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European Journal of Medicinal Chemistry 87 (2014) 735e744

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis of cyclic 1,9-acetal derivatives of forskolin and theirbioactivity evaluation

Devendar Ponnam a, Singh Shilpi b, K.V.N.S. Srinivas a, *, Luqman Suiab b, Sarfaraz Alam c,Zehra Amtul d, Niranjan Kumar Arigari a, Kotesh Kumar Jonnala a, *, Lubna Siddiqui b,Vijaya Dubey b, Ashok Kumar Tiwari d, Sridhar Balasubramanian e, Feroz Khan c

a Natural Product Chemistry, CSIR-Central Institute of Medicinal and Aromatic Plants- Research Centre, Boduppal, Hyderabad 500092, Telangana, Indiab Molecular Bioprospection Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Kukrail Picnic Spot Road, Lucknow 226015, UP, Indiac Metabolic & Structural Biology Department, CSIR-Central Institute of Medicinal and Aromatic Plants, Kukrail Picnic Spot Road, Lucknow 226015, UP, Indiad Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, Telangana, Indiae CSIR-Centre for X-ray Crystallography, Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, Telangana, India

a r t i c l e i n f o

Article history:Received 10 June 2014Received in revised form2 October 2014Accepted 6 October 2014Available online 7 October 2014

Keywords:ForskolinAcetal derivativesCeric ammonium nitrateAnticancer activitya-Glucosidase activity

* Corresponding authors.E-mail addresses: kvnssrinivas@yahoo.co.in (K.V.N

yahoo.com, koteshnpp@yahoo.co.in (K.K. Jonnala).

http://dx.doi.org/10.1016/j.ejmech.2014.10.0130223-5234/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

A new series of 1,9-acetals of forskolin were synthesized by treating with aromatic and aliphatic alde-hydes using Ceric ammonium nitrate as catalyst and evaluated for anticancer and a-glucosidase inhi-bition activities. Among the synthesized compounds 2a, 2b and 3a showed potential cytotoxic activitytowards human cancer cell lines MCF-7 (Human Breast Adenocarcinoma), MDA-MB (Human BreastCarcinoma), HeLa (Human Cervix Adenocarcinoma), A498 (Human Kidney Carcinoma), K562 (HumanErythromyeloblastoid leukemia), SH-SY5Y (Human Neuroblastoma), Hek293 (Human Embryonic Kidney)and WRL68 (Human Hepatic) with IC50 values ranging between 0.95 and 47.96 mg/ml. Osmotic fragilitytest revealed compound 3a as non-toxic to human erythrocytes at the tested concentrations of 50 and100 mg/ml. Compounds 1g (IC50 value 0.76 mg/ml) and 1p (IC50 value 0.74 mg/ml) significantly inhibited a-glucosidase in in vitro system. In silico based docking, ADME and toxicity risk assessment studies alsoshowed discernible a-glucosidase activity for compounds 1g, 1p compared to standard acarbose.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Generally, synthesis of natural products proceeds via protectionand deprotection sequences [1e3]. In the recent years, muchattention has been focused on the synthesis of acetal protectedcompounds [4e6], due to their significant biological activity. Manysemi synthetic derivatives of natural products having the acetalmoiety showed either enhanced or a different activity altogether.For example, semisynthetic acetal derivatives of Podophyllotoxin[7,8], andrographolide [9] and taxanes [10] (Fig. 1), are currentlyused in the treatment of a variety of malignancies.

Forskolin 1, is a highly oxygenated major labdane type diter-penoid present in the roots of Coleus forskohlii [11,12]. Forskolinexerts most of its biological activity by stimulation of adenylatecyclase by increasing cellular concentrations of the secondmessenger, cyclic AMP [13]. It also displays a wide variety of

.S. Srinivas), koteshkumarj@

served.

physiological activities such as branchospasmolytic [14], antihy-pertensive, inotropic [15,16], antiglaucoma [17], cardiovascular [18],antiobesity [19] and anticancer [20,21] activity etc. 7-deacetylforskolin 2, another major phytochemical of the plant,displays a lowering of blood pressure although less intense thanthat of forskolin, but equipotent in spontaneously hypertensive rats[14,15]. The third major phytoconstituent, isoforskolin 3 was foundto stimulate cyclic AMP and therefore, decrease blood pressure andproduce an inotropic effect lesser than that produced by forskolin[15,22,23]. Considering the various biological potentials exhibitedby forskolins (1, 2 and 3) and their high natural abundance in theplant, authors felt to synthesize their 1,9-acetal derivatives andscreen them for in vitro anticancer and antidiabetic potentials.

The most commonly used methods [24] for the synthesis ofacetals involves the catalytic condensation of either 1,2 or 1,3-diolswith aldehydes or dimethyl/diethyl acetals in the presence orabsence of dehydrating agents. These methods suffer harsh reac-tion conditions, use of toxic catalysts, acidic reaction conditions andcompetitive products formation by elimination, dehydration,isomerization, decomposition etc. Hence, these methods are not

Fig. 1. Semisynthetic derivatives of Podophyllotoxin, Andrographolide and Taxanes.

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744736

applicable for natural products especially with acid sensitivefunctional groups. Ceric ammonium nitrate (CAN) [25] is the mostnotable one electron oxidant and has been utilized extensively for abroad variety of oxidative transformations in synthetic chemistry.Additional advantages such as excellent solubility in water, inex-pensiveness, ecofriendly nature and high reactivity makes CAN apotent catalyst in organic syntheses. CAN is also used as a powerfulcatalyst in oxidation [26], nitration [27], 1,3-dipolar cycloaddition[28], thiocyanation [29], esterification [30], 1,4-addition [31] and inthe biginelli reaction [32]. In the present work, we have synthe-sized a series of novel 1,9-acetal derivatives of forskolins (1, 2 and 3)isolated from C. forskohlii, using CAN as a catalyst, and evaluated forin vitro anticancer and a-glucosidase inhibition activity. Com-pounds exhibiting potent cytotoxic activity against human cancercell lines were further evaluated for their toxicity on humanerythrocytes by performing osmotic fragility test and compoundsshowing potential a-glucosidase inhibition were also confirmed bydocking and ADMET studies.

2. Result and discussion

2.1. Chemistry

The synthetic strategy was as depicted in Scheme 1, forskolin 1was treated with a series of aliphatic/aromatic aldehydes aep inthe presence of CAN (30 mol %) in acetonitrile at room temperaturefor 12e24 h and, as envisaged, the reactions proceeded smoothly toafford the corresponding forskolin-1,9-acetals (1ae1p) in excellentyields. Acetonitrile was the best solvent for acetal formation

amongst methanol, ethyl acetate, chloroform or water. Althoughthe amount of catalyst has been optimized to 30 mol % but, lesseramounts like 5, 10 or 20 mol % also worked with longer reactiontimes. The reaction was chemoselective for the aldehydes whencarried out as a mixture of benzaldehyde a and acetophenoneduring the synthesis of compound 1a. Also, the reaction was highlyregioselective, when 7-deacetylforskolin 2 was reacted with a andb, at same reaction conditions, compounds 2a and 2bwere formedby protecting cis-1,9 diol without affecting cis-6,7 or trans-7,9-diols.Similarly, when isoforskolin 3 was treated with a and b, underabove conditions, compounds 3a and 3b were formed majorlywithout affecting trans-7,9 diol. The catalyst CAN's ability ofregioselectivity was also proved when 1a and 3awere deacetylatedwith methanolic KOH to get compound 2a in good yields (Scheme2).

