Synthesis and anticancer activity of novel fused pyrimidine hybrids of myrrhanone C, a bicyclic triterpene of Commiphora mukul gum resin

Mol DiversDOI 10.1007/s11030-015-9621-3

FULL-LENGTH PAPER

Synthesis and anticancer activity of novel fused pyrimidinehybrids of myrrhanone C, a bicyclic triterpene of Commiphoramukul gum resin

Uppuluri Venkata Mallavadhani1 · Madasu Chandrashekhar1 ·Vadithe Lakshma Nayak2 · Sistla Ramakrishna2

Received: 10 February 2015 / Accepted: 19 July 2015© Springer International Publishing Switzerland 2015

Abstract MyrrhanoneC [8(R)-3-oxo-8-hydroxypolypoda-13E,17E,21-triene], a bicyclic triterpene isolated from thegum resin of Commiphora mukul, has been chemically trans-formed to synthesize a series of ten novel pyrimidine hybridsin good to excellent yields. The synthesized compounds (2–22) were evaluated for their anticancer potential against apanel of six cancer cell lines, namely A-549 (lung), Hela(cervical), MCF-7 (breast), ACHN (renal), Colo-205 (colon)and B-16 (mouse melanoma) by employing the MTT assay.In general, the synthesized compounds showed significantanticancer activity against all the cancer cell lines tested.Interestingly, the pyrimidine hybrids 18 and 19 showedgood activity against the A-549, MCF-7, B-16, Colo-205and ACHN cancer cell lines with IC50 values between 7.7–37.8 µM. Most significantly, compounds 19 (IC50: 7.7 µM)and 18 (IC50: 9.5 µM) showed about five- and six-foldenhanced activities, respectively, compared to the parentmyrrhanone C (1) against A-549 cell line. Flow cytometricanalysis revealed that compounds 18 and 19 induced apopto-sis in A-549 cells and arrested the cell growth in the G0/G1phase.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11030-015-9621-3) contains supplementarymaterial, which is available to authorized users.

B Uppuluri Venkata [email protected]; [email protected]

B Sistla [email protected]

1 Natural Products Chemistry Division, CSIR-Indian Instituteof Chemical Technology, Hyderabad 500007, India

2 Department of Medicinal Chemistry and Pharmacology,CSIR-Indian Institute of Chemical Technology, Hyderabad500007, India

Graphical Abstract

2 Steps

Synthesis and an�cancer ac�vity of novel fused pyrimidine hybrids of myrrhanone C, a bicyclic triterpene of Commiphora mukul gum resin

IC50 35.70 (MCF-7); 44.90 (A-549); 56.70 (B -16) 18. R=R2 =R3=H, R1=Cl : IC50 12.50 (MCF-7); 9.50 (A-549); 20.50 (B -16)

19. R=R2 =R3=H, R1=Br : IC50 16.00 (MCF-7); 7.70 (A-549); 14.70 (B -16)

Uppuluri Venkata Mallavadhania*, Madasu Chandrashekhara, Vadithe Lakshma Nayakb, Sistla Ramakrishnab*

Ten novel myrrhanone based fused pyrimidine hybrids were synthesized and evaluated for their anticancer potential against six cancer cell lines.

Keywords Myrrhanone C · Pyrimidines · Hybrids ·Anticancer activity · Apoptosis · Cell cycle arrest

Introduction

Nature provides a diverse array of products, mostly sec-ondarymetabolic in nature, with or without biological activi-ties. In general, these natural products serve as good scaffoldsin the development of potent drugs [1]. In most of the cases,it is necessary to modify and optimize their structural fea-tures to increase potency, selectivity and eliminate or reducetoxicity [2]. Among the various classes of natural products,triterpenoids form an important group of secondary metabo-lites, which are ubiquitous to the plant kingdom, especially,in vegetable and fruit bearing plants. These metabolites haveinteresting skeletal features, exhibit a wide range of biolog-ical activities and most importantly, they are non-toxic [3].Among the most interesting and structurally diverse triter-pene skeletons are the myrrh group of triterpenes, which arebicyclic polypodanes. These compounds are highly specificto the Commiphora and Burseraceae families. Especially,

123

Mol Divers

they accumulate in significant numbers and in abundance inthe gumresin ofCommiphora mukul (ver: guggul). Themajormyrrh triterpenes reported from C. mukul are myrrhanonesA, B, C and myrrhanols A, B, C [4,5]. Interestingly, thesemyrrh triterpenes were reported to exhibit significant anti-inflammatory and anticancer activities [6–8]. Heterocyclicpyrimidine moieties play an important role in medicinalchemistry and serve as key pharmacophores for the devel-opment of various therapeutic agents. Further, pyrimidinederivatives have been reported to exhibit a wide range ofbiological activities including anti-inflammatory [9], anti-HIV [10] and anticancer [11] activities. With this back-ground, and in continuation of our interest on chemicalmodification of natural scaffolds for potent anticancer leadmolecules [12,13], we have carried out chemical modifica-tions in ring A of myrrhanone C (1), isolated from the gumresin ofCommiphora mukul, to synthesise some fused pyrim-idine hybrids. Compound (1) has three major functionalitiessuch as C-3 keto of ring A, C-8 tertiary hydroxyl of ring Band 3 side-chain double bonds. Among these, the C-3 ketofunctionality is highly useful as it confers active methyleneproperties to the C-2, which in turn can be substituted tointroduce a benzylidene moiety. The resultant α-benzylideneketones, with an exocyclic enone system, can be exploitedto synthesize a variety of pyrimidine hybrid structures bycondensing them with guanidine. We report herein the syn-thesis of ring A fused pyrimidine hybrids of myrrhanone Cand their in vitro cytotoxic evaluation including cell cycleanalysis.

Results and discussion

Chemistry

Myrrhanone C (1) was isolated from the n-hexane extract ofthe gum resin of Commiphora mukul in 0.15 %. The pro-tocols adopted for the synthesis of the pyrimidine hybridsof myrrhanone C are outlined in Scheme 1. Compound (1),when treated with different substituted benzaldehydes inpresence of ethanolic potassium hydroxide, afforded the ben-zylidene compounds (2–12) in 65–86 % yields. While themethoxy substituents afforded higher yields of benzylidenecompounds (11: 86 %; 4: 81 %), the nitro and carboxy sub-stituents gave the products in moderate yields (5: 65 %; 12:62%). The benzylidene compounds with an α,β-unsaturatedcarbonyl functionality, on treatment with guanidine in pres-ence KOH in ethanol at reflux temperature, afforded thepyrimidines hybrids (13–22) in 60–75%yield [14–17]. Also,in this case the high yielding analogues are those contain-ing methoxy groups (22: 76 %; 15: 75 %). Surprisingly, thenitro-substituted benzylidene compound (12) did not yieldany product, but underwent decomposition even under var-

ied experimental conditions. All the synthesized compounds(2–22) were characterized by their spectroscopic data (IR,1H and 13CNMR,Mass).While the benzylidene compounds(2–12) showed the characteristic α,β-unsaturated carbonylfunctionality as sharp signal in the range 1660–1678 cm−1 inIR, singlets in the range δ 7.00–7.80 in the 1H NMR spectracorrespond to the olefinic proton, and δ 206–207, 130–133and 135–137 in the 13C NMR spectrum correspond to thecarbonyl and olefinic carbons. The pyrimidine compounds(13–22) showed the characteristic carbon signals at δ 160–161, 164–167 and 174–175 corresponding to C-2′, C-4′ andC-3, respectively, in their 13C NMR spectra.

Biological evaluation

In vitro anticancer activity

The synthesized myrrhanone C-pyrimidine hybrids (13–22)along with their precursor benzylidene compounds (2–12)and the parent myrrhanone C (1) were evaluated for anti-cancer activity against a panel of six cancer cell lines includ-ing A-549 (lung), Hela (cervical), MCF-7 (breast), ACHN(renal), Colo-205 (colon) and B-16 (mouse melanoma) byemploying an MTT assay [18]. Doxorubicin was used asthe reference. The results are summarized in Table 1 andare expressed as IC50 values. The screening results revealedthat the synthesized compounds in general showed promisinganticancer activity against all the cell lines tested. Among thetested compounds, the 4′′chloro pyrimidine hybrid (18) and4′′bromo pyrimidine hybrid (19) showed potent anticanceractivity againstMCF-7,A-549, B-16 andColo-205 cell lines.Interestingly, compound 18 was found to be the most potenton MCF-7 and A-549 cell lines, whereas compound 19 onA-549 and B-16 cell lines. Significantly, these compoundsshowed highly potent activity against A-549 (lung) with IC50

values 9.5±0.21 and 7.7±1.32µM, respectively, which arealmost five and six times higher than the parent compound (1)(IC50: 44.9± 2.75). In the case of MCF-7, these compoundsshowed about three and two times more activity, respec-tively, than the parent compound (1) (IC50: 35.7 ± 1.02).For B-16 and Colo-205 cell lines, compound 18 showedthree and two times more activity, and compound 19 showedfour and two times more activity than the parent compound(1) (B-16: IC50: 56.7 ± 3.85; Colo-205: IC50: 53.9 ± 2.05).From these observations, it can be concluded that the pyrim-idine hybrids substituted with chlorine or bromine atomsenhanced the anticancer activity of myrrhanone C signifi-cantly, especially against A-549 lung cancer cell line. Basedon these results, the most potent pyrimidine hybrids 18 and19were selected for apoptosis and cell cycle studies inA-549cells.

