker of virus infection. The infectivity of HIV-1 subtype B NL4-3 virus or HIV-1 subtype C K3016 virus produced in the presence of 5% DMSO in water (-), in the absence of the carboxyphenyl porphyrins, served as the treatment control for the experiment. The porphyrins c-PB2(OH)2 and PB(OH)3 reduced the infectivity of produced HIV-1 NL4-3 virus by more than 60%. The reference compounds TCPP, THPP, and PB3OH rendered about 50% of the viruses non-infectious (Figure 2a). Similarly, c-PB2(OH)2 reduced the infectivity of the HIV-1 K3016 virus by 50%, followed by THPP and PB(OH)3. (Figure 2b). These results indicate that compared to other carboxyphenyl porphyrins, the cis-conformation in c-PB2(OH)2 and the additional three hydroxyl groups in PB(OH)3 might have a role in their enhanced antiviral activity.
c). Carboxyphenyl porphyrins strongly restricted HIV-1 entry and infection under non-PDT conditions
The impact of carboxyphenyl derivatives on virus entry, a preliminary stage of the HIV-1 life cycle, was examined to evaluate their anti-HIV-1 activity. TZM-bl cells were used for entry inhibition assays as per the methods section. Briefly, TZM-bl cells were infected with 10 ng of HIV-1 p24 equivalent NL4-3 or K3016 virus, followed by the addition of different concentrations of compounds (100 nM to 5 µM) during virus infection as described in methods. After washing, the infected cells were incubated at 37 °C for 48 h. After 48 h, relative luciferase activity was assessed and compared to the control (-). In this case, "control" refers to NL4-3 or K3016 virus-infected cells not exposed to test compounds. Enfuvirtide (T20), an HIV fusion inhibitor, was employed as a positive control.
We observed that these carboxyphenyl porphyrins inhibited the virus entry in a dose-dependent manner. At 5 µM maximal concentration, compounds c-PB2(OH)2 and PB(OH)3 significantly reduced the entry of HIV-1 subtype B NL4-3 virus by 70%, followed by a 60% entry inhibition by t-PB2(OH)2. The positive control, T20, prevented the virus entry by 80% at 0.5 µM concentration. The reference compounds TCPP and THPP blocked the virus entry by nearly 60%, while the precursor PB3OH showed a lower inhibition of 30% (Figure 3a). The EC50 values for c-PB2(OH)2, t-PB2(OH)2, and PB(OH)3 were determined to be 2.622 µM,4.098 µM, and 2.44 µM, respectively. Similarly, in the case of HIV-1 subtype C K3016 virus, compounds PB(OH)3, and c-PB2(OH)2, t-PB2(OH)2 prevented its entry by 50%,40% and 30%, respectively. The positive control, T20, and the reference compounds TCPP and THPP restricted the virus entry by 40% and 30%, respectively (Figure 3c). For the HIV-1 subtype C K3016 virus, the EC50 value of these porphyrins was found to be more than 5 µM. Also, these compounds effectively block the entry of the HIV-1 subtype B virus (NL4-3) compared to the HIV-1 subtype C (K3016). Next, we wanted to study the effect of these compounds on the post-entry stages of the virus life cycle. For this, TZM-bl cells were infected with the HIV-1 NL4-3 virus or HIV-1 K3016 virus for 2 h. The infected cells were washed and incubated for 48 h with the carboxyphenyl derivatives. We observed that none of the compounds could effectively reduce HIV-1 NL4-3 or K3016 virus production post-entry to the cells. (Figure 3 b, d). These results suggested the role of carboxyphenyl derivatives in inhibiting the early stages of HIV-1 entry under non-PDT conditions.
d). Carboxyphenyl porphyrins restricted the virus entry in T cells
Carboxyphenyl porphyrins were also examined for their ability to reduce HIV-1 NL4-3 or K3016 virus entry in HUT-R5 cells (Human T-cell line). These cells were infected with 10 ng of HIV-1 p24 equivalent NL4-3 or K3016 virus for 2 h at 37 °C in the presence (5 µM) or absence of test compounds. HIV-1 p24 ELISA was used to quantify the virus. When added during infection, only the derivative c-PB2(OH)2 could inhibit the entry of HIV-1 NL4-3 virus and K3016 virus by 50% and 30%, respectively (Figure S23 a, c). No reduction in virus infection was observed in both HIV-1 subtypes when the compounds were added post-virus entry in the HuTR5 cells (Figure S23 b, d). Hence, the carboxyphenyl porphyrins also inhibited virus entry in T cells when introduced early in the infection.
e). Carboxyphenyl derivatives did not bind cellular CD4 receptors or co-receptors.