Structures of all the derivatives were elucidated by 1H, 13C NMR,MS and IR spectral data. Further, the structure and stereochemistryof compounds 2a and 3b were also confirmed by single crystal X-ray diffraction studies. The perceptive view of 2a and 3b given inFig. 2 and the new stereo centre formed in cyclic 1,9-acetals isconfirmed as (S) conformation.

2.2. Biology

2.2.1. In vitro anticancer screeningForskolins and all acetal derivatives were screened for in vitro

cytotoxic activity by performing MTT assay against various humancancer cell lines includingMCF-7 (Human Breast Adenocarcinoma),MDA-MB (Human Breast Carcinoma), HeLa (Human Cervix

Scheme 1. Synthesis of 1,9-acetal derivatives of forskolins.

Scheme 2. Deacetylation of 1a and 3a to yield 2a.

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744 737

Adenocarcinoma), A498 (Human Kidney Carcinoma), K562 (HumanErythromyeloblastoid Leukemia), SH-SY5Y (Human Neuroblas-toma), HEK293 (Human Embryonic Kidney), WRL68 (Human He-patic) and NRK (Normal Rat Kidney). Taxol (Paclitaxel) andPodophyllotoxin were used as reference compounds (Table 1).Among all the derivatives, compounds 2a (IC50 1.16 in HEK293, 1.26in K562, 3.89 in HeLa and 9.79 mg/ml inWRL68), 2b (IC50 0.95 mg/mlin HeLa) and 3a (IC50 1.15 in MCF-7, 1.28 in MDA-MB and 1.26 mg/mlin K562) have showed significant cytotoxic activity and compounds

3b, 1f, 1p and 1m displayed moderate activity in the tested celllines. From the Table 1, it is very clear that the starting compoundsforskolin 1, 7-deacetylforskolin 2 and isoforskolin 3, were noncytotoxic (at the higher tested concentrations of 100 mg/ml)whereas benzylidene derivatives of 2, 3 (2a, 3a) and ethylidenederivative of 2 (2b) were found to be significantly active. Consid-ering these results, it is inferred that the forskolins without 7-acetylgroup along with 1,9-hydroxyl protection by either benzaldehydeor acetaldehyde are key functionalities to induce cytotoxicity in the

Fig. 2. Single crystal X-ray structures (ORTEP drawing) of compounds 2a and 3b with thermal displacement ellipsoids drawn at the 30% probability.

Table 1Anticancer and a-glucosidase activity of forskolins and their derivatives.a

Compound a-glucosidase inhibition, (IC50mg/ml) Anticancer activity by MTT assay, (IC50 mg/ml)

MCF-7 MDA-MB HeLa Hek293 A498 K562 SH-SY5Y WRL68 NRK

1a 2.81 >100 >100 >100 >100 >100 >100 >100 >100 >1001b 5.87 >100 >100 >100 >100 >100 >100 >100 >100 >1001f >100 >100 >100 46.17 >100 >100 >100 >100 >100 >1001g 0.76 >100 >100 >100 >100 >100 >100 >100 >100 >1001m >100 >100 >100 41.97 40.15 >100 >100 >100 >100 >1001n 4.96 >100 >100 >100 >100 >100 >100 >100 >100 >1001p 0.74 >100 >100 >100 38.54 >100 >100 >100 >100 >1002a >100 >100 20.75 3.89 1.167 20.44 1.263 21.57 9.791 23.692b 4.23 45.68 24.48 0.95 24.84 40.62 25.37 34.69 29.74 47.963a 4.14 1.15 1.28 9.22 14.29 16.7 1.262 16.59 16.59 20.123b 6.40 39.23 >100 17.4 >100 41.6 >100 >100 >100 >100Paclitaxel NA 0.01 0.026 1.18 0.008 7.53 0.018 7.30 0.004 e

Podophyllotoxin NA 3.52 0.017 3.14 0.120 0.034 0.013 1.05 1.989 e

Acarbose 0.45 NA NA NA NA NA NA NA NA NA

a Data represents mean value of two independent determination in duplicates.

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744738

respective compounds. Rest of the compounds were found to beinactive against almost all the cell lines.

Compounds 2a, 2b and 3a exhibiting significant cytotoxic ac-tivity against various human cancer cell lines were also evaluatedfor erythrocyte osmotic fragility (Fig. 3 and Table 2). Among these,compound 3a showing most potent activity against MCF-7 wasfound to be non-toxic to human erythrocytes whereas compounds2a and 2b increased the haemolysis of erythrocytes, hence thesemay be considered toxic at higher concentrations.

Fig. 3. Erythrocyte osmotic fragility curve of forskolin derivatives, dimethylsulphoxide(DMSO), Curcumin and Podophyllotoxin (PDT).

2.2.2. Intestinal a-glucosidase inhibitory activityThe results of all the forskolins and its derivatives screened for

Intestinal a-glucosidase inhibitory activity were showed in Table 1and Fig. 4. Compounds 1g (IC50 value 0.76 mg/ml) and 1p (IC50 value0.74 mg/ml) showed significant inhibition whereas compounds 1a(IC50 2.81 mg/ml), 3a (IC50 4.14 mg/ml), 2b (IC50 4.23 mg/ml),1n (IC504.96 mg/ml), 1b (IC50 5.87 mg/ml) and 3b (IC50 6.40 mg/ml) showedmoderate activity compared to control Acarbose (IC50 0.45 mg/ml).The activity results infer that the protection of 1,9-diols of forskolin1 with 4-nitrobenzaldehyde (1g) and trans-cinnamaldehyde (1p)enhanced the a-glucosidase inhibition comparable to that of stan-dard Acarbose whereas substitutions like bromo, methyl, methoxyand cyano groups at 4-position of respective benzaldehydes did notshow any activity.

2.2.3. Molecular docking studies on intestinal a-glucosidaseMolecular docking on target protein human intestinal a-gluco-

sidase (PDB: 2QMJ) showed docking score (LibDock) of 72.751 and74.350 for compounds 1g and 1p respectively compared to that ofstandard acarbose 144.845 (Table 3). The docking result shows thatboth the compounds 1g and 1p have a single hydrogen bond for-mation with asparagines (ASN-14). The molecular binding of acar-bose exhibited three hydrogen bonds with lysine (LYS513) and

Table 2Mean Erythrocyte Fragility (MEF) of active derivatives of forskolins.

Samples Concentrations MEF50

2a 100 mg/mL 0.6950 mg/mL 0.67

2b 100 mg/mL 0.6950 mg/mL 0.85

3a 100 mg/mL 0.5750 mg/mL 0.53

Control e 0.61DMSO 0.5% 0.62Curcumin 100 mg/mL 0.53Podophyllotoxin 100 mg/mL 0.69

Fig. 4. Rat intestinal a-glucosidase inhibitory activity shown by forskolins and itsderivatives (at 100 mg/mL concentration). Each data represents mean, SD of triplicatereadings.

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744 739

single hydrogen bond with each of the arginine (ARG520), valine(VAL779) lysine (LYS534), glutamate (GLU510), aspartate (ASP777)and threonine (THR775) residues. The results of molecular dockingindicate that the candidate compound bound well within thebinding pocket of a-glucosidase, and showed a good binding af-finity. The 2D diagram of molecular docking for compounds(Fig. S1; refer in supplementary) showed interaction of a-glucosi-dase amino acid residues with bound ligands as represented bydifferent colours e.g., purple indicates a covalent bond, pink in-dicates electrostatic interaction and green colour indicates Vander-Waals molecular interaction.

