Bioorganic & Medicinal Chemistry Letters 24 (2014) 302–307
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Synthesis, anti-HIV activity, integrase enzyme inhibition and molecular modeling of catechol, hydroquinone and quinol labdane analogs Rohan Pawar a, , Tiyasa Das b, , Sanjay Mishra a, Nutan b, Boskey Pancholi b, Satish K. Gupta b,⇑, Sujata V. Bhat a,⇑ a b
Laboratory for Advanced Research in Natural and Synthetic Chemistry, V.G. Vaze College, Mumbai University, Mithagar Road, Mulund (East), Mumbai 400 081, India National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
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Article history: Received 24 July 2013 Revised 11 October 2013 Accepted 9 November 2013 Available online 18 November 2013 Keywords: Labdane analogs o-Quinol–catechol–hydroquinone moiety Diels–Alder reaction Anti-HIV-1 activity Computational study HIV-1 integrase inhibition
a b s t r a c t Labdane analogs with o-quinol, catechol and hydroquinone moiety have been synthesized using Diels– Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate, 3,4-dioxocyclohexa-1,5-dienecarboxylic acid and 3,6-dioxocyclohexa-1,4-dienecarboxylic acid with mono terpene 1,3-dienes, namely ocimene and myrcene. The resulting molecules and their derivatives were evaluated for their anti-HIV-1 activity using TZM-bl cell based virus infectivity assay. Two molecules 13 and 18 showed anti-HIV activity with IC50 values 5.0 (TI = 11) and 4.6 (TI = 46) lM, respectively. The compounds 17, 18 and 20 showed efficacy against HIV-1 integrase activity and showed inhibition with IC50 13.4, 11.1 and 11.5 lM, respectively. The HIV-1 integrase inhibition activity of these synthetic molecules was comparable with integric acid, the natural fungal metabolite. Molecular modeling studies for the HIV-1 integrase inhibition of these active synthetic molecules indicated the binding to the active site residues of the enzyme. Ó 2013 Elsevier Ltd. All rights reserved.
Acquired immune deficiency syndrome (AIDS) caused by the infection with human immunodeficiency virus (HIV) represents a major health problem worldwide, especially in the developing countries.1–3 Highly active anti-retroviral therapy (HAART), mainly consisting of HIV-1 protease and reverse transcriptase inhibitors, introduced in the late 1990s, has largely improved patients’ lives in the developed countries. However, drug resistance and toxicity are the main problems during HIV treatment.4 Although understanding of the pathogenesis and transmission dynamics of HIV infection has advanced, protective vaccine remains elusive.5 Therefore, there is a continuous need for novel and effective drugs against HIV-1.6 There are several cyclic natural terpenes with o-quinol or catechol moiety, which exhibit interesting biological activities. For example, the eremophilane class of sesquiterpene 1 displayed phytotoxic activity.7,8 Members of quassinoid family 2 exhibit a gamut Abbreviations: MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; TI, therapeutic index; RT, room temperature; IBD, iodoso benzene diacetate; ACN, acetonitrile; IN CCD, integrase catalytic core domain. ⇑ Corresponding authors. Tel.: +91 1126741249; fax: +91 11 26742125 (S.K.G.); tel.: +91 22 21631421x113; fax: +91 022 21634262 (S.V.B.). E-mail addresses: [email protected] (S.K. Gupta), [email protected] (S.V. Bhat). Equal contribution. 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.014
of biological activities, which include anti-HIV, anti-tumor, antimalarial and anti-inflammatory activities.9 The quinonemethide, taxodione 3 from Tadodium disticum displayed anti-tumor and anti-microbial activities.9 Some molecules with o-keto-enol functionality displayed anti-HIV activity due to HIV-1 integrase inhibition. For example, cytosporic acid 4, a fungal metabolite produced by a Cytospora sp., displayed HIV-1 integrase inhibitory activity due to inhibition of strand-transfer reaction of HIV-integrase.10,11 Integric acid 5,12–14 an eremophilane sesquiterpenoid from Xylaria sp. inhibited 30 -end processing, strand transfer and disintegration reactions catalyzed by HIV-1 integrase enzyme (Fig. 1).15–19 o-Quinones are active molecules that can be readily generated in situ by oxidation of catechols.20 They exhibit properties of both diene and dienophile. It has been shown that o-quinones bearing an electron withdrawing substituent at the 2nd and 4th position are sufficiently reactive as dienophiles in cycloadditions with reactive acyclic 1,3-dienes.21 We report herein the synthesis and evaluation of anti-HIV-1 activity of the labdane analogs, Some of these molecules displayed good anti-HIV-1 and HIV-1 integrase inhibition activities. Labdane analogs with o-quinol moiety were synthesized by Diels–Alder reaction of methyl 3,4-dioxocyclohexa-1,5-dienecarboxylate (generated in situ by the oxidation of methyl 3, 4-dihydroxy-benzoate using Ag2O) with monoterpene 1,3-dienes
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O
O
14
O
OMe OH
HO
O
O
O
2
O
HOOC
CHO
O
O
9
6
O
OR 1
14, R = -CH2OH 15, R = -CHO
OH
16, R1 = -Ac O
Scheme 2. Diels–Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate with ocimene and SeO2 oxidation of adduct. Reagents and conditions: (a) toluene:ether, Ag2O, 0 °C for 3 h, RT for 20 h (b) SeO2, t-BuOOH, RT for 15 h (c) Ac2O, pyridine, RT for 18 h.
