Derivatives of grindelic acid: From a non-active natural diterpene to synthetic antitumor derivatives

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Title:

Derivatives of grindelic acid: from a non-active natural diterpene to synthetic antitumor

derivatives

Authors:

a

b,c,

c

Guillermo F. Reta, Alejandra I. Chiaramello, Celina García, * Leticia G. León, Víctor

S. Martín, José M. Padrón, Carlos E. Tonn, and Osvaldo J. Donadel *

b,c

c

a

INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad

Nacional de San Luis, Chacabuco y Pedernera -5700- San Luis, Argentina

b

Departamento de Química Orgánica, Universidad de La Laguna

Instituto Universitario de Bio-Orgánica Antonio González (IUBO-AG), Centro de

c

Investigaciones Biomédicas de Canarias (CIBICAN), Universidad de La Laguna, Avda.

Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain.

Corresponding author:

Dr. Osvaldo J. Donadel,

e-mail: odonadel@gmail.com

INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad

Nacional de San Luis, Chacabuco y Pedernera -5700- San Luis, Argentina,

Phone +54 266 4439909

1

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1

. Introduction

The plant kingdom comprise a relatively small group within biodiversity, with about

50,000 species, of which near 6%, has been studied for their biological activity, and

2

only about 15% has been studied in a phytochemical way. Arguably, the plant kingdom

is the largest source of structural diversity in organic compounds, forming a reservoir of

chemical structures which is not possible to find in another environment. In this sense,

a large number of natural products isolated from plants (or their transformed products)

are known, to have relevant biological activities showing an important applicative

potential [1].

The quest for new chemical structures with antiproliferative properties, which can be

used in the future as chemotherapeutic agents, is an active area of research stimulated

by the discovery of new biological targets and the possibility of arising new drugs

without undesirable side effects. In the past years, there has been an increasing

interest in the development of new pharmaceuticals based on a nucleus present in

natural products along with the increase of life-threatening diseases such as AIDS,

cancer, hepatitis, etc. [2]. It is noteworthy that some semi synthetic compounds derived

from a natural framework sometimes show higher bioactivity than the original natural

product. Taking into account their broad range of biological activities, such as enzyme

inducing, modulation of immune cell function, and cytotoxic against human tumor cell

lines, [3-6] labdane diterpenes have attracted scientific interest.

Analyzing the structure of molecules used in cancer therapy, most of them show

nitrogenated functional groups in their framework like amide and carbamate moieties

[

7]. Recently, it has been reported a series of disubstituted 1,2,3-triazole exhibiting

potent cytotoxicity in the nanomolar range and tubulin inhibitory activity in the low

micromolar range [8]. This kind of compounds is considered an interesting unit in the

design of anticancer drugs. Such heterocycle may act due to their dipolar character,

rigidity, ability to form hydrogen bridge bonds or as simple connectors [9].

Synthetic organic chemists have gained great interest for 1,2,3-triazoles in the

development of new biologically active molecules [10]. The triazole moiety does not

occur in nature, but 1,2,3-triazole cores may form the basis of small-molecule

pharmaceutical leads. Molecules containing this heterocyclic nucleus have being

reported having anti-HIV, antimicrobial, anti-allergic, antifungal and antitumor activities

[

10].

Grindelic acid (1) is a labdane-type diterpene that was reported as the main secondary

metabolite from Grindelia chiloensis Cabr and G. pulchella Dunal var. pulchella

(

Asteraceae). This compound possesses a suitable C-14-side-chain for inserting

functionalities carrying nitrogen and oxygen [11]. In a previous work, we have reported

about the enhancement of the cytotoxicity of some sesquiterpenes and iridoids when

their lipophilicity was increased by the introduction of alkyl and/or aryl-silyl

functionalities on the natural framework [12].

In consideration of the above mentioned factors, herein we describe our findings aimed

to the synthesis and cytotoxic evaluation of oxygenated and oxy-nitrogenated

derivatives from grindelic acid (1). It should be emphasized that this diterpene can be

isolated in significant quantities from the aforementioned natural sources. Furthermore,

2

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these plants have a wide distribution in the Patagonian steppe zones of Argentina and

Chile [13].

2

. Result and Discussion

.1. Chemistry

2

From Grindelia pulchella Dunal var. pulchella, we obtained the metabolites grindelic

acid (1) and 1α-hydroxygrindelic acid (2), as reported earlier (Figure 1) [13].

Insert Figure 1

In preliminary tests, compound 1 (main secondary metabolite) exhibited greater

bioactivity than 2. Therefore, it was chosen as a starting material for the preparation of

a series of thirty-six derivatives. Firstly, we prepared several oxygenated compounds 3-

1

0 taking advantage of the presence of the carboxylic acid group (Scheme 1). Methyl

ester derivative 3 was obtained from 1 by direct esterification under standard

conditions. Subsequent reduction of the ester group at the side chain, with LiAlH gave

4

alcohol 4 in high yields. The oxidation of 4 under Parikh-Doering conditions and

subsequent Wittig reaction using ethyl 2-(triphenylphosphoranylidene)-propanoate, led

to the α,β-unsaturated ester 5 that after reduction with DIBAL-H afforded in good yields

alcohol

6.

Following

the

same

sequence

with

ethyl(triphenyl-

phosphoranylidene)acetate, compounds 7 and 8, were obtained from 4. Epoxides 9

and 10 were achieved by Katsuki-Sharpless asymmetric epoxidation of the allylic

alcohol 8 using R,R-(+)-DET and S,S-(-)-DET, respectively.

Insert Scheme 1

Insert Legend Scheme 1

Cycloaddition and multicomponent reactions are convergent procedures of high

synthetic utility combining chemical and atomic efficiency. Within the first type, azide-

alkyne Huisgen cycloaddition is a powerful tool to prepare 1,2,3-triazoles in a

straightforward manner [14]. Multicomponent Reactions (MCRs) are convergent

reactions in which three or more starting materials react to form a product where,

basically, all or most of the atoms contribute to the newly formed product. In MCRs a

product is assembled according to a cascade of elementary chemical reactions. Thus,

there is a network of reaction equilibrium which all finally flow into an irreversible step

yielding the product. In this work, it was prepared a series of oxy-nitrogenated

derivatives, compounds 11-22 by Ugi reaction, 27-32 and 37 by Huisgen

cycloadditions, and 23-26 and 33-36 were prepared by classical chemistry.

Noteworthy, in all proposals the natural product stereochemistry remained intact.

Derivatives 11-22 were obtained by the Ugi reaction using 1 as carboxylic acid and

acetone as a source of carbonyl group. Benzylamine (compounds 11-17), and aniline

(

compounds 18-22) were used as amines varying the corresponding isocyanides

Table 1).

Insert Table 1

3

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One of the most popular reactions within the click chemistry paradigm is the Cu(I)-

catalyzed 1,3-dipolar Huisgen cycloaddition of alkynes and azides. This reaction

proceeds with great efficiency and selectivity in aqueous media and yields a triazole

moiety [15]. Our first objective was to obtain azide 23 and alkyne 26, using as starting

material alcohol 4 (see Scheme 2).

Insert Scheme 2

Insert Legend Scheme 2

The coupling of azide 23 and various commercial alkynes, under Huisgen conditions,

provided the 1,2,3-triazoles 27-28 in good yields. In a similar manner, the quasi

symmetrical hybrid 29 was prepared from azide 23 and alkyne 26 (Table 2).

Insert Table 2

Using Huisgen conditions and adjusting 1 equivalent of the suitable di-alkyne per 2

equivalents of the azide 23, the dimers 30-32 were achieved (see Table 3). Alkynes

used for the preparations of compounds 30 and 31 are commercially available, and the

alkyne used to arrive to compound 32 was synthesized according to the procedure

described in the literature [16].

Insert Table 3

The final set of derivatives was prepared with the aim to introduce the maximum of

structural diversity at the final hybrid compounds. Thus, carbonylimidazole 33 was

prepared by the reaction of alcohol 4 with carbonyldiimidazole (CDI) in CH Cl .

2 2

Derivatives 34-35 were obtained from compound 33 by treatment with diverse amines.

The use of ethylenediamine provided dimeric carbamate 36. Finally, Huisgen coupling

of 35 with azide 23 yielded the hybrid triazol 37.

Insert Scheme 3

Insert Legend Scheme 3

2

.2. Antiproliferative activity

The in vitro antiproliferative activity was evaluated using the National Cancer Institute

NCI) protocol after 48 h of drug exposure using the sulforhodamine B (SRB) assay.

(

The results, expressed as GI50 values, are shown in Table 4. Derivatives 20-22, 31,

and 36 were not evaluated due to their low solubility in DMSO under the testing

conditions.

From the growth inhibition results we can depict some preliminary structure-activity

relationships. The group of oxygenated derivatives (3-10) is more active than the

natural products (1-2) where they derive from, having GI50 values in all cell lines in the

range 16-37 µM. In this series, compounds 5 and 7, both bearing an ethyl ester in the

side chain, gave higher GI50 values (>30 µM) in the most resistant cell lines T-47D and

4

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WiDr. When compared to the methyl ester analog 3, one could speculate with the

bulkiness of the ester group on the side chain and its reverse effect on the activity.

However, when the side chain was elongated through an Ugi reaction (compounds 11-

22) a clear enhancement in the antiproliferative activity was observed. In fact,

compound 17 resulted the most active compound of the whole study with GI₅₀values in

the range 0.95-1.9 µM. In this second group of compounds, a relationship between

substituents and biological activity could not be established. When considering the

derivatives of the Huisgen reaction 27-29, no improvement was observed in the activity

when compared to the most active derivative 17. Finally, for the analogs bearing two

grindelic scaffolds (29-32 and 36-37), the activity profile was variable. Thus, derivatives

32 and 37 were inactive (GI50 >100 µM), whilst 30 and 34 showed GI50 values <10 µM

against all cell lines tested.

Insert Table 4

3. Conclusion

Given these results, the use of a natural product can be a viable strategy to prepare

new active molecules. With adequate quantities of a natural product, and using

accessible synthetic routes, it has been possible to obtain new structures exhibiting

interesting bioactivities as antitumor agents. Grindelic acid (1) was the starting material

for the transformation into several oxygenated, and oxy-nitrogenated moieties at the C-

1

4 side chain by means of diverse reactions. Our strategy allowed to reach thirty-five

new compounds including functionalities such as epoxides, diamides, carbamates and

,2,3-triazoles, among others. A significant number of these synthetic derivatives

1

showed higher activity than the natural product. It is noteworthy that the oxy-

nitrogenated and nitrogenated systems were more active than oxygenated ones, being

the diamides the most active compounds. The N-benzyl-N-(1-(benzylamino)-2-methyl-

1-oxopropan-2-yl) amide of grindelic acid (17) proved to be the most active product in

all cell lines tested.

Finally, this work aims to demonstrate the possibility of achieving new antitumor

leaders from molecular frameworks provided by nature, getting sometimes a dramatic

enhance of the bioactivity through usual chemical transformations.

4

. Experimental

.1. Chemistry

4

Unless stated otherwise, all solvents were purified by standard techniques. Reactions

requiring anhydrous conditions were performed under argon. Anhydrous magnesium

sulfate was used for drying solutions. Reactions were monitored by thin layer

chromatography (TLC) on silica gel plates (60 F254), and were visualized by use of

UV light, 2.5% phosphomolybdic acid in ethanol or vanillin with acetic and sulfuric

acid in ethanol with heating [17]. Purification was performed by column

5

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chromatography on silica gel (230-400 mesh) using n-hexane and ethyl acetate

1

13

gradient as solvent. H NMR spectra were recorded on a Bruker 500 or 400 MHz,

NMR spectra were recorded at 100 MHz, and chemical shifts are reported relative to

internal Me Si (δ = 0). Melting points were determined by using an Electrothermal

C

4

IA9000 melting point apparatus, are reported in degrees Celsius and are uncorrected.