2.2.4. Bioavailability, drug likeness and toxicity screeningThe ADMET is one of the keymeasures which influence the drug

levels and kinetics of drug and influence the pharmacological ac-tivity of the compound as a drug. In the present study, the aqueoussolubility prediction (defined in water at 25 �C) showed a lowsoluble agent. Besides, no predictive hepatotoxicity was observedduring ADME screening (Table S1; refer in supplementary). Thepredicted ADME results of lead compounds were found comparedto standard range. The compounds 1g and 1p were highly (�90%)bound to carrier proteins in the blood. These binding shows theefficiency of drugs to be absorbed by the intestine when orallyadministered and hence both the compounds can be absorbed bythe intestine. The hit compound is found to be non-inhibitors ofcytochrome P450 2D6 (CYP2D6). A Plot of PSA versus LogP at the95% and 99% confidence limit ellipses corresponding to thebloodebrain barrier and intestinal absorption models showed thatthe lead compounds 1g and 1p are within the limit (Figure S2; refer

in supplementary). The toxic effect of compounds 1g, 1p andstandard acarbose were calculated. The candidate compound didn'tshow any carcinogenicity, genotoxicity, hepatotoxicity and neph-rotoxicity effect. The Maximum Recommended Therapeutic Doses(MRTD) are �0.31 for lead compounds 1g and 1p. The values are inlog mg/kg-bm/day. The results of these toxicities along with MRTDare given in Table 4.

3. Conclusion

The usefulness of this methodology lies in the fact that the re-actions are carried out rapidly under mild conditions to give a totalof 20 novel products (1ae1p, 2ae2b and 3ae3b) in excellent yields.Regioselectivity and chemoselctivity are the salient features of thismethodology and the method is also compatible with substratesubstituents such as halogens, methyl, methoxy, methylenedioxy,cyano, nitro etc. Furthermore, the reaction conditions are mildenough to perform these reactions with acid sensitive aldehydes(such as furfural, 2-thiophenecarboxaldehyde and trans-cinna-maldehyde) without any decomposition or polymerization, andwith enolizable aldehydes (such as acetaldehyde, prenal) in excel-lent yields. Thus, this methodology may provide broad applicationin synthetic/semisynthetic organic chemistry. Undoubtedly, 1,9-acetal protection has introduced anticancer and antidiabetic ac-tivity in forskolins. Anticancer results indicated that the forskolinswithout 7-acetyl group along with 1,9-hydroxyl protection byeither benzaldehyde (2a and 3a) or acetaldehyde (2b) are keyfunctionalities to induce cytotoxicity in the respective compounds.Erythrocyte osmotic fragility assay confirms the non-toxic nature ofcompound 3a at concentrations where it showed anticancer ac-tivity. The a-glucosidase inhibitory activity results infer that theprotection of 1,9-diol of forskolin 1 with 4-nitrobenzaldehyde (1g)and trans-cinnamaldehyde (1p) enhanced the a-glucosidase inhi-bition comparable to that of standard acarbose. In silico docking andADMET studies of the lead compounds (1g and 1p) against thetarget a-glucosidase confirms their drug likeness when comparedto standard acarbose.

4. Experimental section

4.1. Materials and instruments

Evaporations were conducted under reduced pressure. Allnonaqueous reactions were carried out under N2 atmosphere inflame-dried glassware. All reagents were procured from SigmaAldrich and were used as received. Kieselgel 60 F254 (E. Merck) wasused for TLC. Visualization of the developed TLC was performed byUV light or 5% H2SO4 in MeOH stain. Melting points were measuredusing A. KRUSS OPTRONIC and are uncorrected. NMR spectra wererecorded on Bruker Avance 400/500 MHz in CDCl3 using TMS asinternal standard. IR data are given only for compounds with sig-nificant functions (OH, C]O) and were recorded as KBr plate andare reported in wave number (cm�1).

4.2. General procedure for the synthesis of compounds 1aep, 2a,2b, 3a and 3b (Scheme 1)

To a well stirred solution of compound 1/2/3 (2 mmol) inacetonitrile (10 mL) were added aldehyde aep (10 mmol) and Cericammonium nitrate (30 mol %). The whole mixture was stirred atroom temperature for 12e24 h, diluted with water (30 mL),extracted with ethyl acetate (3 � 10 mL). The combined organiclayers were washed with water, brine solution. After drying overanhydrous Na2SO4, solvent was evaporated under reduced pressureto give the crude. The product was purified through neutral

Table 3Details of LibDock score and hydrogen bonds revealed through molecular dockingof compound 1g and 1p along with Acarbose on human intestinal a-glucosidase(PDB: 2QMJ).

S.No.

Compounds LibDockscore

H-Bonding analysis

1 1g 72.7516 ASN-142 1p 74.3505 ASN-143 Acarbose

(control)144.845 LYS513 (3 H-bonds), ARG520, VAL779, LYS534,

GLU510, ASP777, THR775

Table

4Pred

iction

oftoxiceffect

ofco

mpou

nds1g

,1pan

dAcarbose.

Sample

AMES

Anem

iaCarcinog

enicityCarcinog

enicitymou

sefemale

Carcinog

enicitymou

semale

Carcinog

enicityrat

female

Carcinog

enicityrat

male

Cardiotoxicity

Gen

otox

icityHep

atotox

icityMRTD

Nep

hrotoxicity

Neu

rotoxicity

1g0.81

0.21

0.33

0.20

0.16

0.35

0.22

0.76

0.17

0.50

�0.31

0.49

0.67

1p0.27

0.28

0.12

0.15

0.11

0.08

0.13

0.83

0.12

0.35

�0.31

0.46

0.81

Acarbose0.55

0.09

0.16

0.20

0.17

0.09

0.17

0.86

0.31

0.59

0.98

0.36

0.25

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744740

alumina column chromatography (ethyl acetateepetroleumether) to afford the desired product 1aep/2a/2b/3a/3b.

4.2.1. 1,9-Benzylideneforskolin (1a)Yield: 75 mg (75%), white solid, m.p. 142e144 �C; IR (KBr):

3513, 2922, 1714, 1270, 1106, 1019 cm�1; 1H NMR (400 MHz,CDCl3): d (ppm) 7.47 (m, 2H), 7.34 (m, 3H), 5.73 (dd, J ¼ 17.2 and10.4 Hz, 1H), 5.42 (d, J ¼ 4 Hz, 1H), 5.39 (s, 1H), 5.14 (d, J ¼ 16.8 Hz,1H), 4.74 (d, J ¼ 10.4 Hz, 1H), 4.48 (brs, 1H), 4.23 (brs, 1H), 2.92 (d,J¼ 16.8 Hz, 1H), 2.49 (d, J¼ 16.4 Hz, 1H), 2.43 (d, J¼ 2 Hz, 1H), 2.14(s, 3H), 2.09 (m, 1H), 1.80e1.77 (m, 2H), 1.73 (s, 3H), 1.62e1.59 (m,1H), 1.56 (s, 3H), 1.34 (s, 3H), 1.28 (s, 3H), 1.11e1.08 (m, 1H), 1.03 (s,3H); 13C NMR (125 MHz, CDCl3): d (ppm) 205.72, 169.52, 145.84,138.09, 128.77, 128.09, 128.09, 126.31, 126.31, 110.55, 96.66, 85.27,80.63, 76.54, 76.35, 75.19, 70.08, 50.08, 43.04, 39.16, 36.55, 34.47,33.05, 31.48, 24.69, 23.65, 23.41, 21.15, 17.55; ESI-MS: positive ionmode: m/z 499 [MþH]þ, 516 [MþNH4]þ (calculated mass forC29H38O7 is 498).