Figure 1. Bioactive terpenoids.
namely myrcene and ocimene. Similarly, labdane analogs with catechol and hydroquinone moiety were synthesized by Diels–Alder reaction of 3,4-dioxocyclohexa-1,5-diene-carboxylic acid and of 3,6-dioxocyclohexa-1,4-dienecarboxylic acid (generated in situ by the oxidation of 3,4-dihydroxy-benzoic acid and 2,5-dihydroxybenzoic acid using IBD) with myrcene and ocimene. o-Quinones being unstable were prone to rapid dimerization. To circumvent this problem and to increase yield in the cycloaddition reaction, an excess of the diene was used and the reaction was conducted at 0 °C to avoid dimerization. Thus, a mixture of monoterpene diene myrcene or ocimene (5 mM), methyl 3,4-dihydroxy-benzoate (1 mM) and freshly prepared Ag2O (1 mM) was stirred at 0 °C for 3 h, then at RT for 20 h and the reaction mixture was subjected to the usual work-up to yield the adducts 10 and 13, respectively (Schemes 1 and 2). The Diels–Alder reaction of methyl 3,4-dihydroxybenzoate 6 with myrcene 8 yielded a mixture of regio-isomers (9:1) (Scheme 1), which were further separated to obtain major para adduct 10 (with respect to carbomethoxy group), by preparative thin layer chromatography (PTLC), the stereochemistry of the compound was confirmed by NMR ROESY experiments (Fig. 2 and Supporting information). Similarly, the stereochemistry of the compound 13 was also confirmed by NMR ROESY experiments. To obtain adduct 17 and 18 or 19 and 20 a mixture of terpenic diene myrcene or ocimene (5 mM), 3,4-dihydroxy-benzoic acid or 2,5-dihydroxybenzoic acid (1 mM) and freshly prepared IBD (1 mM) was stirred at 0 °C for 1 h, the reaction mixture was subjected to the usual work-up (Schemes 3 and 4). The Diels–Alder reaction of 3,4-dihydroxybenzoic acid 7a and 2,5-dihydroxybenzoic acid 7b with myrcene 8 yielded only para adducts 17 and 19, respectively (Schemes 3 and 4). The excess diene recovered at the end of the reaction, has been characterized by spectral data. Similarly, reactions of acids 7a and 7b with ocimene 9 yielded single orthoadducts 18 and 20, respectively (Schemes 3 and 4). Structures of adducts were supported on the basis of spectral data.21 The IR spectra of cyclo-adducts 10 and 13 showed bands at 3412, 1736 and 1649 cm1 indicating the presence of hydroxyl, ester and conjugated carbonyl group, respectively, whereas the adducts 17–20 showed bands at 3400 and 1595 cm1 indicating the presence of phenolic OH and aromatic C@C stretching, respectively. The absence of peak around 1700–1650 cm1, indicated that these adduct underwent decarboxylation and
COOMe
4
COOMe
2
1
9
OH 10
16
6
10
12 8
7
11 13
COOMe
b
5
3
O
b
7
13, R 1= -H
COOH
5
a
1 9 8
16
6
10 2
MeOOC
c
HO 4
8
O
O
+
5 11
3
OH
3
OH
a
+
HO
R
15
12
4
1
OH 6
MeOOC
MeO O
HO
13
COOMe
14 15
O
R OH 11, R = -CH2OH 12, R = -CHO
Scheme 1. Diels–Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate with myrcene and SeO2 oxidation of adduct. Reagents and conditions: (a) toluene:diethyl ether, Ag2O, 0 °C for 3 h, RT for 20 h (b) SeO2, t-BuOOH, RT, 15 h.
H
H H RH H H O
H H
OH H H H H H 10
H
H H
H H
H R H
H O
R= -COOMe
H
OH H H 13
R= -COOMe
Figure 2. Correlation of protons obtained from ROESY spectrum of compounds 10 and 13.
12 11 10 4 3 2
HO
1
4a
13
COOH
9 6
5
3
a
8
OH
6
13 10
2
7
8a
5
4 4a
14
HO
a
HO
1
8a
8
OH
OH
9
12 11
14
17
7a
18
7
Scheme 3. Diels–Alder reaction of 3,4-dioxocyclohexa-1,5-diene-carboxylic acid with ocimene and myrcene. Reagents and conditions: (a) diisopropyl ether, ACN, IBD, 0 °C for 1 h.
12 11
OH 4
10
4a
3 2
13
OH
OH
5 9
COOH
14 6
1
8a
OH 20
8
7
1 8a
2
a
OH 7b
a
8
7
13 10
3 4
4a
OH
5
6 9
12 11
14
19
Scheme 4. Diels–Alder reaction of 3,6-dioxocyclohexa-1,4-dienecarboxylic acid with ocimene and myrcene. Reagents and conditions: (a) diisopropyl ether, ACN, IBD, 0 °C for 1 h.
