Simplification of antitumoral phenanthroindolizidine alkaloids: Short synthesis of cytotoxic indolizidinone and pyrrolidine analogs

DOI: 10.1016/j.ejmech.2013.06.009

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European Journal of Medicinal Chemistry p. 540 - 554 (2013)

Update date:2022-09-26

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Authors:

Miguélez, Javier

Boto, Alicia

Marín, Raquel

Díaz, Mario

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Article abstract of DOI:10.1016/j.ejmech.2013.06.009

Hydroxylated seco-analogs of cytotoxic phenanthroindolizidine alkaloids were prepared in good yields from inexpensive 4-hydroxyproline derivatives, in just two steps. Thus, a sequential oxidative radical scission - oxidation was used for the direct conversion of the proline derivative into a 2-(2-aryl-oxoethyl) pyrrolidine with a variety of aryl and heteroaryl groups. The 4R-stereogenic center allowed ready isomer separation, and stereocontrol in the introduction of new chains (interestingly, the 2,4-cis isomers predominated). In the second step, a cyclization reaction afforded alkaloid analogs with an indolizidinone core; a partial isomerization took place but the isomers were readily purifi ed. Then the cytotoxic activity of the bicyclic indolizidinones and the simpler pyrrolidine derivatives was compared against tumorogenic human neuronal SHSY-5Y and breast cancer MCF7 cells. All the biphenyl derivatives displayed a potent activity (one derivative caused >80% cell death in both tumor lines at micromolar dosis), being comparable in the pyrrolidine and indolizidinone series.

Full text of DOI:10.1016/j.ejmech.2013.06.009

European Journal of Medicinal Chemistry 66 (2013) 540e554

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Original article

Simpliﬁcation of antitumoral phenanthroindolizidine alkaloids:

Short synthesis of cytotoxic indolizidinone and pyrrolidine analogs

,d,

Javier Miguélez ^a, Alicia Boto ^a, Raquel Marín ^c, Mario Díaz ^c

^aInstituto de Productos Naturales y Agrobiología del CSIC, Avda. Astrofísico Francisco Sánchez 3, 38206 La Laguna, Tenerife, Spain

^bLaboratorio de Neurobiología Celular, Departamento de Fisiología, Facultad de Medicina, Campus de Ofra s/n, Universidad de La Laguna,

38071 La Laguna, Tenerife, Spain

^cInstituto Canario de Investigación del Cáncer (ICIC), Canary Islands, Spain

^dLaboratorio de Fisiología y Biofísica de Membranas, Departamento de Biología Animal, Facultad de Biología, Universidad de La Laguna,

Avda. Astrofísico Francisco Sánchez s/n, 38206 La Laguna, Tenerife, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

Hydroxylated seco-analogs of cytotoxic phenanthroindolizidine alkaloids were prepared in good yields

from inexpensive 4-hydroxyproline derivatives, in just two steps. Thus, a sequential oxidative radical

scissioneoxidation was used for the direct conversion of the proline derivative into a 2-(2-aryl-oxoethyl)

pyrrolidine with a variety of aryl and heteroaryl groups. The 4R-stereogenic center allowed ready isomer

separation, and stereocontrol in the introduction of new chains (interestingly, the 2,4-cis isomers pre-

dominated). In the second step, a cyclization reaction afforded alkaloid analogs with an indolizidinone

core; a partial isomerization took place but the isomers were readily puriﬁed. Then the cytotoxic activity

of the bicyclic indolizidinones and the simpler pyrrolidine derivatives was compared against tumoro-

genic human neuronal SHSY-5Y and breast cancer MCF7 cells. All the biphenyl derivatives displayed a

potent activity (one derivative caused >80% cell death in both tumor lines at micromolar dosis), being

comparable in the pyrrolidine and indolizidinone series.

Received 8 April 2013

Received in revised form

3 June 2013

Accepted 6 June 2013

Available online 18 June 2013

Keywords:

Cytotoxicity

Indolizidine

Alkaloid

Synthetic methodology

Sequential process

1. Introduction

[4,5]. The seco-analog 3 was highly active against human A549 lung

cancer cells (IC₅₀¼ 0.16

mM), comparable with potent antineoplasic

drugs such as etoposide [6,7]. Another seco-analog, the indolizidi-

The modiﬁcation of cytotoxic phenanthroindolizidine or qui-

nolizidine alkaloids [1] (such as tylophorine 1, Fig. 1) [2] has pro-

vided new drug candidates with potent and selective cytotoxic

activity [3]. For instance, the hydroxylated analog 2 (DCB-3503)

downregulates pro-oncogenic and pro-survival proteins such as

none

(0.39

(NSC 707904) [8e10], presents submicromolar IC₅₀

M against HCT-116) and is selective against the colon and

leukemia cancer lines [8]. Interestingly, the minor enantiomer

(8aR)-4 was the most active one [9].

cyclin D1,

-catenin, p53 and p21, while displaying low toxicity

The hydroxylated analogs 5 were prepared by Georg as pre-

cursors of the pure (8aS)-4 or (8aR)-4 enantiomers [9]. However,

the activity of these hydroxylated indolizidinones is also worth

studying. Thus, many phenanthroindolizidine alkaloids with potent

antitumoral activity also present important side-effects, such as

CNS toxicity [11]. The development of polar analogs which are

unable to cross the bloodebrain barrier would minimize this

problem [12]. In fact, hydroxylated compound 2 was developed as a

more selective tylophorine and tylocrebrine analog. Its hydroxyl

group increases its polarity with respect to the parent compounds,

and greatly reduces its CNS side-effects. Compound 3 also presents

polar hydroxy and secondary amino groups, which could improve

its bioavailability and pharmacological activity.

Abbreviations: CNS, central nervous system; Ar, aryl; Ar-M, aryl-metal; Boc,

CO₂CMe₃; Ph, phenyl; dr, diastereomeric ratio; NOESY, nuclear Overhauser effect

spectroscopy; DIB, (diacetoxyiodo)benzene; OTMS, trimethylsilyloxy; TMS, tetra-

methylsilane; Nu, nucleophile; 80 W, 80 watts; EtOAc, ethyl acetate; SAR, struc-

tureeactivity relationships; NMR, nuclear magnetic resonance; TLC, thin-layer

chromatography; MHz, megahertz; Ts, toluenesulfonyl (TsOH is toluenesulfonic

acid); p, probability; VEH, vehicle; br, broad signal (e.g. br s ¼ broad singlet, br

d ¼ broad doublet, br b ¼ broad band); mp, melting point; HRMS, high-resolution

mass spectrometry; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; DMEM,

Dubelcco’s modiﬁed Eagle medium.

* Corresponding author. Fax: þ34 922 260135.

** Corresponding author. Laboratorio de Fisiología y Biofísica de Membranas,

Departamento de Biología Animal, Facultad de Biología, Universidad de La Laguna,

Avda. Astrofísico Francisco Sánchez s/n, 38206 La Laguna, Tenerife, Spain.

E-mail addresses: alicia@ipna.csic.es (A. Boto), madiaz@ull.es (M. Díaz).

The ﬁrst reported procedure to prepare hydroxylated indolizi-

dinones 6 (Scheme 1) used the base-promoted cyclization of the

(2S)-keto amides 7 (for Ar ¼ C₆H₄-p-OMe). Partial epimerization of

http://dx.doi.org/10.1016/j.ejmech.2013.06.009

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

541

studied against the human neuroblastoma SHSY-5Y and human

breast cancer MCF-7 cell lines. Many of these alkaloids displayed

cytotoxic activities at micromolar doses [13a].

The results of this preliminary study supported that hydroxyl-

ated derivatives of cytotoxic indolizidinones could be promising

antitumoral candidates. Besides, the alcohol derived from 9 (for

Ar ¼ C₆H₄-p-Ph) displayed a potent cytotoxic activity, comparable

or superior to the corresponding indolizidinone. Therefore, two

new objectives were set: (a) To complete the study on the cyto-

toxicity of hydroxylated indolizidinones 6, introducing different

aryl and heteroaryl Ar groups; (b) To determine whether a

simpliﬁed, more ﬂexible structure could retain or even improve the

cytotoxic activity, by preparing a variety of hydroxylated pyrroli-

dines and performing SAR studies.

In the present paper, the complete preparation and character-

ization of indolizidinones 6, their precursors 9, and a variety of

hydroxylated pyrrolidines is described. Besides, the cytotoxicity of

indolizidinones versus hydroxylated pyrrolidines is compared, to

determine whether the ﬂexible analogs of the rigid indolizidinone

drug leads are able to elicit a pharmacological response.

Fig. 1. Cytotoxic alkaloids and synthetic analogs.

2. Results and discussion

2.1. Chemistry

the stereogenic center at C-2 was observed in this step. The keto

amides 7 were obtained from an unusual

b-amino acid derivative,

The preparation of the keto amides 9 from 4-acetoxy-L-a-pro-

N-Boc-4-hydroxy- -homoproline 8, whose carboxyl group was

L-b

line amide 10 (Scheme 1) was carried out using a one-pot decar-

boxylationealkylation process [13,14], where an oxidative radical

decarboxylation generates an acyliminium ion 10a. This interme-

diate adopts an envelope conformation with a pseudoaxial OR

substituent, which maximizes the electrostatic interactions be-

tween the oxygen lone electron pairs and the iminium ion [16]. In

this conformation, the nucleophile adds mainly from the inner face,

avoiding eclipsing interactions on the formation of the product,

yielding compound cis-9 with the unusual (2R)-conﬁguration as

the major product.

The sequential procedure yielded compounds 9aeg in good to

excellent yields (83e96%), with cis:trans ratios between 9:2 and 7:1.

Both isomers could be readily separated, and their stereochemis-

tries were determined according to the ¹H NMR coupling constants

and the NOESY spatial interactions [17].

The decarboxylationealkylation process was studied under

different conditions. In general, for the scission step the substrate

10 (Scheme 2) was treated with (diacetoxyiodo)benzene (DIB) and

iodine, at 26 ^ꢀC and under irradiation with visible light (sunlight or

tungsten-ﬁlament lamps). It was noted that the amount of iodine

was important for the reaction outcome. The decarboxylation did

not take place without iodine, but a stoichiometric amount gave

lower yields (about 50%) than 0.5 eq (about 80%).

transformed into an activated N-methoxycarboxamide, followed by

the addition of organometallic (Ar-M) reagents to give the aryl

ketones. Subsequent Boc-cleavage and amidation provided the

(2S)-keto amides 7 [9].

In a preliminary work, we described an alternative strategy,

where the indolizidinones 6 were obtained from (2R)-keto amides

9 via a one-pot saponiﬁcationecyclization reaction, where partial

epimerization also took place [13]. The keto amides 9 were pre-

pared from readily available 4-hydroxy-L-a-proline amides 10

(Scheme 1), whose carboxyl group was removed [13,14] and

replaced in situ by an alkyl chain [13e15]. Interestingly, the one pot

scissionealkylation process produced inversion of conﬁguration at

C-2, giving the (2R)-product as the major isomer.

Then, the cytotoxic activity of some hydroxylated alkaloid ana-

logs 6 and one simpliﬁed analog, derived from the substituted

pyrrolidine 9 (for Ar ¼ C₆H₄-p-Ph) by acetate hydrolysis, was

After disappearance of the starting material, the reaction

mixture was cooled and the Lewis acid and the nucleophile were

added, to give the keto amides 9. Besides, different Lewis acids

(TiCl₄, BF₃$OEt₂, TMSOTf) and addition temperatures were tried;

the best results were obtained with BF₃$OEt₂at either ꢁ78 / 0 ^ꢀC

or 0 / 26 ^ꢀC.

A variety of aromatic and heteroaromatic groups of different

volumes and polarities were introduced, such as phenyl rings with

different electron-donor or electron-withdrawing substituents (in

compounds 9aed), the bulky naphthyl group (product 9e) and

heteroaromatic rings (furan and pyrrole derivatives 9f and 9g).

Interestingly, the halogenated derivative 9c and the furyl com-

pound 9f can be used as precursors of many other alkaloid analogs.

In the ﬁrst case, allyl or aryl groups can be introduced with radical

or sp²espⁿcouplings [18]; in the second case, the furyl ring can

undergo oxidation, reduction or DielseAlder reactions, to give

Scheme 1. Strategy toward the synthesis of indolizidinones 6 [Ar ¼ aryl, heteroaryl;

Ac ¼ acetyl; Ar-M ¼ aryl-metal, Boc ¼ CO₂CMe₃].

542

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

lactones, furan, or carbocyclic rings [19]. Finally, the heteroaryl

rings in derivatives 9f and 9g can modify their potency, bioavail-

ability and interaction with the biological targets [20] with respect

to aryl-substituted indolizidinones.