123

Mol Divers

COMPOUNDS R R1 R2 R3 2, 13 H H H H 3, 14 H CH3 H H 4, 15 H OCH3 H H 5, 16 H COOH H H 6, 17 H F H H 7, 18 H Cl H H 8, 19 H Br H H 9, 20 H H CF3 H

10, 21 H OCH3 Br H 11, 22 OCH3 OCH3 H OCH3

12 -- H NO2 H H Scheme 1 Reagents and conditions. a Substituted benzaldehydes (2–12), KOH, ethanol, rt, benzene, 2 h; b NH2C (NH) NH2 ·HCl, KOH, ethanol,reflux

Hoechst staining

One of the important pathways of cell death is apoptosis,which is characterized by condensation of chromatin andnuclear fragmentation. Compounds 18 and 19were evaluatedat a concentration of 5µMfor 48 h, for their apoptosis induc-ing effect in lung cancer cell line A549 [19]. The apoptoticcells were quantified based on cytoplasmic condensation andrelative fluorescence of the test compounds (18 and 19),which revealed that there was a significant increase of apop-totic cells (Fig. 1)

Cell cycle analysis

Cell cycle analyses were performed in order to understandthe mechanisms responsible for cytotoxic effects of the testcompounds 18 and 19 [20]. The study involves incubationof test compounds at 5 and 10 µM concentrations for 48 hrswith A-549 cells in a cell sorter. Cell cycle analysis indicatedthat the percentage of cancer cells in G0/G1 phase signifi-cantly decreased, and there was a significant increase in theaccumulation of cells in sub G1 phase after 48 h of treatment,which indicates onset of apoptosis in the A549 lung cancercells (Fig. 2).

123

Mol Divers

Table 1 IC50 valuesa (µM) of compounds

Compound Helab MCF-7c A-549d Colo-205e ACHNf B-16g

1 55.7 ± 3.76 35.7 ± 1.02 44.9 ± 2.75 53.9 ± 2.05 71.6 ± 6.36 56.7 ± 3.85

2 118.6 ± 5.71 47.0 ± 0.94 62.3 ± 1.11 80.2 ± 1.24 56.1 ± 2.71 50.8 ± 2.57

3 84.4 ± 3.15 145.7 ± 2.51 73.9 ± 2.58 112.5 ± 9.76 57.0 ± 0.97 57.6 ± 4.01

4 47.9 ± 2.05 45.6 ± 1.13 53.8 ± 3.27 85.7 ± 4.52 43.3 ± 1.43 100.2 ± 7.35

5 114.9 ± 2.19 91.4 ± 0.72 553.9 ± 9.83 116.1 ± 2.01 55.1 ± 0.24 89.1 ± 6.24

6 576.1 ± 4.6 91.2 ± 2.73 70.6 ± 2.34 913.6 ± 9.42 94.7 ± 3.54 288.6 ± 9.94

7 70.4 ± 1.37 54.4 ± 4.21 47.5 ± 1.54 55.9 ± 0.62 62.4 ± 1.75 47.2 ± 1.53

8 50.1 ± 1.25 103.3 ± 1.06 43.1 ± 3.26 59.8 ± 1.32 82.1 ± 8.52 117.4 ± 9.99

9 74.3 ± 0.14 132.5 ± 3.75 264.5 ± 4.87 66.4 ± 5.67 66.8 ± 1.24 58.7 ± 2.07

10 84.4 ± 2.32 98.4 ± 9.63 60.1 ± 2.56 64.7 ± 2.12 124.1 ± 0.31 70.1 ± 3.52

11 321.3 ± 2.81 40.4 ± 2.43 34.4 ± 1.54 58.3 ± 7.82 48.4 ± 1.11 35.4 ± 1.15

12 78.3 ± 1.03 41.6 ± 3.87 23.9 ± 0.95 32.3 ± 1.02 38.7 ± 0.94 34.7 ± 3.65

13 224.6 ± 1.22 277.9 ± 8.54 236.9 ± 3.25 88.9 ± 4.75 440.6 ± 7.52 97.2 ± 7.01

14 84.1 ± 2.54 29.6 ± 3.02 42.8 ± 2.65 46.0 ± 1.32 271.3 ± 4.05 44.8 ± 0.95

15 73.3 ± 5.37 37.1 ± 1.25 33.1 ± 1.51 33.0 ± 0.35 31.1 ± 2.43 23.5 ± 1.09

16 46.7 ± 0.36 64.0 ± 2.65 84.8 ± 2.76 51.4 ± 1.24 38.1 ± 0.65 43.9 ± 2.95

17 86.9 ± 2.01 52.7 ± 6.87 48.8 ± 0.62 67.7 ± 5.42 60.2 ± 5.72 57.5 ± 2.08

18 58.0 ± 1.77 12.5 ± 0.82 9.5 ± 0.21 22.8 ± 1.06 37.8 ± 2.65 20.5 ± 1.13

19 48.7 ± 1.21 16.0 ± 0.34 7.7 ± 1.32 28.6 ± 7.42 37.7 ± 2.45 14.7 ± 1.04

20 115.2 ± 1.46 50.6 ± 1.13 42.0 ± 3.76 49.5 ± 1.07 62.4 ± 1.06 81.5 ± 11.5

21 147.6 ± 3.82 73.8 ± 1.97 39.1 ± 5.47 116.9 ± 0.96 116.9 ± 3.27 74.1 ± 8.22

22 69.0 ± 1.56 39.5 ± 4.65 40.6 ± 0.44 60.6 ± 4.14 53.5 ± 2.76 55.8 ± 2.31

Doxorubicin 3.9 ± 0.96 2.2 ± 0.24 4.1 ± 0.72 3.1 ± 0.53 4.0 ± 0.25 5.4 ± 0.63

Highly potent compounds are in bolda 50 % Inhibitory concentration and the values are average of three individual experiments after 48 h of drug treatmentb Cervical cancerc Breast cancerd Lung cancere Colon cancerf Renal cell carcinomagMouse melanoma

Fig. 1 Hoechst staining inA-549 lung cancer cell line. aA-549 control cells, bcompound 18 at 5 µM and ccompound 19 at 5 µMconcentration

123

Mol Divers

Fig. 2 Flow cytometric analysis in A-549 lung cancer cell lines after treatment for 48 h. a Control: A-549, b compound 18 : 5 µM, c compound18: 10 µM, d compound 19: 5 µM and e compound 19: 10 µM

Caspase-3 activity

Caspase-dependent apoptotic pathway plays an importantrole in the study of antiproliferative activity of the testcompounds. Caspases, a family of cysteine proteases, areimportant mediators of apoptosis. Caspase-3 is an execu-tioner caspase which plays a vital role in apoptosis [21].Lung cancer (A549) cells were treated with compounds18 and 19 at two different concentrations (5 and 10 µM)for 48 h. The cells were examined for the activation ofcaspase-3 activity. Results indicated that there was nearlya four- to eight-fold high induction in caspase-3 levels

in the treated cells compared to control untreated cells(Fig. 3).

DNA fragmentation

Apoptosis or programmed cell death of cells is characterizedby the ability of a compound to induce oligo nucleoso-mal DNA fragmentation or DNA ladder [22]. During theprocess of apoptosis, DNA is cleaved into small fragments byendonucleases. These fragments are usually observed as lad-ders in gel electrophoresis. DNA fragmentation studies weredone on A-549 cells, which were treated with compounds 18

123

Mol Divers

Fig. 3 Caspase 3 activity assay

Fig. 4 DNA laddering assay: track 1: control (untreated cells), track2: 18 (5 µM), track 3: 18 (10 µM), track 4: 100 bp marker, track 5: 19(5 µM) and track 6: 19 (10 µM)

and 19 at 5 and 10 µM concentrations for 48 h. The DNAwas isolated from these cells and then run on 2% agarose gelelectrophoresis, stained with ethidium bromide and visual-ized under UV illumination. Significant DNA fragmentation(Fig. 4) was observed for compounds 18 and 19 at 10 µMcompared to 5μMconcentration and this indicates apoptosisin A549 cells by the test compounds at 10µM (Fig. 4).