aromatization to give catechol analogs 17, 18 and hydroquinone analogs 19 and 20. The 1H NMR spectra of the compounds 10 and 13 displayed a pair of doublets around d 6.95 and 6.40 (1H each, J = 10 Hz) for C-3, C-4 protons. The ring olefinic protons (C6 or C-7) and side-chain olefinic protons resonated at d 5.5 (broad triplet) and d 5.1 (triplet) (1H, J = 5.5 Hz), respectively. Thus, these products are formed through Diels–Alder reaction followed by enolization of the resulting o-diketone to the tautomeric diosphenol. This was confirmed not only by the 1H NMR spectrum but also by making an acetate derivative of the enol. The formation of the enol acetate (16, Scheme 2) was supported by the 1H NMR spectrum of the compound 16, which showed the presence of a acetate peak at d 2.33 (3H, singlet) and also by the appearance of additional band at 1748 cm1 in the IR spectrum. Adducts 10 and 13 were subjected to oxidation with catalytic amount of selenium dioxide in the presence of t-butyl hydroperoxide to yield allylic
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alcohols 11 or 14 and conjugated aldehydes 12 or 15, respectively. The 1H NMR spectra of the compounds 17, 18 and 20 displayed a pair of doublets d 6.69 and 6.51 (1H each, J = 8 Hz) and compound 19 displayed at 6.50 (singlet, 2H) for aromatic protons. The ring olefinic protons at C-6 resonated at d 5.6 (broad triplet) (1H) and the side chain olefinic protons resonated at d 5.1 (broad triplet) (1H). The resulting labdane analogs with o-quinol, catechol or hydroquinone functionality namely molecules 10–20 were evaluated for anti-HIV activity. The inhibitory activity of the compounds is sometimes, a result of their toxic effects and consequently might resemble an erroneous conclusion.11 Prior to the analysis of anti-HIV activity, compounds 10–20 were tested in vitro for their cytotoxicity on TZMbl cells using MTT assay.22,23 The results are summarized in (Table 1). CC50 value indicates the cytotoxic concentration of the compound leading to 50% reduction in cell viability. Out of the 11 compounds evaluated by MTT assay, seven compounds 10–12, 14–16 and 18 showed CC50 values of more than 200 lM. However, compound 13 showed a CC50 value of 56 ± 5.0 lM whereas 17 and 20 showed that of 139 ± 12 and 140 ± 23 lM, respectively. Compounds 10–20 were evaluated for in vitro inhibition of HIV-1 infection by reporter-gene based cell assay system. Compounds 13 and 18 showed IC50 (concentration of the compound leading to 50% inhibition of HIV-1 infection) values of 5.0 ± 1.0 and 4.6 ± 1.0 lM, respectively (Table 1). The compound 17 and 20 showed an IC50 value of 12.3 ± 4.0 and 12.4 ± 2.0 lM, respectively. In these experiments Raltegravir showed an IC50 of 0.5 lM. The IC50 values for compounds 14, 15 and 16 ranged from 36.7 ± 3.0 to 72.7 ± 18.0 lM. Other compounds showed IC50 value of more than 100 lM. Compound 19 was inactive at 50 lM. The integration inhibition properties of these compounds at 50 lM were subsequently studied, using a commercially available kit (Express Biotech International, Maryland, USA) following the manufacturer’s instructions. Sodium azide and Raltegravir were used as positive controls at 50 lM (Table 2). Based on their efficacy against HIV-1 integrase activity, compounds 17, 18 and 20 were further evaluated for a dose-dependent inhibition. IC50 values of compounds which were found to be 13.4 ± 0.77, 11.1 ± 0.34 and 11.5 ± 0.97 lM, respectively (Table 2). Subsequently, these new labdane analogs were subjected to the molecular modeling studies by docking with HIV-1 integrase enzyme (IN). HIV-1 integrase is a 32 kDa enzyme that carries out DNA integration in a two-step reaction. In the first step, called 30 processing, two nucleotides are removed from each 30 end of the viral DNA. In the next step, called DNA strand transfer, a pair of transesterification reactions integrates the ends of the viral DNA into the host Table 1 Cytotoxic concentration and anti-HIV-1 activity of compounds 10–20 Compounds
CC50a (lM)
IC50b (lM)
TIc
10 11 12 13 14 15 16 17 18 19 20 Raltegravir
693.0 ± 32.0 382.8 ± 69.0 625.4 ± 49.0 56.4 ± 5.0 928.3 ± 55.0 626.9 ± 40.0 677.2 ± 45.0 138.5 ± 12.0 211.3 ± 13.0 NA 140 ± 23 855.6 ± 90.0
170.7 ± 58.0 284.6 ± 147.0 171.5 ± 67.0 5.0 ± 1.0 72.7 ± 18.0 60.7 ± 20.0 36.7 ± 3.0 12.3 ± 4.0 4.6 ± 1.0 — 12.4 ± 2.0 0.5 ± 0.1
4 1 4 11 13 10 18 11 46 — 11 1711
NA: not active. a CC50: the cytotoxic concentration of the compounds leading to 50% reduction in cell viability. b IC50: inhibitory concentration of the compounds leading to 50% inhibition in HIV-1 infection. c TI: therapeutic index, CC50/IC50.
Table 2 HIV-1 integrase inhibition activity of compounds 10–20 and Raltegravir Compounds
Percent inhibition of HIV-1 integrase activity (50 lM)
IC50 (lM)
10 11 12 13 14 15 16 17 18 19 20 Raltegravir NaN3 (1.5%)
38 0 0 24 18 19 9 78 88 0 82 100 65
NT NT NT NT NT NT NT 13.4 ± 0.77 11.1 ± 0.34 NT 11.5 ± 0.97 0.12 ± 0.86 NT
NT: not tested.
genome. Extensive mutagenesis studies mapped the catalytic site to the core domain (residues 50–212, IN CCD), which contains the catalytic residues ASP64, ASP116, and GLU152.24 Site-directed mutagenesis and photo-cross-linking experiments have identified several residues near the active site, including LYS156, LYS159, GLN148, and TYR143, that are critical for binding viral DNA substrate.25 The compounds 10–20 were docked into IN CCD to study molecular interactions.26,27 The docking score function of the program ranked the compounds in the same general order observed experimentally. Compounds 17, 18 and 20 exhibited significant hydrogen-bonding interactions with the active site of IN CCD. Docking results were compared with well-known integric acid, which has been reported to be an integrase inhibitor through binding to active site of IN CCD. Integric acid binds through, the one hydrogen bond between its carbonyl C@O and H–N of LYS156 (C@O—HAN: 2.20 Å). Another hydrogen bond between aldehyde carbonyl C@O and H–O of THR66 (C@O—HAO: 2.20 Å) (Fig. 3). Compound 10 was found to bind through two hydrogen bonds between its C-1 O–H, O@C (OAH—O@C: 2.01 Å) of CYS65 and C-2 H– O, H–N of HIS67 (H–O—H–N: 2.08 Å), however these residues are not active site residues. Compound 17 was binding through two hydrogen bonds between its C-1 O–H and O@C of GLU152 (O– H—O: 2.23 Å), C-2 H–O and H–N (H–O—H–N: 2.59 Å) of LYS156 (Fig. 3). Thus docking results show that compound 17 binds to the active site residues (GLU152 and LYS156). Hence compound 17 is more active than compound 10 (both the compounds contain myrcene side chain) (comparative binding Fig. 4a). Compound 13 was binding through one hydrogen bond between its C-1 H–O and H–N of CYS65 (H–O—H–N: 2.02 Å). The most potent compound 18 was nicely accommodated within IN CCD (Fig. 3). Its binding conformation exhibited two hydrogen bonds between its C-2 O–H, O@C (O–H—O: 1.89 Å) and C-2 H–O, N–H (H–O—H–N: 1.93 Å) of GLN148 (Fig. 3) which is active site residue. This may be reason for more potency of compound 18 compared to compound 13 (both compounds contain the ocimene side chain) (Fig. 4b). Compound 20 was also binding through two hydrogen bonds between its C-1 H–O, H–N (H–O—H–N: 2.06 Å) of LYS 159 and C-4 O–H, O@C (O–H—O: 2.12 Å) of GLU 152 (Table 3). Inactive compound 19 was docked in to the IN CCD, which binding through one hydrogen bond between its C-1 O–H, O@C (HAO—O@C: 1.96 Å) of CYS 63, which is not active site residue (Fig. 3). Docking results were compared with well-known Raltegravir, which has been reported to be an integrase inhibitor. Similar molecular docking studies on Raltegravir showed that it binds to the active site of the integrase enzyme. Raltegravir was binding through two hydrogen bonds between its side chain N–H and O@C (NAH—O@C: 2.09 Å) of ASP64 and its cyclic H–N, H–N of LYS159 (H–N—H–N:
R. Pawar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 302–307
(10)
(13)
(17)
(19)
Raltegravir
305
(18)
(20)
Integric acid
Figure 3. Binding of compounds 10, 13, 17, 18 and 20, integric acid and raltegravir in the IN CCD (only main surrounding residue are shown).