A model study which used hydroxyproline carbamate 11 [21] as

a substrate (Scheme 3), suggested that partial epimerization can

take place during acid-catalyzed alkylation. Thus, compound 11

underwent the oxidative radical scission, followed by the addition

of allyltrimethylsilane or 1-phenyl(trimethylsiloxy)ethene, in the

presence of a Lewis acid. The allyl derivative 12 was obtained in

good yields (89%) with an excellent dr (cis:trans >98:2) [22]. Using

the same conditions with the other nucleophile, 1-phenyl-1-

Scheme 3. Direct conversion of the hydroxyproline derivative 11 into the 2-allyl de-

rivative 12 and the -amino ketone 13, and stereochemical outcome

[TMS ¼ trimethylsilyl; Nu ¼ nucleophile; Ph ¼ phenyl; Ac ¼ acetyl].

trimethylsiloxyethene, the dr considerably decreased (78% yield

for ketone 13, cis:trans dr 10:3). The bulkier nucleophile could ac-

count in part for this result, but the large difference in diaster-

eoselectivity suggests that once cis-13 is formed, partial in situ

isomerization of the cis to the trans isomer takes place, via a retro-

Michael reaction followed by a second cyclization reaction [20].

This isomerization is not possible in the case of the allyl derivative

12, which lacks acid protons for the retro-Michael reaction. As

commented later, the epimerization can take place not only under

acid conditions but also in base-promoted reactions, and will have

an important role in the cyclization outcome.

The one-pot conversion of the 2-substituted pyrrolidines 9 into

the indolizidinones 6 (Scheme 4 and Table 1), involving the

Scheme 2. One-pot oxidative radical decarboxylationealkylation process. [a] Global

yield, the yields are given for puriﬁed products [OTMS

trimethylsilyloxy;

Scheme 4. One-pot conversion of the 2-substituted pyrrolidines 9 into the indolizi-

DIB ¼ (diacetoxyiodo)benzene; Nu ¼ nucleophile; Ph ¼ phenyl; Ar ¼ aryl, heteroaryl;

dinones 6, and stereochemical outcome [Ph

Ac ¼ acetyl].

phenyl; Ar

aryl, heteroaryl;

Ac ¼ acetyl].

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

543

Table 1

Table 2

Direct conversion of keto amides 9 into indolizidinones 6 using a one-pot acetate

hydrolysisecyclization [Ph ¼ phenyl; Ar ¼ aryl, heteroaryl].

Generation of hydroxypyrrolidine derivatives 16 by saponiﬁcation of acetate de-

rivatives 9 [Ph ¼ phenyl; Ar ¼ aryl, heteroaryl].

Entry

Substrate

Products (%)

Overall

yield (%)

cis-9a

(8aR)-6a (59), (8aS)-6a (25)

(8aR)-6a (57), (8aS)-6a (34)

(8aR)-6b (55), (8aS)-6b (26)

(8aR)-6c (48), (8aS)-6c (32)

trans-9a

cis-9b

cis-9c

C₆H₄-p-OMe

C₆H₄-p-Br

cis-9d

(8aR)-6d (53), (8aS)-6d (33)

cis-9e

(8aR)-6e (50), (8aS)-6e (29)

(8aR)-6e (54), (8aS)-6e (29)

(8aR)-6f (55), (8aS)-6f (30)

trans-9e

cis-9f

entry

Substrate

Products (%)

cis-9b

trans-9b

cis-9c

C₆H₄-p-OMe

cis-16b (98)

trans-16b (96)

cis-16c (99)

C₆H₄-p-Br

trans-9c

trans-16c (99)

cis-9g

(8aR)-6g (66), (8aS)-6g (20)

cis-9d

cis-16d (96)

trans-16d (95)

cis-16f (96)

trans-9d

cis-9f

hydrolysis of the acetate group and the generation of the indolizi-

dine core, was carried out according to reported procedures (5%

methanolic KOH, reﬂux) [8a,9,20]. Initially, the cyclization was tried

with the major scission-addition products, compounds cis-9aeg

(Table 1, entries 1, 3e6, 8 and 9). The reaction generated the

indolizidinones (8aR)- and (8aS)-6aeg as a separable mixture

(dr from 3:2 to 3:1), generally in good yields.

Surprisingly, the spectroscopic data showed that the major

products were the 8a,2-trans isomers. For instance, the indolizidine

(8aR)-6a (Ar ¼ Ph) presented J_8a,8¼ 5.0, 13.7 Hz; J_8a,1¼ 5.2, 12.0 Hz;

J_1,2¼ 0, 4.2 Hz and J_2,3¼ 0, 4.3 Hz, which matched the theoretical

coupling constants for the trans isomer. On the other side, the

indolizidine (8aS)-6a (Ar ¼ Ph) presented J_8a,8¼ 4.4, 14.1 Hz;

J_8a,1¼ 6.3, 6.8 Hz; J_1,2¼ 6.5, 7.8 Hz and J_2,3¼ 5.2, 6.1 Hz, which were

similar to the theoretical coupling constants for the cis isomer [23].

The NOESY experiments also supported the assigned stereochemis-

tries [24]. The cyclization of two trans compounds (Table 1, entries 2

and7), trans-9a(Ar¼Ph)andtrans-9e(Ar¼2-naphthyl),alsoyielded

the (8aR)-derivatives as the major products [(8aR)-6a:(8aS)-6a, 9:5,

91% overall yield and (8aR)-6e:(8aS)-6e, 9:5, 83% overall yield].

These results can be explained as shown in Scheme 4. During

trans-9f

cis-9g

trans-16f (98)

cis-16g (97)

trans-16g (98)

trans-9g

be valuable to determine the importance of conformational re-

strictions on the biological activity, as will be commented later.

A summary of this Section is given in Scheme 5. Since hydrox-

ylated derivatives of cytotoxic alkaloids often present fewer side-

effects, especially on the CNS, different (hydroxy)pyrrolidine and

indolizidinone analogs were prepared. Interestingly, these hy-

droxylated compounds would be valuable to create libraries of

alkaloid analogs, in order to perform SAR studies. Thus, the elimi-

nation of the OR group or its replacement by other functional

groups (halo, amino, alkyl) would increase the product diversity,

affording derivatives with different volume, lipophilicity, ability to

form hydrogen bonds, etc. Alternatively, the OR group could also be

used to anchor probes, in order to study the biological targets and

the mechanisms of pharmacological action.

The indolizidinones are formed by cyclization of 2-substituted

pyrrolidines. The latter are generated from hydroxyproline am-

ides, using a one-pot decarboxylationealkylation process, which

allows the introduction of a variety of aryl and heteroaryl rings. The

stereogenic center with the acyloxy substituent allows stereo-

control of the addition step, and an easier separation of the

resulting diastereomers. Noteworthy, while in the addition step the

(2R) isomer is favored, in the cyclization step an epimerization (via

a retro-Michael reaction) takes place and the (8aR) isomer pre-

dominates. Since different methodologies allow the conversion of

inexpensive 4R-hydroxyproline into its 4S epimer [27], the opposite

series of alkaloid enantiomers could also be synthesized.

the base-promoted cyclization, due to the similar pK_aof the

protons of methyl aryl ketones (20e25) and aryl acetamides (25e

27) [25], non-selective enolization takes place. In addition to the

desired enolate from the aryl acetamide (not shown), the enolate of

the methyl ketone 14 is produced. A retro-Michael reaction follows

[20], yielding the unsaturated ketone 15, then a Michael addition

produces the 2-alkyl-4-(hydroxy)pyrrolidines 16, predominating

the thermodynamically favored 2,4-trans isomer. Subsequent

cyclization gives the indolizidinone (8aR)-6 as the major product.

The partial epimerization can be minimized under milder basic

conditions, as those needed just for acetate hydrolysis. Thus, on

treatment of the keto amides cis-9 (Table 2, entries 1, 3, 5, 7 and 9)

with methanolic potassium carbonate at 26 ^ꢀC, only the desired

(2R,4R)-products cis-16 were obtained. In a similar way, the keto

amides trans-9 (Table 2, entries 2, 4, 6, 8 and 10) provided solely the

(2S,4R)-products trans-16 [26].

The hydroxy pyrrolidines 16 are ﬂexible analogs of the rigid

indolizidinones 6, and the comparison of their cytotoxicities would

544

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

derivatives (compounds 16f and 16g) were screened for cyto-

toxicity against MCF-7 (Fig. 3, above) and SHSY-5Y (Fig. 3,

below) cell lines.

In these experiments, cells were exposed to the different alka-

loid derivatives under two different conditions: (a) 10

mM for 24 h;

(b) 30 M for 48 h. As shown in Fig. 3, in both cell lines, the biphenyl

derivatives cis- and trans-16d displayed a potent dose-dependent

cytotoxicity, which was comparable or even higher than the ac-

tivity of their indolizidinone analogs (at 30

isomers caused ꢂ80% cell mortality). Even at the lower dose

(10 M/24 h) considerable cytotoxicity was observed for the

biphenyl derivatives cis- and trans-16d, which was comparable to

the toxicity of (8aR)-6d in MCF7 cells at 30 M. On the contrary, the

mM in MCF7 cells, both

cytotoxicity of the p-methoxyphenyl (16b) and p-bromophenyl

(16c) derivatives was considerably lower than the activity of their

indolizidinone analogs, indicating that in those cases more struc-

tural ﬂexibility is detrimental for activity. In the case of the biphenyl

derivatives 16d, additional interactions (p-stacking, more perfect

ﬁt of the bulkier biphenyl group in receptor pockets) might account

for the difference in results.

With respect to the furan and pyrrole derivatives, the pyrroli-

dines 16f and 16g displayed lower cytotoxicity than their indolizi-

dinone analogs. Surprisingly, while in the indolizidinone series only

the pyrrole derivatives displayed signiﬁcant toxicity, in the pyrro-

lidine series the furan derivatives 16f outperformed the activity of

pyrrole analogs 16g. It must be said that while the furan compound

trans-16f presented signiﬁcant dose-dependent toxicity, its isomer

cis-16f and the pyrrole derivative trans-16g caused increased

mortality compared to vehicle, but the effects were not dose-

dependent, likely due to detrimental effects on the rate of cell

proliferation.

Scheme 5. Summary of the chemical section.

2.2. Evaluation of the biological activity

Finally, the effects of the most potent indolizidinone and pyr-

rolidine derivatives (the biphenyl compounds (8aS)-6d and cis-16d,

respectively) on the morphology of MCF7 and SHSY-5Y cells were

studied. Thus, both cell lines were exposed to indolizidinone (8aS)-

The cytotoxicity of the new indolizidinones was studied in the

human neuroblastoma SHSY-5Y and the human breast cancer MCF-

7 cell lines. Cells were exposed to the different alkaloid derivatives

under different conditions. Three treatments were initially used:

6d, at 30 mM for 48 h, and compared with cells treated under non-

toxic conditions in the presence of the vehicle. As shown in Figs. S1

and S2 in the Supplementary Information, besides the high cell

mortality described for these compounds, dramatic changes in the

morphology of the cells were also observed. These changes were

more evident in MCF-7 cells, where treatment with the pyrrolidine

(a) 10

the different treatments, cells were trypsinized, and the cell

viability was quantiﬁed. At the lower doses (10 and 20 M) during

mM for 24 h; (b) 20 mM for 24 h; (c) 30 mM for 48 h. Following

24 h, the effects of compounds on cell culture viability showed a

certain degree of toxicity but the results were not statistically sig-

niﬁcant. Therefore, we performed assays at the higher concentra-

tion of 30 mM and for a longer preincubation time, 48 h (Fig. 2)

observing a highly signiﬁcant speciﬁc toxicity with some of these

compounds.

cis-16d at a dose of 30 mM caused time-dependent morphological

changes in cell and nuclear volume, nucleolar refringency, and

cytoplasm vacuolization. These effects are evident after 6 h treat-

ment and reaches the height of its expression 48 h after exposure

(shown at 12 h, Supplementary Fig. S2). These early changes in

morphological features together with the cell swelling triggered by

the treatment indicate that compound cis-16d provokes cell death

through cell necrosis rather than apoptosis. Similar, although less

dramatic, effects were also observed with the SHSY-5Y tumor line

(Supplementary Fig. S2).

In summary, the indolizidinone analogs of cytotoxic phenan-

throindolizidine alkaloids also present a potent cytotoxic activity

against MCF7 human breast cancer cells and SHSY-5Y neuroblas-

toma cells. In general, breast cancer cells are more sensitive than

neuronal cells as reﬂected by their higher absolute mortality. The

biphenyl derivatives 6d were the most active ones, with (8aS)-6d

causing around 80% cell death in both tumor lines at micromolar

doses.