Optical rotations were recorded in a Perkin Elmer polarimeter 343. High resolution ESI

mass spectra were obtained from a Fourier transform ion cyclotron resonance (FT-ICR)

mass spectrometer, an RF-only hexapole ion guide, and an external electrospray ion

source. HRMS spectra were obtained on a mass spectrometer Micromass AutoSpec.

4

.1.1. Preparation of grindelic acid methyl ester (3).

Grindelic acid (1) (10 g, 31.2 mmol) was dissolved in 124 ml of dry Et

was cooled at 0 ºC. A solution of CH in Et O (1M) was added dropwise until the

formation of N disappear. Reaction was checked by TLC. A few drops of acetic acid

2

O (0,25 M) and

2

N

2

were added to quench the excess of diazomethane. Solvent was evaporated and the

crude material was purified by silica gel chromatography to obtain 9.85 g, as yellow oil

2

0

-1

of 3, (29.46 mmol, 94% yield). [α]

2

D

= -117.2 (c 8.1, MeOH); IR

ν

max, cm (KBr): 3030,

1

922, 1749 (C=O), 1672 (C=C), 991 (=C-H); H NMR (500MHz, CDCl

H-7), 3.67 (s, 3H, OCH ), 2.75 (d, J =14, 1H, H-14 proR), 2.64 (d, J =14, 1H, H-14

proS), 2.22 (m, 1H, H-12A), 2.07 (m, 2H, H-6A and H-11A), 1.87 (m, 3H, H-1 and H-

3

): δ 5.52 (s, 1H,

3

1

3

2B), 1.84 (m, 2H, H-6B and H-11B), 1.79 (s, 3H, H-17), 1.66 (m, 3H, H-3 and H-5),

.39 (m, 2H, H-2), 1.36 (s, 3H, H-16), 0.92 (s, 3H, H-19), 0.89 (s, 3H, H-18), 0.84 (s,

13

3

H, H-20); C NMR (100MHz, CDCl ): δ 171.9 (C-15), 134.9 (C-8), 126.5 (C-7), 90.6

(

C-9), 81.6 (C-13), 51.4 (OMe), 47.9 (C-14), 42.7 (C-5), 42.0 (C-3), 40.7 (C-10), 38.2

C-12), 33.2 (C-4), 32.9 (C-18), 32.8 (C-1), 28.5 (C-11), 27.3 (C-16), 24.2 (C-6), 22.4

+

C-19), 21.3 (C-17), 18.7 (C-2), 16.6 (C-20); HRMS-ES (m/z): calcd for C21

H

34

O

3

[M] :

334.2508, found 334.2560. All data are consistent with literature [13].

4.1.2. Preparation of grindelic alcohol (4).

4

A solution of 5 gr of 3 (14.94 mmol) and 2.83 gr (74.74 mmol) of LiAlH in dry THF (150

mL, 0.1 M) was refluxed during 2 h under Ar pressure. When reaction was completed

the mixture was cooled and ethyl acetate was added until complete elimination of

LiAlH residues. The crude was filtered and solids washed with 100 mL Et O. Water

4

2

(

100 mL) was added and extracted with Et

2

O (3 x 50mL). Organic layers were dried

4

with MgSO and resulting crude was purified by silica gel chromatography. 4.18 gr

2

0

(

13.64 mmol, 91% yield) of 4 was recovered as a white solid, mp 64-66 ºC. [α]

D

= -

1

3

31.7 (c 1.25, CHCl ); H NMR (400MHz, CDCl ): δ 5.51 (s, 1H), 3.97 (t, J = 11 Hz,

H), 3.69 (dd, J = 8.8 and 3 Hz, 1H), 2.10-1.95 (m, 4H), 1.91-1.72 (m, 3H), 1.82 (s,

H), 1.65-1.50 (m, 6H), 1.48-1.36 (m, 2H), 1.31 (s, 3H), 1.25 (m, 1H), 0.91 (s, 3H), 0.87

s, 3H), 0.82 (s, 3H); C NMR (100MHz, CDCl ): δ 134.5, 126.9, 91.1, 84.4, 60.0, 43.7,

3

1

3

(

4

2.9, 41.9, 40.7, 40.5, 33.2, 32.9, 32.5, 27.5, 26.6, 24.1, 22.2, 21.1, 18.7, 16.6; HRMS-

+

ES (m/z): calcd for C20

with literature [13].

34 2

H O [M] : 306.2559, found 306.2548. All data are consistent

4

.1.3. Preparation of (E)-ethyl 2-methyl-4-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)but-2-enoate

(

5).

6

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2

(

.32 g of 4 (7.58 mmol), 6.31 ml of Et

triphenyl-phosphoranylidene)propanoate (15.17 mmol) were stirred with 30 ml of dry

CH Cl (0,25 M) and 6.1 ml of DMSO (0.8 ml/mmol of alcohol). When phosphorane

was completely dissolved 3.62 g of SO .Py complex (22.75 mmol) was added.

3

N (4.6 g, 45.51 mmol) and 5.28 g of ethyl 2-

2

3

Reaction was monitored by TLC. When reaction was completed, 20 ml of HCl (5%)

was added. The layers were partitioned, and the aqueous phase was extracted with

Et

saturated solution (1 x 20 ml), dried with MgSO

was purified by silica gel column chromatography to give 5 (2.18 g, 5.6 mmol, 74%

2

O (3 x 20 ml). Organic layers were washed with water (3 x 20 ml) and aqueous NaCl

4

, filtered and concentrated. The crude

2

0

1

yield). [α]

D

3

= -36.1 (c 2.81, MeOH); H NMR (400MHz, CDCl ): δ 6.77 (t, J = 7 Hz,

1

3

4

1

H), 5.52 (s, 1H), 4.21 (q, J = 7 Hz, 2H), 2.61(dd, J = 14 and 8 Hz, 1H), 2.46 (dd, J =

4 and 8 Hz, 1H), 2.15-1.95 (m 4H), 1.90-1.65 (m, 6H), 1.86 (s, 3H), 1.78 (s, 3H), 1.60-

.35 (m, 4H), 1.31 (t, J = 7 Hz, 3H), 1.22 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.83 (s,

13

3

H); C NMR (100MHz, CDCl ): δ 168.2, 138.8, 135.0, 129.3, 126.5, 90.5, 83.4, 60.4,

3.0, 42.6, 42.1, 40.7, 38.2, 33.2, 32.9, 32.8, 28.4, 27.5, 24.2, 22.4, 21.3, 18.8, 16.7,

+

4.3, 12.6; HRMS-ES (m/z): calcd for C H O [M] : 388.2977, found 388.2992.

2

5

40

3

4

.1.4. Preparation of (E)-2-methyl-4-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)but-2-en-1-ol

(

6).

8

96 mg of 5 (2.39 mmol) was dissolved in 24 ml of dry Et O at 0 ºC. 5.26 ml of DIBAL-

2

H solution (0.1 M in cyclohexane, 5.26 mmol) was added dropwise. When reaction was

completed 5 ml of water was added and reaction was stirred for 15 minutes at room

4

temperature. Reaction mixture was dried with MgSO , filtered through celite and

purified by silica gel column chromatography to give 705 mg of 6 (2.12 mmol, 89%

2

0

1

yield). [α]

td, J = 7 and 2 Hz, 1H), 4.04 (s, 2H), 2.48 (dd, J = 14 and 8 Hz, 1H), 2.34 (dd, J = 14

and 8 Hz, 1H), 2.13-1.92 (m, 4H), 1.88-1.80 (m, 2H), 1.79 (s, 3H), 1.71-1.68 (m, 2H),

D

3

= -28.2 (c 1.06, MeOH); H NMR (400MHz, CDCl ): δ 5.51 (s, 1H), 5.45

(

1

.69 (s, 3H), 1.60-1.50 (m, 2H), 1.48-1.35 (m, 2H), 1.28-1.22 (m, 2H), 1.20 (s, 3H),

1

3

0.92 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H); C NMR (100MHz, CDCl ): δ 136.4, 135.3,

1

26.3, 122.9, 90.3, 83.9, 69.0, 42.6, 42.0, 41.9, 40.8, 38.0, 33.2, 32.9, 32.8, 28.4, 27.0,

+

24.2, 22.3, 21.2, 18.8, 16.7, 14.0; HRMS-ES (m/z): calcd for C H O [M] : 346.2872,

23

38

2

found 346.2872.

4

.1.5. Preparation of (E)-ethyl 4-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)but-2-enoate

(

7).

9

95 mg of 4 (3.25 mmol), 2.72 ml of Et N (1.97 g, 19.5 mmol) and 2.26 g of

3

ethyl(triphenyl-phosphoranylidene)acetate (6.5 mmol) were stirred with 13 ml of dry

CH Cl (0,25 M) and 2.6 ml of DMSO (0.8 ml/mmol of alcohol). When phosphorane

2

was completely dissolved, 1.55 g of SO

was monitored by TLC. When reaction was completed, 20 ml of HCl (5%) was added.

The layers were partitioned, and the aqueous phase was extracted with Et O (3 x 20

ml). Organic layers were washed with water (3 x 20 ml) and aqueous NaCl saturated

solution (1 x 20 ml), dried with MgSO , filtered and concentrated. Crude material was

purified by silica gel column chromatography yielding 1.14 g of 7 (3.05 mmol, 94%

3

.Py complex (9.74 mmol) was added. Reaction

2

4

20

1

yield). [α]

D

3

= -121.76 (c 1.71, MeOH); H NMR (500MHz, CDCl ): δ 6.95 (m, 1H), 5.87

(

d, J = 12 Hz, 1H), 5.52 (s, 1H), 4.21 (q, J = 7 Hz, 2H), 2.60 (m, 1H), 2.47 (m, 1H),

7

ACCEPTED MANUSCRIPT

2

3

1

3

.21-1.98 (m, 4H), 1.88-1.79 (m, 3H), 1.77 (s, 3H), 1.72-1.38 (m, 6H) 1.31 (t, J = 7 Hz,

H), 1.22 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H); C NMR (100MHz, CDCl ): δ

3

66.6, 146.1, 134.9, 126.6, 123.6, 90.6, 82.6, 60.2, 46.8, 42.6, 42.0, 40.7, 38.2, 33.2,

13

2.9, 32.8, 28.4, 27.5, 24.2, 22.4, 21.3, 18.8, 16.7, 14.3; HRMS-ES (m/z): calcd for

+

C H O [M] : 374.2821, found 374.2838.

2

4

38

3

4

.1.6. Preparation of (E)-2-methyl-4-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)but-2-en-1-ol

(

8).

4

26.2 mg of 7 (1.13 mmol) was dissolved in 12 ml of dry Et O at 0 ºC. 2.5 ml of DIBAL-

2

H (0.1 M in cyclohexane, 2.5 mmol) was added dropwise. When reaction was

completed 3 ml of water was added and reaction was stirred for 15 minutes at room

temperature. Reaction mixture was dried with MgSO , filtered through celite and

4

purified by silica gel column chromatography to yield 8 (308.8 mg, 0.93 mmol, 82%

2

D

0

1

yield). [α]

3

= -75.3 (c 6.22, MeOH); H NMR (500MHz, CDCl ): δ 5.66 (m, 2H), 5.49

(

bs, 1H), 4.09 (bs, 2H), 2.44 (m, 1H), 2.32 (m, 1H), 2.11-1.92 (m, 4H), 1.88-1.70 (m,

2

0

1

2

3

H), 1.76 (s, 3H), 1.70-1.60 (m, 4H), 1.58-1.45 (m, 2H), 1.44-1.20 (m, 2H), 1.18 (s, 3H),

1

3

.89 (s, 3H), 0.87 (s, 3H), 0.80 (s, 3H); C NMR (100MHz, CDCl ): δ 135.0, 131.6,

29.4, 126.2, 90.3, 83.1, 62.4, 46.6, 42.5, 41.9, 40.6, 37.9, 33.1, 32.8, 32.7, 28.3, 27.0,

+

4.1, 22.3, 21.2, 18.7, 16.6; HRMS-ES (m/z): calcd for C22

32.2718.