Conclusion

Myrrhanone C, a natural bicyclic triterpene, was chemi-cally modified and a series of ten novel pyrimidine hybridswere synthesized. The compounds were evaluated for theircytotoxic potential against six cancer cell lines. Some ofthe synthesized compounds were found to be promisinglead compounds against a majority of the cancer cell lines,

exhibiting the IC50 values much lower than those of parentmyrrhanoneC. Especially, the chloro- and bromo-substitutedpyrimidine hybrids (18 and 19) showed about five- and six-fold enhanced activities against the lung cancer cell line(A-549). Mechanistic studies revealed that both the com-pounds induce apoptosis and arrest the cell cycle in theG0/G1 phase. The chloro- and bromo-substituted pyrimidinehybrids seem to be important for enhanced anticancer activityof myrrhanone C.

Experimental

Chemistry

All reagents used for chemical synthesis were purchasedfrom Sigma-Aldrich and used as such. Solvents were dis-tilled before use. The progress of all reactions was monitoredby TLC on silica gel 60 F254 plates (Merck). Purification ofthe products was carried out by column chromatography. Allproducts were characterized by 1H NMR and 13C NMR. IRspectra were recorded in KBr discs on a Nicollet 740 FT-IRspectrophotometer. 1H and 13C NMR spectra were recordedon a Bruker AVANCE-I 300 (1H: 300 MHz, 13C: 75 MHz)or Bruker AVANCE-III 500 (1H: 500 MHz, 13C: 125 MHz)NMR spectrometers; chemical shifts (δ scale) are expressedin parts per million with TMS as an internal standard andcoupling constants are expressed inHz. The following abbre-viations were used in NMR data: s = singlet, d =doublet, t = triplet,m = multiplet, J = coupling constant.Mass spectra were recorded on an Agilent ESI-QTOF instru-ment and HRMS experiments were performed using anORBITRAP by thermo-scientific instrument.

Isolation of myrrhanone C (1)

Gum resin of Commiphora mukul was collected throughBaidyanath Ayurvedic Pharmacy, Patna. The resin was dried,

123

Mol Divers

cut into small pieces and powdered in a mixer. The powderedmaterial (1.0 kg) was exhaustively extracted with n-hexane(3.0 l) for 24 h in a soxhlet extractor. The n-hexane solubleson concentration under reduced pressure afforded an yellow-ish brown gummy extract (190 g). This material (180 g) waspurified by column chromatography over silica gel using n-hexane-ethyl acetate (9.5:0.5) as eluent to yield myrrhanoneC (1) (1.5 g, yield 0.15 %) as a colourless semi-solid. Rf : 0.5[n-hexane–ethyl acetate (80:20)]. The identity of this com-pound was confirmed by its spectral data (1H NMR, 13CNMR, HRMS and IR) and by comparing with the reportedvalues [23]. IR (KBr, cm−1): 3446, 2938, 1703, 1455, 1384,1079, 925, 588; 1H NMR: (300MHz, CDCl3): δ 0.94 (3H, s,Me-25), 1.02 (3H, s, Me-24), 1.09 (3H, s, Me-23), 1.19 (3H,s, Me-26), 1.25–1.54 (8H, m), 1.59 (9H, s, 3xMe-27,28,29),1.67 (3H, s, Me-30), 1.93-2.13 (12H, m), 2.39 (1H, m, CH-2), 2.59 (1H, m, CH-2), 5.07–5.18 (3H, m, 3xCH-13,17,21);13CNMR (125 MHz, CDCl3): δ 14.74 (C25), 15.97(C28),16.18 (C27), 17.61 (C29), 21.27 (C6), 23.53 (C26), 25.63(C30), 25.70 (C11), 26.21 (C20), 26.54 (C23), 26.68 (C16),31.10 (C12), 33.88 (C2), 38.20 (C1), 38.52 (C10), 39.60 (C19),39.67 (C15), 43.67 (C7), 47.40 (C4), 55.03 (C5), 60.23 (C9),73.55 (C8), 124.03 (C17), 124.27 (C13), 124.54 (C21), 131.15(C22), 134.92 (C18), 135.43 (C14), 216.87 (C3). ESI-HRMScalcd for C30H50O2[M + Na]+ 465.3703, found 465.3697.

General procedure for the preparation of compounds(2–12)

The key intermediates were prepared by the reaction of com-pound 1 (1 equiv) with substituted benzaldehydes (1.5 equiv)in 2ml absolute ethanol and stirred in the presence of KOH atroom temperature until consumption of the startingmaterialsas indicated by TLC. The reaction mixture was neutralizedwith aq. HCl (1:1) and extracted with ethyl acetate. The com-bined organic layers was dried over anhydrous Na2SO4 andevaporated under reduced pressure on a rotavapor. The cruderesidue was purified by column chromatography with ethylacetate:hexane (1:4, 2:3) to give the title compounds withgood yields.

2-(Benzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one(2)

Yield73%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.5; IR (KBr, cm−1): 3470, 2933, 1675, 1594,1449, 1383, 1257, 1169, 1130, 1078, 932, 765, 695, 519;1H-NMR (300 MHz, CDCl3): δ 0.75 (s, 3H, Me), 1.15 (s,6H, 2xMe), 1.15 (s, 3H, Me), 1.33–1.52 (m, 6H), 1.59 (s,6H, 2xMe), 1.64 (s, 3H, Me), 1.67 (s, 3H, Me), 1.90–2.24(m, 12H), 2.40 (d, J = 16.2 Hz, 1H), 3.09 (d, J = 16.2 Hz,1H), 5.09 (t, J = 6.8 Hz, 1H), 5.11 (t, J = 6.6 Hz, 1H), 5.20(t, J = 6.9 Hz, 1H), 7.46 (m, 5H), 7.573 (s, 1H); 13C-NMR

(125 MHz, CDCl3): δ 14.56, 16.01, 16.27, 17.66, 22.36,22.51, 23.06, 25.57, 25.68, 26.64, 26.74, 29.67, 31.31, 38.19,39.73, 43.31, 43.43, 45.24, 52.81, 58.95, 73.54, 124.07,124.30, 124.57, 128.41, 128.57, 130.25, 131.24, 133.16,135.06, 135.56, 135.76, 137.82, 207.21. ESI-HRMS m/zcalcd for C37H54O2[M + Na]+ 553.4016, found 553.4048.

2-(4′′-Methylbenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (3)

Yield78%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.51; IR (KBr, cm−1): 3485, 2926, 1673, 1592,1452, 1383, 1257, 1169, 1130, 1080, 1020, 811, 771, 521;1H-NMR (300 MHz, CDCl3): 0.75 (s, 3H, Me), 1.18 (s, 3H,Me), 1.25 (s, 6H, 2xMe), 1.28–1.46 (m, 6H), 1.59 (s, 6H,2xMe), 1.60 (s, 6H, 2xMe), 1.91–2.12 (m, 12H), 2.37 (s,3H, Me), 2.41 (d, J = 16.0 Hz, 1H), 3.08 (d, J = 16.0 Hz,1H), 5.09 (t, J = 7.0 Hz, 1H), 5.12 (t, J = 6.8 Hz,1H), 5.21 (t, J = 6.4 Hz, 1H), 7.20 (d, J = 7.9 Hz,2H), 7.36 (d, J = 7.9 Hz, 2H), 7.55 (s, 1H); 13C-NMR(125 MHz, CDCl3): δ 14.55, 16.00, 16.25, 17.66, 21.35,22.36, 22.50, 23.01, 25.53 25.67, 26.64, 26.72, 29.67, 31.29,38.14, 39.72, 43.31, 45.14, 52.70, 58.93, 73.55, 124.05,124.28, 124.62, 129.16, 130.38, 131.24, 132.23, 132.90,135.05, 135.53, 137.95, 138.84, 207.21. ESI-HRMS calcdfor C38H56O2[M + Na]+ 567.4172, found 567.4167.