As observed in Fig. 2, in the case of MCF-7 cells, most com-

pounds exhibited a signiﬁcant level of toxicity under these exper-

imental conditions, while compounds (8aR)-6b, (8aR)-6c, (8aR)-

and (8aS)-6d, (8aR)-6e, (8aR)-6f, (8aR)- and (8aS)-6g were toxic in

SHSY-5Y cells. Interestingly, the p-methoxyphenyl 6b, p-bromo-

phenyl 6c, biphenyl 6d and pyrrole (8aS)-6g derivatives were toxic

at different levels in both cell lines.

The highest toxicities in both cell lines were obtained with the

para-substituted phenyl derivatives (6bed), particularly with the

biphenyl derivatives (8aS)-6d and (8aR)-6d. The lowest levels of

toxicity were observed with the phenyl derivatives (8aR)- and

(8aS)-6a, and the naphthyl derivatives (8aR)- and (8aS)-6e. With

respect to the heterocycle-substituted alkaloid analogs, the furan

derivatives (8aR)- and (8aS)-6f were among the less cytotoxic

alkaloid analogs, while the isomers of the pyrrole derivatives,

compounds (8aR)- and (8aS)-6g, displayed differential effects in

both cell lines.

The pyrrolidine series displayed cytotoxicity with respect to the

vehicle, but their activities were considerably lower than those

observed for their indolizidinone counterparts, suggesting that

more structural ﬂexibility decreases activity. An exception is the

biphenyl derivatives 16g, whose activity remained similar or even

increased with respect to indolizidinones 16d. Therefore, the bulky

Then, the pyrrolidine analogs of the most active indolizidi-

nones (compounds 16bed) and the furan and pyrrole

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

545

Fig. 2. Evaluation of the cytotoxicity of indolizidinones 6. (Above) Analyses of cell toxicity in MCF-7 cultures following exposure at 30

*p < 0.05; **p < 0.01; **p < 0.005 compared to vehicle. (Below) Analyses of cell toxicity in SHSY-5Y cultures following exposure at 30

*p < 0.05; **p < 0.01; ***p < 0.005 compared to vehicle [p ¼ probability; VEH ¼ vehicle].

M for 48 h to the different compounds.

biphenyl group must be responsible of additional interactions

which favor toxicity.

2-substituted pyrrolidines with a variety of aryl and heteroaryl

rings of different volumes and lipophilicities. Unlike other meth-

odologies, the 2R isomers were obtained preferentially. In a second

step, a cyclization generated the indolizidinones in excellent yields,

although a partial epimerization took place to afford the (8aR)-

isomers as the major products. In both cases, the isomers could be

readily separated. This is the shortest route available to prepare

these analogs, and proceeds with high efﬁciency.

3. Conclusion

A two-step procedure for the preparation of simpliﬁed, more

polar analogs of cytotoxic phenanthroindolizidine alkaloids is

described herein. Thus, hydroxylated indolizidinones and pyrroli-

dines were obtained from inexpensive hydroxyproline N-amides. A

one-pot decarboxylationealkylation reaction efﬁciently generated

The cytotoxic activity of the alkaloid analogs was studied against

the human neuroblastoma SHSY-5Y and human breast cancer MCF-

546

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

Fig. 3. Evaluation of the cytotoxicity of the hydroxylated pyrrolidines 16. (Above) Analyses of cell toxicity in MCF-7 cultures following exposure at 30

to the different compounds. #, ## and & indicate p < 0.05, p < 0.01 and p < 0.005 compared to vehicle at the corresponding dose and length of treatment. *, **p < 0.05 and p < 0.01

compared to 10 M/24 h. (Below) Analyses of cell toxicity in SHSY-5Y cultures following exposure at 30 M for 48 h and 10 M for 24 h to the different compounds. #, ## and &

mM for 48 h and 10 mM for 24 h

indicate p < 0.05, p < 0.01 and p < 0.005 compared to vehicle at the corresponding dose and length of treatment. *, ***p < 0.05 and p < 0.005 compared to 10

[p ¼ probability; VEH ¼ vehicle].

mM/24 h

7 cell lines. Most of the hydroxylated indolizidinones displayed

cytotoxic activities in both cell lines at 30 M for 48 h, particularly

4. Experimental section

the bulky biphenyl derivatives (8aS)-6d and (8aR)-6d; the former

caused around 80% cell death in both tumor lines at micromolar

doses. The activities of the pyrrolidine derivatives were in general

lower, indicating that more structural ﬂexibility is detrimental for

activity. However, the activities of the biphenyl derivatives cis-16d

and trans-16d were an exception, being comparable or superior to

those of the indolizidinones 6d. The SAR studies suggest that the

hydroxylated indolizidinones and the biphenyl-substituted pyrro-

lidines could be useful cytotoxic agents, with better hydrosolubility

(and possibly bioavailability) than the alkaloid leads.

4.1. Chemistry section

General Methods. General Remarks: Commercially available re-

agents and solvents were analytical grade or were puriﬁed by

standard procedures prior to use. All reactions involving air- or

moisture-sensitive materials were carried out under nitrogen at-

mosphere. Three alternative spray reagents for TLC analysis were

used: (a) 0.5% vanillin in H₂SO₄eEtOH (4:1); (b) 0.25% ninhydrin

in ethanol and (c) Fleet’s reagent [Ce(SO₄)₂(0.5 g) and ammonium

phosphomolybdate hydrate (2.5 g) in H₂SO₄(5 mL) and water

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

547

(65 mL)]. Once sprayed, the TLC was heated until development of

color. Merck silica gel 60 PF₂₅₄and 60 (0.063e0.2 mm) were used

for rotatory chromatography and column chromatography,

respectively. Melting points were determined with a hot-stage

apparatus and are uncorrected. Optical rotations were measured

at the sodium line at ambient temperature (26 ^ꢀC). IR spectra were

carried out using chloroform solutions of the products in 0.2 mm

cells. NMR spectra were determined at 500 or 400 MHz for ¹H and

125.7 or 100 MHz for ¹³C, using CDCl₃as solvent in the presence of

TMS as internal standard, at 25 ^ꢀC, unless otherwise stated. ¹H

4.1.1.3. (2S,4R)-4-(Acetoxy)-1-(2-phenylacetyl)proline (10). To a so-

lution of the benzyl ester 18 (930 mg, 2.44 mmol) in ethyl acetate

(30 mL) was added palladium (10% on activated carbon,150 mg). The

mixture was stirred under hydrogen atmosphere for 12 h, then was

ﬁltered through Celite and concentrated under vacuum, giving the

acid 10 (710 mg, 99%) as a colorless oil (two rotamers at 26 ^ꢀC; one

rotamer at 70 ^ꢀC); [

]

ꢁ30 (c 0.53, CH₃OH); IR

¼ 3018, 1744,

1705 cm^ꢁ1; ¹H NMR (500 MHz, 70 ^ꢀC, CDCl₃with TMS):

¼ 1.85 (s,

3H), 2.24 (ddd, J ¼ 2.0, 8.8,10.0 Hz,1H), 2.36 (ddd, J ¼ 6.7, 7.2,14.2 Hz,

1H), 3.58 (br d, J ¼ 12.1 Hz,1H), 3.62 (s, 2H), 3.65 (dd, J ¼ 4.6,11.8 Hz,

1H), 4.60 (dd, J ¼ 7.9, 8.2 Hz, 1H), 5.17 (m, 1H), 7.14e7.17 (m, 3H),

7.20e7.23 (m, 2H), 9.20 (br b, 1H); ¹³C NMR (125.7 MHz, 70 ^ꢀC):

NMR references: CDCl₃

77.0).

(d (d

7.26); ¹³C NMR references: CDCl₃

¼ 20.6 (CH₃), 33.9 (CH₂), 41.9 (CH₂), 53.1 (CH₂), 58.2 (CH), 72.4 (CH),

4.1.1. Preparation of proline precursor (10)

4.1.1.1. Benzyl (2S,4R)-4-(hydroxy)-1-(2-phenylacetyl)proline (17).

mixture of commercial trans- -4-hydroxyproline (1.5 g,

127.1 (CH), 128.8 (2ꢃ CH), 128.9 (2ꢃ CH), 133.5 (C), 170.1 (C), 172.0

(C), 172.5 (C); MS (EI): m/z 291 (M^þ, <1), 247 (M^þþ H ꢁ COOH, 15),

231 (M^þꢁ HOAc,12), 91 ([PhCH₂]^þ,100); HRMS calcd for C₁₅H₁₇NO₅

[M^þ] 291.1107, found 291.1120. Anal. Calcd for C₁₅H₁₇NO₅: C, 61.85;

H, 5.88; N, 4.81; Found: C, 61.96; H, 5.87; N, 4.91.

11.4 mmol), TsOH$H₂O (2.2 g, 11.6 mmol), and benzyl alcohol

(9 mL) in toluene (9 mL) was heated under reﬂux overnight. Then it

was cooled to room temperature (26 ^ꢀC) and the solvents were

evaporated under vacuum. The crude residue was dissolved in THF

(30 mL), and added to a saturated aqueous NaHCO₃solution

(25 mL). The mixture was cooled to 0 ^ꢀC and then 2-phenylacetyl

chloride (3.8 mL, 28.7 mmol) was added dropwise. After stirring

at room temperature overnight, the mixture was treated with

aqueous 10% HCl until pH ¼ 5, and extracted with EtOAc. The

organic layer was dried over sodium sulfate, ﬁltered and evapo-

rated under vacuum. The residue was puriﬁed by column chro-

matography (hexanes:EtOAc, 4:6), affording the phenyl acetamide

17 (2.21 g, 57%) as a white solid (two rotamers at 26 ^ꢀC; one

4.1.2. Preparation of the nucleophiles (silyl enol ethers) for the

scissionealkylation process

One of the nucleophiles (1-phenyl-1-(trimethylsiloxy)ethene)

was commercial, but the others were prepared from their corre-

sponding methyl aryl ketones. Thus, a solution of TMSOTf (0.67 mL,

3.5 mmol) in dichloromethane (3 mL) was added dropwise to a

solution of the starting methyl ketone (3.5 mmol) and triethyl-

amine (1.1 mL) in dichloromethane (15 mL), cooled at 0 ^ꢀC. The

mixture was stirred at 0e5 ^ꢀC for 30 min, and then was allowed to

reach 26 ^ꢀC and stirred for 1 h. Then it was diluted with hexanes,

and washed with saturated aqueous NaHCO₃solution and water.

The organic layer was dried over anhydrous sodium sulfate, ﬁltered

and evaporated under vacuum to afford the silyl enol ether, which

was used without further puriﬁcation in the next step.

The starting amounts for each ketone were: methyl 4-bromo-

phenyl ketone (677 mg, 3.5 mmol), methyl 4-methoxyphenyl ke-

tone (525 mg, 3.5 mmol), methyl 4-(phenyl)phenyl ketone (700 mg,

3.5 mmol), methyl 2-naphthalenyl ketone (600 mg, 3.5 mmol),

methyl 2-furyl ketone (0.35 mL, 3.5 mmol), methyl N-methyl-2-

pyrrolyl ketone (0.41 mL, 3.5 mmol).

rotamer at 70 ^ꢀC); mp 78e80 ^ꢀC; [

]

;

ꢁ63 (c 0.53, CH₃OH); IR

¼ 3613, 3423, 3018, 1744, 1645 cm^ꢁ1

¹H NMR (500 MHz, 70 ^ꢀC,

CDCl₃with TMS):

¼ 1.96 (m, 1H), 2.14 (m, 1H), 3.10 (br b, 1H), 3.41

(br d, J ¼ 11 Hz, 1H), 3.57 (dd, J ¼ 4.7, 10.8 Hz, 1H), 3.60 (br s, 2H),

4.35 (m, 1H), 4.62 (dd, J ¼ 7.6, 7.8 Hz, 1H), 5.09 (d, J ¼ 12.3 Hz, 1H),

5.13 (d, J ¼ 12.4 Hz, 1H), 7.10e7.32 (m, 10H); ¹³C NMR (125.7 MHz,

70 ^ꢀC):

¼ 37.7 (CH₂), 41.9 (CH₂), 55.4 (CH₂), 58.0 (CH), 67.0 (CH₂),

70.3 (CH), 126.9 (2ꢃ CH), 128.0 (2ꢃ CH), 128.1 (CH), 128.5 (CH),

128.7 (2ꢃ CH), 129.0 (2ꢃ CH), 134.4 (C), 135.9 (C), 170.1 (C), 171.9

(C); MS (EI): m/z 339 (M^þ, 9), 204 (M^þꢁ COOBn, 32), 91 ([PhCH₂]^þ,

80), 86 (M^þþ H ꢁ [COOBn þ COCH₂Ph], 100); HRMS calcd for

₂₀H₂₁NO₄[M^þ] 339.1471; found 339.1460. Anal. Calcd for

₂₀H₂₁NO₄: C, 70.78; H, 6.24; N, 4.13; Found: C, 70.84; H, 6.20; N,

4.1.3. General procedure for the scissionealkylation process

A solution of acid 10 (200 mg, 0.7 mmol) in dry dichloromethane

(10 mL) was treated with DIB (437 mg, 1.36 mmol) and iodine

(87 mg, 0.34 mmol), and was stirred under nitrogen at 26 ^ꢀC for 3 h,

under irradiation with visible light (80 W tungsten-ﬁlament lamp).