36 2

H O [M] : 332.2715, found

4

2

.1.7.

Preparation

of

((2S,3S)-3-(((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)methyl)oxiran-

-yl)methanol (9).

In a reaction flask containing powered activated 4 Å molecular sieves (30 mg), 130 mg

of 8 (0.39 mmol) was dissolved in 4 ml of dry CH Cl (0.1 M) and cooled at -20 ºC

under argon atmosphere. 138.5 ꢀl of Ti(OPr-i) (133.46 mg, 0.47 mmol) and 93.8 ꢀl of

R,R)-(+)-DET (112.98 mg, 0.55 mmol) were added sequentially. The mixture was

stirred at the same temperature for 20 min, and TBHP (0.14 ml, 5.5 M in isooctane,

.78 mmol) was added slowly. After the addition, the reaction was maintained with

2

4

(

0

stirring for 12 h at low temperature. Tartaric acid aqueous solution (15%w/v) was

added and the stirring was continued at room temperature for 1 h. The phases were

separated and the aqueous phase was extracted with CH

organic phase were washed with brine, concentrated, diluted with Et

2

Cl

2

(3 x 3 ml). The combined

O, and treated

2

with precooled, at 0 ºC, 15%(w/v) NaOH aqueous solution. The two-phase mixture was

stirred vigorously for 15 minutes at 0 ºC. The organic phase was separated, and the

2 2

aqueous phase was extracted with CH Cl (3 x 3 ml). The combined organic phase

were washed with brine, dried, filtered, concentrated, and purified by silica gel column

20

chromatography to yield 9 (96.8 mg, 0.28 mmol, 71% yield). [α]

= -97.3 (c 1.1,

): δ 5.51 (s, 1H), 3.92 (dd, J = 12 and 2 Hz, 1H), 3.66

dd, J = 12 and 4 Hz, 1H), 3.06 (m, 1H), 2.92 (m, 1H), 2.15-2.00 (m, 3H), 1.98-1.80 (m,

D

1

MeOH); H NMR (500MHz, CDCl

3

(

2

H), 1.88-1.77 (m, 2H) 1.78 (s, 3H), 1.71-1.65 (m, 2H), 1.62-1.51 (m, 4H), 1.48-1.35

13

(

m, 1H), 1.30 (s, 3H), 1.28-1.18 (m, 1H) 0.90 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H);

C

3

NMR (100MHz, CDCl ): δ 135.0, 126.5, 90.6, 82.3, 61.7, 58.4, 53.4, 45.4, 42.6, 42.0,

4

0.7, 38.5, 33.2, 32.9, 32.8, 28.3, 28.0, 24.2, 22.4, 21.2, 18.8, 16.7; HRMS-ES (m/z):

+

calcd for C H O [M] : 348.2664, found 348.2672.

22

36

3

8

ACCEPTED MANUSCRIPT

4

2

.1.8.

Preparation

of

((2R,3R)-3-(((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)methyl)oxiran-

-yl)methanol (10).

The procedure used above to obtain 9 was applied again to 8 (64 mg, 0.19 mmol),

using the (S,S)-(-)-DET instead of (R,R)-(+)-DET, to yield 10 (57.0 mg, 0.16 mmol,

2

0

1

8

3

1

5%.yield). [α]

D

3

= -137.6 (c 2.82, MeOH); H NMR (500MHz, CDCl ): δ 5.49 (s, 1H),

.89 (dd, J = 12 and 2 Hz, 1H), 3.63 (dd, J = 12 and 4 Hz, 1H), 3.10 (m, 1H), 2.90 (m,

H), 2.19-1.95 (m, 5H), 1.86-1.73 (m, 2H), 1.74 (s, 3H), 1.72-1.60 (m, 2H), 1.58-1.49

(

0

m, 2H), 1.48-1.35 (m, 2H), 1.32 (s, 3H), 1.28-1.17 (m, 2H) 0.91 (s, 3H), 0.88 (s, 3H),

13

.82 (s, 3H); C NMR (100MHz, CDCl

3

): δ 135.1, 126.3, 90.4, 82.2, 61.8, 58.1, 53.8,

4

5.8, 42.8, 42.0, 40.7, 39.2, 33.2, 33.0, 32.7, 28.4, 27.4, 24.2, 22.4, 21.3, 18.8, 16.7;

+

HRMS-ES (m/z): calcd for C H O [M] : 348.2664, found 348.2672.

22

36

3

4.1.9. General procedure for Ugi reaction. Preparation of 2-(N-benzyl-2-

(

(1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-

spiro[furan-2,1'-naphthalene]-5-yl)acetamido)-N-tert-butyl-2-methylpropanamide (11).

A solution of acetone (0.030 mL, 0.41 mmol), and benzylamine (0.038 mL, 0.35 mmol)

in methanol (0.7 mL, 0.5 M) were stirred for 3 hours. After that, grindelic acid (107.1

mg, 0.35 mmol) and tert-butyl-isocyanide (0.044 mL, 0.38 mmol) were added. Reaction

mixture was stirred for 24 h. The solvent was removed under reduced pressure and the

crude reaction mixture was purified by flash chromatography on silica gel to afford the

20

D

desired product 11 (111 mg, 59% yield) as a white solid: mp 72-74 ºC. [α]

= -137.6

): δ 7.37 (m, 2H, H-3’ and H-5’), 7.29 (m, 3H,

H-2’, H-4’ and H-6’), 5.59 (s, 1H, N-H), 5.46 (s, 1H, H-7), 5.07 (d, J = 14.4 Hz, 1H, H-

1

(

c 2.82, MeOH); H NMR (500MHz, CDCl

3

7’A), 4.45 (d, J = 14.4 Hz, 1H, H-7’B), 2.89 (d, J = 11.6 Hz, 1H, H-14A), 2.56 (d, J =

11.6 Hz, 1H, H-14B), 2.07 (m, 1H, H-11A), 2.04 (m, 3H, H-6A and H-12), 1.86 (m, 1H,

H-6B), 1.73 (m, 1H, H-11B), 1.62 (m, 1H, H-5), 1.56 (s, 3H, H-17), 1.55 (m, 2H, H-1),

.52 (m, 2H, H-2), 1.40 (s, 3H, H-16), 1.38 (s, 6H, H-3’’ and H-4’’), 1.36 (s, 9H, H-2’’’,

H-3’’’ and H-4’’’), 1.18 (m, 1H, H-3A), 1.13 (m, 1H, H-3B), 0.92 (s, 3H, H-19), 0.88 (s,

1

3

1

2

3

H, H-18), 0.81 (s, 3H, H-20); C NMR (100MHz, CDCl ): δ 174.0 (C-1’’), 171.9 (C-

5), 139.0 (C-1’), 135.0 (C-8), 128.8 (2C, C-3 and C-5’), 127.1 (C-7), 126.4 (C-4’),

25.7 (2C, C-2 and C-6’), 90.4 (C-9), 83.1 (C-13), 63.1 (C-2’’), 50.7 (C-1’’’), 48.0 (C-

4), 47.6 (C-7’), 43.0 (C-5), 42.0 (C-3), 40.6 (C-10), 39.7 (C-12), 33.2 (C-4), 33.1 (C-

8), 32.6 (C-1), 28.6 (3C, C-2’’’, C-3’’’ and C-4’’’), 28.3 (C-11), 27.7 (C-16), 24.4 (C-6),

4.3 (2C, C-3’’ and C-4’’), 22.3 (C-19), 21.2 (C-17), 18.7 (C-2), 16.6 (C-20); HRMS-ES

+

(

m/z): calcd for C35

H

54

N O

2 3

[M] : 550.3734, found 550.3722.

4.1.10.

Preparation

of

2-(N-benzyl-2-((1'R,5S,8α'S)-2',5,5',5',8α'-pentamethyl-

4,4α',5,5',6',7',8',8α-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)acetamido)-2-

methyl-N-(2,3,3-trimethylbutan-2-yl)propanamide (12).

This compound was prepared by General procedure for Ugi reaction using

benzylamine and 1,1,3,3-tetramethylbutylisocyanide to obtain 444 mg of 12 (75% yield)

2

D

0

1

as a clear oil color less oil. [α]

3 3

= -66.73 (c 1.2, CHCl ); H NMR (500MHz, CDCl ): δ

7

=

1

6

.38 (m, 2H), 7.30 (m, 3H), 5.63 (s, 1H), 5.45 (s, 1H), 5.00 (d, J = 18 Hz, 1H), 4.48 (d, J

18 Hz, 1H), 2.89 (d, J = 15 Hz, 1H), 2.57 (d, J = 15 Hz, 1H), 2.15-2.00 (m, 5H), 1.91-

.82 (m, 1H), 1.77-1.65, (m, 2H), 1.61 (s, 3H), 1.57 (m, 1H), 1.54-1.49 (m, 2H), 1.45 (s,

H), 1.40 (s, 3H), 1.38 (s, 3H), 1.37(s, 3H), 1.30-1.25 (m, 1H), 1.22-1.14 (m, 1H) 1.01

1

3

(

s, 9H), 0.91 (s, 3H), 0.88 (s, 3H), 0.81 (s, 3H); C NMR (100MHz, CDCl

3

): δ 173.4,

9

ACCEPTED MANUSCRIPT

1

4

2

6

71.9, 138.9, 135.1, 128.8 (2C), 127.1, 126.4, 125.8 (2C), 90.3, 83.2, 63.1, 54.9, 53.0,

8.0, 47.6, 43.0, 42.0, 40.6, 39.5, 33.2, 33.1, 32.6, 31.6 (3C), 31.5, 28.5, 28.4 (2C),

+

7.7, 24.4, 24.2, 22.3, 21.2, 18.7, 16.6; HRMS-ES (m/z): calcd for C38

06.4760, found 606.4756.

H

60

N

2

O

3

[M] :

4.1.11.

Preparation

of

2-(N-benzyl-2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)acetamido)-N-

cyclohexyl-2-methylpropanamide (13).

This compound was prepared by General procedure for Ugi reaction using

benzylamine and cyclohexyl isocyanide to obtain 378 mg of 13 (71% yield) as a white

20

1

solid: mp 81-83 ºC. [α]

D

3 3

= -91.08 (c 1.0, CHCl ); H NMR (500MHz, CDCl ): δ 7.36

(

m, 4H), 7.26 (m, 1H), 5.55 (d, J = 8 Hz,, 1H), 5.48 (s, 1H), 5.10 (d, J = 18 Hz, 1H),

4

2

1

.43 (d, J = 18 Hz, 1H), 3.76 (m, 1H), 2.84 (d, J = 14 Hz, 1H), 2.51 (d, J = 14 Hz, 1H),

.09-1.81 (m, 7H), 1.75-1.60 (m, 10H) 1.54 (bs, 3H), 1.45-1.30 (m, 3H), 1.40 (s, 6H),

13

.37 (s, 3H), 1.28-1.05 (m, 5H) 0.91 (s, 3H), 0.88 (s, 3H), 0.80 (s, 3H); C NMR

): δ 174.0, 172.0, 139.1, 135.0, 128.7 (2C), 127.0, 126.4, 125.7 (2C),

(

100MHz, CDCl

3

90.4, 83.0, 62.5, 48.2, 47.8, 47.4, 43.1, 42.1, 40.6, 39.7, 33.2 (2C), 33.0, 32.6, 28.2,

2

7.8, 25.7, 24.9 (2C), 24.5 (2C), 24.3, 24.2, 22.3, 21.2, 18.7, 16.6; HRMS-ES (m/z):

+

calcd for C37

H

56

N

2

O

3

[M] : 576.4291, found 576.4288.

4.1.12.