2.05 Å), which gave the best fit value. Some of the residues, including LYS156, LYS159 and GLN148, that are critical for binding viral DNA substrate, also are surrounding these molecules. Thus, we have successfully synthesized catechol and quinol labdane analogs to evaluate their anti-HIV activity. The graphical representation of structure verses activity is shown in Figure 5. The structure activity relationship (SAR) with respect to their biological activity and docking results of molecules 10–20 indicated that the
compound 13 with o-quinol functionality displays considerable anti-HIV activity. The acyl protection of hydroxyl group of o-quinol moiety of compound 13 destroys the activity completely. Compounds 13, 18 and 20 are more active than the compounds 10, 17 and 19, respectively, which indicates that the presence of ocimene side chain at position 5 of ortho adducts 13, 18 and 20 increases the anti-HIV activity as compared to myrcene side chain of para adducts 10, 17 and 19. The side chains of compounds 10
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COOMe
Increase in activity
MeOOC
O
O
OH
OH 10 IC 50 = 170.7± 58.0 µM (TI = 4)
13
CH 2OH MeOOC Oxidative modif ications Decrease in activity
IC 50 = 5.9 ± 1.0 µM (TI = 11)
O OH 14 IC 50 = 72.7 ± 18.0 µM (TI = 13) CHO MeOOC
MeOOC
O OAc 16 Inactive
Figure 4a. Comparative docking study of compound 10 (blue) and 17 (pale yellow).
HO
O OH
18 Most active IC 50 = 4.6 ± 1.0 µM (TI = 46)
OH 15 IC 50 = 60.7± 20.0 µM (TI = 10)
Figure 5. Graphical representation of structure verses anti-HIV activity.
In conclusion, eleven new labdane analogs were synthesized using Diels–Alder reaction and evaluated for anti-HIV activity. Compound 18 showed both anti HIV-1 activity and HIV-1 integrase inhibition activity, which were further evaluated for a dosedependent inhibition and computational study. The HIV-1 integrase inhibition activity of molecules 17, 18 and 20 were comparable with integric acid, the natural fungal metabolite. Presently, most regimens for the treatment of HIV infection employ drugs that primarily inhibit HIV-1 specific reverse transcriptase and/or protease. The synthetic labdane analogs described in the present study, which are integrase inhibitors, may have potential as anti-HIV agents and need further exploration. Acknowledgments Figure 4b. Comparative docking study of compound 13 (purple) and 18 (pale yellow).
Table 3 Comparison of IC50 and pIC50 with docking score of compounds 10–20 Compounds
IC50a (lM)
pIC50b
Docking score
10 11 12 13 14 15 16 17 18 19 20 Raltegravir Integric acid
170.7 284.6 171.50 5.0 72.7 60.7 36.7 12.3 4.6 NA 12.4 0.5 10
3.77 3.55 3.77 5.30 4.14 4.22 4.44 4.91 5.34 — 4.91 6.30 5.00
3.32 3.89 3.97 5.36 4.28 4.33 4.68 4.77 5.01 2.56 4.95 6.83 5.21
Authors are grateful to Kelkar Education Trust, Mumbai, Indian Council of Medical Research and Department of Biotechnology, Government of India for financial support. We are also thankful to the Department of Chemistry and Sophisticated Analytical Instrumentation Facility, Indian Institute of Technology, Mumbai and Tata Institute of Fundamental Research, Mumbai for NMR spectral data. We would also like to acknowledge NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH for providing us the molecular clone of HIV-1 (NL4.3) and Raltegravir. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11. 014. References and notes
a
IC50: inhibitory concentration of the compounds leading to 50% inhibition in HIV-1 infection. b pIC50: negative logarithm of IC50.
and 13 were functionalized by SeO2 oxidation to give aldehydes (12 and 15) and hydroxyl derivatives (11 and 14), respectively. However these modifications decreased the activity with respect to compound 10 and 13. Compounds 17, 18 and 20 with catechol or hydroquinone moiety showed integrase inhibition activity with IC50 13.4 ± 0.77, 11.1 ± 0.34 and 11.5 ± 0.97 lM, respectively. Catechol and hydroquinone functionality increase the activity may be due to phenolic hydroxyl groups. The catechol moiety (17 and 18) is superior to the hydroquinone moiety (19 and 20) as found from biological data.