The solution was cooled to 0 ^ꢀC, and the silyl enol ether (3.5 mmol)

4.15.

4.1.1.2. Benzyl (2S,4R)-4-(acetoxy)-1-(2-phenylacetyl)proline (18).

A solution of the 4-(hydroxy)proline derivative 17 (900 mg,

2.65 mmol) in dry pyridine (10 mL) was cooled to 0 ^ꢀC and treated

with acetic anhydride (3 mL). The solution was allowed to reach

room temperature and stirred for 12 h; then it was extracted and

concentrated as commented before. The residue was puriﬁed by

column chromatography (hexanes:EtOAc, 6:4), to give the proline

derivative 18 (930 mg, 92%) as a colorless oil (two rotamers at 26 ^ꢀC;

and boron triﬂuoride (170 mL, 1.35 mmol) was added. The mixture

was stirred for 90 min and then was poured into a 1:1 mixture of

aqueous 10% Na₂S₂O₃and saturated aqueous NaHCO₃solution.

After extraction with CH₂Cl₂, the organic layer was dried over

anhydrous Na₂SO₄, ﬁltered and evaporated under vacuum. The

residue was puriﬁed by ﬂash chromatography (dichloromethane/

EtOAc) to give the products.

one rotamer at 70 ^ꢀC); [

]

ꢁ23 (c 0.24, CHCl₃); IR

¼ 3033, 1742,

1649 cm^ꢁ^{1 1}H NMR (500 MHz, 70 ^ꢀC):

;

¼ 1.85 (s, 3H), 2.06 (ddd,

J ¼ 5.8, 7.6, 13.9 Hz, 1H), 2.25 (dd, J ¼ 10.0, 12.3 Hz, 1H), 3.51 (br d,

J ¼ 11.6 Hz, 1H), 3.57 (s, 2H), 3.65 (dd, J ¼ 4.7, 11.5 Hz, 1H), 4.58 (dd,

J ¼ 7.9, 8.2 Hz, 1H), 5.05 (d, J ¼ 12.1 Hz, 1H), 5.09 (d, J ¼ 12.5 Hz, 1H),

5.14 (m, 1H), 7.09e7.25 (m, 10H); ¹³C NMR (125.7 MHz, 70 ^ꢀC):

4.1.3.1. (2R,4R)-4-(Acetoxy)-2-(2-oxo-2-phenylethyl)-1-(2-phenylace-

tyl)pyrrolidine (cis-9a) and (2S,4R)-4-(acetoxy)-2-(2-[4-

methoxyphenyl]-2-oxoethyl)-1-(2-phenylacetyl)pyrrolidine (trans-9a).

Obtained from acid 10 according to the General Procedure, using

¼ 20.9 (CH₃), 34.7 (CH₂), 42.0 (CH₂), 52.8 (CH₂), 57.8 (CH), 67.1

commercial 1-phenyl-1-(trimethylsilyloxy)ethylene (710

mL,

(CH₂), 72.9 (CH), 126.9 (2ꢃ CH), 128.2 (2ꢃ CH), 128.3 (CH), 128.5

(CH), 128.7 (2ꢃ CH), 128.9 (2ꢃ CH), 134.1 (C), 135.8 (C), 169.9 (C),

170.0 (C),171.4 (C); MS (EI): m/z 381 (M^þ, 3), 321 (M^þꢁ HOAc, 6), 91

([PhCH₂]^þ, 100); HRMS calcd for C₂₂H₂₃NO₅[M^þ] 381.1576; found

381.1568. Anal. Calcd for C₂₂H₂₃NO₅: C, 69.28; H, 6.08; N, 3.67;

Found: C, 69.42; H, 6.22; N, 3.79.

3.5 mmol) as the nucleophile. After column chromatography

(CH₂Cl₂/EtOAc, 9:1), compounds cis-9a (194 mg, 77%) and trans-9a

(42 mg, 17%) were isolated (94% overall yield, 236 mg, dr 5:1).

Compound cis-9a was obtained as a crystalline solid; mp 99e

100 ^ꢀC (EtOAc/hexane); [

]

_Dþ26 (c 0.53, CHCl₃); IR

¼ 3017, 1737,

1682,1639 cm^ꢁ1; ¹H NMR (500 MHz) A 7:1 mixture of rotamers was

548

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

observed, only the major rotamer is described:

¼ 2.06 (s, 3H), 2.07

₂₂H₂₂NO₄⁷⁹Br [M^þ] 443.0732, found 443.0751. Anal. Calcd for.

₂₂H₂₂NO₄Br: C, 59.47; H, 4.99; N, 3.15; Found: C, 59.15; H, 4.83; N,

(br d, J ¼ 14.5 Hz, 1H), 2.30 (ddd, J ¼ 6.0, 8.9, 14.8 Hz, 1H), 3.04 (dd,

J ¼ 10.7,15.5 Hz,1H), 3.62 (br d, J ¼ 12.3 Hz,1H), 3.66 (s, 2H), 3.78 (dd,

J ¼ 5.3,12 Hz,1H), 4.01 (dd, J ¼ 3.2,15.5 Hz,1H), 4.69 (m,1H), 5.29 (m,

1H), 7.28 (d, J ¼ 6.9 Hz, 2H), 7.34 (dd, J ¼ 7.2, 7.4 Hz, 2H), 7.46 (dd,

J ¼ 7.5, 7.9 Hz, 2H), 7.55 (dd, J ¼ 7.4, 7.4 Hz,1H), 8.04 (d, J ¼ 7.9 Hz, 2H);

2.86.

Compound trans-9c was obtained as a crystalline solid; mp

105e106 ^ꢀC (EtOAc/hexane); [

ꢁ19 (c 0.30, CHCl₃); IR ¼ 3017,

1739, 1681, 1641 cm^ꢁ^{1 1}H NMR (500 MHz) A 20:1 mixture of

rotamers was observed, only the major rotamer is described:

]

;

¹³C NMR (125.7 MHz)

d 21.2 (CH₃), 34.9 (CH₂), 42.2 (CH₂), 42.5 (CH₂),

53.1 (CH₂), 54.2 (CH), 73.6 (CH), 127.1 (CH), 128.3 (2ꢃ CH), 128.7 (2ꢃ

CH), 128.8 (2ꢃ CH), 129.1 (2ꢃ CH), 133.3 (CH), 134.2 (C), 136.7 (C),

169.8 (C), 170.2 (C), 198.6 (C); MS (EI): m/z ¼ 365 (M^þ, 3), 246

(M^þꢁ CH₂COPh, 14), 186 (M^þꢁ [CH₂COPh þ HOAc], 28), 105

([PhCO]^þ, 92), 91 ([PhCH₂]^þ, 100); HRMS calcd for C₂₂H₂₃NO₄[M^þ]

365.1627, found 365.1619. Anal. Calcd for. C₂₂H₂₃NO₄: C, 72.31; H,

6.34; N, 3.83; Found: C, 72.36; H, 6.47; N, 3.96.

¼ 1.91 (s, 3H), 2.05 (ddd, J ¼ 5.1, 7.6, 13.8 Hz, 1H), 2.34 (brdd,

J ¼ 7.8, 14.5 Hz, 1H), 2.98 (dd, J ¼ 8.9, 15.8 Hz, 1H), 3.63 (br s, 2H),

3.64e3.72 (m, 2H), 3.88 (dd, J ¼ 3.2,15.8 Hz,1H), 4.59 (dddd, J ¼ 2.6,

8.2, 8.2, 8.2 Hz,1H), 5.21 (m, 1H), 7.22e7.26 (m, 3H), 7.31 (dd, J ¼ 7.0,

7.6 Hz, 2H), 7.59 (d, J ¼ 8.8 Hz, 2H), 7.87 (d, J ¼ 8.2 Hz, 2H); ¹³C NMR

(125.7 MHz):

¼ 20.9 (CH₃), 36.4 (CH₂), 42.1 (CH₂), 42.7 (CH₂), 52.8

(CH₂), 53.3 (CH), 72.5 (CH), 126.9 (CH), 128.5 (C), 128.7 (2ꢃ CH),

128.8 (2ꢃ CH), 129.8 (2ꢃ CH), 131.9 (2ꢃ CH), 134.3 (C), 135.3 (C),

170.1 (C), 170.4 (C), 197.3 (C); MS (EI): m/z 443/445 (M^þ, 3/3), 324/

326 (M^þꢁ COCH₂Ph, 20/20), 183/185 ([4-Br-C₆H₄CO]^þ, 61/62), 91

([PhCH₂]^þ, 100); HRMS calcd for C₂₂H₂₂NO₄⁸¹Br [M^þ] 445.0712,

Compound trans-9a was obtained as a colorless gum; [

]

_Dꢁ12 (c

0.77, CHCl₃); IR

¼ 3010, 1735, 1681, 1639, 1425 cm^ꢁ1

;

¹H NMR

(500 MHz) A 20:1 mixture of rotamers was observed, only the

major rotamer is described:

¼ 1.92 (s, 3H), 2.06 (m, 1H), 2.36 (m,

1H), 3.05 (dd, J ¼ 9.0, 16 Hz, 1H), 3.64 (s, 2H), 3.65 (d, J ¼ 11 Hz, 1H),

3.70 (dd, J ¼ 4.1, 12 Hz,1H), 3.94 (dd, J ¼ 3.1, 16.1 Hz,1H), 4.63 (dddd,

J ¼ 3.1, 8.1, 8.2, 8.6 Hz, 1H), 5.21 (m, 1H), 7.23 (m, 1H), 7.24 (d,

J ¼ 8.1 Hz, 2H), 7.31 (dd, J ¼ 7.5, 8.0 Hz, 2H), 7.46 (dd, J ¼ 7.6, 7.8 Hz,

2H), 7.56 (dd, J ¼ 7.3, 7.5 Hz, 1H), 7.99 (d, J ¼ 7.8 Hz, 2H); ¹³C NMR

found 445.0697; calcd for C

₂₂H₂₂NO₄⁷⁹Br 443.0732, found

443.0750. Anal. Calcd for. C₂₂H₂₂NO₄Br: C, 59.47; H, 4.99; N, 3.15;

Found: C, 59.65; H, 4.83; N, 3.10.

4.1.3.4. (2R,4R)-4-(Acetoxy)-2-(2-[p-biphenyl]-2-oxoethyl)-1-(2-

phenylacetyl)pyrrolidine

(2-[p-biphenyl]-2-oxoethyl)-1-(2-phenylacetyl)pyrrolidine

9d). Obtained from acid 10 according to the General Procedure,

using 1-[4-(phenyl)phenyl]-1-(trimethylsilyloxy)ethylene as the

nucleophile. After column chromatography (CH₂Cl₂/EtOAc, 9:1),

compounds cis-9d (223 mg, 74%) and trans-9d (51 mg, 17%) were

isolated (91% overall yield, 274 mg, dr 4:1).

(125.7 MHz):

¼ 20.8 (CH₃), 36.5 (CH₂), 42.1 (CH₂), 42.8 (CH₂), 53.1

(cis-9d)

and

(2S,4R)-4-(acetoxy)-2-

(trans-

(CH₂), 53.5 (CH), 72.7 (CH), 126.9 (CH), 128.2 (2ꢃ CH), 128.6 (2ꢃ

CH), 128.7 (2ꢃ CH), 128.8 (2ꢃ CH), 133.2 (CH), 134.4 (C), 136.7 (C),

170.1 (C), 170.4 (C), 198.4 (C); MS (EI): m/z ¼ 365 (M^þ, 7), 246

(M^þꢁ CH₂COPh, 16), 186 (M^þꢁ [CH₂COPh þ HOAc], 31), 105

([PhCO]^þ, 99), 91 ([PhCH₂]^þ, 100); HRMS calcd for C₂₂H₂₃NO₄[M^þ]

365.1627, found 365.1616. Anal. Calcd for. C₂₂H₂₃NO₄: C, 72.31; H,

6.34; N, 3.83; Found: C, 72.40; H, 6.42; N, 3.91.