Preparation

of

2-(N-benzyl-2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)acetamido)-N-

(

2,6-dimethylphenyl)-2-methylpropanamide (14).

This compound was prepared by General procedure for Ugi reaction using

benzylamine and 2,6-dimethylphenyl isocyanide to obtain 350 mg of 14 (63% yield) as

2

0

1

a white solid: mp 181-183 ºC. [α]_D= -74.63 (c 1.023, CHCl ); H NMR (500MHz,

3

CDCl

3

): δ 7.29 (m, 5H), 7.00 (m, 3H), 5.41 (bs, 1H), 5.37 (s, 1H), 5.07 (d, J = 19 Hz,

H), 4.54 (d, J = 19 Hz, 1H), 2.89 (d, J = 15 Hz, 1H), 2.78 (d, J = 15 Hz, 1H ), 2.56 (d, J

15 Hz, 1H), 2.45 (d, J = 15 Hz, 1H), 2.28 (bs, 6H), 2.15-1.90 (m, 4H), 1.59 (s, 3H),

1

=

1

3

.54 (s, 3H), 1.49-1.18 (10H), 1.37 (s, 3H), 0.82 (s, 3H), 0.81 (s, 3H), 0.78 (s, 3H);

): δ 175.1, 172.1, 138.8, 134.9, 128.7 (2C), 128.5 (2C), 127.8

2C), 127.2, 127.0, 126.6, 125.7 (2C), 82.9, 62.5, 47.6, 47.5, 43.8, 43.2, 42.1, 40.5,

C

NMR (100MHz, CDCl

3

(

3

9.7, 39.6, 33.2 (2C), 32.6, 28.2, 28.1, 27.7, 24.5, 24.3 (2C), 22.2, 21.3, 19.5, 18.7,

+

1

6.6; HRMS-ES (m/z): calcd for C39

.1.13. Preparation of 2-(N-benzyl-2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)acetamido)-N-

54 2 3

H N O [M] : 598.4134, found 598.4149.

4

butyl-2-methylpropanamide (15).

This compound was prepared by General procedure for Ugi reaction using

benzylamine and butyl isocyanide to obtain 438 mg of 15 (79% yield) as a white solid:

20

1

mp 70-72 ºC. [α]_D= -78.53 (c 1.34, CHCl ); H NMR (500MHz, CDCl ): δ 7.35 (m,

3

5

(

5

(

H), 5.77 (bs, 1H), 5.51 (s, 1H), 5.16 (d, J = 19 Hz, 1H), 4.41 (d, J = 19 Hz, 1H), 3.32

m, 1H), 3.23 (m, 1H), 2.83 (d, J = 14 Hz, 1H), 2.49 (d, J = 14 Hz, 1H), 2.23-2.00 (m,

H), 2.00-1.97 (m, 2H), 1.92-1.80 (m, 4H), 1.80-1.60 (m, 2H), 1.60-1.30 (m 4H), 1.56

bs, 3H), 1.42 (bs, 3H), 1.41 (bs, 3H), 1.36 (s, 3H), 0.93 (t, J = 6 Hz 3H), 0.92 (s, 3H),

13

0

3

.89 (s, 3H), 0.81 (s, 3H); C NMR (100MHz, CDCl ): δ 175.1, 172.1, 138.9, 134.9,

128.7 (2C), 127.0, 126.6, 125.7 (2C), 90.5, 82.9, 62.4, 47.7, 47.6, 43.3, 42.1, 40.6,

10

ACCEPTED MANUSCRIPT

3

9.8, 39.7, 33.2 (2C), 32.5, 31.8, 28.1, 27.7, 24.5, 24.4, 24.3, 22.2, 21.3, 20.2, 18.7,

+

1

6.6, 13.8; HRMS-ES (m/z): calcd for C35

.1.14. Preparation of

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)acetamido)-2-

H

54

N

2

O

3

[M] : 550.4134, found 550.4132.

4

2-(N-benzyl-2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

methyl-N-(2-morpholinoethyl)propanamide (16).

This compound was prepared by General procedure for Ugi reaction using

benzylamine and 2-morpholinoethyl isocyanide to obtain 106.8 mg of 16 (79% yield) as

20

1

a white solid: mp 164-166 ºC. [α]

D

3

= -87.98 (c 1.32, CHCl ); H NMR (500MHz,

CDCl ): δ 7.27 (m, 4H), 7.18 (m, 1H), 6.20 (bs, 1H), 5.41 (s, 1H), 5.04 (d, J = 19 Hz,

3

1

2

3

0

1

H), 4.29 (d, J = 19 Hz, 1H), 3.65 (m, 4H), 3.30 (m, 2H), 2.73 (d, J = 14 Hz, 1H), 2.50-

.41 (m, 6H), 2.38 (d, J = 14 Hz, 1H), 2.10-1.90 (m, 4H), 1.85-1.50 (m, 4H), 1.47 (bs,

H), 1.45-1.09 (m, 4H), 1.35 (bs, 3H), 1.28 (bs, 3H), 1.25 (s, 3H), 1.22-1.03 (m, 2H)

1

3

.83 (s, 3H), 0.80 (s, 3H), 0.73 (s, 3H); C NMR (100MHz, CDCl ): δ 175.4, 172.1.

39.1, 134.9, 128.7 (2C), 126.9, 126.6, 125.7 (2C), 90.4, 82.9, 67.1, 62.3, 56.9, 53.3

(

2

3C), 47.5, 47.2, 43.1, 42.2, 41.9, 40.5, 39.7, 36.1, 33.2 (2C), 28.1, 27.8, 24.8, 24.3,

+

3.4, 22.2, 21.3, 18.7, 16.6; HRMS-ES (m/z): calcd for C H N O [M+H] : 608.4427,

37 58 3 4

found 608.4443.

4

.1.15. Preparation of N-benzyl-2-methyl-2-(2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)-N-

phenylacetamido)propanamide (17).

This compound was prepared by General procedure for Ugi reaction using

benzylamine and benzylisocyanide to obtain 528 mg of 17 (97% yield) as a white solid:

2

D

0

1

mp 76-78 ºC. [α]

3 3

= -72.91 (c 1.18, CHCl ); H NMR (500MHz, CDCl ): δ 7.25 (m,

1

2

0H), 6.94 (m, 1H), 5.41 (s, 1H), 5.04 (d, J = 19 Hz, 1H), 4.50 (dd, J = 19 and 6 Hz,

H), 4.34 (m, 2H), 2.76 (d, J = 14 Hz, 1H), 2.41 (d, J = 19 Hz, 1H), 2.15-1.59 (m, 6H),

.58-1.10 (m, 6H), 1.47 (bs, 3H), 1.37 (bs, 3H), 1.36 (bs, 3H), 1.22 (s, 3H), 1.00 (m,

1

3

H), 0.81 (s, 3H), 0.75 (s, 3H), 0.73 (s, 3H); C NMR (100MHz, CDCl ): δ 175.1,

3

72.1, 138.8 (2C), 134.9, 128.7 (2C), 128.5 (2C), 127.8 (2C), 127.2, 127.0, 126.6,

25.7 (2C), 90.4, 82.9, 62.5, 47.6, 47.5, 43.8, 43.2, 42.1, 40.5, 39.7, 33.2 (2C), 32.6,

8.2, 27.7, 24.5, 24.3 (2C), 22.2, 21.3, 18.7, 16.6; HRMS-ES (m/z): calcd for

+

C H N O [M] : 584.3978, found 584.3956.

3

8

52

2

3

4.1.16. Preparation of N-tert-butyl-2-methyl-2-(2-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-

pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)-

N-phenylacetamido)propanamide (18).

This compound was prepared by General procedure for Ugi reaction using aniline and

tert-butyl-isocyanide to obtain 389.4 mg of 18 (78% yield) as a white solid: mp 74-76

2

0

1

ºC. [α]

m, 2H), 5.69 (s, 1H), 5.38 (s, 1H), 2.45 (d, J = 15 Hz, 1H), 2.24 (d, J = 15 Hz, 1H),

.15 (m, 1H), 1.99 (m, 3H), 1.82-1.54 (m, 4H), 1.53 (bs, 3H), 1.51-1.42 (m, 3H), 1.38

s, 9H), 1.33 (s, 3H), 1.32 (s, 3H), 1.22 (s, 3H), 1.20-1.08 (m, 2H), 0.87 (s, 3H), 0.81 (s,

D

3 3

= -65.36 (c 11.37, CHCl ); H NMR (500MHz, CDCl ): δ 7.36 (m, 3H), 7.21

(

2

(

3

1

3

1

3

H), 0.77 (s, 3H); C NMR (100MHz, CDCl ): δ 174.0, 170.8, 139.9, 135.2, 130.6,

30.2, 129.4 (2C), 128.4, 126.0, 123.9, 90.0, 83.5, 63.0, 50.8, 48.1, 42.8, 42.0, 40.7,

8.8, 33.0, 32.9, 28.7, 28.6 (3C), 27.6, 25.9, 24.8, 24.1, 22.4, 21.2, 18.7, 16.7; HRMS-

ES (m/z): calcd for C34

+

H

52

N

2

O

3

[M] : 536.3978, found 536.3961.

11

ACCEPTED MANUSCRIPT

4.1.17.

Preparation

of

N-(2,6-dimethylphenyl)-2-methyl-2-(2-((1'R,5S,8a'S)-

2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-

naphthalene]-5-yl)-N-phenylacetamido)propanamide (19).

This compound was prepared by General procedure for Ugi reaction using aniline and

2

9

7

6

3

1

2

,6-dimethylphenyl isocyanide to obtain 140 mg of 19 (26% yield) as a white solid: mp

2

0

1

0-92 ºC. [α]

D

3 3

= -62.58 (c 10.47, CHCl ); H NMR (500MHz, CDCl ): δ 7.41 (m, 3H),

.30 (m, 3H), 7.20 (s, 1H), 7.10 (m, 2H), 5.40 (s, 1H), 2.49 (d, J = 18 Hz, 1H), 2.32 (bs,

H), 2.30-2.15 (m, 4H), 2.12-1.89 (m, 4H), 1.80-1.58 (m, 6H) 1.53 (bs, 6H), 1.48 (bs,

13

H), 1.32 (s, 3H), 0.86 (s, 3H), 0.79 (s, 3H), 0.76 (s, 3H); C NMR (100MHz, CDCl ): δ

3

72.9, 171.0, 139.9, 139.7, 135.3, 130.7, 130.4, 129.5 (2C), 128.6, 128.1 (2C), 126.9,

26.0, 124.3, 118.0, 90.0, 83.5, 63.0, 47.9, 42.8, 41.9, 40.7, 38.7, 33.2, 33.0, 32.9,

8.7, 27.6, 26.1, 25.5, 24.1, 22.4, 21.3, 18.7, 18.5 (2C), 16.7; HRMS-ES (m/z): calcd

+

for C38

H

52

N

2

O

3

[M] : 584.3978, found 584.3971.

4

.1.18 Preparation of N-butyl-2-methyl-2-(2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)-N-

phenylacetamido)propanamide (20).

This compound was prepared by General procedure for Ugi reaction using aniline and

butyl isocyanide to obtain 115 mg of 20 (23% yield) as a white solid: mp 187-189 ºC.