4.2.2. 1,9-Ethylideneforskolin (1b)Yield: 60 mg (68%), white solid, m.p. 168e170 �C; IR (KBr):

3460, 2969, 1742, 1372, 1226, 1099, 1055 cm�1; 1H NMR (400MHz,CDCl3): d (ppm) 5.85 (dd, J¼ 17.2 and 10.8 Hz,1H), 5.37 (d, J¼ 4 Hz,1H), 5.21 (d, J ¼ 17.2 Hz, 1H), 4.88 (d, J ¼ 10.8 Hz, 1H), 4.57 (q,5.2 Hz, 1H), 4.44 (brs, 1H), 4.02 (brs, 1H), 2.75 (d, J ¼ 17.6 Hz, 1H),2.49 (d, J ¼ 17.6 Hz, 1H), 2.28 (brs, 1H), 2.15 (s, 3H), 2.07e2.00 (m,1H), 1.76e1.72 (m, 2H), 1.66 (s, 3H), 1.52e1.51 (m, 1H), 1.46 (s, 3H),1.31 (s, 3H), 1.29 (d, J ¼ 5.2 Hz, 3H), 1.25 (s, 3H), 1.01 (m, 4H); 13CNMR (125 MHz, CDCl3): d (ppm) 204.99, 169.66, 146.62, 110.23,94.45, 84.36, 80.16, 76.6, 75.77, 74.62, 70.03, 49.94, 42.86, 38.68,36.39, 34.37, 33.06, 31.95, 24.65, 23.55, 23.22, 21.21, 21.21, 17.50;ESI-MS: positive ion mode: m/z 437 [MþH]þ, 454 [MþNH4]þ

(calculated mass for C24H36O7 is 436).

4.2.3. 1,9-(3-methl-2-butenylidene)forskolin (1c)Yield: 57 mg (60%), white solid, m.p. 149e151 �C; IR (KBr):

3539, 2946, 1719, 1370, 1260, 1080, 1012 cm�1; 1H NMR (400 MHz,CDCl3): d (ppm) 5.84 (dd, J ¼ 17.2 and 10.4 Hz, 1H), 5.36 (d,J¼ 3.6 Hz, 1H), 5.31 (m,1H), 5.13 (dd, J¼ 16.8 and 0.8 Hz, 1H), 5.07(d, J ¼ 7.2 Hz, 1H), 4.78 (dd, J ¼ 10.4 and 1.2 Hz, 1H), 4.45 (brs, 1H),4.11 (brs, 1H), 2.79 (d, J¼ 17.6 Hz,1H), 2.51 (d, J¼ 17.6 Hz, 1H), 2.32(d, J ¼ 1.6 Hz, 1H), 2.14 (s, 3H), 2.08e2.00 (m, 1H), 1.78e1.73 (m,5H), 1.67 (s, 3H), 1.61 (s, 3H), 1.56e1.52 (m,1H), 1.47 (s, 3H), 1.31 (s,3H), 1.25 (s, 3H), 1.07e1.06 (m, 1H), 1.02 (s, 3H); 13C NMR(125 MHz, CDCl3): d (ppm) 205.07, 169.63, 146.16, 138.35, 122.27,110.28, 93.96, 84.44, 80.13, 76.5, 75.89, 74.52, 69.99, 49.98, 42.79,38.69, 36.39, 34.37, 33.03, 31.50, 25.68, 24.66, 23.55, 23.28, 21.17,18.81, 17.60; ESI-MS: positive ion mode: m/z 477 [MþH]þ, 494[MþNH4]þ (calculated mass for C27H40O7 is 476).

4.2.4. 1,9-(p-florobenzylidene)forskolin (1d)Yield: 70 mg (68%), white solid, m.p. 161e163 �C; IR (KBr):

3500, 2927, 1713, 1374, 1271, 1107, 1018 cm�1; 1H NMR (400 MHz,

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744 741

CDCl3): d (ppm) 7.44 (m, 2H), 7.03 (m, 2H), 5.71 (dd, J ¼ 17.2 and10.8 Hz, 1H), 5.40 (d, J ¼ 4.0 Hz, 1H), 5.36 (s, 1H), 5.13 (dd, J ¼ 17.2and 1.2 Hz, 1H), 4.73 (dd, J¼ 10.8 and 1.2 Hz, 1H), 4.47 (d, J¼ 1.6 Hz,1H), 4.22 (brs, 1H), 2.87 (d, J¼ 16.4 Hz, 1H), 2.53 (d, J¼ 16.8 Hz, 1H),2.38 (brs, 1H), 2.14 (s, 3H), 2.12e2.08 (m, 1H), 1.80e1.75 (m, 2H),1.73 (s, 3H), 1.64e1.60 (m, 1H), 1.57 (s, 3H), 1.34 (s, 3H), 1.28 (s, 3H),1.12e1.08 (m, 1H), 1.02 (s, 3H); ESI-MS: positive ion mode: m/z 517[MþH]þ, 534 [MþNH4]þ (calculated mass for C29H37FO7 is 516).

4.2.5. 1,9-(o-chlorobenzylidene)forskolin (1e)Yield: 77mg (72%), white solid, m.p. 136e138 �C; IR (KBr): 3525,

2924, 1717, 1371, 1265, 1102, 1017 cm�1; 1H NMR (400 MHz, CDCl3):d (ppm) 7.68 (d, 1H), 7.31 (m, 3H), 5.78 (s, 1H), 5.61 (dd, J ¼ 17.2 and10.8 Hz, 1H), 5.39 (d, J ¼ 4.0 Hz, 1H), 4.94 (dd, J ¼ 17.2 and 1.2 Hz,1H), 4.53 (d, J ¼ 2.0 Hz, 1H), 4.46 (dd, J ¼ 10.8 and 1.2 Hz, 1H), 4.30(brs, 1H), 3.00 (d, J¼ 18.0 Hz, 1H), 2.53 (d, J¼ 18.0 Hz,1H), 2.49 (brs,1H), 2.11e2.07 (m, 1H), 2.14 (s, 3H), 1.81e1.76 (m, 2H), 1.71 (s, 3H),1.62 (m, 1H), 1.57 (s, 3H), 1.29 (s, 3H), 1.29 (s, 3H), 1.13 (m, 1H), 1.08(s, 3H); ESI-MS: positive ion mode: m/z 533 [MþH]þ, 550[MþNH4]þ (calculated mass for C29H37ClO7 is 532).

4.2.6. 1,9-(p-bromobenzylidene)forskolin (1f)Yield: 72 mg (62%), white solid, m.p. 130e132 �C; IR (KBr): 3497,

2925, 1715, 1373, 1268, 1103, 1010, 822 cm�1; 1H NMR (400 MHz,CDCl3): d (ppm) 7.48 (d, J ¼ 8.4 Hz, 2H), 7.35 (d, J ¼ 8.4 Hz, 2H), 5.72(dd, J ¼ 17.2 and 10.4 Hz, 1H), 5.39 (d, J ¼ 4.0 Hz, 1H), 5.34 (s, 1H),5.15 (dd, J ¼ 17.2 and 1.2 Hz, 1H), 4.77 (dd, J ¼ 10.4 and 1.2 Hz, 1H),4.46 (brs, 1H), 4.22 (brs, 1H), 2.86 (d, J ¼ 16.8 Hz, 1H), 2.50 (d,J ¼ 16.8 Hz, 1H), 2.35 (brs, 1H), 2.14 (s, 3H), 2.11e2.08 (m, 1H), 1.75(m, 2H), 1.73 (s, 3H), 1.64e1.59 (m, 1H), 1.55 (s, 3H), 1.34 (s, 3H), 1.28(s, 3H), 1.11e1.07 (m, 1H), 1.01 (s, 3H); ESI-MS: positive ion mode:m/z 577 [MþH]þ, 579 [Mþ2þH]þ, 594 [MþNH4]þ, 596[Mþ2þNH4]þ (calculated mass for C29H37BrO7 is 576).