2-(4′′-Methoxybenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (4)

Yield81%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.36; IR (KBr, cm−1): 3478, 2930, 1670, 1588,1511, 1453, 1383, 1256, 1171, 1131, 1080, 1034, 830, 533;1H-NMR (500 MHz, CDCl3): δ 0.68 (s, 3H, Me), 1.07 (s,3H, Me), 1.08 (s, 3H, Me), 1.11 (s, 3H, Me), 1.18–1.47 (m,6H), 1.52 (s, 6H, 2xMe), 1.60 (s, 3H, Me), 1.61 (s, 3H, Me),1.83–2.17 (m, 12H), 2.34 (d, J = 16.0 Hz, 1H), 2.99 (d,J = 16.0 Hz, 1H), 3.76 (s, 3H, Me), 5.01 (t, J = 6.8 Hz,1H), 5.04 (t, J = 6.8 Hz, 1H), 5.15 (t, J = 6.8 Hz, 1H),6.85 (d, J = 9.0 Hz, 2H), 7.36 (d, J = 9.0 Hz, 2H),7.47 (s, 1H); 13C-NMR (125 MHz, CDCl3): δ 14.58, 16.00,16.31, 17.66, 22.40, 22.47, 23.03, 25.59, 25.67, 26.66, 26.73,29.67, 31.36, 38.12, 39.72, 39.77, 43.44, 43.50, 45.06, 52.65,55.26, 59.00, 73.57, 113.92, 124.03, 124.29, 124.65, 128.42,130.92, 131.25, 132.15, 135.10, 135.52, 137.73, 159.90,207.10. ESI-HRMS calcd for C38H57O3[M+H]+ 561.4302,found 561.4303.

2-(4′′-Carboxybenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (5)

Yield65%; light yellowsemi-solid;TLC: (60%hexane:ethylacetate) Rf = 0.54; IR (KBr, cm−1): 3432, 2927, 1603, 1564,

123

Mol Divers

1451, 1384, 1259, 1176, 1113, 1080, 1019, 919, 772, 521;1H-NMR (300 MHz, CDCl3): δ 0.77 (s, 3H, Me), 1.16 (s,3H, Me), 1.17 (s, 3H, Me), 1.19 (s, 3H, Me), 1.27–1.54 (m,6H), 1.57 (s, 3H, Me), 1.58 (s, 3H, Me), 1.64 (s, 3H, Me),1.65 (s, 3H, Me), 1.92–2.24 (m, 12H), 2.42 (d, J = 16.0 Hz,1H), 3.06 (d, J = 16.0 Hz, 1H), 5.07 (t, J = 5.6 Hz, 1H),5.10 (t, J = 5.9 Hz, 1H), 5.19 (t, J = 6.4 Hz, 1H), 7.52(d, J = 8.3 Hz, 2H), 7.57 (s, 1H), 8.12 (d, J = 8.2 Hz,2H);13C-NMR (125 MHz, CDCl3): δ 14.60, 15.96, 16.27,17.62, 22.29, 22.50, 22.99, 25.56, 25.64, 26.58, 26.71, 29.55,31.23, 38.25, 39.68, 43.26, 43.31, 45.37, 52.82, 58.76, 73.67,124.01, 124.31, 124.37, 128.99, 129.98, 130.16, 131.17,135.03, 135.46, 135.71, 136.31, 140.40, 170.77, 207.09.ESI-HRMS calcd for C38H54O4[M+Na]+ 597.3914, found597.3930.

2-(4′′-Fluorobenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (6)

Yield73%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.43; IR (KBr, cm−1): 3478, 29247, 1676,1599, 1507, 1453, 1382, 1224, 1160, 1130, 1079, 1018, 833,772, 526; 1H-NMR (500 MHz, CDCl3): δ 0.75 (s, 3H, Me),1.15 (s, 6H, 2xMe), 1.19 (s, 3H Me), 1.27-1.53 (m, 6H),1.59 (s, 6H, 2xMe), 1.64 (s, 3H, Me), 1.66 (s, 3H, Me),1.91–2.23 (m, 12H), 2.39 (d, J = 16.1 Hz, 1H), 3.02 (d,J = 16.2 Hz, 1H), 5.09 (t, J = 6.8 Hz, 1H), 5.11 (t,J = 6.8 Hz, 1H), 5.19 (t, J = 6.8, 1H), 7.08 (t, J = 8.5,2H), 7.43 (t, J = 8.5 Hz, 2H), 7.52 (s, 1H); 13C-NMR (125MHz, CDCl3): δ 14.57, 16.00, 16.24, 17.65, 22.35, 22.49,23.09, 25.56, 25.66, 26.63, 26.74, 29.68, 31.30, 38.20, 39.72,43.26, 43.42, 45.24, 52.78, 58.91, 73.53, 115.45, 115.63,124.01, 124.31, 124.51, 131.25, 132.06, 132.12, 132.89,135.13, 135.62, 136.63, 163.56, 207.06. ESI-HRMS calcdfor C37H53FO2[M + Na]+ 571.3921, found 571.3930.

2-(4′′-Chlorobenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (7)

Yield73%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.43; IR (KBr, cm−1): 3478, 2924, 1677, 1594,1490, 1453, 1383, 1255, 1168, 1093, 1014, 915, 821, 772,519; 1H-NMR (500 MHz, CDCl3): δ 0.75 (s, 3H, Me), 1.08(s, 6H, 2xMe), 1.11 (s, 3H, Me), 1.28–1.50 (m, 6H), 1.52 (s,3H,Me), 1.57 (s, 6H, 2xMe), 1.59 (s, 3H,Me), 1.84-2.14 (m,12H), 2.28 (d, J = 16.2 Hz, 1H), 2.93 (d, J = 16.2 Hz, 1H),5.01 (t, J = 6.8 Hz, 1H), 5.04 (t, J = 5.8 Hz, 1H), 5.12 (t,J = 6.4 Hz, 1H), 7.28 (m, 4H), 7.43 (s, 1H); 13C-NMR(125 MHz, CDCl3): δ 14.56, 16.00, 16.21, 17.65, 22.32,22.49, 23.05, 25.57, 25.67, 26.61, 26.72, 29.63, 31.27, 38.18,39.72, 43.24, 43.35, 45.25, 52.75, 58.83, 73.51, 124.00,124.29, 124.46, 128.66, 131.37, 131.20, 133.71, 134.15,

134.18, 135.09, 135.64, 136.42, 207.06. ESI-HRMS calcdfor C37H53O35

2 Cl[M+Na]+ 587.3626, found 587.3611, Iso-topic signal for C37H53O37

2 Cl [M + Na]+ 589.3576.

2-(4′′-Bromobenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (8)

Yield71%; light yellow semi-solid; TLC: (20%hexane:ethylacetate) Rf = 0.43; IR (KBr, cm−1): 3468, 2923, 1677, 1593,1486, 1452, 1383, 1251, 1168, 1128, 1076, 1009, 968, 934,812, 772, 517; 1H-NMR (500 MHz, CDCl3): δ 0.74 (s, 3H,Me), 1.10 (s, 6H, 2xMe), 1.11 (s, 3H,Me), 1.17-1.44 (m, 6H),1.54 (s, 3H, Me), 1.55 (s, 3H, Me), 1.59 (s, 3H, Me), 1.62 (s,3H,Me), 1.86-2.15 (m, 12H), 2.28 (d, J = 16.1Hz, 1H), 2.97(d, J = 16.1 Hz, 1H), 5.04 (t, J = 7.1 Hz, 1H), 5.08 (t, J =7.1 Hz, 1H), 5.15 (t, J = 6.8 Hz, 1H), 7.25 (d, J = 8.6 Hz,2H), 7.47 (d, J = 8.6 Hz, 2H), 7.43 (s, 1H); 13C-NMR(125 MHz, CDCl3): δ 14.56, 16.03, 16.23, 17.67, 22.31,22.50, 23.06, 25.56, 25.67, 26.63, 26.73, 29.66, 31.26, 38.20,39.73, 43.25, 43.37, 45.27, 52.79, 58.82, 73.51, 122.81,124.02, 124.31, 124.46, 131.23, 131.59, 131.62, 133.86,134.61, 135.10, 135.66, 136.45, 206.99. ESI-HRMS calcdfor C37H53O79

2 Br [M+Na]+ 631.3121, found 631.3142, Iso-topic signal for C37H53O81

2 Br [M + Na]+ 631.3133.

2-(5′′-Trifluoromethylbenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (9)

Yield72%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.43; IR (KBr, cm−1): 3456, 2920, 1678, 1601,1455, 1382, 1330, 1219, 1166, 1130, 1075, 1020, 772, 698;1H-NMR (500 MHz, CDCl3): δ 0.76 (s, 3H, Me), 1.15 (s,3H, Me), 1.16 (s, 6H, 2xMe), 1.32–1.50 (m, 6H), 1.59 (s,9H, 3xMe), 1.66 (s, 3H, Me), 1.91–2.12 (m, 12H), 2.43 (d,J = 15.8 Hz,1H), 3.05 (d, J = 16.6 Hz, 1H), 5.08–5.16 (m,3H), 7.53–7.67 (m, 4H), 7.48 (s, 1H); 13C-NMR (125 MHz,CDCl3): δ 14.56, 15.94, 16.12, 17.62, 22.28, 22.50, 23.00,25.53, 25.62, 26.64, 26.73, 29.53, 31.12, 38.28, 39.67, 39.70,43.05, 43.30, 45.36, 52.84, 58.83, 73.44, 123.45, 124.07,124.31, 125.00, 126.49, 128.90, 129.96, 131.21, 133.12,134.85, 135.02, 135.77, 135.94, 136.45, 207.00. ESI-HRMScalcd for C38H53F3O2[M+Na]+ 621.3889, found 621.3882.