20

1

[

α]

D

= -52.86 (c 1.17, CHCl

3 3

); H NMR (500MHz, CDCl ): δ 7.38 (m, 3H), 7.27 (m,

2H), 5.87 (t, J = 6 Hz, 1H), 5.41 (s, 1H), 3.32 (m, 2H), 2.47 (d, J = 15 Hz, 1H), 2.26 (d,

J = 15 Hz, 1H), 2.15 (m, 1H), 2.05-1.90 (m, 3H), 1.85-1.72 (m, 1H), 1.70-1.60 (m, 4H)

1

.60-1.45 (m, 4H), 1.52 (bs, 3H), 1.45-1.25 (m, 3H), 1.35 (s, 3H), 1.32 (s, 3H), 1.31 (s,

1

3

H), 1.15 (m, 1H) 0.95 (t, J = 7 Hz, 3H), 0.89 (s, 3H), 0.84 (s, 3H), 0.79 (s, 3H);

): δ 174.9, 171.0, 140.0, 135.2, 130.7, 130.3, 129.4 (2C), 128.4,

26.0, 90.1, 83.5, 62.5, 47.9, 42.9, 42.0, 40.7, 39.7, 38.9, 33.1, 33.0, 32.9, 31.7, 28.6,

7.6, 25.9, 25.1, 24.1, 22.4, 21.3, 20.2, 18.7, 16.7, 13.8; HRMS-ES (m/z): calcd for

C

NMR (100MHz, CDCl

1

2

3

+

C H N O [M] : 536.3978, found 536.3962.

3

4

52

2

3

4

.1.19. Preparation of N-benzyl-2-methyl-2-(2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)-N-

phenylacetamido)propanamide (21).

This compound was prepared by General procedure for Ugi reaction using aniline and

benzylisocyanide to obtain 229 mg of 21 (43% yield) as a white solid: mp 194-196 ºC.

20

1

[

α]

D

3 3

= -57.56 (c 1.16, CHCl ); H NMR (500MHz, CDCl ): δ 7.37 (m, 7H), 7.27 (m,

3

1

2

1

H), 6.14 (t, J = 6 Hz, 1H), 5.41 (s, 1H), 4.60 (dd, J = 15 and 6 Hz, 1H), 4.49 (dd, J =

5 and 6 Hz, 1H), 2.47 (d, J = 15 Hz, 1H), 2.26 (d, J = 15 Hz, 1H), 2.20-2.08 (m, 1H),

.07-1.81 (m, 3H), 1.75-1.55 (m, 5H) 1.53 (bs, 3H), 1.51-1.42 (m, 4H) 1.37 (s, 6H),

13

.30 (s, 3H), 1.15 (m, 1H), 0.89 (s, 3H), 0.82 (s, 3H), 0.79 (s, 3H); C NMR (100MHz,

CDCl ): δ 174.9, 171.1, 139.8, 138.7, 135.2, 130.7, 130.4, 129.5 (2C), 128.6 (2C),

3

128.5, 127.8 (2C), 127.3, 126.0, 90.1, 83.5, 62.6, 47.9, 43.9, 42.9, 42.0, 40.6, 38.8,

3

3.2, 33.0, 32.9, 28.7, 27.6, 25.7, 25.2, 24.1, 22.5, 21.3, 18.7, 16.7; HRMS-ES (m/z):

+

calcd for C37

H

51

N

2

O

3

[M+H] : 571.3900, found 571.3872.

4.1.20. Preparation

of

N-cyclohexyl-2-methyl-2-(2-((1'R,5S,8a'S)-2',5,5',5',8a'-

pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)-

N-phenylacetamido)propanamide (22).

12

ACCEPTED MANUSCRIPT

This compound was prepared by General procedure for Ugi reaction using aniline and

cyclohexyl isocyanide to obtain 481 mg of 22 (92% yield) as a white solid: mp 222-224

2

0

1

ºC. [α]

H), 5.68 (d, J = 8 Hz, 1H), 5.40 (s, 1H), 3.79 (m, 1H), 2.45 (d, J = 15 Hz, 1H), 2.25 (d,

J = 15 Hz, 1H), 2.12-1.98 (m, 1H), 1.95-1.78 (m, 5H), 1.75-1.50 (m, 9H), 1.53 (bs, 3H),

D

3 3

= -60.91 (c 1.33, CHCl ); H NMR (500MHz, CDCl ): δ 7.38 (m, 3H), 7.23 (m,

2

1

3

1

4

2

5

.42-1.30 (m, 3H), 1.33 (s, 3H), 1.31 (s, 3H), 1.28 (s, 3H), 1.27-1.02 (m, 5H), 0.87 (s,

H), 0.82 (s, 3H), 0.77 (s, 3H); C NMR (100MHz, CDCl ): δ 173.9, 170.9, 140.0,

3

35.2, 130.6, 130.3, 129.4 (2C), 128.4, 125.9, 90.0, 83.5, 62.6, 48.4, 48.0, 42.8, 42.0,

13

0.6, 38.7, 33.2, 33.0 (2C), 32.9, 29.7, 28.7, 27.6, 25.8, 25.7, 25.1, 24.9 (2C), 24.1,

+

2.47, 21.2, 18.7, 16.7; HRMS-ES (m/z): calcd for C36

62.4127.

H

54

N

2

O

3

[M] : 562.4134; found

4.1.21. Preparation of (1'R,4a'S,5S,8a'S)-5-(2-azidoethyl)-2',5,5',5',8a'-pentamethyl-

4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene] (23).

2 2

Alcohol 4 (2 g, 6.53 mmol) was dissolved in dry CH Cl (65 mL) and the solution was

cooled to 0 ºC. Triethylamine (1.8 mL, 13.06 mmol) was added via syringe followed by

methanesulfonyl chloride (758 ꢀL, 9.78 mmol) in one portion. After 4.5 h, the reaction

2 2 2

mixture was diluted with CH Cl (20 mL), washed with H O (30 mL) and brine (30 mL),

dried with MgSO , filtered, and evaporated under reduced pressure to leave a yellow

4

residue. The crude residue was purified by flash-chromatography column on silica gel,

using n-hexane/ethyl acetate as the eluent. The mesylate obtained (2.26 g, 5.8 mmol,

9

0% yield) was dissolved in dry DMF (37 mL, 0.16 M). Sodium azide (764 mg, 11.7

mmol) was added and the mixture was stirred at 40 ºC. After 24 h, the reaction mixture

was diluted with Et O (40 mL), washed with H O (3 x 20 mL) and brine (30 mL), dried

with MgSO , filtered, and concentrated under reduced pressure. The crude oil was

purified by column chromatography on silica gel, using n-hexane/ethyl acetate as the

2

4

2

0

eluent, to afford 23 as an oil (1.55 g, 80% yield). [α] = -140.8 (c 1.15, CHCl ); IR ν_m_a_x

,

D

3

-1

cm (KBr): 2968, 2924, 2870, 2098 (azide), 1460, 1377, 1315, 1267, 1003, 874, 677.;

H NMR (500MHz, CDCl ): δ 5.50 (s, 1H, H-7), 3.42 (m, 1H, H-15A), 3.32 (m, 1H, H-

3

1

15B), 2.07 (m. 1H, H-11A), 2.05 (m, 1H, H-6A), 1.98 (m, 1H, H-12A), 1.97 (m, 1H, H-

14A), 1.86 (m, 1H, H-14B), 1.83 (m, 1H, H-6B), 1.81 (m, 1H, H-11B), 1.77 (m, 1H, H-

12B), 1.76 (s, 3H, H-17), 1.69 (m, 1H, H-5), 1.65 (m, 1H, H-1A), 1.57 (m, 2H, H-2), 1.41

(

m, 1H, H-1B), 1.39 (m, 1H, H-3A), 1.23 (s, 3H, H-16), 1.22 (m, 1H, H-3B), 0.91 (s, 3H,

1

3

H-19), 0.89 (s, 3H, H-18), 0.82 (s, 3H, H-20); C NMR (100MHz, CDCl ): δ 135.0 (C-

3

8

3

1

), 126.4 (C-7), 90.4 (C-9), 81.5 (C-13), 48.2 (C-15), 42.6 (C-5), 42.1 (C-14), 42.0 (C-

), 40.7 (C-10), 39.5 (C-12), 33.2 (C-4), 33.0 (C-18), 32.8 (C-1), 28.2 (C-11), 26.9 (C-

6), 24.2 (C-6), 22.3 (C-19), 21.2 (C-17), 18.8 (C-2), 16.8 (C-20); HRMS-ES (m/z):

+

calcd for C20

H

34

N

3

O [M+H] : 332.2702, found 332.2709.

4.1.22. Preparation of 3-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-

octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)propanal (24).

To an oxalyl chloride solution (60 ꢀL, 0.622 mmol) in CH Cl (8 mL) at -78 ºC was

2

added dropwise DMSO (90 ꢀL, 1.24 mmol), and the resulting mixture was stirred for 15

min. A solution of 4 (150.5 mg, 0.49 mmol) in CH Cl (2 mL) was added dropwise at -

8 ºC. The mixture was stirred for 30 min. Triethylamine (0.7 ml, 4.9 mmol) was added

dropwise and the resultant mixture was gradually warmed to room temperature and

stirred for 7h. The mixture was quenched with water (10 mL) and CH Cl (10 mL). The

organic layer was washed with brine, dried over MgSO , ﬁltered and concentrated in

2

7

2

4

13

ACCEPTED MANUSCRIPT

vacuum. Puriﬁcation of the residue by silica gel column chromatography (n-

25

hexane/ethyl acetate, 85:15) gave the aldehyde 24 (148 mg, 99%). [α]

D

= -72.14 (c

): δ 9.82 (s, 1H), 5.52 (s, 1H), 2.80 (dd, J = 13

and 2 Hz, 1H), 2.60 (dd, J = 13 and 2 Hz, 1H), 2.08 (m, 4H), 1.87 (m, 3H), 1.76 (bs,

1

3 3

0.14, CHCl ); H NMR (500MHz, CDCl

3

H), 1.57 (m, 5H), 1.33 (s, 3H), 1.22 (m,1H), 0.91 (s, 3H), 0.88 (s, 3H), 0.83 (s, 3H);

1

3

C NMR (100MHz, CDCl ): δ 202.8, 134.6, 126.8, 80.9, 57.1, 42.7, 42.0, 40.7, 39.4,

3

3.2, 33.0, 32.8, 28.3, 28.1, 24.2, 22.3, 21.3, 18.8, 18.7, 16.7; HRMS-ES (m/z): calcd

+

for C20

H

32

O

2

[M] : 304.24022, found 304.2412.

.1.23. Preparation of (1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-5-(prop-2-ynyl)-

,4a',5,5',6',7', 8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene] (26).

Cl (5 mL, 0.1 M) at 0 ºC

under argon atmosphere. Triphenylphosphine (514 mg, 1.96 mmol), triethylamine (0.3

mL, 1.96 mmol) and CBr (325 mg, 0.98 mmol) were added. The mixture was stirred for

5 min. and the work-up was carried out by addition of NaHCO saturated solution. The

product was extracted with Et O (3 x 25 mL), washed with brine, dried over MgSO

4

Aldehyde 24 (148 mg, 0.48 mmol) was dissolved in dry CH

2

4

1

3

2

4

,

ﬁltered and concentrated in vacuum. The dibromoolefin 25 obtained was used without

purification in the next reaction. The crude material was dissolved in THF (5 mL, 0.1 M)

and cooled at -78 ºC under argon with stirring. A solution of n-BuLi (0.36 mL, 2.7 M in

heptane) was added using triphenylmethane as indicator. After 30 min. a NH

4

Cl

saturated solution was added and the product was extracted with Et O (3 x 25 mL).

2

4

The organic layer was washed with brine, dried over MgSO , ﬁltered and concentrated

in vacuum. The residue was purified by column chromatography on silica gel, using a

mixture of n-hexane-ethyl acetate as eluent, to afford 26 as a clear oil (78 mg, 53%

25

1

total yield). [α]_D= -77.5 (c 0.48, CHCl ); H NMR (500 MHz, CDCl ): δ 5.53 (s, 1H),

3

2

1

.57 (dd, J = 8 and 2 Hz, 1H), 2.71 (t, J = 8 and 2 Hz, 1H), 2.19 (m, 3H), 2.00 (s, 1H),

.91-1.78 (m, 2H), 1.79 (bs, 3H), 1.75-1.62 (m, 1H), 1.58-1.45 (m, 2H), 1.41 (s, 3H),

13

.32-1.23 (m, 2H), 0.92 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.84 (s, 3H); C NMR (100

): δ 134.9, 126.6, 91.2, 82.9, 82.5, 69.3, 42.6, 42.0, 40.9, 38.1, 33.4, 33.2,

32

2.9, 32.8, 28.3, 26.8, 24.2, 22.4, 21.3, 18.8, 16.7; HRMS-ES (m/z): calcd for C21H O

MHz, CDCl

3

+

[

M] : 300.2453, found 300.2453.