4.2.7. 1,9-(p-nitrobenzylidene)forskolin (1g)Yield: 100 mg (92%), yellow solid, m.p. 157e159 �C; IR (KBr):

3532, 2919, 1710, 1348, 1212, 1106, 1040 cm�1; 1H NMR (400 MHz,CDCl3): d (ppm) 8.21 (d, J ¼ 8.8 Hz, 2H), 7.67 (d, J ¼ 8.8 Hz, 2H), 5.74(dd, J¼ 17.2 and 10.8 Hz,1H), 5.45 (s, 1H), 5.40 (d, J¼ 4 Hz, 1H), 5.20(dd, J ¼ 17.2 and 1.2 Hz, 1H), 4.82 (dd, J ¼ 10.8 and 1.2 Hz, 1H), 4.46(brs, 1H), 4.27 (brs, 1H), 2.87 (d, J¼ 17.2 Hz, 1H), 2.55 (d, J¼ 16.8 Hz,1H), 2.31 (brs,1H), 2.15 (s, 3H), 2.11e2.10 (m,1H), 1.77e1.63 (m, 6H),1.57 (s, 3H), 1.36 (s, 3H), 1.29 (s, 3H), 1.14e1.10 (m, 1H), 1.01 (s, 3H);ESI-MS: positive ion mode: m/z 544 [MþH]þ, 566 [MþNa]þ

(calculated mass for C29H37NO7 is 543).

4.2.8. 1,9-(p-methylbenzylidene)forskolin (1h)Yield: 72 mg (70%), white solid, m.p. 160e162 �C; IR (KBr): 3498,

2926, 1712, 1377, 1265, 1097,1022 cm�1; 1H NMR (400MHz, CDCl3):d (ppm) 7.36 (d, J¼ 8 Hz, 2H), 7.15 (d, J¼ 8 Hz, 2H), 5.74 (dd, J¼ 17.2and 10.8 Hz, 1H), 5.41 (d, J ¼ 3.6 Hz, 1H), 5.36 (s, 1H), 5.15 (dd,J ¼ 17.2 and 1.2 Hz, 1H), 4.75 (dd, J ¼ 10.4 and 1.2 Hz, 1H), 4.46 (brs,1H), 4.20 (br, 1H), 2.91(d, J ¼ 16.4 Hz, 1H), 2.47 (d, J ¼ 16.4 Hz, 1H),2.42 (brs, 1H), 2.14 (s, 3H), 2.11e2.07 (m, 1H), 1.83e1.78 (m, 5H),1.64e1.60 (m, 1H), 1.56 (s, 3H), 1.34 (s, 3H), 1.28 (s, 3H), 1.09e1.06(m, 1H), 1.01 (s, 3H); ESI-MS: positive ion mode: m/z 513 [MþH]þ,535 [MþNa]þ (calculated mass for C30H40O7 is 512).

4.2.9. 1,9-(p-methoxybenzylidene)forskolin (1i)Yield: 69 mg (65%), white solid, m.p. 98e100 �C; IR (KBr): 3500,

2925, 1711, 1381, 1256, 1094,1025 cm�1; 1H NMR (500MHz, CDCl3):d (ppm) 7.40 (d, J ¼ 6.8 Hz, 2H), 6.89 (d, J ¼ 7.2 Hz, 2H), 5.75 (dd,J ¼ 17.0 and 11 Hz, 1H), 5.43 (d, J ¼ 4.0 Hz, 1H), 5.36 (s, 1H), 5.14 (dd,J ¼ 17.0 and 1.5 Hz, 1H), 4.76 (dd, J ¼ 10.5 and 1.2 Hz, 1H), 4.49 (brs,1H), 4.21 (brs, 1H), 3.83 (s, 3H), 2.92 (d, J ¼ 17.0 Hz, 1H), 2.50 (d,

J ¼ 16.5 Hz, 1H), 2.44 (d, J ¼ 2.5 Hz, 1H), 2.16 (m, 4H), 1.82e1.79 (m,1H), 1.75 (s, 3H), 1.63 (m, 1H), 1.58 (s, 3H), 1.36 (s, 3H), 1.28 (s, 3H),1.12e1.10 (m, 1H), 1.01 (s, 3H); ESI-MS: positive ion mode: m/z 529[MþH]þ, 551 [MþNa]þ, 567 [MþK]þ (calculated mass for C30H40O8is 528).

4.2.10. 1,9-(3,4,5-trimethoxybenzylidene)forskolin (1j)Yield: 71 mg (60%), white solid, m.p. 64e66 �C; IR (KBr): 3506,

2927, 1716, 1463, 1389, 1236, 1128, 1108, 1049 cm�1; 1H NMR(400 MHz, CDCl3): d (ppm) 6.73 (s, 2H), 5.79 (dd, J ¼ 17.2 and10.4 Hz, 1H), 5.49 (d, J¼ 4 Hz, 1H), 5.34 (s, 1H), 5.19 (dd, J ¼ 17.2 and1.2 Hz, 1H), 4.76 (dd, J ¼ 10.4 and 1.2 Hz, 1H), 4.46 (brs, 1H), 4.21(brs, 1H), 3.87 (s, 6H), 3.85 (s, 3H), 2.93 (d, J ¼ 16 Hz, 1H), 2.46 (d,J¼ 16.4 Hz,1H), 2.39 (d, J¼ 2 Hz,1H), 2.15 (s, 3H), 2.10e2.09 (m,1H),1.76e1.75 (m, 5H), 1.67e1.62 (m, 1H), 1.57 (s, 3H), 1.36 (s, 3H), 1.29(s, 3H), 1.11e1.08 (m, 1H), 1.01 (s, 3H); ESI-MS: positive ion mode:m/z 611.27 [MþNa]þ (calculated mass for C32H44O9 is 588.69).

4.2.11. 1,9-(p-cyanobenzylidene)forskolin (1k)Yield: 91 mg (87%), white solid, m.p. 136e138 �C; IR (KBr): 3495,

2900, 1714, 1375, 1271, 1097, 1033 cm�1; 1H NMR (400 MHz, CDCl3):d (ppm) 7.65 (d, J¼ 8 Hz, 2H), 7.60 (d, J¼ 8 Hz, 2H), 5.73 (dd, J¼ 16.8and 10.8 Hz, 1H), 5.41 (s, 1H), 5.38 (d, J ¼ 3.6 Hz, 1H), 5.17 (d,J ¼ 16.8 Hz, 1H), 4.78 (d, J ¼ 10.4 Hz, 1H), 4.45 (brs, 1H), 4.25 (brs,1H), 2.85 (d, J¼ 16.8 Hz, 1H), 2.53 (d, J¼ 16.8 Hz, 1H), 2.31 (brs, 1H),2.10e2.15 (m, 4H), 1.81e1.72 (m, 5H), 1.64e1.56 (m, 4H),1.35 (s, 3H),1.28 (s, 3H), 1.12e1.09 (m, 1H), 1.00 (s, 3H); 13C NMR (125 MHz,CDCl3): d (ppm) 204.94, 169.54, 145.85, 142.68, 132.02, 132.02,127.13, 127.13, 118.64, 112.64, 110.53, 95.30, 85.38, 80.52, 76.58,76.18, 75.06, 69.96, 49.79, 43.06, 39.21, 36.47, 34.47, 33.00, 31.80,24.60, 23.59, 23.37, 21.12, 17.52; ESI-MS: positive ion mode: m/z546.20 [MþNa]þ (calculated mass for C30H37NO7 is 523.62).