2-(5′′-Bromo-4′′-methoxybenzylidine)-8–hydroxyl-13,17,21-polypodatriene-3-one (10)

Yield78%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.43; IR (KBr, cm−1): 3476, 2927, 1672, 1589,1495, 1454, 1383, 1266, 1170, 1079, 1054, 1021, 917, 809,772, 582; 1H-NMR (500 MHz, CDCl3): δ 0.75 (s, 3H, Me),1.14 (s, 6H, 2xMe), 1.18 (s, 3H, Me), 1.28–1.51 (m, 6H),

123

Mol Divers

1.59 (s, 6H, 2xMe), 1.66 (s, 6H, 2xMe), 1.91–2.28 (m, 12H),2.38 (d, J = 16.0 Hz, 1H), 3.04 (d, J = 16.0 Hz, 1H), 3.96(s, 3H, OMe), 5.09 (t, J = 6.6 Hz, 1H), 5.11 (t, J = 6.6 Hz,1H), 5.25 (t, J = 6.6 Hz, 1H), 6.91–7.46 (m, 3H), 7.70(s, 1H); 13C-NMR (125 MHz, CDCl3,): δ 14.58, 16.00,16.39, 17.65, 22.35, 22.45, 22.99, 25.51, 25.66, 26.68, 29.68,31.23, 38.14, 39.70, 43.36, 45.13, 52.65, 56.23, 58.88, 73.43,111.48, 111.73, 124.04, 124.28, 124.55, 129.68, 131.14,131.23, 132.22, 135.03, 135.75, 136.06, 155.99, 206.87.ESI-HRMS calcd for C38H55O79

3 Br[M + Na]+ 661.3226,found 661.3220, Isotopic signal for C38H55O81

3 Br[M+Na]+663.3201.

2-(4′′-Trimethoxybenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (11)

Yield86%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.48; IR (KBr, cm−1): 3482, 2932, 1670,1604, 1458, 1332, 1227, 1205, 1155, 1126, 1061, 1020, 812,771, 755; 1H-NMR (500 MHz, CDCl3): δ 0.68 (s, 3H, Me),1.03 (s, 3H, Me), 1.07 (s, 3H, Me), 1.12 (s, 3H, Me), 1.18–1.42 (m, 6H), 1.52 (s, 6H, 2xMe), 1.60 (s, 3H, Me), 1.61 (s,3H, Me), 1.82–2.03 (m, 12H), 2.26 (d, J = 15.1 Hz,1H),2.48 (d, J = 15.1 Hz, 1H), 3.71 (s, 6H, 2xOMe), 3.76 (s,3H, OMe), 5.01–5.23 (m, 3H), 6.04 (s, 2H), 7.41 (s, 1H);13C-NMR (125 MHz, CDCl3): δ 14.58, 16.00, 16.31, 17.61,22.16, 22.86, 23.05, 25.41, 25.62, 26.58, 26.69, 29.12, 31.25,38.20, 39.59, 39.67, 43.09, 43.55, 45.38, 53.44, 55.21, 55.43,59.39, 73.20, 90.29, 106.70, 124.03, 124.24, 124.66, 131.19,132.10, 134.10, 134.95, 135.21, 158.89, 161.73, 206.21. ESI-HRMS calcd for C40H60O5[M + Na]+ 643.4333, found643.4306.

2-(4′′-Nitrobenzylidine)-8-hydroxy-13,17,21-polypodatriene-3-one (12)

Yield62%; light yellowsemi-solid;TLC: (20%hexane:ethylacetate) Rf = 0.33; IR (KBr, cm−1): 3491, 2925, 1680,1595, 1521, 1452, 1383, 1344, 1219, 1169, 1111, 1018,851, 772; 1H-NMR (300 MHz, CDCl3): δ 0.69 (s, 3H, Me),1.09 (s, 3H, Me), 1.10 (s, 3H, Me), 1.11 (s, 3H Me), 1.21–1.46 (m, 6H), 1.51 (s, 6H, 2xMe), 1.54 (s, 3H, Me), 1.59(s, 3H, Me), 1.85–2.02 (m, 12H), 2.33 (d, J = 15.3 Hz,1H), 2.93 (d, J = 15.3 Hz, 1H), 5.03 (t, J = 6.8 Hz,1H), 5.06 (t, J = 6.8 Hz, 1H), 5.12 (t, J = 6.8 Hz, 1H),7.50 (s, 1H), 7.52 (d, J = 8.7 Hz, 2H), 8.20 (d, J =8.7 Hz, 2H); 13C-NMR (125 MHz, CDCl3): δ 14.56, 15.94,16.17, 17.59, 22.22, 22.48, 23.04, 25.54, 25.62, 26.67, 26.50,29.67, 31.21, 38.22, 39.65, 43.22, 45.41, 52.77, 58.67, 73.42,123.55, 123.87, 124.24, 128.41, 130.52, 131.20, 134.83,135.10, 135.71, 136.67, 142.23, 206.78. ESI-HRMS calcdfor C37H53NO4[M + Na]+ 598.3866, found 598.3883.

General procedure for the preparation of ring A fusedpyrimidine compounds (13–22)

The target compounds were prepared by the reaction of1 equiv. of appropriate intermediate (2–12) with guani-dine hydrochloride (2 equiv.) in absolute ethanol was addedpotassium hydroxide (2 equiv.) to the reaction mixture. Thesolution of contents was refluxed for 3–12 h till comple-tion of reaction (monitored by TLC analysis). After thereaction mixture was neutralized with aq HCl (1:1), it wasextracted with ethyl acetate (three times). The combinedorganic layerwas dried over anhydrousNa2 SO4 and concen-trated under reduced pressure to give crude product, whichwas purified by column chromatography (silica gel) usingethyl acetate:hexane (1:4, 3:2) mixture eluent to afford thedesired pure compounds (13–22) in 60–76 % yields.

2,3-(2′-Amino-4′′-phenyl)-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (13)

Yield70%; light yellowsemi-solid;TLC: (40%hexane:ethylacetate) Rf = 0.30; IR (KBr, cm−1): 3358, 2924, 1618, 1552,1451, 1380, 1219, 771, 701; 1H-NMR (500MHz, CDCl3): δ0.59 (s, 3H,Me), 1.09 (s, 3H,Me), 1.18 (s, 6H, 2xMe), 1.28–1.32 (m, 6H), 1.38 (s, 3H, Me), 1.52 (s, 6H, 2xMe), 1.60 (s,3H, Me), 1.86–2.01 (m, 12H), 2.26 (d, J = 15.1 Hz, 1H),2.57 (d, J = 15.1 Hz, 1H), 5.03–5.10 (m, 3H), 7.43 (m, 5H);13C-NMR (125 MHz, CDCl3): δ 14.28, 16.02, 16.12, 17.68,22.20, 23.07, 23.75, 25.27, 25.69, 26.62, 26.74, 29.68, 31.53,38.01, 39.63, 39.67, 39.72, 41.42, 43.63, 52.64, 59.32, 73.63,114.21, 124.11, 124.30, 124.55, 128.23, 128.42, 128.76,131.29, 135.00, 135.45, 138.69, 161.03, 167.12, 174.22. ESI-HRMS calcd for C38H56ON3 [M + H]+ 570.4417, found570.4402.