4.1.24. General procedure for Huisgen reaction. Preparation of (4-decyl-1-(2-

(

(1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-

spiro[furan-2,1'-naphthalene]-5-yl)ethyl)-1H-1,2,3-triazole (27).

The azide 23 (53.7 mg, 0.16 mmol) and 1-dodecyne (35µL, 0.16 mmol) were

suspended in a 1:1 mixture of water and ethanol (0.1 M). Sodium ascorbate (9.6 mg,

0

.05 mmol) and copper(II) sulfate pentahydrate (4 mg, 0.02 mmol) were added. The

resulting mixture was stirred at room temperature for 24 h. Then, the reaction was

diluted with water, extracted with ethyl acetate (10 mL×3), dried over MgSO , filtered,

4

and concentrated in vacuum. The residue was purified by column chromatography on

silica gel using a mixture of n-hexane-ethyl acetate as eluent, to obtain the pure

2

5

1

product 27 (38.6 mg, yield 48%) as a clear oil. [α]

500MHz, CDCl ): δ 7.32 (s, 1H), 5.51 (s, 1H), 4.46 (t, J = 8 Hz, 2H), 2.71 (t, J = 8 Hz,

H), 2.19 (m, 3H), 2.15-1.78 (m, 5H), 1.74 (bs, 3H), 1.71-1.49 (m, 5H), 1.45-1.08 (m,

D

3

= -64.35 (c 0.20, CHCl ); H NMR

(

2

1

3

1

3

8H) 1.28 (s, 3H), 0.92 (t, J = 7 Hz, 3H), 0.91 (s, 3H), 0.90 (s, 3H), 0.82 (s, 3H);

): δ 148.3, 134.9, 126.6, 120.6, 90.6. 81.3, 68.0, 47.0, 43.7, 42.7,

2.1, 40.7, 39.7, 33.2, 33.0, 32.8, 31.9, 29.6, 29.5 (2C), 29.3, 28.1, 26.9, 25.7, 24.3,

C

NMR (100MHz, CDCl

3

4

14

ACCEPTED MANUSCRIPT

+

2

2.7, 22.2, 21.3, 18.8, 18.4, 16.7, 14.1; HRMS-ES (m/z): calcd for C32

55 3

H N O [M] :

497.4345, found 497.4329.

4.1.25.

Preparation

of

4-(3-ethynylphenyl)-1-(2-((1'R,5S,8a'S)-2',5,5',5',8a'-

pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-

yl)ethyl)-1H-1,2,3-triazole (28).

This compound was prepared by General procedure for Huisgen reaction using 50 mg

of azide 23 (0.15 mmol) and 9.5 mg of 1,3-diethynilbenzene (0.075 mmol) to obtain 54

2

0

mg of 28 (78% yield) as a white solid: mp 129-131 ºC. [α]

D

= -175.6 (c 1.57, CHCl

3

);

-1

IR

νmax, cm (KBr): 3311 (Csp-H), 3130 (=C-H), 2966, 2916, 2866, 2114 (Csp- Csp),

479, 1373, 1232, 1001, 804, 656, 619; H NMR (500MHz, CDCl

1

3

): δ 7.92 (s, 1H, H-

5’), 7.86 (m, 2H, H-9’ and H-11’), 7.46 (d, J = 6.3 Hz, 1H, H-7’), 7.39 (t, J = 7.6 Hz, 1H,

H-10’), 5.50 (s, 1H, H-7), 4.57 (t, J = 7.6 Hz, 2H, H-15), 3.12 (s, 1H, H-13’), 2.25 (t, J =

.6 Hz, 2H, H-14), 2.01 (m, 1H, H-6A) 1.99 (m, 1H, H-11A), 1.98 (m, 1H, H-12A), 1.83

m, 2H, H-11B and H-12B), 1.78 (m, 1H, H-6B), 1.74 (s, 3H, H-17), 1.69 (m, 1H, H-5),

7

(

1

3

2

1

.63 (m, 1H, H-1A), 1.56 (m, 2H, H-2), 1.44 (m, 1H, H-1B), 1.39 (m, 1H, H-3A), 1.30 (s,

H, H-16), 1.22 (m, 1H, H-3B), 0.91 (s, 3H, H-19), 0.87 (s, 3H, H-18), 0.82 (s, 3H, H-

1

3

0); C NMR (100MHz, CDCl

3

): δ 146.6 (C-4’), 134.8 (C-8), 131.5 (C-5’), 131.2 (C-6’),

29.2 (C-9’), 128.8 (C-7’), 126.7 (C-7), 126.0 (C-10’), 122.6 (C-8’), 120.2 (C-11’), 90.8

(

C-9), 83.3 (C-12’), 81.4 (2-C, C-13’ and C-13), 47.2 (C-15), 43.5 (C-14), 42.8 (C-5),

42.1 (C-3), 40.7 (C-10), 40.1 (C-12), 33.2 (C-4), 33.0 (C-18), 32.8 (C-1), 28.1 (C-6),

2

7.0 (C-16), 24.2 (C-11), 22.3 (C-19), 21.2 (C-17), 18.8 (C-2), 16.7 (C-20); HRMS-ES

+

(

m/z): calcd for C H N O [M] : 457.3093, found 457.3088.

3

0

39

3

4.1.26.

Preparation

of

1-(2-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl)-4-

(

((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-

spiro[furan-2,1'-naphthalene]-5-yl)methyl)-1H-1,2,3-triazole (29).

This compound was prepared by General procedure for Huisgen reaction using 48.0

mg of azide 23 (0.14 mmol) and 44.3 mg of alquine 26 (0.14 mmol) to obtain 58.5 mg

2

5

1

of 29 (64% yield) as a clear oil. [α]

CDCl ): δ 7.39 (s, 1H), 5.51 (s, 2H), 4.44 (t, J= 8 Hz, 2H), 3.00 (s, 2H), 2.18 (m, 4H),

.06 (m, 6H), 1.98-1.73 (m, 6H), 1.74 (bs, 3H), 1.69 (s, 3H), 1.68-1.47 (m, 6H), 1.41

m, 5H), 1.25 (s, 3H), 1.20 (m, 4H), 1.18 (s, 3H), 0.91 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H),

D

3

= -103.33 (c 0.12, CHCl ); H NMR (500MHz,

3

2

(

13

0

1

3

2

6

3

.88 (s, 3H), 0.81 (s, 3H); C NMR (100MHz, CDCl ): δ 144.9, 135.5, 134.8, 126.6,

26.2, 122.7, 90.6, 90.5, 82.7, 81.3, 60.4, 47.0, 43.6, 42.7, 42.6, 42.1 (2C), 40.8, 40.7,

9.6, 39.5, 38.4, 33.3, 33.2, 33.1, 33.0, 32.8, 29.7, 28.3, 28.1, 27.2, 26.9, 24.3 (2C),

2.3 (2C), 21.3, 21.0, 18.8, 16.7, 14.2; HRMS-ES (m/z): calcd for C41

+

H

65

N

3

O

2

[M] :

31.5077, found 631.5073.

4.1.27. Preparation of 1,4-bis(1-(2-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl)-1H-

1,2,3-triazol-4-yl)butane (30).

This compound was prepared by General procedure for Huisgen reaction using 27.0

mg of azide 23 (0.08 mmol) and 5 µL of 1,7-octadiyne (0.04 mmol) to obtain 22.4 mg of

25

1

30 (64% yield) as a clear oil, yield 64%. [α]

D

3

= -84.57 (c 0.31, CHCl ); H NMR

(

500MHz, CDCl ): δ 7.34 (s, 2H, H-5’), 5.50 (s, 2H, H-7), 4.46 (t, J = 7.6 Hz, 4H, H-15),

3

15

ACCEPTED MANUSCRIPT

2.74 (t, J = 7.14 Hz, 4H, H-6’), 2.18 (m, 4H, H-14), 2.03 (m, 2H, H-11A), 1.95 (m, 2H,

H-12A), 1.85 (m, 2H, H-11B), 1.80 (m, 8H, H-6 and H-7’), 1.79 (m, 2H, H-12B), 1.73 (s,

6

1

0

1

4

H, H-17), 1.70 (m, 2H, H-5), 1.63 (m, 2H, H-1A), 1.61 (m, 4H, H-2), 1.43 (m, 2H, H-

B), 1.41 (m, 2H, H-3A), 1.26 (s, 6H, H-16), 1.21 (m, 2H, H-3B), 0.91 (s, 6H, H-19),

13

.89 (s, 6H, H-18), 0.81 (s, 6H, H-20); C NMR (100MHz, CDCl ): δ 147.7 (2C, C-4’),

3

34.8 (2C, C-8), 126.3 (2C, C-7), 120.6 (2C, C-5’), 90.8 (2C, C-9), 81.3 (2C, C-13),

7.1(2C, C-15), 43.7 (2C, C-14), 42.7 (2C, C-5), 42.1 (2C, C-3), 40.7 (2C, C-10), 39.8

(

2C, C-12), 33.2 (2C, C-4), 33.0 (2C, C-18), 32.8 (2C, C-1), 28.5 (2C, C-6), 28.2 (2C,

C-7’), 26.9 (2C, C-16), 25.2 (2C, C-6’), 24.3 (2C, C-11), 22.3 (2C, C-19), 21.3 (2C, C-

17), 18.8 (2C, C-2), 16.7 (2C, C-20).

4

1

.1.28. Preparation of 1,3-bis(1-(2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl)-1H-

,2,3-triazol-4-yl)benzene (31).

This compound was prepared by General procedure for Huisgen reaction using 87.0

mg of azide 23 (0.26 mmol) and 17 µL of 1,3-diethynylbenzene (0.13 mmol) to obtain

2

0

5

1.0 mg of 31 (51% yield) as a white solid: mp 101-103 ºC. [α]

D

= -144.5 (c 8.8,

-1

CHCl

3

); IR

ν

max, cm (KBr): 3136 (=C-H), 2962, 2926, 2870, 1699, 1460, 1379, 1261,

101, 1020, 800; H NMR (500MHz, CDCl3): δ 8.28 (s, 1H), 7.91 (s, 2H), 7.82 (dd, J =

and 2Hz, 2H), 7.49 (t, J = 8 Hz, 1H), 5.52 (s, 2H), 4.58 (m, 4H), 2.28 (m, 4H), 2.17-

.92 (m, 8H), 1.89-1.78 (m, 6H), 1.77 (s, 6H), 1.74-1.49 (m, 6H), 1.46-1.38 (m, 4H),

.32 (s, 6H), 1.29-1.10 (m, 2H), 0.92 (s, 6H), 0.88 (s, 6H), 0.83 (s, 6H); C NMR

): δ 134.7 (2C), 131.4 (2C), 126.7 (2C), 125.2 (2C), 122.8 (2C), 120.0

2C), 90.7 (2C), 81.3 (2C), 77.2 (2C), 47.3 (2C), 43.7 (2C), 42.7 (2C), 42.1 (2C), 40.7

2C), 39.9 (2C), 33.2 (2C), 33.0 (2C), 32.8 (2C), 28.1 (2C), 26.9 (2C), 24.2 (2C), 22.3

2C), 21.3 (2C), 18.8 (2C), 16.7 (2C); HRMS-ES (m/z): calcd for C50

1

8

1

3

(

100MHz, CDCl

3

+

H

72

N

6

O

2

[M+H] :

+

7

8

88.5717, found 788.5724. HRMS-ESI (m/z): calcd for C50

11.5614, found 811.5606.