Compound cis-9d was obtained as a colorless oil; [

]

þ21 (c

0.30, CHCl₃); IR

¼ 3016, 2929,1736,1644 cm^ꢁ1; ¹H NMR (500 MHz)

4.1.3.2. (2R,4R)-4-(Acetoxy)-2-(2-[4-methoxyphenyl]-2-oxoethyl)-1-

A 4:1 mixture of rotamers was observed, only the major rotamer is

(2-phenylacetyl)pyrrolidine (cis-9b) and (2S,4R)-4-(acetoxy)-2-(2-[4-

described:

¼ 2.06 (s, 3H), 2.10 (br d, J ¼ 15.1 Hz, 1H), 2.30 (ddd,

methoxyphenyl]-2-oxoethyl)-1-(2-phenylacetyl)pyrrolidine

(trans-

J ¼ 5.7, 8.8, 14.5 Hz, 1H), 3.06 (dd, J ¼ 10.8, 15.2 Hz, 1H), 3.64 (d,

J ¼ 12.0 Hz, 2H), 3.67 (s, 2H), 3.80 (dd, J ¼ 5.7, 12.0 Hz, 1H), 4.04 (dd,

J ¼ 3.2, 15.8 Hz, 1H), 4.72 (m, 1H), 5.30 (m, 1H), 7.27e7.30 (m, 3H),

7.34 (dd, J ¼ 7.0, 7.3 Hz, 2H), 7.39 (dd, J ¼ 7.5, 7.6 Hz, 1H), 7.46 (dd,

J ¼ 7.6, 7.6 Hz, 2H), 7.62 (d, J ¼ 7.0 Hz, 2H), 7.69 (d, J ¼ 8.2 Hz, 2H),

9b). Obtained from acid 10 according to the General Procedure,

using 1-(4-methoxyphenyl)-1-(trimethylsilyloxy)ethylene as the

nucleophile. After column chromatography (CH₂Cl₂/EtOAc, 8:2),

compounds cis-9b (214 mg, 79%) and trans-9b (38 mg, 14%) were

isolated (93% overall yield, 252 mg, dr 5:1). Products cis-9b and

trans-9b were previously reported [9].

8.12 (d, J ¼ 8.2 Hz, 2H); ¹³C NMR (125.7 MHz):

¼ 21.1 (CH₃), 34.8

(CH₂), 42.2 (CH₂), 42.4 (CH₂), 53.1 (CH₂), 54.3 (CH), 73.6 (CH), 127.0

(2ꢃ CH), 127.2 (2ꢃ CH), 127.3 (2ꢃ CH), 128.2 (CH), 128.7 (2ꢃ CH),

128.9 (3ꢃ CH), 129.0 (2ꢃ CH), 134.1 (C),135.3 (C), 139.8 (C), 145.9 (C),

169.9 (C), 170.3 (C), 198.3 (C); MS (EI): m/z 441 (M^þ, 5), 322

(M^þꢁ COCH₂Ph, 19), 262 (M^þꢁ [COCH₂Ph þ AcOH], 29), 196

([CH₂COC₆H₄Ph þ H]^þ, 47), 181 ([CO-C₆H₄-Ph]^þ, 90), 153 ([C₆H₄-

Ph]^þ, 23), 152 ([C₆H₄-Ph ꢁ H]^þ, 34), 91 ([CH₂Ph]^þ, 100); HRMS calcd

for C₂₈H₂₇NO₄[M^þ] 441.1940, found 441.1961. Anal. Calcd for.

4.1.3.3. Preparation of (2R,4R)-4-(acetoxy)-2-(2-[4-bromophenyl]-2-

oxoethyl)-1-(2-phenylacetyl) pyrrolidine (cis-9c) and (2S,4R)-4-

(acetoxy)-2-(2-[4-bromophenyl]-2-oxoethyl)-1-(2-phenylacetyl)

pyrrolidine (trans-9c). Obtained from acid 10 according to the

General Procedure, using 1-(4-bromophenyl)-1-(trimethylsilyloxy)

ethylene as the nucleophile. After column chromatography

(CH₂Cl₂/EtOAc, 95:5), compounds cis-9c (246 mg, 81%) and trans-9c

(37 mg, 12%) were isolated (93% overall yield, 283 mg, dr 6:1).

C₂₈H₂₇NO₄: C, 76.17; H, 6.16; N, 3.17; Found: C, 76.18; H, 6.15; N, 3.06.

Compound trans-9d was obtained as a colorless oil; [

0.44, CHCl₃); IR

A 15:1 mixture of rotamers was observed, only the major rotamer is

described:

_Dꢁ13 (c

Compound cis-9c was obtained as a colorless gum; [

]

þ31 (c

¼ 3019,1735,1644,1418 cm^ꢁ1; ¹H NMR (500 MHz)

0.66, CHCl₃); IR

¼ 3018,1740,1681,1642 cm^ꢁ1; ¹H NMR (500 MHz)

A 7:1 mixture of rotamers was observed, only the major rotamer is

¼ 1.92 (s, 3H), 2.09 (ddd, J ¼ 5.4, 7.5, 14.5 Hz, 1H), 2.37

described:

¼ 2.05 (s, 3H), 2.06 (br d, J ¼ 14.5 Hz, 1H), 2.28 (ddd,

(brdd, J ¼ 7.9, 14.2 Hz, 1H), 3.07 (dd, J ¼ 9.2, 15.8 Hz, 1H), 3.62e3.75

(m, 2H), 3.66 (br s, 2H), 3.96 (br d, J ¼ 15.8 Hz,1H), 4.65 (m,1H), 5.22

(m, 1H), 7.24 (m, 1H), 7.25 (d, J ¼ 7.3 Hz, 2H), 7.31 (dd, J ¼ 7.3, 7.6 Hz,

2H), 7.39 (dd, J ¼ 7.3, 7.6 Hz,1H), 7.47 (dd, J ¼ 7.3, 7.9 Hz, 2H), 7.62 (d,

J ¼ 5.7, 8.9, 14.5 Hz, 1H), 2.98 (dd, J ¼ 10.9, 15.5 Hz, 1H), 3.62 (br d,

J ¼ 12.0 Hz, 1H), 3.65 (br s, 2H), 3.78 (dd, J ¼ 5.1, 12.0 Hz, 1H), 3.97

(dd, J ¼ 3.2, 15.2 Hz, 1H), 4.64 (m, 1H), 5.29 (m, 1H), 7.21e7.30 (m,

3H), 7.34 (dd, J ¼ 7.6, 7.6 Hz, 2H), 7.60 (d, J ¼ 8.2 Hz, 2H), 7.91 (d,

J ¼ 7.3 Hz, 2H), 7.68 (d, J ¼ 8.2 Hz, 2H), 8.07 (d, J ¼ 8.2 Hz, 2H); ¹³

J ¼ 8.8 Hz, 2H); ¹³C NMR (125.7 MHz):

¼ 21.1 (CH₃), 34.8 (CH₂),

NMR (125.7 MHz):

¼ 20.9 (CH₃), 36.4 (CH₂), 42.1 (CH₂), 42.7 (CH₂),

42.2 (CH₂), 42.4 (CH₂), 53.1 (CH₂), 54.1 (CH), 73.5 (CH), 127.0 (CH),

128.7 (C), 128.7 (2ꢃ CH), 129.0 (2ꢃ CH), 129.9 (2ꢃ CH), 132.0 (2ꢃ

CH), 134.0 (C), 135.3 (C), 169.9 (C), 170.2 (C), 197.7 (C); MS (EI): m/

z ¼ 443/445 (M^þ, 2/2), 324/326 (M^þꢁ COCH₂Ph, 14/15), 183/185

([4-Br-C₆H₄CO]^þ, 47/45), 91 ([PhCH₂]^þ, 100); HRMS calcd for

52.9 (CH₂), 53.5 (CH), 72.6 (CH), 126.9 (2ꢃ CH), 127.23 (2ꢃ CH),

127.26 (2ꢃ CH), 128.2 (CH), 128.7 (2ꢃ CH), 128.80 (2ꢃ CH), 128.84

(CH), 128.9 (2ꢃ CH), 134.3 (C), 135.3 (C), 139.8 (C), 145.9 (C), 170.2

(C), 170.5 (C), 197.9 (C); MS (EI): m/z 441 (M^þ, 9), 322

(M^þꢁ COCH₂Ph, 33), 262 (M^þꢁ [COCH₂Ph þ AcOH], 52), 196

([CH₂COC₆H₄Ph þ H]^þ, 58), 181 ([CO-C₆H₄-Ph]^þ, 100), 153 ([C₆H₄-

₂₂H₂₂NO₄⁸¹Br [M^þ] 445.0712, found 445.0696; calcd for

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

549

Ph]^þ, 33), 91 ([CH₂Ph]^þ, 80); HRMS calcd for C₂₈H₂₇NO₄[M^þ]

441.1940, found 441.1923. Anal. Calcd for. C₂₈H₂₇NO₄: C, 76.17; H,

6.16; N, 3.17; Found: C, 76.15; H, 6.32; N, 2.86.

5.29 (m, 1H), 6.52 (dd, J ¼ 1.7, 3.6 Hz, 1H), 7.23e7.30 (m, 3H), 7.33

(dd, J ¼ 7.6, 7.6 Hz, 2H), 7.40 (d, J ¼ 3.6 Hz, 1H), 7.58 (d, J ¼ 1.7 Hz,

1H); ¹³C NMR (125.7 MHz)

¼ 20.9 (CH₃), 34.6 (CH₂), 41.9 (CH₂),

42.2 (CH₂), 52.9 (CH₂), 53.9 (CH), 73.3 (CH), 112.0 (CH), 118.0 (CH),

126.7 (CH),128.5 (2ꢃ CH),128.7 (2ꢃ CH),133.8 (C),146.4 (CH),152.2

(C), 169.6 (C), 170.0 (C), 187.0 (C); MS (EI): m/z 355 (M^þ, 7), 236

(M^þꢁ COCH₂Ph, 22), 176 (M^þꢁ [COCH₂Ph þ AcOH], 35), 91

([CH₂Ph]^þ, 100); HRMS calcd for C₂₀H₂₁NO₅[M^þ] 355.1420, found

355.1407. Anal. Calcd for. C₂₀H₂₁NO₅: C, 67.59; H, 5.96; N, 3.94;

Found: C, 67.52; H, 6.34; N, 4.08.

4.1.3.5. (2R,4R)-4-(Acetoxy)-2-(2-[naphthalen-2-yl]-2-oxoethyl)-1-

(2-phenylacetyl)pyrrolidine (cis-9e) and (2S,4R)-4-(acetoxy)-2-(2-

[naphthalen-2-yl]-2-oxoethyl)-1-(2-phenylacetyl)pyrrolidine (trans-

9e). Obtained from acid 10 according to the General Procedure,

using 1-(2-naphthalenyl)-1-(trimethylsilyloxy)ethylene as the

nucleophile. After column chromatography (CH₂Cl₂/EtOAc, 9:1),

compounds cis-9e (231 mg, 81%) and trans-9e (31 mg, 11%) were

isolated (92% overall yield, 262 mg, dr 7:1).

Compound trans-9f was obtained as a colorless oil; [

]

ꢁ10 (c

0.31, CHCl₃); IR

¼ 3019,1737,1640, 1466 cm^ꢁ1; ¹H NMR (500 MHz)

Compound cis-9e was obtained as a colorless oil; [

]

þ26 (c

A 20:1 mixture of rotamers was observed, only the major rotamer is

0.35, CHCl₃); IR

¼ 3022, 1738, 1675, 1640 cm^ꢁ1

;

¹H NMR

described:

¼ 1.91 (s, 3H), 2.13 (ddd, J ¼ 5.7, 8.2, 14.5 Hz, 1H), 2.33

(500 MHz) A 7:1 mixture of rotamers was observed, only the major

(m, 1H), 3.02 (dd, J ¼ 8.2, 15.8 Hz, 1H), 3.60e3.70 (m, 3H), 3.63 (br s,

2H), 4.59 (m, 1H), 5.20 (m, 1H), 6.51 (dd, J ¼ 1.9, 3.8 Hz, 1H), 7.20e

7.26 (m, 3H), 7.28e7.33 (m, 3H), 7.57 (d, J ¼ 1.8 Hz, 1H); ¹³C NMR

rotamer is described:

¼ 2.05 (s, 3H), 2.12 (br d, J ¼ 14.5 Hz, 1H),

2.32 (ddd, J ¼ 5.7, 8.8, 14.8 Hz, 1H), 3.17 (dd, J ¼ 10.7, 15.5 Hz, 1H),

3.64 (d, J ¼ 12.0 Hz, 1H), 3.69 (br s, 2H), 3.80 (dd, J ¼ 5.3, 12.0 Hz,

1H), 4.15 (dd, J ¼ 3.2, 15.5 Hz, 1H), 4.76 (m, 1H), 5.30 (m, 1H), 7.28

(m,1H), 7.29 (d, J ¼ 7.3 Hz, 2H), 7.34 (dd, J ¼ 7.3, 7.6 Hz, 2H), 7.54 (dd,

J ¼ 7.4, 7.9 Hz, 1H), 7.59 (dd, J ¼ 7.3, 8.2 Hz, 1H), 7.86 (d, J ¼ 8.2 Hz,

1H), 7.89 (d, J ¼ 8.8 Hz, 1H), 7.99 (d, J ¼ 8.0 Hz, 1H), 8.06 (dd, J ¼ 1.9,

(125.7 MHz):

¼ 20.9 (CH₃), 36.2 (CH₂), 41.6 (CH₂), 42.7 (CH₂), 52.9

(CH₂), 53.2 (CH), 72.6 (CH), 112.0 (CH), 118.1 (CH), 126.8 (CH), 128.7

(2ꢃ CH), 128.8 (2ꢃ CH), 134.3 (C), 146.6 (CH), 152.4 (C), 170.0 (C),

170.4 (C), 186.9 (C); MS (EI) m/z 355 (M^þ, 3), 236 (M^þꢁ COCH₂Ph,

7), 176 (M^þꢁ [COCH₂Ph þ AcOH], 13), 91 ([CH₂Ph]^þ, 100); HRMS

calcd for C₂₀H₂₁NO₅[M^þ] 355.1420, found 355.1422. Anal. Calcd for.