4.2.12. 1,9-(a-napthylidene)forskolin (1l)Yield: 93 mg (85%), white solid, m.p. 171e173 �C; IR (KBr): 3510,

2923,1719,1374, 1262, 1106,1045 cm�1; 1H NMR (400MHz, CDCl3):d (ppm) 8.00 (d, J ¼ 9.2 Hz, 1H), 7.86 (m, 3H), 7.49 (m, 3H), 6.00 (s,1H), 5.59 (m, 2H), 5.06 (dd, J ¼ 17.2 and 1.2 Hz, 1H), 4.56 (d,J¼ 1.6 Hz, 1H), 4.50 (dd, J¼ 10.8 and 1.2 Hz, 1H), 4.43 (brs, 1H), 2.78(d, J¼ 16.8 Hz,1H), 2.53 (s, 1H), 2.45 (d, J¼ 16.8 Hz,1H), 2.18 (s, 3H),2.14e2.10 (m, 1H), 1.79e1.66 (m, 6H), 1.62 (s, 3H), 1.32 (s, 3H), 1.29(s, 3H), 1.04e1.03 (m, 1H), 1.02 (s, 3H); ESI-MS: positive ion mode:m/z 549 [MþH]þ, 571 [MþNa]þ (calculated mass for C33H40O7 is548).

4.2.13. 1,9-(2-thiophenylidene)forskolin (1m)Yield: 61 mg (60%), white solid, m.p. 138e140 �C; IR (KBr): 3535,

2926, 1740, 1370, 1210, 1112, 1007 cm�1;1H NMR (400 MHz, CDCl3):d (ppm) 7.29 (d, J ¼ 5.2 Hz, 1H), 7.09 (d, J ¼ 3.2 Hz, 1H), 6.96 (dd,J¼ 4.8 and 4 Hz, 1H), 5.79 (dd, J¼ 17.2 and 10.4 Hz, 1H), 5.62 (s, 1H),5.40 (d, J ¼ 3.6 Hz, 1H), 5.16 (d, J ¼ 17.2 Hz, 1H), 4.78 (d, J ¼ 10.8 Hz,1H), 4.47 (brs, 1H), 4.23 (brs, 1H), 2.91 (d, J ¼ 16.8 Hz, 1H), 2.50 (d,J ¼ 16.8 Hz, 1H), 2.41 (brs, 1H), 2.13 (s, 3H), 2.10e2.06 (m, 1H),1.84e1.76 (m, 2H), 1.72 (s, 3H), 1.64e1.63 (m, 1H), 1.11e1.07 (m, 1H),1.54 (s, 3H),1.34 (s, 3H),1.28 (s, 3H),1.03 (s, 3H); 13C NMR (125MHz,CDCl3): d (ppm) 205.4, 169.45, 145.86, 141.29, 126.29, 125.77, 125.16,110.65, 93.78, 85.31, 80.54, 76.5, 76.40, 75.21, 70.03, 50.21, 42.98,39.09, 36.40, 34.44, 33.02, 31.34, 24.65, 23.54, 23.38, 21.13, 17.48;ESI-MS: positive ion mode: m/z 505 [MþH]þ, 522 [MþNH4]þ

(calculated mass for C27H6O7S is 504).

4.2.14. 1,9-(2-furanylidene)forskolin (1n)Yield: 62 mg (63%), white solid, m.p. 156e158 �C; IR (KBr): 3533,

2927, 1714, 1368, 1259, 1116, 1005 cm�1; 1H NMR (400MHz, CDCl3):d (ppm) 7.38 (d, J ¼ 2.0 Hz, 1H), 6.46 (d, J ¼ 3.2 Hz, 1H), 6.35 (dd,

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744742

J ¼ 3.2 and 2.0 Hz, 1H), 5.80 (dd, J ¼ 17.2 and 10.4 Hz, 1H), 5.44 (s,1H), 5.39 (d, J¼ 4 Hz,1H), 5.13 (dd, J¼ 17.2 and 1.2 Hz,1H), 4.77 (dd,J ¼ 10.8 and 1.2 Hz, 1H), 4.47 (d, J ¼ 1.6 Hz, 1H), 4.20 (brs, 1H), 2.90(d, J ¼ 17.2 Hz, 1H), 2.49 (d, J ¼ 16.8 Hz, 1H), 2.39 (brs, 1H), 2.14 (s,3H), 2.10e2.05 (m,1H),1.82e1.71 (m, 5H),1.66e1.61 (m,1H),1.53 (s,3H), 1.34 (s, 3H), 1.27 (s, 3H), 1.10e1.06 (m,1H), 1.05 (s, 3H); ESI-MS:positive ion mode: m/z 489 [MþH]þ, 511 [MþNa]þ, 527 [MþK]þ

(calculated mass for C27H36O8 is 488).

4.2.15. 1,9-(3,4-methylenedioxybenzylidene)forskolin (1o)Yield: 81 mg (75%), white solid, m.p. 131e133 �C; IR (KBr): 3462,

2969, 1741, 1370, 1223, 1101 cm�1; 1H NMR (400 MHz, CDCl3):d (ppm) 6.98 (s, 1H), 6.92 (d, J¼ 8 Hz,1H), 6.76 (d, J¼ 8 Hz,1H), 5.96(s, 2H), 5.74 (dd, J ¼ 17.2 and 10.8 Hz, 1H), 5.39 (d, J ¼ 3.2 Hz, 1H),5.29 (s, 1H), 5.14 (d, J ¼ 16.8 Hz, 1H), 4.77 (d, J ¼ 10.8 Hz, 1H), 4.47(brs, 1H), 4.20 (brs, 1H), 2.88 (d, J¼ 16.4 Hz,1H), 2.49 (d, J¼ 16.8 Hz,1H), 2.39 (brs, 1H), 2.14 (s, 3H), 2.11e2.07 (m, 1H), 1.78e1.60 (m,6H), 1.55 (s, 3H), 1.34 (s, 3H), 1.28 (s, 3H), 1.11e1.03 (m, 1H), 1.03 (s,3H); 13C NMR (125 MHz, CDCl3): d (ppm) 205.60, 169.49, 147.88,147.45, 145.85, 132.22, 120.10, 110.62, 107.78, 106.95, 101.04, 96.45,85.23, 80.56, 76.48, 76.30, 75.13, 70.02, 50.04, 43.05, 39.05, 36.56,34.45, 33.07, 31.46, 24.66, 23.62, 23.38, 21.15, 17.52; ESI-MS: posi-tive ion mode: m/z 543 [MþH]þ, 560 [MþNH4]þ (calculated massfor C30H38O9 is 542).