2,3-[2′-Amino-4′-(4′′-methyl-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (14)

Yield72%; light yellowsemi-solid;TLC: (40%hexane:ethylacetate) Rf = 0.31; IR (KBr, cm−1): 3345, 3200, 2925,1616, 1553, 1448, 1359, 1219, 1186, 772; 1H-NMR (300MHz, CDCl3): δ 0.58 (s, 3H, Me), 1.08 (s, 3H, Me), 1.17(s, 3H, Me), 1.18(s, 3H, Me), 1.21–1.45 (m, 6H), 1.38 (s,3H, Me), 1.59 (s, 6H, 2xMe), 1.60 (s, 3H, Me), 1.89–2.02(m, 12H), 2.25 (d, J = 15.1 Hz 1H), 2.31(s, 3H), 2.57 (d,J = 15.8 Hz, 1H), 5.00–5.08 (m, 3H), 7.16 (t, J = 8.3 Hz,2H), 7.31(t, J = 8.3Hz, 2H); 13C-NMR (125MHz, CDCl3):δ 14.21, 15.92, 15.99, 17.65, 21.28, 22.15, 23.03, 23.67,25.28, 25.65, 26.60, 26.71, 29.65, 31.50, 37.97, 39.58, 39.67,39.71, 41.47, 43.58, 52.64, 59.38, 73.62, 114.00, 124.07,124.27, 124.64, 128.42, 128.83, 131.25, 134.97, 135.26,135.49, 138.60, 161.04, 166.98, 174.15. ESI-HRMS calcdfor C39H58ON3 [M + H]+ 584.4574, found 584.4555.

123

Mol Divers

2,3-[2′-Amino-4′-(4′′-methoxy-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (15)

Yield 75% light yellow semi-solid; TLC: (40%hexane:ethylacetate) Rf = 0.31; IR (KBr, cm−1): 3354, 3202, 2926, 1609,1552, 1450, 1359, 1250, 1177, 1034, 837, 772; 1H-NMR(300 MHz, CDCl3): δ 0.63 (s, 3H, Me), 1.14 (s, 3H, Me),1.24 (s, 6H, 2xMe), 1.27-1.51 (m, 6H), 1.48 (s, 3H, Me),1.58 (s, 6H, 2xMe), 1.66 (s, 3H, Me), 1.91–2.07 (m, 12H),2.30 (d, J = 15.3 Hz, 1H), 2.65 (d, J = 15.2, 1H), 3.82, (s,3H,OMe), 5.05–5.11 (m, 3H), 6.95 (d, J = 8.7Hz, 2H), 7.46(d, J = 8.7 Hz, 2H);13C-NMR (125 MHz, CDCl3): δ 14.22,15.98, 16.06, 17.67, 22.16, 23.07, 23.70, 25.29, 25.66, 26.62,26.72, 29.65, 31.51, 38.02, 39.59, 39.70, 41.68, 43.63, 52.65,55.22, 59.36, 73.62, 113.54, 113.95, 124.08, 124.29, 124.65,130.90, 130.92, 131.29, 135.00, 135.36, 160.03, 161.03,166.55, 174.09. ESI-HRMScalcd forC39H58O2N3 [M+H]+600.4523, found 600.4506.

2,3-[2′-Amino-4′-(4′′-carboxy-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (16)

Yield65%; light yellowsemi-solid;TLC: (60%hexane:ethylacetate) Rf = 0.41; IR (KBr, cm−1): 3338, 3209, 2925,1696, 1552, 1450, 1359, 1272, 1180, 865, 772; 1H-NMR(300 MHz, CDCl3): δ 0.66 (s, 3H, Me), 1.15 (s, 3H, Me),1.26 (s, 6H, 2xMe), 1.32 (s, 3H, Me), 1.27–1.51 (m, 6H),1.38 (s, 3H, Me), 1.57 (s, 6H, 2xMe), 1.66 (s, 3H, Me),1.90–2.04 (m, 12H), 2.26 (d, J = 15.8, 1H), 2.54 (d,J = 15.1 Hz, 1H), 5.01–5.10 (m, 3H), 7.53 (d, J =8.3, 2H), 8.09 (d, J = 8.3, 2H);13C-NMR (125 MHz,CDCl3): δ 14.31, 15.97, 17.69, 22.19, 23.08, 23.77, 25.28,25.69, 26.56, 26.75, 29.69, 31.54, 38.04, 39.64, 39.73, 39.91,41.17, 43.59, 52.65, 59.19, 73.59, 114.19, 124.16, 124.37,124.65, 128.65, 129.88, 130.80, 131.25, 135.03, 135.58,142.02, 161.03, 165.23, 169.41, 175.89. ESI-HRMS calcdfor C39H56O3N3 [M + H]+ 614.4316, found 614.4318.

2,3-[2′-Amino-4′-(4′′-fluoro-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (17)

Yield69%; light yellowsemi-solid;TLC: (40%hexane:ethylacetate) Rf = 0.31 IR (KBr, cm−1): 3351, 3206, 2924, 1605,1554, 1450, 1360, 1220, 842, 772; 1H-NMR (300 MHz,CDCl3): δ 0.57 (s, 3H, Me), 1.08 (s, 3H, 2xMe), 1.18 (s,6H, 2xMe), 1.21-1.38 (m, 6H), 1.39 (s, 3H, Me), 1.52 (s,6H, 2xMe), 1.60 (s, 3H, Me), 1.87–2.02 (m, 12H), 2.21 (d,J = 15.1, 1H), 2.52 (d, J = 14.3 Hz, 1H), 5.01–5.08 (m,3H), 7.04 (t, J = 8.3 Hz, 2H), 7.40 (t, J = 8.3, 2H);13C-NMR (125 MHz, CDCl3): δ 14.26, 16.00, 17.67, 22.68,23.10, 23.73, 25.34, 25.69, 26.59, 26.73, 29.68, 31.54, 38.02,39.67, 39.72, 41.48, 43.58, 52.68, 59.32, 73.69, 114.13,

115.18, 115.35, 124.06, 124.31, 124.48, 130.44, 130.51,131.26, 135.05, 135.51, 160.99, 162.02, 163.98, 174.54. ESI-HRMS calcd for C38H55ON3F[M + H]+ 588.4323, found588.4319.

2,3-[2′-Amino-4′-(4′′-chloro-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (18)

Yield69%; light yellowsemi-solid;TLC: (40%hexane:ethylacetate) Rf = 0.31; IR (KBr, cm−1): 3355, 3210, 2920, 1657,1551, 1452, 1384, 1260, 1186, 1090, 800, 772; 1H-NMR(300 MHz, CDCl3): δ 0.59 (s, 3H, Me), 1.10 (s, 3H, Me),1.22 (s, 3H, Me), 1.27 (s, 3H, Me), 1.23–1.50 (m, 6H), 1.41(s, 3H, Me), 1.53 (s, 6H, 2xMe), 1.60 (s, 3H, Me), 1.87–2.02(m, 12H), 2.25 (d, J = 15.7 Hz, 1H), 2.54 (d, J = 15.6 Hz,1H), 5.01–5.06 (m, 3H) 7.36–7.41 (m, 4H); 13C-NMR (125MHz, CDCl3): δ 14.25, 15.90, 16.00, 17.66, 22.16, 23.12,23.71, 25.38, 25.66, 26.62, 26.75, 29.66, 31.50, 38.02,39.68, 39.72, 41.41, 43.58, 52.67, 59.37, 73.59, 114.07,124.11, 124.34, 124.46, 128.49, 129.94, 131.20, 135.01,135.50, 136.88, 160.94, 165.91, 174.53. ESI-HRMS calcdfor C38H55ON35

3 Cl[M+H]+ 604.4034, found 604.4029, Iso-topic signal for C38H55ON37

3 Cl[M + H]+ 606.4004.

2,3-[2′-Amino-4′-(4′′-bromo-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (19)

Yield67%; light yellowsemi-solid;TLC: (40%hexane:ethylacetate) Rf = 0.31; IR (KBr, cm−1): 3351, 3210, 2921,1620, 1550, 1451, 1360, 1219, 1186, 1072, 772; 1H-NMR(500 MHz, CDCl3): δ 0.57 (s, 3H, Me), 1.09 (s, 3H, Me),1.18 (s, 6H, 2xMe), 1.24-1.37 (m, 6H), 1.39 (s, 3H, Me),1.52 (s, 6H, 2xMe), 1.60 (s, 3H, Me), 1.86–2.04 (m, 12H),2.23 (d, J = 14.9 Hz, 1H), 2.52 (d, J = 14.9 Hz, 1H),5.01–5.06 (m, 3H), 7.29 (d, J = 8.3 Hz, 2H), 7.50, (d, J =8.3 Hz, 2H);13C-NMR (75 MHz, CDCl3): δ 14.28, 15.97,16.05, 17.71, 22.19, 23.12, 23.73, 25.39, 25.71, 26.64, 26.74,29.12, 31.53, 38.05, 39.70, 39.76, 41.39, 43.58, 52.65, 59.40,73.69, 114.09, 123.29, 124.16, 124.36, 124.45, 130.21,131.46, 131.25, 135.07, 135.59, 161.94, 165.72, 174.96.ESI-HRMS calcd for C38H55ON79

3 Br[M + H]+ 648.3517,found 648.3523, isotopic signal for C38H55ON81

3 Br[M+H]+650.3497.

2,3-[2′-Amino-4′-(5′′-trifluoromethyl-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (20)