1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
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Synthesis, anti-HIV activity, integrase enzyme inhibition and molecular modeling of catechol, hydroquinone and quinol labdane analogs Rohan Pawar a, , Tiyasa Das b, , Sanjay Mishra a, Nutan b, Boskey Pancholi b, Satish K. Gupta b,⇑, Sujata V. Bhat a,⇑ a b
Laboratory for Advanced Research in Natural and Synthetic Chemistry, V.G. Vaze College, Mumbai University, Mithagar Road, Mulund (East), Mumbai 400 081, India National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
a r t i c l e
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Article history: Received 24 July 2013 Revised 11 October 2013 Accepted 9 November 2013 Available online 18 November 2013 Keywords: Labdane analogs o-Quinol–catechol–hydroquinone moiety Diels–Alder reaction Anti-HIV-1 activity Computational study HIV-1 integrase inhibition
a b s t r a c t Labdane analogs with o-quinol, catechol and hydroquinone moiety have been synthesized using Diels– Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate, 3,4-dioxocyclohexa-1,5-dienecarboxylic acid and 3,6-dioxocyclohexa-1,4-dienecarboxylic acid with mono terpene 1,3-dienes, namely ocimene and myrcene. The resulting molecules and their derivatives were evaluated for their anti-HIV-1 activity using TZM-bl cell based virus infectivity assay. Two molecules 13 and 18 showed anti-HIV activity with IC50 values 5.0 (TI = 11) and 4.6 (TI = 46) lM, respectively. The compounds 17, 18 and 20 showed efficacy against HIV-1 integrase activity and showed inhibition with IC50 13.4, 11.1 and 11.5 lM, respectively. The HIV-1 integrase inhibition activity of these synthetic molecules was comparable with integric acid, the natural fungal metabolite. Molecular modeling studies for the HIV-1 integrase inhibition of these active synthetic molecules indicated the binding to the active site residues of the enzyme. Ó 2013 Elsevier Ltd. All rights reserved.
Acquired immune deficiency syndrome (AIDS) caused by the infection with human immunodeficiency virus (HIV) represents a major health problem worldwide, especially in the developing countries.1–3 Highly active anti-retroviral therapy (HAART), mainly consisting of HIV-1 protease and reverse transcriptase inhibitors, introduced in the late 1990s, has largely improved patients’ lives in the developed countries. However, drug resistance and toxicity are the main problems during HIV treatment.4 Although understanding of the pathogenesis and transmission dynamics of HIV infection has advanced, protective vaccine remains elusive.5 Therefore, there is a continuous need for novel and effective drugs against HIV-1.6 There are several cyclic natural terpenes with o-quinol or catechol moiety, which exhibit interesting biological activities. For example, the eremophilane class of sesquiterpene 1 displayed phytotoxic activity.7,8 Members of quassinoid family 2 exhibit a gamut Abbreviations: MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; TI, therapeutic index; RT, room temperature; IBD, iodoso benzene diacetate; ACN, acetonitrile; IN CCD, integrase catalytic core domain. ⇑ Corresponding authors. Tel.: +91 1126741249; fax: +91 11 26742125 (S.K.G.); tel.: +91 22 21631421x113; fax: +91 022 21634262 (S.V.B.). E-mail addresses: [email protected] (S.K. Gupta), [email protected] (S.V. Bhat). Equal contribution. 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.014
of biological activities, which include anti-HIV, anti-tumor, antimalarial and anti-inflammatory activities.9 The quinonemethide, taxodione 3 from Tadodium disticum displayed anti-tumor and anti-microbial activities.9 Some molecules with o-keto-enol functionality displayed anti-HIV activity due to HIV-1 integrase inhibition. For example, cytosporic acid 4, a fungal metabolite produced by a Cytospora sp., displayed HIV-1 integrase inhibitory activity due to inhibition of strand-transfer reaction of HIV-integrase.10,11 Integric acid 5,12–14 an eremophilane sesquiterpenoid from Xylaria sp. inhibited 30 -end processing, strand transfer and disintegration reactions catalyzed by HIV-1 integrase enzyme (Fig. 1).15–19 o-Quinones are active molecules that can be readily generated in situ by oxidation of catechols.20 They exhibit properties of both diene and dienophile. It has been shown that o-quinones bearing an electron withdrawing substituent at the 2nd and 4th position are sufficiently reactive as dienophiles in cycloadditions with reactive acyclic 1,3-dienes.21 We report herein the synthesis and evaluation of anti-HIV-1 activity of the labdane analogs, Some of these molecules displayed good anti-HIV-1 and HIV-1 integrase inhibition activities. Labdane analogs with o-quinol moiety were synthesized by Diels–Alder reaction of methyl 3,4-dioxocyclohexa-1,5-dienecarboxylate (generated in situ by the oxidation of methyl 3, 4-dihydroxy-benzoate using Ag2O) with monoterpene 1,3-dienes
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O
O
14
O
OMe OH
HO
O
O
O
2
O
HOOC
CHO
O
O
9
6
O
OR 1
14, R = -CH2OH 15, R = -CHO
OH
16, R1 = -Ac O
Scheme 2. Diels–Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate with ocimene and SeO2 oxidation of adduct. Reagents and conditions: (a) toluene:ether, Ag2O, 0 °C for 3 h, RT for 20 h (b) SeO2, t-BuOOH, RT for 15 h (c) Ac2O, pyridine, RT for 18 h.