8.8 Hz, 1H), 8.61 (br s, 1H); ¹³C NMR (125.7 MHz):

¼ 21.1 (CH₃),

34.9 (CH₂), 42.3 (CH₂), 42.5 (CH₂), 53.1 (CH₂), 54.4 (CH), 73.6 (CH),

123.8 (CH), 126.7 (CH), 127.0 (CH), 127.7 (CH), 128.5 (2ꢃ CH), 128.7

(2ꢃ CH), 128.9 (2ꢃ CH), 129.7 (CH), 130.2 (CH), 132.6 (C), 134.0 (C),

134.1 (C), 135.6 (C), 169.9 (C), 170.3 (C), 198.6 (C); MS (EI): m/z 415

(M^þ, 4), 296 (M^þꢁ COCH₂Ph, 17), 155 ([CO-naphthyl]^þ, 100); HRMS

calcd for C₂₆H₂₅NO₄[M^þ] 415.1784, found 415.1791. Anal. Calcd for.

C₂₀H₂₁NO₅: C, 67.59; H, 5.96; N, 3.94; Found: C, 67.39; H, 6.34; N,

4.24.

4.1.3.7. (2R,4R)-4-(Acetoxy)-2-(2-[1-methyl-1H-pyrrol-2-yl]-2-

oxoethyl)-1-(2-phenylacetyl)pyrrolidine (cis-9g) and (2S,4R)-4-(ace-

toxy)-2-(2-[1-methyl-1H-pyrrol-2-yl]-2-oxoethyl)-1-(2-

phenylacetyl) pyrrolidine (trans-9g). Obtained from acid 10 ac-

cording to the General Procedure, using 1-(N-methyl-2-pyrrolyl)-1-

(trimethylsilyloxy)ethylene as the nucleophile. After column

chromatography (CH₂Cl₂/EtOAc, 9:1), compounds cis-9g (176 mg,

70%) and trans-9g (34 mg, 13%) were isolated (83% overall yield,

210 mg, dr 5:1).

₂₆H₂₅NO₄: C, 75.16; H, 6.06; N, 3.37; Found: C, 75.31; H, 6.11; N,

3.00.

Compound trans-9e was obtained as a colorless oil; [

;

]

_Dꢁ12 (c

0.39, CHCl₃); IR

¼ 3015, 1735, 1675, 1637, 1427 cm^ꢁ1

¹H NMR

(500 MHz) A 20:1 mixture of rotamers was observed, only the

major rotamer is described:

¼ 1.91 (s, 3H), 2.12 (ddd, J ¼ 5.1, 7.6,

14.5 Hz, 1H), 2.39 (brdd, J ¼ 8.2, 14.5 Hz, 1H), 3.18 (dd, J ¼ 8.9,

15.8 Hz, 1H), 3.66 (br s, 2H), 3.67 (br d, J ¼ 12 Hz, 1H), 3.72 (dd,

J ¼ 3.8, 12.0 Hz, 1H), 4.07 (dd, J ¼ 3.2, 15.8 Hz, 1H), 4.70 (dddd,

J ¼ 3.2, 8.2, 8.2, 8.5 Hz,1H), 5.23 (m,1H), 7.23e7.27 (m, 3H), 7.31 (dd,

J ¼ 7.0, 7.6 Hz, 2H), 7.55 (dd, J ¼ 7.0, 8.2 Hz, 1H), 7.60 (dd, J ¼ 7.0,

8.2 Hz, 1H), 7.86 (d, J ¼ 8.2 Hz, 1H), 7.88 (d, J ¼ 8.8 Hz, 1H), 7.98 (d,

J ¼ 8.2 Hz, 1H), 8.03 (dd, J ¼ 1.5, 8.8 Hz, 1H), 8.57 (br s, 1H); ¹³C NMR

Compound cis-9g was obtained as a colorless oil; [

]_Dþ15 (c 0.40,

CHCl₃); IR

¼ 3010,1738,1640,1408 cm^ꢁ1; ¹H NMR (500 MHz) A 4:1

mixture of rotamers was observed, only the major rotamer is

described:

¼ 2.07 (s, 3H), 2.12 (br d, J ¼ 14.0 Hz, 1H), 2.23 (m, 1H),

2.80 (dd, J ¼ 11.4,14.5 Hz,1H), 3.60 (d, J ¼ 12.0 Hz,1H), 3.66 (br s, 2H),

3.71e3.80 (m, 2H), 3.91 (s, 3H), 4.67 (m, 1H), 5.30 (m, 1H), 6.12 (d,

J ¼ 2.6 Hz, 1H), 6.79 (br s, 1H), 7.21 (d, J ¼ 2.5 Hz, 1H), 7.22e7.35 (m,

(125.7 MHz)

¼ 20.9 (CH₃), 36.5 (CH₂), 42.2 (CH₂), 42.9 (CH₂), 53.0

5H); ¹³C NMR (125.7 MHz)

¼ 21.1 (CH₃), 34.7 (CH₂), 37.5 (CH₃), 42.4

(CH₂), 53.6 (CH), 72.7 (CH), 123.7 (CH), 126.7 (CH), 126.9 (CH), 127.7

(CH), 128.5 (2ꢃ CH), 128.9 (2ꢃ CH), 129.0 (2ꢃ CH), 129.8 (CH), 130.3

(CH), 132.6 (C), 134.1 (C), 134.4 (C), 135.7 (C), 170.1 (C), 170.5 (C),

198.3 (C); MS (EI) m/z 415 (M^þ, 4), 296 (M^þꢁ COCH₂Ph, 14), 155

([CO-naphthyl]^þ, 100); HRMS calcd for C₂₆H₂₅NO₄[M^þ] 415.1784,

found 415.1770. Anal. Calcd for. C₂₆H₂₅NO₄: C, 75.16; H, 6.06; N,

3.37; Found: C, 75.15; H, 6.19; N, 3.06.

(CH₂), 42.8 (CH₂), 53.1 (CH₂), 55.1 (CH), 73.6 (CH), 108.2 (CH), 120.2

(CH), 126.9 (CH), 128.7 (2ꢃ CH), 129.0 (2ꢃ CH), 130.7 (C), 131.2 (CH),

134.1 (C),169.7 (C),170.3 (C),189.0 (C); MS (EI) m/z 368 (M^þ,12), 249

(M^þꢁ COCH₂Ph, 18), 189 (M^þꢁ [COCH₂Ph þ AcOH], 40), 108 ([CO-

(N-methylpyrrolyl)]^þ, 100), 91 ([CH₂Ph]^þ, 87); HRMS calcd for

₂₁H₂₄N₂O₄[M^þ] 368.1736, found 368.1737. Anal. Calcd for.

₂₁H₂₄N₂O₄: C, 68.46; H, 6.57; N, 7.60; Found: C, 68.12; H, 6.84;

N, 7.21.

4.1.3.6. (2R,4R)-4-(Acetoxy)-2-(2-[furan-2-yl]-2-oxoethyl)-1-(2-

phenylacetyl)pyrrolidine (cis-9f)and(2S,4R)-4-(acetoxy)-2-(2-[furan-2-

Compound trans-9g was obtained as a colorless oil; [

]

_Dꢁ8.3 (c

0.40, CHCl₃); IR

¼ 3013, 1736, 1639, 1406 cm^ꢁ1

;

¹H NMR

yl]-2-oxoethyl)-1-(2-phenylacetyl)pyrrolidine

(trans-9f). Obtained

(500 MHz) A 10:1 mixture of rotamers was observed, only the

from acid 10 according to the General Procedure, using 1-(2-furyl)-1-

(trimethylsilyloxy)ethylene as the nucleophile. After column chro-

matography (CH₂Cl₂/EtOAc, 9:1), compounds cis-9f (201 mg, 82%)

and trans-9f (33 mg, 14%) were isolated (96% overall yield, 234 mg, dr

6:1).

major rotamer is described:

¼ 1.90 (s, 3H), 2.14 (ddd, J ¼ 5.1, 6.9,

13.9 Hz, 1H), 2.27 (m, 1H), 2.76 (dd, J ¼ 9.5, 14.5 Hz, 1H), 3.58e3.66

(m, 2H), 3.64 (br s, 2H), 3.68 (dd, J ¼ 3.2, 14.5 Hz, 1H), 3.89 (s, 3H),

4.59 (m, 1H), 5.20 (m, 1H), 6.11 (dd, J ¼ 2.4, 3.8 Hz, 1H), 6.79 (br s,

1H), 7.15 (dd, J ¼ 1.9, 3.7 Hz, 1H), 7.21e7.26 (m, 3H), 7.30 (dd, J ¼ 7.8,

Compound cis-9f was obtained as a colorless oil; [

]

þ75 (c

8.2 Hz, 2H); ¹³C NMR (125.7 MHz):

¼ 20.8 (CH₃), 36.2 (CH₂), 37.5

0.19, CHCl₃); IR

¼ 3019, 1737, 1640, 1426 cm^ꢁ1; ¹H NMR (500 MHz)

(CH₃), 42.6 (CH₂), 42.7 (CH₂), 52.7 (CH₂), 54.1 (CH), 72.6 (CH), 108.2

(CH), 120.3 (CH), 126.8 (CH), 128.6 (2ꢃ CH), 128.8 (2ꢃ CH), 130.7 (C),

131.2 (CH), 134.4 (C), 169.9 (C), 170.4 (C), 188.7 (C); MS (EI) m/z 368

(M^þ, 5), 249 (M^þꢁ COCH₂Ph, 9),189 (M^þꢁ [COCH₂Ph þ AcOH], 26),

108 ([CO-(N-methylpyrrolyl)]^þ, 100), 91 ([CH₂Ph]^þ, 88); HRMS

A 5:1 mixture of rotamers was observed, only the major rotamer is

described:

¼ 2.06 (s, 3H), 2.11 (br d, J ¼ 14.5 Hz, 1H), 2.27 (ddd,

J ¼ 5.7, 8.5, 14.5 Hz, 1H), 2.94 (dd, J ¼ 10.2, 14.8 Hz, 1H), 3.60 (d,

J ¼ 12.0 Hz, 1H), 3.65 (br s, 2H), 3.72e3.80 (m, 2H), 4.64 (m, 1H),

550

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

calcd for C₂₁H₂₄N₂O₄368.1736, found 368.1722; calcd for

₁₃H₁₇N₂O₃249.1239, found 249.1232; calcd for C₁₁H₁₃N₂O [M^þ]

189.1028, found 189.1022. Anal. Calcd for. C₂₁H₂₄N₂O₄: C, 68.46; H,

6.57; N, 7.60; Found: C, 68.39; H, 6.70; N, 7.27.

12.8 Hz,1H), 2.71 (dd, J ¼ 13.7, 16.4 Hz,1H), 2.80 (dd, J ¼ 5.0,16.4 Hz,

1H), 3.65 (d, J ¼ 13.5 Hz, 1H), 3.72 (dd, J ¼ 4.3, 13.5 Hz, 1H), 4.19 (m,

1H), 4.47 (m, 1H), 6.97e7.01 (m, 2H), 7.04e7.10 (m, 2H), 7.14e7.18

(m, 6H); ¹³C NMR (125.7 MHz):

¼ 37.3 (CH₂), 42.3 (CH₂), 53.4 (CH),

54.3 (CH₂), 69.1 (CH), 126.9 (CH), 127.5 (2ꢃ CH), 127.6 (CH), 128.0

(2ꢃ CH), 128.3 (2ꢃ CH), 131.1 (2ꢃ CH), 133.0 (C), 135.7 (C), 140.1 (C),

145.9 (C), 164.1 (C); MS (EI) m/z 305 (M^þ, 71), 304 (M^þꢁ H, 100),

220 ([COeC(Ph)]C(Ph)eCH₂]^þ, 55), 192 ([PheC]C(Ph)eCH₂]^þ,

36); HRMS calcd for C₂₀H₁₉NO₂[M^þ] 305.1416, found 305.1415.