The HIV-1 virus infects the host cells through specific binding of HIV-1 envelope (Env) glycoproteins gp120 with cellular CD4 receptors and chemokine co-receptors CXCR4 or CCR5.60 It prompts the viral gp41 transmembrane protein to mediate the fusion of the viral and cell membranes, enabling the virus to enter and deliver its genetic material inside the cells. We hypothesized that carboxyphenyl porphyrins inhibited the early stages of virus entry. We further assessed whether this inhibitory effect of these compounds relied on their specific interactions with viral envelope proteins or host-cell receptors/co-receptors. TZM-bl cells were pre-incubated with the compounds before infection with HIV-1 NL4-3 or K3016 virus for 2 h at 37 °C. It ensured that all the receptors were saturated before adding the virus. We observed that pre-incubation of cells with carboxyphenyl porphyrins did not restrict virus infection (Figure S24 a, b). The lack of entry inhibition of the HIV-1 NL43 or K3016 virus in the pre-treated TZM-bl cells suggested that these porphyrins do not interact with the host cell receptors or coreceptors. These results indicate that the carboxyphenyl porphyrins inhibit the virus entry in the target cells by possible interactions with the viral envelope proteins instead of host cell receptors and co-receptors.
f). Carboxyphenyl porphyrins were most effective in blocking the HIV-1 virus entry when added early during infection.
To better understand the mode of action and the key targets, carboxyphenyl porphyrins were introduced to the host cells at different time points. It sought to establish how soon after the virus was added, the compounds may prevent its entry into the cells. 10 ng of p24 equivalent HIV-1 NL4-3 virus was used to infect TZM-bl cells. 5 µM of carboxyphenyl porphyrins were added to the cells at four-time points post-virus addition: 0 minutes, 30 minutes, 1 h, and 2 h. Here, control cells were the infected cells treated with 5% DMSO in water (-) without compounds. We observed that the extent of virus entry inhibition was time-dependent. As the addition of compounds progressed to the later time points, their ability to inhibit virus entry was impaired. It means that the carboxyphenyl porphyrins were most potent when added during the HIV-1 NL4-3 virus infection. (Figure 4a). The compounds c-PB2(OH)2 and PB(OH)3 inhibited virus entry by nearly 70%, followed by 60% inhibition by t-PB2(OH)2 when combined with the virus. Delaying the addition of compounds for 30 minutes after virus addition resulted in 50-60% restriction in virus entry compared to 40% inhibition by t-PB2(OH)2. These results implied that the carboxyphenyl porphyrins prevented virus infection maximally when added during the early stages of virus entry.