Figure 1. Bioactive terpenoids.
namely myrcene and ocimene. Similarly, labdane analogs with catechol and hydroquinone moiety were synthesized by Diels–Alder reaction of 3,4-dioxocyclohexa-1,5-diene-carboxylic acid and of 3,6-dioxocyclohexa-1,4-dienecarboxylic acid (generated in situ by the oxidation of 3,4-dihydroxy-benzoic acid and 2,5-dihydroxybenzoic acid using IBD) with myrcene and ocimene. o-Quinones being unstable were prone to rapid dimerization. To circumvent this problem and to increase yield in the cycloaddition reaction, an excess of the diene was used and the reaction was conducted at 0 °C to avoid dimerization. Thus, a mixture of monoterpene diene myrcene or ocimene (5 mM), methyl 3,4-dihydroxy-benzoate (1 mM) and freshly prepared Ag2O (1 mM) was stirred at 0 °C for 3 h, then at RT for 20 h and the reaction mixture was subjected to the usual work-up to yield the adducts 10 and 13, respectively (Schemes 1 and 2). The Diels–Alder reaction of methyl 3,4-dihydroxybenzoate 6 with myrcene 8 yielded a mixture of regio-isomers (9:1) (Scheme 1), which were further separated to obtain major para adduct 10 (with respect to carbomethoxy group), by preparative thin layer chromatography (PTLC), the stereochemistry of the compound was confirmed by NMR ROESY experiments (Fig. 2 and Supporting information). Similarly, the stereochemistry of the compound 13 was also confirmed by NMR ROESY experiments. To obtain adduct 17 and 18 or 19 and 20 a mixture of terpenic diene myrcene or ocimene (5 mM), 3,4-dihydroxy-benzoic acid or 2,5-dihydroxybenzoic acid (1 mM) and freshly prepared IBD (1 mM) was stirred at 0 °C for 1 h, the reaction mixture was subjected to the usual work-up (Schemes 3 and 4). The Diels–Alder reaction of 3,4-dihydroxybenzoic acid 7a and 2,5-dihydroxybenzoic acid 7b with myrcene 8 yielded only para adducts 17 and 19, respectively (Schemes 3 and 4). The excess diene recovered at the end of the reaction, has been characterized by spectral data. Similarly, reactions of acids 7a and 7b with ocimene 9 yielded single orthoadducts 18 and 20, respectively (Schemes 3 and 4). Structures of adducts were supported on the basis of spectral data.21 The IR spectra of cyclo-adducts 10 and 13 showed bands at 3412, 1736 and 1649 cm1 indicating the presence of hydroxyl, ester and conjugated carbonyl group, respectively, whereas the adducts 17–20 showed bands at 3400 and 1595 cm1 indicating the presence of phenolic OH and aromatic C@C stretching, respectively. The absence of peak around 1700–1650 cm1, indicated that these adduct underwent decarboxylation and
COOMe
4
COOMe
2
1
9
OH 10
16
6
10
12 8
7
11 13
COOMe
b
5
3
O
b
7
13, R 1= -H
COOH
5
a
1 9 8
16
6
10 2
MeOOC
c
HO 4
8
O
O
+
5 11
3
OH
3
OH
a
+
HO
R
15
12
4
1
OH 6
MeOOC
MeO O
HO
13
COOMe
14 15
O
R OH 11, R = -CH2OH 12, R = -CHO
Scheme 1. Diels–Alder reaction of methyl 3,4-dioxocyclohexa-1,5-diene-carboxylate with myrcene and SeO2 oxidation of adduct. Reagents and conditions: (a) toluene:diethyl ether, Ag2O, 0 °C for 3 h, RT for 20 h (b) SeO2, t-BuOOH, RT, 15 h.
H
H H RH H H O
H H
OH H H H H H 10
H
H H
H H
H R H
H O
R= -COOMe
H
OH H H 13
R= -COOMe
Figure 2. Correlation of protons obtained from ROESY spectrum of compounds 10 and 13.
12 11 10 4 3 2
HO
1
4a
13
COOH
9 6
5
3
a
8
OH
6
13 10
2
7
8a
5
4 4a
14
HO
a
HO
1
8a
8
OH
OH
9
12 11
14
17
7a
18
7
Scheme 3. Diels–Alder reaction of 3,4-dioxocyclohexa-1,5-diene-carboxylic acid with ocimene and myrcene. Reagents and conditions: (a) diisopropyl ether, ACN, IBD, 0 °C for 1 h.
12 11
OH 4
10
4a
3 2
13
OH
OH
5 9
COOH
14 6
1
8a
OH 20
8
7
1 8a
2
a
OH 7b
a
8
7
13 10
3 4
4a
OH
5
6 9
12 11
14
19
Scheme 4. Diels–Alder reaction of 3,6-dioxocyclohexa-1,4-dienecarboxylic acid with ocimene and myrcene. Reagents and conditions: (a) diisopropyl ether, ACN, IBD, 0 °C for 1 h.