H

72

N

6

O

2

Na [M+Na] :

4

1

.1.29.

Preparation

of

1,3-bis((1-(2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl)-1H-

,2,3-triazol-4-yl)methoxy)benzene (32).

This compound was prepared by General procedure for Huisgen reaction using 100.0

mg of azide 23 (0.30 mmol) and 28.0 mg of 1,3-bis(prop-2-yn-1-yloxy)benzene (0.15

2

D

0

mmol) to obtain 59.0 mg of 32 (46% yield) as a white solid: mp 75-77 ºC. [α]

= -

): δ 7.67 (bs, 2H), 7.20 (t, J = 8 Hz,

H), 6.63 (s, 1H), (6.62 (d, J = 8 Hz, 2H), 5.49 (bs, 2H), 5.19 (s, 4H), 4.51 (m, 4H), 2.21

3 3

123.4 (c 1.08, CHCl ); 1H NMR (500MHz, CDCl

1

(

t, J = 8, 4H), 2.15-1.90 (m, 8H), 1.90-1.75 (m, 6H), 1.72 (s, 6H), 1.71-1.46 (m, 8H),

1

6

1

.45-1.30 (m, 2H) 1.28 (s, 6H), 1.28-1.15 (m, 2H), 0.91 (s, 6H), 0.89 (s, 6H), 0.81 (s,

H); 13C NMR (100MHz, CDCl ): δ 159.5 (2C), 144.0 (2C), 134.7, 130.0, 126.6 (2C),

3

22.9 (2C), 107.5 (2C), 102.2 (2C), 90.7 (2C), 81.2 (2C), 62.1 (2C), 47.4 (2C), 43.6

(

2C), 42.7 (2C), 42.1 (2C), 40.7 (2C), 39.9 (2C), 33.2 (2C), 33.0 (2C), 32.7 (2C), 28.0

2C), 26.9 (2C), 24.3 (2C), 22.3 (2C), 21.3 (2C), 18.8 (2C), 16.7 (2C); HRMS-ESI (m/z):

+

calcd for C H N O Na [M+Na] : 871.5825, found 871.5832.

52

76

6

4

4.1.30.

Preparation

of

2-((1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl-1H-

imidazole-1-carboxylate (33).

16

ACCEPTED MANUSCRIPT

2 2

To a solution of alcohol 4 (1 g, 3.26 mmol) in CH Cl (65 mL, 0.05 M) was added

carbonyl diimidazole (2.64 g, 16.4 mmol). The reaction mixture was heated to 40 ºC

and stirred for 4.5 h. Solvent was removed and the residue subjected to a flash

chromatography to provide carbonyl imidazole 33 (927 mg, yield 71%) as clear oil.

2

0

-1

[

(

α]

D

= -117.90 (c 15.54, CHCl

3

); IR

ν

max, cm (KBr): 3124, 2970, 1747 (C=O), 1672

1

C=C), 1466, 1379,1271, 1005, 872, 758; H NMR (500MHz, CDCl

3

): δ 8.11 (s, 1H, H-

2’), 7.41 (s, 1H, H-4’), 7.05 (s, 1H, H-5’), 5.46 (bs, 1H, H-7), 4.56 (m, 2H, H-15), 2.16

(

dt , J = 14 and 7 Hz, 1H, H-14A), 2.07 (m, 1H, H-11A), 2.03 (m, 1H, H-14B), 2.00 (m,

1

2

H, H-12A), 1.96 (m, 1H, H-6A), 1.79 (m, 2H, H-11B and H-12B), 1.76 (s, 3H, H-17),

.76 (m, 1H, H-6B), 1.67 (m, 1H, H-5), 1.61 (m, 2H, H-3), 1.51 (m, 2H, H-2), 1.40 (m,

H, H-1), 1.28 (s, 3H, H-16), 0.89 (s, 3H, H-19), 0.85 (s, 3H, H-18), 0.80 (s, 3H, H-20);

1

3

C NMR (100MHz, CDCl ): δ 137.1 (C-2’), 134.8 (C-8), 130.5 (C-4’), 126.6 (C-7),

1

4

1

17.1 (C-5’), 90.6 (C-9), 81.3 (C-13), 65.9 (C-15), 42.6 (C-5), 42.0 (C-3), 41.8 (C-14),

0.7 (C-10), 40.0 (C-12), 33.2 (C-4), 33.0 (C-18), 32.7 (C-1), 27.1 (C-16), 24.2 (2C, C-

1 and C-6), 22.3 (C-19), 21.3 (C-17), 18.7 (C-2), 16.7 (C-20); HRMS-ES (m/z): calcd

+

for C24

H

37

N

2

O

3

[M+H] : 401.2799, found 401.2791.

4.1.31. General procedure for synthesis of carbamates derivatives. Preparation of 2-

(

(1'R,4a'S,5S,8a'S)-2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-

spiro[furan-2,1'-naphthalene]-5-yl)ethyl-3-(dimethylamino)propylcarbamate (34).

A solution of the carbonyl imidazole 33 (24.2 mg, 0.06 mmol) was dissolved in

acetonitrile (0.12 mL, 0.5 M). Methyl iodide (0.75 mL, 12 mmol) was added and the

solution was heated to 60 °C for 2 h. The volatiles were removed by evaporation and

the residue was redissolved in methylene chloride (1.5 mL, 0.04M), 3-(dimethylamino)-

1-propylamine (0.15 mL, 1.2 mmol) was added, and the resulting reaction mixture was

stirred 4h. The volatiles were removed by evaporation and the crude residue was

2

5

purified by flash chromatography to give 20.7 mg of 34 (79% yield) as a clear oil. [α]

D

1

=

-38 (c 0.1, CHCl

3 3

); H NMR (500MHz, CDCl ): δ 5.53 (bs, 1H), 5.49 (bs, 1H), 4.16

(

bs, 2H), 3.26 (m, 2H), 2.58 (m, 2H), 2.40 (bs, 6H), 2.06 (m, 6H), 1.78 (m, 1H), 1.76 (s,

3

0

4

2

H), 1.69 (m, 4H), 1.51 (m, 2H), 1.40 (m, 2H), 1.23 (s, 3H), 0.90 (s, 3H), 0.88 (s, 3H),

13

3

.81 (s, 3H); C NMR (100MHz, CDCl ): δ 156.9, 135.2, 126.2, 90.2, 81.9, 62.6, 56.8,

4.4, 42.6, 42.4, 42.0, 40.7, 39.2 (2C), 33.2, 32.9, 32.8, 29.7, 28.4, 27.0, 26.3, 24.2,

+

46 2 3

2.4, 21.2, 18.8, 16.7; HRMS-ESI (m/z): calcd for C26H N O Na [M+Na] : 457.3406,

found 457.3407.

4.1.32. Preparation of 2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-

octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl prop-2-ynylcarbamate (35).

This compound was prepared by General procedure for synthesis of carbamates

derivatives using 97.0 mg carbonyl imidazole 33 (0.24 mmol) and 0.35 mL of

propargylamine (5.52 mmol) to obtain 78 mg of 35 (83% yield) as a white solid: mp

2

0

1

3

16-118 ºC. [α]

D

3 3

= -113.7 (c 0.918, CHCl ); H NMR (500MHz, CDCl ): δ 5.49 (bs,

H), 4.83 (bs, 1H), 4.20 (m, 2H), 3.98 (bs, 2H), 2.25 (m, 1H), 2.10-1.88 (m, 4H), 1.78-

.60 (m, 4H), 1.76 (s, 3H), 1.59-1.45 (m, 3H), 1.40-1.35 (m, 2H), 1.28-1.20 (m, 2H),

.23 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.82 (s, 3H); C NMR (100MHz, CDCl ): δ

3

35.2, 134.2, 126.3, 90.4, 81.8, 80.8, 71.5, 63.2, 42.6, 42.3, 42.0, 40.7, 39.3, 33.2,

2.9, 32.8, 29.7, 28.4, 27.0, 24.2, 22.4, 21.2, 18.8, 16.7; HRMS-ES (m/z): calcd for

1

3

+

C

24

H37NO

3

[M] : 387.2773, found 387.2761.

17

ACCEPTED MANUSCRIPT

4

1

.1.33.

Preparation

of

bis(2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-

,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl) ethane-

,2-diyldicarbamate (36).

This compound was prepared by General procedure for synthesis of carbamates

derivatives using 100.0 mg carbonyl imidazole 33 (0.25 mmol) and 0.38 mL of

ethylenediamine (5.75 mmol) to obtain 46 mg of 36 (51% yield) as a white solid: mp

20

1

4

70-172 ºC. [α]

.99 (bs, 2H), 4.17 (m, 4H), 3.30 (bs, 4H), 2.09-1.80 (m, 8H), 1.79-1.62 (m, 2H), 1.77

D

3 3

= -126.7 (c 1.12, CHCl ); H NMR (500MHz, CDCl ): δ 5.50 (bs, 2H),

(

1

s, 6H), 1.60-1.52 (m, 4H), 1.51-1.38 (m, 10H), 1.35-1.22 (m, 4H), 1.20-1.09 (m, 2H),

1

3

.23 (s, 6H), 0.91 (s, 6H), 0.88 (s, 6H), 0.82 (s, 6H); C NMR (100MHz, CDCl

3

): δ

78.8 (2C), 135.2 (2C), 126.2 (2C), 115.0 (2C), 90.6 (2C), 81.8 (2C), 62.9 (2C), 56.5

(

2C), 42.6 (2C), 42.3 (2C), 42.0 (2C), 40.7 (2C), 33.2 (2C), 33.0 (2C), 32.8 (2C), 28.4

2C), 27.0 (2C), 24.2 (2C), 22.4 (2C), 21.3 (2C), 18.8 (2C), 16.7 (2C); HRMS-ESI (m/z):

+

calcd for C44

H

72

N

2

O

6

Na [M+Na] : 747.5294, found 747.5262.

4

.1.34. Preparation of 2-((1'R,5S,8a'S)-2',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-

octahydro-3H,4'H-spiro[furan-2,1'-naphthalene]-5-yl)ethyl(1-(2-((1'R,5S,8a'S)-

',5,5',5',8a'-pentamethyl-4,4a',5,5',6',7',8',8a'-octahydro-3H,4'H-spiro[furan-2,1'-

2

naphthalene]-5-yl)ethyl)-1H-1,2,3-triazol-4-yl)methylcarbamate (37).

This compound was prepared by General procedure for Huisgen reaction using 81.0

mg of azide 23 (0.24 mmol) and 43.0 mg of propynylcarbamate 35 (0.11 mmol) to

20

obtain 51 mg of 37 (64% yield) as a white solid, mp 69-71 ºC. [α]

CHCl ); H NMR (500MHz, CDCl

3 3

(

D

= -137.1 (c 1.022,

): δ 7.58 (s. 1H), 5.49 (bs, 2H), 5.27 (bs, 1H), 4.46

m, 4H), 4.16 (m, 2H), 2.19 (m, 2H), 2.12-1.86 (m, 7H), 1.85-1.70 (m, 8H), 1.75 (s, 3H),

1

.73 (s, 3H), 1.71-1.45 (m, 6H), 1.42-1.32 (m, 4H), 1.28-1.20 (m, 3H), 1.27 (s, 3H),

13

1

.21 (s, 3H), 0.91 (s, 3H), 0.90 (s, 6H), 0.89 (s, 3H), 0.81 (s, 6H); C NMR (100MHz,

): δ 176.2, 156.6, 135.2, 134.7, 126.7 (2C), 126.2 (2C), 122.1, 90.7, 90.2, 81.8,

1.2, 62.9, 47.3, 43.6, 42.6 (2C), 42.3, 42.1 (2C), 40.7 (2C), 39.9, 39.2, 36.3, 33.2,

3.0, 32.9, 32.8, 32.7, 28.4, 28.0, 27.0, 26.9, 24.3, 24.2, 22.4, 21.3 (2C), 18.8 (2C),

CDCl

3

8

3

1

7

+

6.7 (2C); HRMS-ESI (m/z): calcd for C44

41.5292.