g). Carboxyphenyl porphyrins operate as a post-binding inhibitor of HIV-1 entry
The interaction of viral gp120 with cellular CD4 receptors has been reported to occur at 4 °C, but the fusion of the viral and cell membranes requires 37 °C.61 We performed a temperature arrest assay at 4 °C and 37 °C in the presence or absence of carboxyphenyl porphyrins to determine whether they prevent the viral entry at the binding or post-binding stages. TZM-bl cells were infected with 10 ng of HIV-1 equivalent NL4-3 or K3016 virus at 4 °C in the presence or absence of the test compounds. The cells were washed and incubated at 37 °C for 48 h in the presence or absence of the compounds. The relative luciferase activity of infected cells was compared to those treated with 5% DMSO in water (-) without compounds (negative control). If the porphyrins prevented the virus's attachment to cellular receptors at 4 °C, then incubating cells at 37 °C in their absence would prevent virus entry. However, our findings demonstrated that HIV-1 NL4-3 or K3016 viral entry was blocked substantially only when cells were incubated at 37°C in the presence of the compounds. At 37 °C, T20, c-PB2(OH)2, and PB(OH)3 blocked the HIV-1 subtype B NL4-3 entry by 70%. TCPP, THPP, and t-PB2(OH)2 exhibited more than 50% entry inhibition, followed by 30% inhibition by PB3OH (Figure 4b). The HIV-1 subtype C K3016 virus entry was inhibited by 50% by PB(OH)3. T20 and c-PB2(OH)2, t-PB2(OH)2 and THPP, TCPP, and PB3OH restricted the virus entry by nearly 40%, 30%, and 20%, respectively (Figure 4c). At 4 °C, no inhibition of virus entry for any porphyrin in both the HIV-1 subtypes was observed. These findings revealed that porphyrins did not prevent the viral envelope glycoprotein gp120 from interacting or attaching to the cellular CD4 receptor. Hence, these carboxyphenyl derivatives were considerably more active during the post-binding events of the HIV-1 virus entry.
h). Carboxyphenyl porphyrins strongly restricted HIV-1 entry and infection under PDT conditions
We investigated the effect of test compounds on virus entry under PDT conditions, as stated in the methods. Under PDT conditions, 10ng of HIV-1 p24 equivalent NL4-3 or K3016 virus was pre-incubated with the carboxyphenyl derivatives. The pre-treated virus was then utilized to infect TZM-bl cells. Infected cells were washed and cultured at 37 °C for 48 h. The relative luciferase activity was calculated concerning the control. The term "control" refers to virus-infected cells treated with 5% DMSO in water (-) in the absence of compounds. Enfuvirtide (T20) was utilized as a positive control. The carboxyphenyl porphyrins effectively prevented HIV-1 NL4-3 or K3016 viral entry at the higher concentrations in a dose-dependent manner at concentrations ranging from 10nM to 5 µM. The maximum concentrations used for the compounds TCPP, THPP, and PB3OH were 500 nM and 1 µM, respectively, because they were found to be cytotoxic at higher concentrations in PDT conditions.
Under PDT conditions at 500 nM concentration, the reference porphyrin TCPP strongly restricted HIV-1 subtype B virus entry by 96%, followed by 90 to 95% inhibition by c-PB2(OH)2, PB3OH, and THPP. More than 85% of virus entry inhibition was observed by the porphyrins t-PB2(OH)2 and PB(OH)3. The porphyrin PB3OH blocked the virus entry by 97% at 1 µM, followed by more than 98% virus entry inhibition by c-PB2(OH)2, t-PB2(OH)2 and PB(OH)3 at 5 µM. These compounds showed anti-HIV activity in a dose-dependent manner. (Figure 5 a, b). Post-infection, at 1 µM concentration, PB3OH reduced the virus production by 95%. The porphyrins c-PB2(OH)2, t-PB2(OH)2 and PB(OH)3 effectively blocked HIV-1 production by 90 to 95% at 5 µM. (Figure 5 c, d). The porphyrins exhibited EC50 value <50 nM under PDT conditions for HIV-1 NL43 virus. The carboxyphenyl porphyrin PB(OH)3 inhibited HIV-1 subtype C K3016 virus entry by 70%, while the reference porphyrins TCPP and THPP inhibited entry by nearly 50% at 500 nM. At 1 µM, PB3OH restricted the virus entry by more than 90%, followed by more than 85% reduction at 5 µM for the compounds c-PB2(OH)2 and PB(OH)3. (Figure 6 a, b). The EC50 value of the compounds for HIV-1 subtype C virus was nearly 500 nM or more. The porphyrins were not active against HIV-1 subtype C virus post-infection at 500 nM under the PDT conditions (Figure 6c). However, upon increasing the concentration to 1 µM for PB3OH and 5 µM for c-PB2(OH)2, t-PB2(OH)2, and PB(OH)3, the virus production was decreased by more than 60% and 80%, respectively (Figure 6d).