aromatization to give catechol analogs 17, 18 and hydroquinone analogs 19 and 20. The 1H NMR spectra of the compounds 10 and 13 displayed a pair of doublets around d 6.95 and 6.40 (1H each, J = 10 Hz) for C-3, C-4 protons. The ring olefinic protons (C6 or C-7) and side-chain olefinic protons resonated at d 5.5 (broad triplet) and d 5.1 (triplet) (1H, J = 5.5 Hz), respectively. Thus, these products are formed through Diels–Alder reaction followed by enolization of the resulting o-diketone to the tautomeric diosphenol. This was confirmed not only by the 1H NMR spectrum but also by making an acetate derivative of the enol. The formation of the enol acetate (16, Scheme 2) was supported by the 1H NMR spectrum of the compound 16, which showed the presence of a acetate peak at d 2.33 (3H, singlet) and also by the appearance of additional band at 1748 cm1 in the IR spectrum. Adducts 10 and 13 were subjected to oxidation with catalytic amount of selenium dioxide in the presence of t-butyl hydroperoxide to yield allylic
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alcohols 11 or 14 and conjugated aldehydes 12 or 15, respectively. The 1H NMR spectra of the compounds 17, 18 and 20 displayed a pair of doublets d 6.69 and 6.51 (1H each, J = 8 Hz) and compound 19 displayed at 6.50 (singlet, 2H) for aromatic protons. The ring olefinic protons at C-6 resonated at d 5.6 (broad triplet) (1H) and the side chain olefinic protons resonated at d 5.1 (broad triplet) (1H). The resulting labdane analogs with o-quinol, catechol or hydroquinone functionality namely molecules 10–20 were evaluated for anti-HIV activity. The inhibitory activity of the compounds is sometimes, a result of their toxic effects and consequently might resemble an erroneous conclusion.11 Prior to the analysis of anti-HIV activity, compounds 10–20 were tested in vitro for their cytotoxicity on TZMbl cells using MTT assay.22,23 The results are summarized in (Table 1). CC50 value indicates the cytotoxic concentration of the compound leading to 50% reduction in cell viability. Out of the 11 compounds evaluated by MTT assay, seven compounds 10–12, 14–16 and 18 showed CC50 values of more than 200 lM. However, compound 13 showed a CC50 value of 56 ± 5.0 lM whereas 17 and 20 showed that of 139 ± 12 and 140 ± 23 lM, respectively. Compounds 10–20 were evaluated for in vitro inhibition of HIV-1 infection by reporter-gene based cell assay system. Compounds 13 and 18 showed IC50 (concentration of the compound leading to 50% inhibition of HIV-1 infection) values of 5.0 ± 1.0 and 4.6 ± 1.0 lM, respectively (Table 1). The compound 17 and 20 showed an IC50 value of 12.3 ± 4.0 and 12.4 ± 2.0 lM, respectively. In these experiments Raltegravir showed an IC50 of 0.5 lM. The IC50 values for compounds 14, 15 and 16 ranged from 36.7 ± 3.0 to 72.7 ± 18.0 lM. Other compounds showed IC50 value of more than 100 lM. Compound 19 was inactive at 50 lM. The integration inhibition properties of these compounds at 50 lM were subsequently studied, using a commercially available kit (Express Biotech International, Maryland, USA) following the manufacturer’s instructions. Sodium azide and Raltegravir were used as positive controls at 50 lM (Table 2). Based on their efficacy against HIV-1 integrase activity, compounds 17, 18 and 20 were further evaluated for a dose-dependent inhibition. IC50 values of compounds which were found to be 13.4 ± 0.77, 11.1 ± 0.34 and 11.5 ± 0.97 lM, respectively (Table 2). Subsequently, these new labdane analogs were subjected to the molecular modeling studies by docking with HIV-1 integrase enzyme (IN). HIV-1 integrase is a 32 kDa enzyme that carries out DNA integration in a two-step reaction. In the first step, called 30 processing, two nucleotides are removed from each 30 end of the viral DNA. In the next step, called DNA strand transfer, a pair of transesterification reactions integrates the ends of the viral DNA into the host Table 1 Cytotoxic concentration and anti-HIV-1 activity of compounds 10–20 Compounds
CC50a (lM)
IC50b (lM)
TIc
10 11 12 13 14 15 16 17 18 19 20 Raltegravir
693.0 ± 32.0 382.8 ± 69.0 625.4 ± 49.0 56.4 ± 5.0 928.3 ± 55.0 626.9 ± 40.0 677.2 ± 45.0 138.5 ± 12.0 211.3 ± 13.0 NA 140 ± 23 855.6 ± 90.0
170.7 ± 58.0 284.6 ± 147.0 171.5 ± 67.0 5.0 ± 1.0 72.7 ± 18.0 60.7 ± 20.0 36.7 ± 3.0 12.3 ± 4.0 4.6 ± 1.0 — 12.4 ± 2.0 0.5 ± 0.1
4 1 4 11 13 10 18 11 46 — 11 1711
NA: not active. a CC50: the cytotoxic concentration of the compounds leading to 50% reduction in cell viability. b IC50: inhibitory concentration of the compounds leading to 50% inhibition in HIV-1 infection. c TI: therapeutic index, CC50/IC50.
Table 2 HIV-1 integrase inhibition activity of compounds 10–20 and Raltegravir Compounds
Percent inhibition of HIV-1 integrase activity (50 lM)
IC50 (lM)
10 11 12 13 14 15 16 17 18 19 20 Raltegravir NaN3 (1.5%)
38 0 0 24 18 19 9 78 88 0 82 100 65
NT NT NT NT NT NT NT 13.4 ± 0.77 11.1 ± 0.34 NT 11.5 ± 0.97 0.12 ± 0.86 NT
NT: not tested.
genome. Extensive mutagenesis studies mapped the catalytic site to the core domain (residues 50–212, IN CCD), which contains the catalytic residues ASP64, ASP116, and GLU152.24 Site-directed mutagenesis and photo-cross-linking experiments have identified several residues near the active site, including LYS156, LYS159, GLN148, and TYR143, that are critical for binding viral DNA substrate.25 The compounds 10–20 were docked into IN CCD to study molecular interactions.26,27 The docking score function of the program ranked the compounds in the same general order observed experimentally. Compounds 17, 18 and 20 exhibited significant hydrogen-bonding interactions with the active site of IN CCD. Docking results were compared with well-known integric acid, which has been reported to be an integrase inhibitor through binding to active site of IN CCD. Integric acid binds through, the one hydrogen bond between its carbonyl C@O and H–N of LYS156 (C@O—HAN: 2.20 Å). Another hydrogen bond between aldehyde carbonyl C@O and H–O of THR66 (C@O—HAO: 2.20 Å) (Fig. 3). Compound 10 was found to bind through two hydrogen bonds between its C-1 O–H, O@C (OAH—O@C: 2.01 Å) of CYS65 and C-2 H– O, H–N of HIS67 (H–O—H–N: 2.08 Å), however these residues are not active site residues. Compound 17 was binding through two hydrogen bonds between its C-1 O–H and O@C of GLU152 (O– H—O: 2.23 Å), C-2 H–O and H–N (H–O—H–N: 2.59 Å) of LYS156 (Fig. 3). Thus docking results show that compound 17 binds to the active site residues (GLU152 and LYS156). Hence compound 17 is more active than compound 10 (both the compounds contain myrcene side chain) (comparative binding Fig. 4a). Compound 13 was binding through one hydrogen bond between its C-1 H–O and H–N of CYS65 (H–O—H–N: 2.02 Å). The most potent compound 18 was nicely accommodated within IN CCD (Fig. 3). Its binding conformation exhibited two hydrogen bonds between its C-2 O–H, O@C (O–H—O: 1.89 Å) and C-2 H–O, N–H (H–O—H–N: 1.93 Å) of GLN148 (Fig. 3) which is active site residue. This may be reason for more potency of compound 18 compared to compound 13 (both compounds contain the ocimene side chain) (Fig. 4b). Compound 20 was also binding through two hydrogen bonds between its C-1 H–O, H–N (H–O—H–N: 2.06 Å) of LYS 159 and C-4 O–H, O@C (O–H—O: 2.12 Å) of GLU 152 (Table 3). Inactive compound 19 was docked in to the IN CCD, which binding through one hydrogen bond between its C-1 O–H, O@C (HAO—O@C: 1.96 Å) of CYS 63, which is not active site residue (Fig. 3). Docking results were compared with well-known Raltegravir, which has been reported to be an integrase inhibitor. Similar molecular docking studies on Raltegravir showed that it binds to the active site of the integrase enzyme. Raltegravir was binding through two hydrogen bonds between its side chain N–H and O@C (NAH—O@C: 2.09 Å) of ASP64 and its cyclic H–N, H–N of LYS159 (H–N—H–N:
R. Pawar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 302–307
(10)
(13)
(17)
(19)
Raltegravir
305
(18)
(20)
Integric acid
Figure 3. Binding of compounds 10, 13, 17, 18 and 20, integric acid and raltegravir in the IN CCD (only main surrounding residue are shown).