Anal. Calcd for. C₂₀H₁₉NO₂: C, 78.66; H, 6.27; N, 4.59; Found: C,

78.80; H, 6.44; N, 4.75.

4.1.3.8. Preparation of (2S,4R)-4-(acetoxy)-2-allyl-1-(methoxycarbo-

nyl)pyrrolidine (12). Obtained from acid 11 [21] (162 mg, 0.7 mmol)

according to the General Procedure, using allyltrimethylsilane

(0.56 mL, 3.5 mmol) as the nucleophile. After column chromatog-

raphy (hexanes:EtOAc, 8:2), compound 12 (141 mg, 89%) was iso-

lated as a colorless oil (two rotamers at 26 ^ꢀC; one rotamer at 70 ^ꢀC);

[

]

_Dþ39 (c 0.68, CHCl₃); IR

¼ 1735, 1692 cm^ꢁ1; ¹H NMR (500 MHz,

Compound (8aS)-6a was isolated as a colorless oil; [

]

ꢁ51 (c

70 ^ꢀC)

¼ 1.94 (ddd, J ¼ 1.4, 1.5, 13 Hz, 1H), 2.02 (s, 3H), 2.19 (ddd,

0.33, CHCl₃); IR

¼ 3612, 3395, 3015,1642 cm^ꢁ1; ¹H NMR (500 MHz)

J ¼ 6.2, 8.5, 14.2 Hz, 1H), 2.29 (dddd, J ¼ 1.2, 8.4, 8.9, 15 Hz, 1H), 2.67

(m, 1H), 3.39 (ddd, J ¼ 1.1, 2.9, 12.6 Hz, 1H), 3.68 (s, 3H), 3.79 (dd,

J ¼ 6.0, 12.6 Hz, 1H), 3.96 (m, 1H), 5.03 (d, J ¼ 17.2 Hz, 1H), 5.06 (d,

J ¼ 10 Hz, 1H), 5.22 (m, 1H), 5.75 (m, 1H); ¹³C NMR (125.7 MHz,

¼ 1.85 (ddd, J ¼ 6.8, 7.8, 12.6 Hz, 1H), 2.46 (br b, 1H), 2.47 (ddd,

J ¼ 6.3, 6.5, 12.7 Hz, 1H), 2.74 (dd, J ¼ 4.4, 16.4 Hz, 1H), 2.96 (dd,

J ¼ 14.1, 16.4 Hz, 1H), 3.69 (dd, J ¼ 5.2, 12.6 Hz, 1H), 3.73 (dd, J ¼ 6.1,

12.6 Hz,1H), 4.06 (m,1H), 4.48 (m,1H), 6.98e7.00 (m, 2H), 7.01e7.08

70 ^ꢀC):

¼ 20.7 (CH₃), 35.0 (CH₂), 38.7 (CH₂), 52.0 (CH₃), 52.2 (CH₂),

(m, 2H), 7.12e7.16 (m, 6H); ¹³C NMR (125.7 MHz):

¼ 37.7 (CH₂),

56.4 (CH), 72.9 (CH), 117.3 (CH₂), 134.9 (CH), 155.4 (C), 170.1 (C); MS

(EI) m/z 186 (M^þꢁ allyl, 15), 126 (M^þꢁ [allyl þ HOAc], 100); HRMS

calcd for C₈H₁₂NO₄[M^þ] 186.0766, found 186.0768. Anal. Calcd for

41.2 (CH₂), 52.6 (CH₂), 54.1 (CH), 69.4 (CH),126.9 (CH),127.5 (3ꢃ CH),

128.0 (2ꢃ CH), 128.3 (2ꢃ CH), 131.1 (2ꢃ CH), 132.7 (C), 135.8 (C),

140.0 (C), 146.1 (C), 164.1 (C); MS (EI) m/z 306 (M^þþ H, 18), 305 (M^þ,

89), 304 (M^þꢁ H, 100), 220 ([COeC(Ph)]C(Ph)eCH₂]^þ, 94), 192

([PheC]C(Ph)eCH₂]^þ, 65); HRMS calcd for C₂₀H₂₀NO₂[M^þ]

306.1494, found 306.1503. Anal. Calcd for C₂₀H₁₉NO₂: C, 78.66; H,

6.27; N, 4.59; Found: C, 78.74; H, 6.32; N, 4.61.

C₁₁H₁₇NO₄: C, 58.14; H, 7.54; N, 6.16. Found: C, 57.93; H, 7.38; N, 6.29.

4.1.3.9. Preparation of methyl 4-(acetoxy)-2-(2-oxo-2-phenylethyl)

pyrrolidine-1-carboxylate (13). Obtained from acid 11 [21] (162 mg,

0.7 mmol) according to the General Procedure, using commercial 1-

phenyl-1-(trimethylsiloxy)ethylene (710

L, 3.5 mmol) as the

4.1.4.2. (2R,8aR)-2-Hydroxy-6-phenyl-7-(4-methoxyphenyl)-

2,3,8,8a-tetrahydroindolizin-5(1H)-one (8aR)-6b and (2R,8aS)-2-

hydroxy-6-phenyl-7-(4-methoxyphenyl)-2,3,8,8a-tetrahydroindolizin-

5(1H)-one (8aS)-6b. Substrate cis-9b (99 mg, 0.25 mmol) un-

derwent the General Cyclization Procedure, and after work-up

and puriﬁcation of the residue by column chromatography

(EtOAc/MeOH, 95:5), compounds (8aR)-6b (46 mg, 55%) and

(8aS)-6b (22 mg, 26%) were isolated (81% overall yield, 68 mg, dr

2:1). Compounds (8aR)-6b and (8aS)-6b were previously re-

ported [9].

nucleophile. After column chromatography (hexanes:EtOAc, 7:3),

compound 13 (167 mg, 78%) was isolated as a colorless oil (unse-

parable 10:3 mixture of the (2R,4R):(2S,4R) diastereomers); IR

¼ 3019, 1737, 1690, 1453, 1390, 1247 cm^ꢁ1

;

¹H NMR (500 MHz,

70 ^ꢀC) major isomer:

¼ 2.03 (s, 3H), 2.05 (br d, J ¼ 14.7 Hz,1H), 2.38

(ddd, J ¼ 5.8, 8.6, 14.5 Hz, 1H), 3.16 (dd, J ¼ 10.2, 15.7 Hz, 1H), 3.51

(ddd, J ¼ 1.7, 1.7, 12.7 Hz, 1H), 3.70 (s, 3H), 3.76 (dd, J ¼ 5.5, 12.6 Hz,

1H), 3.78 (m, 1H), 4.48 (m, 1H), 5.29 (m, 1H), 7.45 (dd, J ¼ 7.8, 7.9 Hz,

2H), 7.54 (dd, J ¼ 7.4, 7.8 Hz, 1H), 7.98 (d, J ¼ 7.4 Hz, 2H); minor

isomer:

¼ 2.04 (m, 1H), 2.39 (m, 1H), 2.99 (dd, J ¼ 9.0, 16.2 Hz, 1H),

3.60 (dd, J ¼ 4.7, 12.5 Hz,1H), 3.66 (m, 1H), 3.69 (s, 3H), 3.77 (m, 1H),

4.46 (m, 1H), 5.25 (m, 1H), 7.44 (dd, J ¼ 7.8, 7.9 Hz, 2H), 7.53 (dd,

J ¼ 7.7, 7.8 Hz,1H), 7.96 (d, J ¼ 8.0 Hz, 2H); ¹³C NMR (100.6 MHz, 26 ^ꢀC)

4.1.4.3. (2R,8aR)-2-Hydroxy-6-phenyl-7-(4-bromophenyl)-2,3,8,8a-

tetrahydroindolizin-5(1H)-one (8aR)-6c and (2R,8aS)-2-hydroxy-6-

phenyl-7-(4-bromophenyl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

(8aS)-6c. Substrate cis-9c (111 mg, 0.25 mmol) underwent the

General Cyclization Procedure, and after work-up and puriﬁcation

of the residue by column chromatography (EtOAc/MeOH, 5:95),

compounds (8aR)-6c (46 mg, 48%) and (8aS)-6c (31 mg, 32%) were

isolated (80% overall yield, dr 6:4).

Each diastereomer appears as a mixture of two rotamers:

¼ 20.6/

20.7 (CH₃), 35.3/36.5/37.1/38.3 (CH₂), 42.6/43.4/43.7 (CH₂), 51.9/52.6

(CH₂), 52.1 (CH₃), 53.2/53.8 (CH), 71.6/72.2/72.4/73.1 (CH),127.8 (2ꢃ

CH),128.3 (2ꢃ CH),132.9 (CH),136.4 (C),154.7/155.1 (C),169.7/170.0

(C), 197.9/198.3 (C); MS (EI) m/z 305 (M^þ, <1), 186

(M^þꢁ [HOAc þ COOMe], 40), 126 (M^þꢁ [CH₂COPh þ HOAc], 100),

105 ([PhCO]^þ, 97); HRMS calcd for C₁₆H₁₉NO₅[M^þ] 305.1263, found

305.1268. Anal. Calcd for. C₁₆H₁₉NO₅: C, 62.94; H, 6.27; N, 4.59;

Found: C, 63.11; H, 6.54; N, 4.59.

Compound (8aR)-6c was isolated as a colorless gum; [

]

_Dþ33 (c

0.20, CHCl₃); IR

NMR (500 MHz)

¼ 3695, 3609, 2929, 1642, 1603, 1442 cm^ꢁ1

;

¹H

¼ 1.81 (m, 1H), 2.30 (dd, J ¼ 5.1, 12.6 Hz, 1H), 2.75

(dd, J ¼ 13.5, 15.8 Hz, 1H), 2.81 (dd, J ¼ 5.1, 16.4 Hz, 1H), 3.66 (d,

J ¼ 13.3 Hz,1H), 3.82 (dd, J ¼ 4.5,13.9 Hz,1H), 4.30 (m, 1H), 4.62 (dd,

J ¼ 3.8, 4.4 Hz, 1H), 6.88 (d, J ¼ 8.2 Hz, 2H), 7.05e7.09 (m, 2H), 7.16e

4.1.4. General procedure for the cyclization step

4.1.4.1. Preparation of (2R,-8aR)-2-hydroxy-6,7-diphenyl-2,3,8,8a-

tetrahydroindolizin-5(1H)-one (8aR)-6a and (2R,8aS)-2-hydroxy-

7.20 (m, 3H), 7.27 (d, J ¼ 8.9 Hz, 2H); ¹³C NMR (125.7 MHz):

¼ 37.1

(CH₂), 42.4 (CH₂), 53.4 (CH), 54.3 (CH₂), 69.2 (CH), 121.7 (C), 127.1

(CH), 127.7 (2ꢃ CH), 130.0 (2ꢃ CH), 131.0 (2ꢃ CH), 131.2 (2ꢃ CH),

133.7 (C), 135.4 (C), 139.0 (C), 144.2 (C), 163.8 (C); MS (EI) m/z 382/

384 (M^þꢁ H,11/10), 298/300 ([COeC(Ph)]C(C₆H₄-4-Br)eCH₂]^þ, 9/

14), 256/258 ([PheC^CeC₆H₄-p-Br]^þ, 14/2), 55 ([CH₂]CHeCH₂e

CH₂]^þ, 100); HRMS calcd for C₂₀H₁₇NO₂⁸¹Br [M^þꢁ H] 384.0422,

found 384.0416; calcd for C₂₀H₁₇NO₂⁷⁹Br [M^þꢁ H] 382.0443,

found 382.0443. Anal. Calcd for. C₂₀H₁₈NO₂Br: C, 62.51; H, 4.72; N,

3.63; Found: C, 62.28; H, 4.88; N, 3.70.

6,7-diphenyl-2,3,8,8a-tetra-hydroindolizin-5(1H)-one

(8aS)-6a.

The product cis-9a (90 mg, 0.25 mmol) was added to a 5% KOH

methanolic solution (16 mL), and the mixture was heated under

reﬂux for 4 h. Then it was partially evaporated under vacuum,

poured into 10% aqueous HCl (10 mL) and extracted with CH₂Cl₂.

The organic layer was dried, ﬁltered and concentrated as usual, and

the residue was puriﬁed by preparative chromatography on TLC

(EtOAc) to give products (8aR)-6a (44 mg, 59%) and (8aS)-6a

(19 mg, 25%): 84% overall yield, 63 mg, dr 7:3.