Yield68%; light yellowsemi-solid;TLC: (30%hexane:ethylacetate) Rf = 0.30; IR (KBr, cm−1): 3357, 3206, 2924,1616, 1554, 1453, 1324, 1209, 1167, 1131, 1074, 973, 804,772, 704; 1H-NMR (300 MHz, CDCl3): δ 0.66 (s, 3H, Me),

123

Mol Divers

1.15 (s, 3H, Me), 1.25 (s, 6H, 2xMe), 1.25–1.49 (m, 6H),1.41 (s, 3H, Me), 1.59 (s, 6H, 2Me), 1.67 (s, 3H, Me),1.94–2.04 (m, 12H), 2.31 (d, J = 15.3 Hz, 1H), 2.59 (d,J = 15.3 Hz, 1H), 5.03–5.11 (m, 3H), 7.53–7.77 (m, 4H);13C-NMR (125 MHz, CDCl3): δ 14.29, 15.91, 17.63, 22.16,23.04, 23.76, 25.31, 25.65, 26.58, 26.72, 29.66, 31.51, 38.0439.60, 39.73, 41.23, 43.54, 52.62, 59.29, 73.523, 114.06,124.09 124.31, 124.38, 125.57, 128.68, 131.88, 134.97,135.47, 139.29, 161.21, 165.22, 175.12. ESI-HRMS calcdfor C39H55ON3F3 [M + H]+ 638.4291, found 638.4287.

2,3-[2′-Amino-4′-(4′′-methoxy-5′′-bromo-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (21)

Yield72%; light yellowsemi-solid;TLC: (40%hexane:ethylacetate) Rf = 0.40; IR (KBr, cm−1): 3344, 3205, 2921,1602, 1550, 1452, 1359, 1254, 1185, 1055, 809, 772; 1H-NMR (500 MHz, CDCl3): δ 0.57 (s, 3H, Me), 1.08 (s,3H, Me), 1.22 (s, 6H, 2xMe), 1.26–1.41 (m, 6H), 1.43(s, 3H, Me), 1.52 (s, 6H, 2xMe), 1.60 (s, 3H, Me), 1.86–2.00 (m, 12H), 2.26 (d, J = 14.9 Hz, 1H), 2.59 (d,J = 15.3 Hz, 1H), 3.86 (s, 3H, OMe), 5.03–5.08 (m,3H), 6.84–7.68 (m, 3H); 13C-NMR (125 MHz, CDCl3):δ 14.25, 16.00, 16.08, 17.66, 22.66, 23.06, 23.72, 25.30,25.67, 26.66, 26.74, 29.67, 31.52, 38.07, 39.23, 39.72,41.60, 43.61, 52.63, 56.24, 59.36, 73.57, 111.12, 111.57,114.03, 124.09, 124.30, 124.60, 129.08, 131.23, 132.04,133.90, 135.03, 135.50, 156.36, 160.97, 164.98, 174.58. ESI-HRMS calcd for C39H57O2N79

3 Br[M+H]+ 678.3628, found678.3621. Isotopic signal for C39H57O2N81

3 Br[M + H]+680.3613.

2,3-[2′-Amino-4′-(2′′,4′′,6′′-trimethoxy-phenyl)]-pyrimidinyl-8-hydroxy-13,17,21-polypodatriene (22)

Yield76%; light yellowsemi-solid;TLC: (60%hexane:ethylacetate) Rf = 0.43; IR (KBr, cm−1): 3363, 3206, 2928,1609, 1559, 1454, 1359, 1224, 1130, 1054, 813, 772; 1H-NMR (500 MHz, CDCl3): δ 0.69 (s, 3H, Me), 1.15 (s, 3H,Me), 1.24 (s, 3H, Me), 1.26 (s, 3H, Me), 1.28–1.47 (m, 6H),1.35 (s, 3H, Me), 1.60 (s, 6H, 2xMe), 1.67 (s, 3H, Me),1.85.2.08 (m, 12H), 2.06 (d, J = 15.4 Hz, 1H), 2.35 (d,J = 15.4 Hz, 1H), 3.68 (s, 3H, OMe), 3.70 (s, 3H, OMe),3.82 (s, 3H, OMe), 5.03–5.10 (m, 3H), 6.14 (s, 2H); 13C-NMR (125 MHz, CDCl3): δ 14.10, 15.56, 15.85, 17.54,22.02, 22.86, 23.53, 25.33, 25.57, 26.47, 26.61, 29.53, 31.76,37.49, 39.24, 39.53, 39.59, 43.49, 52.62, 55.09, 55.39, 55.62,59.28, 73.51, 90.44, 90.65, 107.98, 114.03, 124.00, 124.16,124.48, 131.14, 134.81, 134.98, 157.41, 160.73, 161.66,172.95, 175.56. ESI-HRMScalcd forC41H62O4N3 [M+H]+660.4734, found 660.4726.

Cytotoxicity assay

Evaluation of in vitro anticancer activity

The cytotoxic activity of the test compounds was determinedin a panel of six cancer cell lines using an MTT assay[18]. The cells were seeded at a concentration of 1 × 104

cells well−1 in 200 ml DMEM, supplemented with 10 %FBS in each well of 96-well micro culture plates and wereincubated for 24 h at 37 ◦C in a CO2 incubator. The test com-pounds were dissolved in DMSO and diluted to the desiredconcentrations in culturemediumandwere added to thewellswith respective vehicle control. After 48 h of incubation, avolume of 10 µl MTT [3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] (5 µM) was added to eachwell. The plates were further incubated for 4 h in a CO2 incu-bator. The supernatant liquid from each well was carefullyremoved, and the obtained formazon crystals were dissolvedin 100 ml of DMSO. The absorbance in each well was readat 540 nm wavelength employing plate reader (Synergy 4,Biotek Inc., USA).

Hoechst staining for morphological analysis of apoptosis

This study was done in A549 lung cancer cells, which wereseeded at a density of 10,000 cells over 18-mm cover slipsand were incubated for 24 h. The medium was replaced after24 h, and the cellswere treatedwith either the test compounds18 and 19 at 4 and 5µMor vehicle (0.001%DMSO) for 24 h.After 24 h treatment, Hoechst 33258 (Sigma-Aldrich) stainwas added to themedium at a concentration of 0.5 µM.Afterfurther incubation for 30 min at 37 ◦C, cells from each wellwere captured from randomly selected fields under fluores-cent microscope (Leica, Germany) to determine qualitativelythe proportion of viable and apoptotic cells based on the rel-ative fluorescence and nuclear fragmentation [19].

Cell cycle analysis

The effect of the potent test compound on various stages ofcell cycle was studied in A549 cells. These cells (1 × 106)were seeded in six-well plates and were treated with the testcompounds 18 and 19 at two different concentrations of 5and 10 µM. After 48 h of incubation, cells were then har-vested, washed and fixed in 70 % ice-cold ethanol at 40 ◦Cfor 2 h. Then, cells were centrifuged, washed with cold PBSand re-centrifuged. Cells were then resuspended in 250 µlPBS andwere stained with 50µMpropidium iodide in hypo-tonic lysis buffer (0.1 % sodium citrate, 0.1 % Triton X-100)containing DNase-free RNase-A for 20 min. Stained cellswere analysed using fluorescence-activated cell sorter cali-bre (Becton Dickinson) [20] to calculate the percentage ofcells in G0/G1, S and G2/M phases.

123

Mol Divers

Caspase-3

Lung cancer cells (A-549) were seeded in six-well plates ata concentration of 2.5× 105 per well. The cells were treatedwith the test compounds 18 and 19 at concentrations of 5 and10 µM for a period of 48 h. After incubating for 48 h, thetreated cellswerewashedwith PBS, scraped in PBS andwerecentrifuged at 2000 rpm for 10 min at 4 ◦C. The obtainedpellet was resuspended in 80 mL of lysis buffer, and thesuspension was incubated on ice for 20–30 min. The lysatewas then centrifuged at 13,200 rpm for 20min at 4 ◦C, and thesupernatantwas transferred to fresh tubes.Amixture of 50mlof 2X assay buffer, 50 ml cell lysate and 2 ml of caspase-3substrate was taken in a 96-well black polystyrene plate. Thereaction was allowed to take place for 1 h. The fluorescencegenerated by the release of the fluorogenic group AFC aftercleavage by caspase-3 was measured by excitation at 400 nmand emission at 505 nm for every 5 min over a period of 1 h.Protein content was estimated by Bradford’s method andwasnormalized [24].