2.05 Å), which gave the best fit value. Some of the residues, including LYS156, LYS159 and GLN148, that are critical for binding viral DNA substrate, also are surrounding these molecules. Thus, we have successfully synthesized catechol and quinol labdane analogs to evaluate their anti-HIV activity. The graphical representation of structure verses activity is shown in Figure 5. The structure activity relationship (SAR) with respect to their biological activity and docking results of molecules 10–20 indicated that the
compound 13 with o-quinol functionality displays considerable anti-HIV activity. The acyl protection of hydroxyl group of o-quinol moiety of compound 13 destroys the activity completely. Compounds 13, 18 and 20 are more active than the compounds 10, 17 and 19, respectively, which indicates that the presence of ocimene side chain at position 5 of ortho adducts 13, 18 and 20 increases the anti-HIV activity as compared to myrcene side chain of para adducts 10, 17 and 19. The side chains of compounds 10
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COOMe
Increase in activity
MeOOC
O
O
OH
OH 10 IC 50 = 170.7± 58.0 µM (TI = 4)
13
CH 2OH MeOOC Oxidative modif ications Decrease in activity
IC 50 = 5.9 ± 1.0 µM (TI = 11)
O OH 14 IC 50 = 72.7 ± 18.0 µM (TI = 13) CHO MeOOC
MeOOC
O OAc 16 Inactive
Figure 4a. Comparative docking study of compound 10 (blue) and 17 (pale yellow).
HO
O OH
18 Most active IC 50 = 4.6 ± 1.0 µM (TI = 46)
OH 15 IC 50 = 60.7± 20.0 µM (TI = 10)
Figure 5. Graphical representation of structure verses anti-HIV activity.
In conclusion, eleven new labdane analogs were synthesized using Diels–Alder reaction and evaluated for anti-HIV activity. Compound 18 showed both anti HIV-1 activity and HIV-1 integrase inhibition activity, which were further evaluated for a dosedependent inhibition and computational study. The HIV-1 integrase inhibition activity of molecules 17, 18 and 20 were comparable with integric acid, the natural fungal metabolite. Presently, most regimens for the treatment of HIV infection employ drugs that primarily inhibit HIV-1 specific reverse transcriptase and/or protease. The synthetic labdane analogs described in the present study, which are integrase inhibitors, may have potential as anti-HIV agents and need further exploration. Acknowledgments Figure 4b. Comparative docking study of compound 13 (purple) and 18 (pale yellow).
Table 3 Comparison of IC50 and pIC50 with docking score of compounds 10–20 Compounds
IC50a (lM)
pIC50b
Docking score
10 11 12 13 14 15 16 17 18 19 20 Raltegravir Integric acid
170.7 284.6 171.50 5.0 72.7 60.7 36.7 12.3 4.6 NA 12.4 0.5 10
3.77 3.55 3.77 5.30 4.14 4.22 4.44 4.91 5.34 — 4.91 6.30 5.00
3.32 3.89 3.97 5.36 4.28 4.33 4.68 4.77 5.01 2.56 4.95 6.83 5.21
Authors are grateful to Kelkar Education Trust, Mumbai, Indian Council of Medical Research and Department of Biotechnology, Government of India for financial support. We are also thankful to the Department of Chemistry and Sophisticated Analytical Instrumentation Facility, Indian Institute of Technology, Mumbai and Tata Institute of Fundamental Research, Mumbai for NMR spectral data. We would also like to acknowledge NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH for providing us the molecular clone of HIV-1 (NL4.3) and Raltegravir. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11. 014. References and notes
a
IC50: inhibitory concentration of the compounds leading to 50% inhibition in HIV-1 infection. b pIC50: negative logarithm of IC50.
and 13 were functionalized by SeO2 oxidation to give aldehydes (12 and 15) and hydroxyl derivatives (11 and 14), respectively. However these modifications decreased the activity with respect to compound 10 and 13. Compounds 17, 18 and 20 with catechol or hydroquinone moiety showed integrase inhibition activity with IC50 13.4 ± 0.77, 11.1 ± 0.34 and 11.5 ± 0.97 lM, respectively. Catechol and hydroquinone functionality increase the activity may be due to phenolic hydroxyl groups. The catechol moiety (17 and 18) is superior to the hydroquinone moiety (19 and 20) as found from biological data.
1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
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