70 4 4

H N O Na [M+Na] : 741.5294, found

4.2. Cells, culture and plating

The human solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573 (non-small

cell lung), T-47D (breast) and WiDr (colon) were used in this study. Cells were

2

maintained in 25 cm culture flasks in RPMI 1640 supplemented with 5% heat

2

inactivated fetal calf serum and 2 mM L-glutamine in a 37 ºC, 5% CO , 95% humidified

air incubator. Exponentially growing cells were trypsinized and resuspended in

antibiotic containing medium (100 units penicillin G and 0.1 mg of streptomycin per

mL). Single cell suspensions displaying >97% viability by trypan blue dye exclusion

were subsequently counted. After counting, dilutions were made to give the appropriate

cell densities for inoculation onto 96-well microtiter plates. Cells were inoculated in a

volume of 100 ꢀL per well at densities of 20,000 (WiDr), 15,000 (T-47D ) and 10,000

(

HeLa, SW1573 and HBL-100) cells per well, based on their doubling times.

4.3. Antiproliferative tests

18

ACCEPTED MANUSCRIPT

Chemosensitivity tests were performed using the SRB assay of the NCI with slight

modifications. Briefly, pure compounds were initially dissolved in DMSO at 400 times

the desired final maximum test concentration. Control cells were exposed to an

equivalent concentration of DMSO (0.25% v/v, negative control). Each agent was

tested in triplicates at different dilutions in the range 1–100 ꢀM. The drug treatment was

started on day 1 after plating. Drug incubation times were 48 h, after which time cells

were precipitated with 25 ꢀL ice-cold 50% (w/v) trichloroacetic acid and fixed for 60 min

at 4 ºC. Then the SRB assay was performed. The optical density (OD) of each well was

measured at 492 nm, using BioTek’s PowerWave XS Absorbance Microplate Reader.

Values were corrected for background OD from wells only containing medium. The

percentage growth (PG) was calculated with respect to untreated control cells (C) at

each of the drug concentration levels based on the difference in OD at the start (T0)

and end of drug exposure (T), according to NCI formulas. Therefore, if T is greater than

or equal to T0 the calculation is 100 × [(T–T0)/(C–T0)]. If T is less than T0 denoting cell

killing the calculation is 100 × [(T–T0)/(T0)]. The effect is defined as percentage of

growth, where 50% growth inhibition (GI ) represents the concentration at which PG is

5

0

+50. With these calculations a PG value of 0 corresponds to the amount of cells

present at the start of drug exposure, while negative PG values denote net cell kill.

Acknowledgments

This research was supported by CONICET (PIP 00628), UNSL (Project 7301 and 2-

1412), co-financed by the EU Research Potential (FP7-REGPOT-2012-CT2012-31637-

IMBRAIN) and the European Regional Development Fund (FEDER), the Spanish

MINECO, (CTQ2011-28417-C02-01/BQU), the Spanish ISCIII (PI11/00840) and the

Canary Islands Government (Proyecto Estructurante en Ciencias Marinas, SE-10/19).

L.G.L. thanks the Spanish MSC-FIS for a postdoctoral contract. G.F.R. thanks to

Monica Ferrari for technical support. This work is part of the doctoral thesis of G.F.R.

References and notes

[

2

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ACCEPTED MANUSCRIPT

Oxide Production from Hedychium coronarium Bioorg. Med. Chem. 10 (2002) 2527-

2

534. (b) N. Nakatani, H. Kikuzaki, H. Yamaji, K. Yoshio, C. Kitora, K. Okada, W.G.

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3

chemistry and biological activity. Stud. Nat. Prod. Chem. 25 (2001) 235-292. (d) P.

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[

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Hansen,Synthesis, biological evaluation and molecular modeling of 1,2,3-triazole

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[

9] P. Singh, R. Raj, V. Kumar, M.P. Mahajan, P.M.S. Bedi, T. Kaur, A.K.

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16] CRC Handbook of Cromatography, 1972, CRC Press Ed, Ohio, Vol 2 p 111.

20

ACCEPTED MANUSCRIPT

Figure 1: Grindelic acid and 1-(α)-hydroxygrindelic acid

ACCEPTED MANUSCRIPT

Scheme 1: Reagents and conditions: a) CH

2

N

2

, Et

)CO Et, CH

P=CHCO Et, CH

, -20 ºC, 71%; g) Ti(OPr-i) , S,S-(-)-DET, TBHP, CH

2

O, rt. 94%; b) LiAlH

Cl , rt, 74%; d) DIBAL-H, Et

Cl , rt, 94%; f) Ti(OPr-i)

Cl , -

4

, THF, 70 ºC,

9

0

1%; c) SO

°C , 82-89%; e) SO

3

.Py, Et

3

N, DMSO, Ph

.Py, Et N, DMSO, Ph

Cl

3

P=C(CH

3

2

O,

3

2

4

,

R,R-(+)-DET, TBHP, CH

0 ºC, 85%.

2

4

2

2 2 3

Scheme 2: Reagents and conditions: a) MsCl, TEA, CH Cl , 0 °C , 12h, 90%; b) NaN ,

DMF, 40 °C , 48h, 80%; c) (COCl) , Et N, DMSO, Cl CH , -78 °C , 30 min, 99%; d)

2

3

2

3 4 3 2 2

PPh , CBr , Et N, Cl CH , 0 °C , 15 min; e) n-BuLi, THF, -78 °C , 30 min, 53% .

Scheme 3: Reagents and conditions: a) CDI, Cl

2

CH

, 4h, rt, 79-83%; c) i: ICH

, 4h, rt, 51%; d) 23, sodium ascorbate, CuSO

2

, 40 °C , 16 h, 71%; b) i: ICH

, acetonitrile, 60 ºC,

.5H O,

3

,

acetonitrile, 60 ºC, 2h, ii: RNH

h, ii: ethylenediamine, Cl CH

H O-EtOH, rt, 64%.

2

, Cl

2

CH

2

3

2

4

2

ACCEPTED MANUSCRIPT

Table 1: Diamides obtained from the

multicomponent Ugi reaction of grindelic acid (1).

a

1

2

b

Compound

R

Yield

11

12

13

59

75

71

14

63

1

5

6

79

1

7

8

97

78

1

19

26

2

0

1

23

43

2

22

92

a

b

All the reactions were carried out using 1 eq of compound 1,

1

eq of the corresponding amine, 1.2 eq of propanone and

.1 eq of the corresponding isocyanide.

Isolated yield.

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Table 2: 1,2,3-triazole obtained by Huisgen

a

reaction using mono-alkynes.

Compound

R

yield

48

27

28

78

29

64

a

All the reactions were carried out using the azide

2

3 (1 equiv), alkyne (1 equiv), sodium ascorbate (0.3

equiv) and copper(II) sulfate pentahydrate (0.12

equiv) in a 1:1 mixture of water and ethanol (0.1 M).

b

Isolated yield.

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Table 3: 1,2,3-triazole obtained by Huisgen reaction

a

using di-alkynes.

Compound

R

yield

30

64

3

1

51

46

32

a

All the reactions were carried out using the azide 23 (1

equiv), di-alkyne (0.5 equiv), sodium ascorbate (0.3

equiv) and copper(II) sulfate pentahydrate (0.12 equiv)

in a 1:1 mixture of water and ethanol (0.1 M).

b

Isolated yield.

ACCEPTED MANUSCRIPT

a

Table 4: In vitro antiproliferative activity against human solid tumor cell lines.

Cell line

HBL-100

breast)

46 (±10)

>100

HeLa

(cervix)

60 (±31)

>100

SW1573

(NSCLC)

49 (±28)

T-47D

WiDr

(colon)

57 (±13)

>100

23 (±3.9)

21 (±0.65)

37 (±0.87)

19 (±0.23)

35 (±0.69)

17 (±1.2)

19 (±0.18)

17 (±0.14)

Comp.

(

(breast)

69 (±5.5)

>100

1

2

3

4

5

6

7

8

9

1

2

3

>72 (±39)

18 (±1.9)

19 (±1.1)

20 (±4.5)

18 (±1.3)

18 (±3.3)

17 (±1.3)

18 (±0.64)

18 (±1.0)

11 (±1.4)

14 (±2.6)

3.6 (±0.71)

5.1 (±1.9)

17 (±2.4)

17 (±2.6)

17 (±4.4)

16 (±2.6)

16 (±3.8)

17 (±1.7)

16 (±2.3)

16 (±1.9)

4.6 (±3.9)

10 (±3.5)

3.7 (±0.31)

8.9 (±2.2)

1.9 (±0.44)

16 (±0.88)

16 (±2.9)

17 (±2.5)

18 (±5.8)

16 (±2.3)

19 (±7.4)

18 (±2.5)

17 (±1.7)

17 (±1.4)

5.3 (±5.6)

30 (±6.9)

4.7 (±0.84)

11 (±1.6)

2.9 (±0.89)

18(±2.0)

19 (±5.4)

16 (±1.8)

32 (±9.1)

15 (±1.7)

32 (±7.8)

17 (±2.0)

16 (±2.0)

15 (±2.5)

0

1

2

3

4

5

6

7

8

9

3

4

6

7

8

9

0

2

3

4

5

7

3.9 (±0.87) 12 (±0.84)

40 (±4.5) 49 (±7.9)

4.4 (±1.0) 5.1 (±0.82)

31 (±5.8)

38 (±7.8)

3.1 (±1.3)

12 (±2.4)

1.8 (±0.51) 3.0 (±0.78)

16 (±3.3) 19 (±2.3)

0.95 (±0.38) 1.6 (±0.46)

1.2 (±0.54) 1.9 (±0.62) 1.8 (±0.81)

5.4 (±2.1)

8.7 (±0.63)

>100

5.7 (±2.6)

22 (±2.3)

>100

5.8 (±2.6)

5.9 (±1.2)

>100

17 (±3.3)

15 (±2.4)

15 (±2.3)

>100

5.3 (±2.5) 6.8 (±0.28)

21 (±3.5)

>100

26 (±8.1)

28 (±10)

44 (±13)

>100

37 (±8.2)

>100

15

4.8

15

>100

n.a.

14 (±3.5)

17 (±1.9)

16 (±5.0)

>100

17 (±2.6)

16 (±1.2)

25 (±6.8)

>100

9.3 (±1.4)

2.9 (±0.49)

>100

17 (±0.82)

2.7 (±0.74)

>100

11 (±0.5)

17 (±1.0)

3.0 (±0.39) 4.3 (±0.63)

1.3

>100

17 (±0.88)

4.3 (±0.98)

16 (±2.4)

>100

13

1.4

12 (±1.9)

>100

26 (±5.3)

1.8 (±0.7)

16 (±0.34)

6.1 (±0.57)

14 (±0.35)

>100

18 (±1.1)

7.8 (±2.9)

14 (±1.6)

>100

17 (±0.85)

8.0 (±6.9)

13 (±2.3)

>100

3.0 (±0.4)

0.25 (±0.12) 2.0 (±0.5)

Cisplatin

Camptothecin 0.23 (±0.05)

1.9 (±0.2)

2.0 (±0.3)

0.6 (±0.4)

15 (±2.3)

a

Values expressed as GI₅₀(50% growth inhibition) are given in µM and are means

of two to four experiments, standard deviation is given in parentheses.

ACCEPTED MANUSCRIPT

Article Doi

Derivatives of grindelic acid: From a non-active natural diterpene to synthetic antitumor derivatives

DOI: 10.1016/j.ejmech.2013.06.013

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2013.06.013

Full text of DOI:10.1016/j.ejmech.2013.06.013

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