DNA laddering assay

A549 Cells were seeded (1 × 106) in six-well plates andwere incubated for 24 h. After incubation, the cells weretreated with compounds 18 and19 at 5 and 10 µM for 48 h.Cells were then collected and centrifuged at 2500 rpm for5 min at 4 ◦C. The pellet was isolated, collected, and washedwith phosphate-buffered saline (PBS). A volume of 100 µlof lysis buffer was added to the pellet, mixed and centrifugedat 3000 rpm for 5 min at 4 ◦C. To the supernatant, a volumeof 10 µl of 10 % SDS and 10 µl of (50 µM) RNase-A wereadded, and the mixture was incubated at 56 ◦C for 2 h. Afterincubation, a volume of 10 µl of (25 µM) of Proteinase Kwas added and was further incubated for 2 h at 37 ◦C, andthen added 65 µl of 10 M ammonium acetate and 500 µlof ice-cold ethanol. The samples were mixed and were incu-bated at 80 ◦C for 1 h. After incubation, the samples werecentrifuged at 12,000 rpm for 20 min at 4 ◦C, and the pel-let was washed with 80 % ethanol, air dried for 10 min atroom temperature. The pellet was dissolved in 50 µl of TEbuffer, and DNA laddering assay was performed by using2 % agarose gel electrophoresis as described elsewhere [25].DNA fragmentation was visualized upon staining gel withethidium bromide (0.5 mg ml−1) and exposed to UV light.The presence of apoptosis was indicated by the appearanceof a ladder of oligonucleosomal DNA fragments.

Acknowledgments We are thankful to Director, CSIR-IICT for sup-port and encouragement. We are also thankful to CSIR, India forproviding fellowship to MC and DBT, Govt. of India for partial fund-ing under the co-ordinated project “Biotechnological Interventions forPharmaceutically Valuable Compounds from Forest Resins” (GAP-0412).

References

1. Daniel AD, Sylvia U, Ute R (2012) A historical overview of naturalproducts in drug discovery. Metabolites 2:303–336. doi:10.3390/metabo2020303

2. Guo Z (2012) Modification of natural products for drug discovery.Yao Xue Xue Bao 47:144–157

3. Yadav VR, Prasad S, Sung Ramaswamy BK, Aggarwal BB (2010)Targeting inflammatory pathways by triterpenoids for preven-tion and treatment of cancer. Toxins 2:2428–2466. doi:10.3390/toxins2102428

4. Hanus LO, Rezanka T, Dembitsky VM, Moussaieff A (2005)Myrrh-commiphora chemistry. Biomed Papers 149:3–28. doi:10.5507/bp.2005.001

5. Matsuda H, Morikawa T, Ando S, Oominami H, MurakamiT, Kimura I, Yoshikawa M (2004) Absolute stereostructures ofpolypodane-type triterpenes,myrrhanolAandmyrrhanoneA, fromguggul-gum resin (the resin of Balsamodendron mukul). ChemPharm Bull 52:1200–1203. doi:10.1248/cpb.52.1200

6. Matsuda H, Morikawa T, Ando S, Oominami H, MurakamiT, Kimura I, Yoshikawa M (2004) Absolute stereostructures ofpolypodane- and octanordammarane-type triterpenes with nitricoxide production inhibitory activity from guggul-gum resins.BioorgMedChem12:3037–3046. doi:10.1016/j.bmc.2004.03.020

7. Samy Morad AF, Claudia S, Berthold B, Bernd S, MichaelW, Tatiana S, Thomas S (2011) (8R)-3β,8-Dihydroxypolypoda-13E,17E,21-triene induces cell cycle arrest and apoptosis intreatment-resistant prostate cancer cells. J Nat Prod 74:1731–1736.doi:10.1021/np200161a

8. Victoriano D, Lidia L, José F, Alejandro F (2013) First synthesis of(+)-myrrhanol C, an anti-prostate cancer lead. Org Biomol Chem11:559–562. doi:10.1039/c2ob26947c

9. Amr AEGE, Sabry NM, Abdulla MM (2007) Synthesis, reactions,and anti-inflammatory activity of heterocyclic systems fused to athiophene moiety using citrazinic acid as synthon. Monatshefte fürChemie 138:699–707. doi:10.1007/s00706-007-0651-0

10. Fujiwara N, Nakajima T, Ueda Y, Fujita H, Kawakami H (2008)Novel piperidinylpyrimidine derivatives as inhibitors ofHIV-1LTRactivation. Bioorg Med Chem 16:9804–9816. doi:10.1016/j.bmc.2008.09.059

11. Wagner E, Al-Kadasi K, Zimecki M, Sawka-Dobrowolska W(2008) Synthesis and pharmacological screening of derivativesof isoxazolo[4,5-d]pyrimidine. Eur J Med Chem 43:2498–2504.doi:10.1016/j.ejmech.2008.01.035

12. Mallavadhani UV, Mahapatra A, Pattnaik B, Vanga NR, Suri N,Saxena KA (2013) Synthesis and anti-cancer activity of somenovel C-17 analogs of ursolic and oleanolic acids. Med Chem Res22:1263–1269. doi:10.1007/s00044-012-0106-y

13. Mallavadhani UV, Vanga NR, Jeengar MK, Naidu VGM (2014)Synthesis of novel ring-A fused hybrids of oleanolic acid withcapabilities to arrest cell cycle and induce apoptosis in breast cancercells. Eur J Med Chem 74:398–404. doi:10.1016/j.ejmech.2013.12.040

14. Selvam TP, Karthick V, Kumar PV, Ali MA (2012)Synthesis and structure–activity relationship study of2-(substitutedbenzylidene)-7-(4-fluorophenyl)-5-(furan-2-yl)-2H -thiazolo[3,2-a]pyrimidin-3(7H )-one derivatives as anticanceragents. Drug Discov Ther 6:198–204. doi:10.5582/ddt.2012.v6.4.198

15. EI-Rayyes NR, Ramadan HM (1987) Synthesis of newbenzo[6,7]cyclohepta[1,2-d] pyrimidine. J Heterocycl Chem24:1141–1147. doi:10.1002/jhet.5570240442

16. Ahluwalia VK, Kaila N, Bala S (1987) Synthesis & antifun-gal & antibacterial activities of some 2-amino-4,6-substituted-

123

Mol Divers

pyrimidines & 4-styryl-6,7-pyranocoumarins. Indian J Chem SecB 26B:700–702

17. Pasha TY, Udipi RH, Bhat AR (2005) Synthesis and antimicro-bial screening of some pyrimidine derivatives. Indian J HeterocyclChem 15:149–152

18. Botta M, Armaroli S, Castagnolo D, Fontana G, Perad P, Bom-bardelli E (2007) Synthesis and biological evaluation of newtaxoids derived from 2-deacetoxytaxinine. Bioorg Med Chem Lett17:1579–1583. doi:10.1016/j.bmcl.2006.12.101

19. Sun SY, Hail N Jr, Lotan RJ (2004) Apoptosis as a novel target forcancer chemoprevention. J Natl Cancer Inst 96:662–672. doi:10.1093/jnci/djh123

20. HotzMA, Gong J, Traganos F, Darzynkiewicz Z (1994) Flow cyto-metric detection of apoptosis: comparison of the assays of in situDNA degradation and chromatin changes. Cytometry 15:237–244.doi:10.1002/cyto.990150309

21. Konstantinov SM, Berger MR (1999) Human urinary bladder car-cinoma cell lines respond to treatment with alkylphosphocholines.Cancer Lett 144:153–160. doi:10.1016/S0304-3835(99)00219-0

22. Henkels PM, Turchi JJ (1999) Cisplatin-induced apoptosis pro-ceeds by caspase-3-dependent and -independent pathways incisplatin-resistant and -sensitive human ovarian cancer cell lines.Cancer Res 59:3077–3083

23. Francis JA, Raja SN, Nair MG (2004) Bioactive terpenoids andguggulusteroids from Commiphora mukul gum resin of potentialanti-inflammatory interest. Chem Biodiver 1:1842–1853. doi:10.1002/cbdv.200490138

24. Kamal A, Ramakrishna G, Lakshma Nayak V, Raju P, SubbaRao AV, Viswanath A, Vishnuvardhan MVPS, Ramakrishna S,Srinivas G (2012) Design and synthesis of benzo[c, d] indolone–pyrrolobenzodiazepine conjugates as potential anticancer agents.Bioorg Med Chem 20:789–800. doi:10.1016/j.bmc.2011.12.003

25. Cosse JP, Sermeus A, Vannuvel K, Ninane N, Raes M, Michiels C(2007) Differential effects of hypoxia on etoposide-induced apop-tosis according to the cancer cell lines. Mol Cancer 6:1–16. doi:10.1186/1476-4598-6-61

123