4.2.16. 1,9-(trans-cinnamylidene)forskolin (1p)Yield: 82 mg (78%), white solid, m.p. 62e64 �C; IR (KBr): 3509,

2948, 1714,1372,1270,1123, 1024 cm�1; 1H NMR (400MHz, CDCl3):d (ppm) 7.42e7.40 (m, 2H), 7.34e7.27 (m, 3H), 6.74 (d, J¼ 16 Hz,1H),6.16 (dd, J ¼ 16 and 4.4 Hz, 1H), 5.83 (dd, J ¼ 17.2 and 10.8 Hz, 1H),5.44 (d, J ¼ 4 Hz, 1H), 5.25 (dd, J ¼ 17.2 and 1.6 Hz, 1H), 5.02 (d,J ¼ 4.4 Hz, 1H), 4.87 (dd, J ¼ 10.8 and 1.6 Hz, 1H), 4.47 (brs, 1H), 4.13(brs, 1H), 2.86 (d, J ¼ 16.8 Hz, 1H), 2.49 (d, J ¼ 17.2 Hz, 1H), 2.35 (d,J¼ 2 Hz, 1H), 2.15 (s, 3H), 2.09e2.05 (m,1H), 1.78e1.74 (m, 2H), 1.71(s, 3H), 1.61e1.57 (m, 1H), 1.52 (s, 3H), 1.34 (s, 3H), 1.27 (s, 3H),1.11e1.04 (m, 1H), 1.03 (s, 3H); 13C NMR (125 MHz, CDCl3): d (ppm)205.41, 169.61, 146.20, 136.04, 133.29, 128.51, 128.51, 128.14, 126.89,126.89, 125.41, 110.43, 96.14, 84.84, 80.46, 76.52, 76.22, 75.08 70.0,50.01, 42.85, 39.13, 36.43, 34.45, 32.96, 31.65, 24.70, 23.61, 23.30,21.18, 17.55; ESI-MS: positive ion mode: m/z 547.26 [MþNa]þ

(calculated mass for C31H40O7 is 524.65).

4.2.17. 1,9-Benzylidene-7-deacetylforskolin (2a)Yield: 63 mg (69%), white solid, m.p. 149e151 �C; IR (KBr): 3550,

3495, 2923, 1704, 1371, 1242, 1110, 1028 cm�1; 1H NMR (500 MHz,CDCl3): d (ppm) 7.46 (m, 2H), 7.33 (m, 3H), 5.85 (dd, J ¼ 17.5 and10.5 Hz, 1H), 5.42 (s, 1H), 5.06 (dd, J ¼ 17.5 and 1.0 Hz, 1H), 4.77 (dd,J ¼ 10.5 and 1.0 Hz, 1H), 4.53 (brs, 1H), 4.27 (brs, 1H), 4.10 (d,J ¼ 3.0 Hz, 1H), 2.95 (d, J ¼ 17 Hz, 1H), 2.56 (d, J ¼ 17 Hz, 1H), 2.50(brs, 1H), 2.38 (d, J ¼ 2 Hz, 1H), 2.23 (m, 2H), 1.82e1.81 (m, 1H), 1.72(s, 3H), 1.57 (m, 4H), 1.32 (s, 3H), 1.28 (s, 3H), 1.15e1.09 (m, 4H); 13CNMR (125 MHz, CDCl3): d (ppm) 205.07, 146.29, 138.27, 128.92,128.16, 128.16, 126.89, 126.29, 126.29, 110.55, 96.76, 84.95, 81.85,76.40, 75.43, 74.67, 70.56, 50.01, 43.14, 39.20, 36.58, 34.37, 33.20,30.73, 24.57, 23.65, 22.92, 17.68; ESI-MS: positive ion mode: m/z457 [MþH]þ, 474 [MþNH4]þ (calculated mass for C27H36O6 is 456).

4.2.18. 1,9-Ethylidene-7-deacetylforskolin (2b)Yield: 51 mg (65%), white solid; m.p. 64e66 �C; IR (KBr): 3595,

3500, 2926, 1701, 1407, 1242, 1126, 1097 cm�1; 1H NMR (400 MHz,CDCl3): d (ppm) 5.98 (dd, J ¼ 17.2 and 10.8 Hz, 1H), 5.16 (dd, J ¼ 17.2and 0.8 Hz,1H), 4.94 (dd, J¼ 10.8 and 0.8 Hz,1H), 4.60 (q, J¼ 5.2 Hz,1H), 4.48 (brs, 1H), 4.03 (brs, 1H), 2.78 (d, J ¼ 17.6 Hz, 1H), 2.54 (d,J ¼ 17.6 Hz, 1H), 2.44 (brs, 1H), 2.18 (d, J ¼ 1.6 Hz, 1H), 2.15 (brs, 1H),2.06e2.01 (brs, 1H), 1.75e1.67 (m, 1H), 1.63 (s, 3H), 1.51e1.47 (m,

1H), 1.44 (s, 3H), 1.39 (s, 3H), 1.28 (d, J ¼ 5.2 Hz, 3H), 1.26 (s, 3H),1.09e1.06 (m, 1H), 1.04 (s, 3H); 13C NMR (125 MHz, CDCl3): d (ppm)204.56,146.90,110.26, 94.26, 84.17, 81.49, 75.80, 75.00, 74.66, 70.55,49.78, 42.90, 38.80, 36.40, 34.29, 33.16, 31.23, 24.56, 23.56, 22.73,21.26,17.63; ESI-MS: positive ionmode: 417.15 [MþNa]þ (calculatedmass for C22H34O6 is 394.50).

4.2.19. 1,9-Benzylideneisoforskolin (3a)Yield: 71 mg (72%), white solid, m.p. 149e151 �C; IR (KBr): 3462,

2970, 1741, 1370, 1224, 1078 cm�1; 1H NMR (500 MHz, CDCl3): 7.45(m, 2H), 7.37 (m, 3H), 5.89 (dd, J ¼ 8.5 and 4.5 Hz, 1H), 5.85 (dd,J ¼ 17.5 and 11.0 Hz), 5.43 (s, 1H), 5.04 (d, J ¼ 17.5 Hz, 1H), 4.75 (d,J¼ 11 Hz,1H), 4.31 (brs,1H), 4.27 (d, J¼ 3.5 Hz,1H), 3.47 (s,1H), 2.96(d, J ¼ 17 Hz, 1H), 2.60e2.57 (m, 2H), 2.33 (brs, 1H), 2.14 (s, 3H),2.09e2.06 (m, 1H), 1.86e1.81 (m, 1H), 1.66 (m, 4H), 1.55 (s, 3H), 1.43(s, 3H), 1.34 (s, 3H), 1.14e1.12 (m, 1H), 1.06 (s, 3H); 13C NMR(125 MHz, CDCl3): d (ppm) 204.93, 170.57, 146.21, 137.96, 129.04,128.21, 128.21, 126.27, 126.27, 110.60, 96.93, 84.67, 81.70, 76.17,75.44, 73.62, 71.19, 50.00, 42.24, 39.38, 36.70, 34.09, 33.12, 30.79,23.75, 23.46, 22.09, 21.57, 17.32; ESI-MS: positive ion mode: m/z521.35 [MþNa]þ (calculated mass for C29H38O7 is 498.61).

4.2.20. 1,9-Ethylideneisoforskolin (3b)Yield: 64 mg (73%), white solid, m.p. 154e156 �C; IR (KBr): 3469,

2969, 1739, 1371, 1225, 1085 cm�1; 1H NMR (400 MHz, CDCl3): 6.03(dd, J ¼ 17.6 and 10.8 Hz, 1H), 5.84 (dd, J ¼ 4.0 and 2.8 Hz, 1H), 5.16(dd, J ¼ 17.6 and 0.8 Hz, 1H), 4.95 (dd, J ¼ 10.8 and 0.8 Hz, 1H), 4.61(q, J ¼ 5.2 Hz, 1H), 4.19 (dd, J ¼ 4.0 and 2.8 Hz, 1H), 4.08 (brs, 1H),2.79 (d, J ¼ 17.6 Hz, 1H), 2.55 (d, J ¼ 18.0 Hz, 1H), 2.37 (d, J ¼ 2.8 Hz,1H), 2.30 (d, J ¼ 2.8 Hz, 3H), 2.10 (s, 3H), 2.03e1.96 (m, 1H),1.79e1.70 (m, 1H), 1.58 (s, 3H), 1.54e1.49 (m, 1H), 1.44 (s, 3H), 1.49(s, 3H), 1.30 (d, J¼ 5.2 Hz,1H), 1.09e1.07 (m,1H), 1.07 (s, 3H), 1.09 (s,3H); 13C NMR (125 MHz, CDCl3): d (ppm) 204.33, 170.56, 146.86,110.29, 94.35, 83.89, 81.35, 75.57, 74.97, 73.51, 71.28, 49.76, 41.99,38.97, 36.51, 33.99, 33.06, 31.30, 23.71, 23.36, 21.87, 21.52, 21.18,17.55; ESI-MS: positive ion mode: m/z 459.30 [MþNa]þ (calculatedmass for C24H36O7 is 436.54).

4.3. General procedure for the synthesis of compounds 2a from 1a/3a (Scheme 2)

A solution of compound 1a/3a (50 mg, 0.1 mmol) and potassiumhydroxide (11.2 mg, 0.2 mmol) in methanol (10 mL) was stirred atroom temperature for 12 h. The reaction mass was diluted withwater (40 mL), was extracted with ethyl acetate (3 � 10 mL).Combined organic layer was washed with brine, dried over anhy-drous sodium sulphate and evaporated to give the crude. Residuewas purified by neutral alumina column chromatography (eluent:ethyl acetate: petroleum ether) to give 2a yields 40 mg (87%),44 mg (95%) respective to 1a/3a.

4.4. X-ray diffraction method

X-ray data of compound 2a and 3b were collected at roomtemperature using a Bruker Smart Apex CCD diffractometer withgraphite monochromated MoKa radiation (l ¼ 0.71073 Å) with u-scan method [33]. Preliminary lattice parameters and orientationmatrices were obtained from four sets of frames. Integration andscaling of intensity data were accomplished using SAINT program[33]. The structures were solved by Direct Methods using SHELXS-97 [34] and refinement was carried out by full-matrix least-squarestechnique using SHELXL-97 [34]. Anisotropic displacement pa-rameters were included for all non-hydrogen atoms. The O bound Hatoms of 2a and 3b were located in a difference density map andrefined isotropically. All other H atoms were positioned

D. Ponnam et al. / European Journal of Medicinal Chemistry 87 (2014) 735e744 743

geometrically and treated as riding on their parent C atoms, withCeH distances of 0.93e0.96 Å, and with Uiso(H) ¼ 1.2Ueq (C) or1.5Ueq for methyl atoms. The atoms C27/C28/C29 of 3a weredisordered over two positions (C27/C271, C28/C281 and C29/C291)and their site-occupancy factors were refined to 0.529(13)/0.471(13). The anisotropic displacement parameters of thesedisordered atomswere restrained to be similar (SIMU instruction inSHELXL-97; Sheldrick, 2008). Distance restraints (DFIX instructionin SHELXL-7; Sheldrick, 2008) were also applied to disorderedatoms. Compound 2a and 3b show a meaningless Flack parameter[35] values of 0.3(6) and �0.1(9). This is due to the molecularstructure having light atoms (<Si) with no significant anomalousscattering effects. In this case, the Flack parameter is indeterminatewith Mo radiation and hence the absolute structure cannot bedetermined. However, the absolute configuration of the procuredmaterial was known in advance.

4.5. Cytotoxicity evaluation

The cell lines used cytotoxicity assay comprises of both adherent(HeLa: Human cervix adenocarcinoma, A498: Human kidney car-cinoma, MCF-7: Human breast adenocarcinoma, WRL-68: Humanhepatic cells, NRK: Normal rat kidney, HEK293: Human embryonickidney) as well as suspended (K562: Human erythromyeloblastoidleukemia) cell lines. Experiments were performed in a 96 well plateformat followed the procedure reported earlier [36] based on themethod of Mosmann [37]. The seeding of cells (1 � 106 to 1 � 105

cells/well) in foetal bovine serum rich media supplemented withantibiotics. After 24 h, compounds were added to the plates fol-lowed by the addition ofMTT-Dye 10 ml/well (5mg/ml in phosphatebuffer saline pH 7.4). The plates were kept in dark for 4 h in anincubator for the formation of formazan crystal. Finally, the dyewasremoved from the plates (not in suspended cells) and 100 ml ofDMSO was added in each well with appropriate shaking for 10 min.The absorbance was read at 570 nm. Percent cytotoxicity wascalculated by the formula:

Percent cytotoxicity : Absorbance of Control� Absorbance of Test Compounds=Absorbance of Control*100

4.6. Erythrocyte osmotic fragility

The erythrocyte osmotic fragility assay was performed in vitro asper previously reported procedure [38]. The osmotic fragility ofhuman erythrocyte was determined by measuring the release ofhaemoglobin from erythrocytes exposed to an environment ofhypotonic solutions of varying concentrations of phosphate buff-ered saline (0.1e0.85 %) followed by incubation at 37 �C for 60 minwith mild shaking and recording the extent of haemolysis spec-trophotometrically at 540 nm. Likewise heparinised blood wasincubated with the effective concentration of compound 50 and100 mg/mL at 37 �C for 60 min and then transferred to the tubescontaining decreasing concentrations of PBS. The cell suspensionswere left to equilibrate for 30 min and then centrifuged. Theabsorbance of the supernatants was read at 540 nm and the percentlysis was calculated by dividing the optical density (OD) of thesupernatant obtained from a particular saline concentration by theOD of the standard representing 100% haemolysis. Osmotic fragilitycurves were constructed by plotting the lysis percentage against

the concentration of the saline solution. The results are expressedin terms of Mean Erythrocyte Fragility (MEF50), which is the level ofhaemolysis at 50% PBS (Table 2). The MEF50 values at standard pHand temperature were then obtained from the standard curve.Curcumin, Podophyllotoxin and DMSO were used as a positivecontrol, standard drug control and vehicle control respectively.

4.7. a-Glucosidase inhibition assay

As reported [39], rat intestinal acetone powder in normal saline(100:1; w/v) was sonicated properly and the supernatant was usedas a source of crude intestinal a-glucosidase after centrifugation. Inbrief, 10 mL of test samples (5 mg/mL DMSO solution) were recon-stituted in 100 mL of 100 mM-phosphate buffer (pH 6.8) in 96-wellmicroplate and incubated with 50 mL yeast a-glucosidase (0.76 U/mL in same buffer) or crude intestinal a-glucosidase for 5 minbefore 50 mL substrate (5 mM, p-nitrophenyl-a-D-glucopyranosideprepared in same buffer) was added. Release of p-nitrophenol wasmeasured at 405 nm spectrophotometrically (SpectraMAx Plus384,Molecular Devices Corporation, Sunnyvale, CA, USA) 5 min afterincubation with substrate. Individual blanks for test samples wereprepared to correct back-ground absorbance where substrate wasreplaced with 50 mL of buffer. Control sample contained 10 mLDMSO in place of test samples. Acarbose was taken as standardreference for a-glucosidase inhibition. All the samples were studiedin triplicate. Percentage of enzyme inhibition was calculated as(1�B/A) � 100 where A represents absorbance of control withouttest samples, and B represents absorbance in presence of testsamples. All the tests were run in duplicate. IC50 values werecalculated applying suitable regression analysis from the meaninhibitory values.

Acknowledgements

Authors thank Director, CSIR-CIMAP, Lucknow, and Scientist-In-Charge, CSIR-CIMAP, Research Centre, Hyderabad, India for theirconstant encouragement and support. One of the authors Devendar

thanks CSIR-New Delhi for granting Senior Research Fellowship.Cytotoxicity experiments were carried out in the laboratory ofLuqman Suiab is partly supported by grants from CSIR, New Delhi(BSC 0121) and SERB-DST, New Delhi.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.10.013.

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