Compound (8aS)-6c was isolated as a crystalline solid; mp 124e

Compound (8aR)-6a was isolated as a colorless oil; [

;

]

þ62 (c

126 ^ꢀC (EtOAc/hexane); [

]

_Dꢁ29 (c 0.23, CHCl₃); IR

2928, 1642, 1604 cm^ꢁ1; ¹H NMR (500 MHz):

¼ 1.85 (ddd, J ¼ 6.4,

8.0,14.2 Hz,1H), 2.49 (ddd, J ¼ 6.0, 7.0, 12.9 Hz,1H), 2.69 (dd, J ¼ 4.4,

¼ 3695, 3610,

0.67, CHCl₃); IR

(500 MHz):

¼ 3691, 3610, 3022, 1642, 1609 cm^ꢁ1

¹H NMR

¼ 1.71 (ddd, J ¼ 4.2, 12.0, 12.5 Hz,1H), 2.22 (dd, J ¼ 5.2,

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

551

16.4 Hz, 1H), 2.95 (dd, J ¼ 14.0, 16.4 Hz, 1H), 3.69e3.72 (m, 2H), 4.06

7.72 (m, 2H); ¹³C NMR (125.7 MHz):

¼ 37.5 (CH₂), 42.4 (CH₂), 53.5

(m, 1H), 4.51 (m, 1H), 6.88 (d, J ¼ 8.5 Hz, 1H), 7.05e7.09 (m, 2H),

(CH), 54.5 (CH₂), 69.2 (CH), 126.2 (CH), 126.3 (CH), 126.4 (CH), 127.0

(CH), 127.3 (CH), 127.5 (CH), 127.6 (2ꢃ CH), 128.1 (CH), 131.2 (2ꢃ

CH), 132.5 (C), 132.9 (C), 133.2 (C), 135.6 (C), 137.6 (C), 145.7 (C),

164.1 (C); MS (EI) m/z 355 (M^þ, 84), 354 (M^þꢁ H, 83), 295 (([4-

7.15e7.20 (m, 3H), 7.27 (d, J ¼ 8.5 Hz, 2H); ¹³C NMR (125.7 MHz):

¼ 37.5 (CH₂), 41.1 (CH₂), 52.6 (CH₂), 54.0 (CH), 69.5 (CH), 121.7 (C),

127.1 (CH), 127.7 (2ꢃ CH), 129.9 (2ꢃ CH), 130.9 (2ꢃ CH), 131.2 (2ꢃ

CH), 133.3 (C), 135.4 (C), 138.9 (C), 144.5 (C), 163.8 (C); MS (EI) m/z

384 (M^þꢁ H, 11), 298/300 ([COeC(Ph)]C(C₆H₄-4-Br)eCH₂]^þ, 2/

12), 256/258 ([PheC^CeC₆H₄-p-Br]^þ, 20/2), 55 ([CH₂]CHeCH₂e

CH₂]^þ, 100); HRMS calcd for C₂₀H₁₇NO₂⁸¹Br [M^þꢁ H] 384.0422,

found 384.0418; calcd for C₂₀H₁₇NO₂⁷⁹Br [M^þꢁ H] 382.0443,

found 382.0445. Anal. Calcd for. C₂₀H₁₈NO₂Br: C, 62.51; H, 4.72; N,

3.63. Found: C, 62.51; H, 4.62; N, 3.50.

naphthalen-2-yl)-3-phenylpyridin-2(1H)-oneeH]^þ,

97),

270

([COeC(Ph)]C(naphthyl)eCH₂]^þ, 100); HRMS calcd for C₂₄H₂₁NO₂

[M^þ] 335.1572, found 335.1567; calcd for C₂₄H₂₀NO₂[M^þꢁ H]

354.1494, found 354.1507. Anal. Calcd for. C₂₄H₂₁NO₂: C, 81.10; H,

5.96; N, 3.94; Found: C, 80.92; H, 6.32; N, 3.97.

Compound (8aS)-6e was isolated as a crystalline solid; mp 124e

126 ^ꢀC (EtOAc/hexane); [

_Dꢁ48 (c 0.16, CHCl₃); IR

¼ 3611, 3382,

3015, 1639, 1444 cm^ꢁ1

;

¹H NMR (500 MHz):

¼ 1.92 (ddd, J ¼ 7.2,

4.1.4.4. (2R,8aR)-2-Hydroxy-6-phenyl-7-(biphenyl-4-yl)-2,3,8,8a-tetr-

ahydroindolizin-5(1H)-one (8aR)-6d and (2R,8aS)-2-hydroxy-6-phe-

nyl-7-(biphenyl-4-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one (8aS)-

6d. Substrate cis-9d (110 mg, 0.25 mmol) underwent the General

Cyclization Procedure, and after work-up and puriﬁcation of the

residue by column chromatography (EtOAc/MeOH, 95:5), com-

pounds (8aR)-6d (51 mg, 53%) and (8aS)-6d (31 mg, 33%) were iso-

lated (86% overall yield, 82 mg, dr 6:4).

13.2,13.5 Hz,1H), 2.55 (ddd, J ¼ 6.3, 6.4,13.5 Hz,1H), 2.89 (dd, J ¼ 4.2,

16.3 Hz, 1H), 3.09 (dd, J ¼ 14.1, 16.2 Hz, 1H), 3.72e3.83 (m, 2H), 4.13

(m, 1H), 4.58 (m, 1H), 7.02 (dd, J ¼ 1.6, 8.6 Hz,1H), 7.09e7.15 (m, 5H),

7.40 (dd, J ¼ 3.9, 5.4 Hz, 1H), 7.43 (dd, J ¼ 3.8, 5.5 Hz, 1H), 7.54 (d,

J ¼ 8.5 Hz, 1H), 7.57 (br s, 1H), 7.65 (m, 1H), 7.70 (m, 1H); ¹³C NMR

(125.7 MHz):

¼ 37.9 (CH₂), 41.2 (CH₂), 52.9 (CH₂), 54.3 (CH), 69.6

(CH), 126.4 (CH), 126.5 (2ꢃ CH), 127.0 (CH), 127.51 (CH), 127.55 (CH),

127.6 (2ꢃ CH), 128.0 (CH), 131.1 (2ꢃ CH), 132.5 (C), 132.9 (C), 135.6

(C), 137.5 (C), 145.9 (C), 164.1 (C); MS (EI) m/z 355 (M^þ, 96), 354

(M^þꢁ H, 90), 270 ([COeC(Ph)]C(naphthyl)-CH₂]^þ, 100); HRMS

calcd for C₂₄H₂₁NO₂[M^þ] 335.1572, found 335.1563; calcd for

Compound (8aR)-6d was isolated as a colorless oil; [

]

þ24 (c

0.27, CHCl₃); IR

(500 MHz):

¼ 3688, 3610, 3015,1640,1603,1440 cm^ꢁ1; ¹H NMR

¼ 1.84 (ddd, J ¼ 3.8, 12.6, 13.2 Hz, 1H), 2.31 (dd, J ¼ 5.1,

13.3 Hz,1H), 2.79 (dd, J ¼ 13.9, 16.4 Hz,1H), 2.90 (dd, J ¼ 4.5, 16.4 Hz,

1H), 3.68 (d, J ¼ 13.3 Hz, 1H), 3.84 (dd, J ¼ 4.0, 13.9 Hz, 1H), 4.32 (m,

1H), 4.64 (m,1H), 7.09 (d, J ¼ 8.8 Hz, 2H), 7.12e7.19 (m, 5H), 7.31 (dd,

J ¼ 7.6, 8.0 Hz,1H), 7.39 (dd, J ¼ 7.5, 7.6 Hz, 4H), 7.52 (d, J ¼ 8.0 Hz, 2H);

₂₄H₂₀NO₂[M^þꢁ H] 354.1494, found 354.1502. Anal. Calcd for.

₂₄H₂₁NO₂: C, 81.10; H, 5.96; N, 3.94. Found: C, 81.21; H, 6.35; N, 4.18.

4.1.4.6. (2R,8aR)-2-Hydroxy-6-phenyl-7-(2-furyl)-2,3,8,8a-tetrahydr-

oindolizin-5(1H)-one (8aR)-6f and (2R,8aS)-2-hydroxy-6-phenyl-7-

(2-furyl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

Substrate cis-9f (89 mg, 0.25 mmol) underwent the General Cycli-

zation Procedure, and after work-up and puriﬁcation of the residue

by column chromatography (EtOAc/MeOH 95:5), compounds (8aR)-

6f (41 mg, 55%) and (8aS)-6f (22 mg, 30%) were isolated (85% overall

yield, 63 mg, dr 6:4).

¹³C NMR (125.7 MHz):

¼ 37.3 (CH₂), 42.5 (CH₂), 53.4 (CH), 54.3

(CH₂), 69.3 (CH), 126.6 (2ꢃ CH), 126.9 (3ꢃ CH), 127.5 (CH), 127.7 (2ꢃ

CH), 128.7 (2ꢃ CH), 128.9 (2ꢃ CH), 131.1 (2ꢃ CH), 133.2 (C), 135.8 (C),

139.0 (C),140.2 (2ꢃ C),145.0 (C),164.1 (C); MS (EI) m/z 381 (M^þ,1), 91

([CH₂]CHeCH]CHeCH₂eC^C]^þ,100); HRMS calcd for C₂₆H₂₃NO₂

[M^þ] 381.1729, found 381.1736. Anal. Calcd for. C₂₆H₂₃NO₂: C, 81.86;

H, 6.08; N, 3.67; Found: C, 81.69; H, 6.38; N, 4.05.

(8aS)-6f.

Compound (8aS)-6d was isolated as a colorless oil; [

;

]

þ18 (c

Compound (8aR)-6f was isolated as a colorless oil; [

]

;

_Dþ178 (c

0.30, CHCl₃); IR

(500 MHz)

¼ 3692, 3610, 3015, 1642, 1441 cm^ꢁ1

¹H NMR

0.253, CHCl₃); IR

(500 MHz):

¼ 3695, 3606, 2929, 1640, 1442 cm^ꢁ1

¹H NMR

¼ 1.90 (ddd, J ¼ 6.3, 7.6, 12.6 Hz, 1H), 2.55 (ddd, J ¼ 6.3,

¼ 1.81 (ddd, J ¼ 4.4,11.4,13.2 Hz,1H), 2.31 (br dd, J ¼ 5.1,

6.4, 12.8 Hz, 1H), 2.82 (dd, J ¼ 4.4, 16.4 Hz, 1H), 3.00 (dd, J ¼ 13.9,

16.4 Hz, 1H), 3.73 (dd, J ¼ 4.4, 12.6 Hz, 1H), 3.78 (dd, J ¼ 5.7, 12.6 Hz,

1H), 4.11 (m, 1H), 4.58 (m, 1H), 7.07 (d, J ¼ 8.2 Hz, 2H), 7.11e7.18 (m,

5H), 7.31 (dd, J ¼ 6.9, 7.0 Hz,1H), 7.39 (dd, J ¼ 7.6, 8.2 Hz, 4H), 7.52 (d,

12.6 Hz,1H), 2.50 (dd, J ¼ 13.8, 16.4 Hz,1H), 3.36 (dd, J ¼ 5.1, 16.8 Hz,

1H), 3.65 (d, J ¼ 13.3 Hz, 1H), 3.76 (dd, J ¼ 5.0, 13.8 Hz, 1H), 4.20 (m,

1H), 4.58 (m,1H), 5.45 (d, J ¼ 3.2 Hz,1H), 6.21 (dd, J ¼ 1.3, 3.2 Hz,1H),

7.20 (d, J ¼ 7.0 Hz, 2H), 7.35e7.40 (m, 4H); ¹³C NMR (125.7 MHz):

J ¼ 7.2 Hz, 2H); ¹³C NMR (125.7 MHz):

¼ 37.8 (CH₂), 41.4 (CH₂), 52.7

¼ 31.7 (CH₂), 42.2 (CH₂), 53.5 (CH), 54.4 (CH₂), 69.1 (CH),112.2 (CH),

(CH₂), 54.1 (CH), 69.7 (CH), 126.6 (2ꢃ CH), 126.9 (3ꢃ CH), 127.5 (CH),

127.7 (2ꢃ CH),128.8 (2ꢃ CH),128.9 (2ꢃ CH),131.1 (2ꢃ CH),133.0 (C),

135.9 (C),138.9 (C),140.3 (2ꢃ C),145.3 (C),164.0 (C); MS (EI) m/z 381

(M^þ,18), 91 ([CH₂]CHeCH]CHeCH₂eC^C]^þ,100); HRMS calcd for

113.5 (CH), 127.6 (CH), 128.6 (2ꢃ CH), 129.7 (2ꢃ CH), 134.3 (C), 136.5

(C), 142.8 (CH), 151.0 (C), 164.0 (C); MS (EI) m/z 295 (M^þ, 100), 210

([COeC(Ph)]C(furyl)eCH₂]^þ, 65); HRMS calcd for C₁₈H₁₇NO₃[M^þ]

295.1208, found 295.1199. Anal. Calcd for. C₁₈H₁₇NO₃: C, 73.20; H,

5.80; N, 4.74; Found: C, 73.56; H, 6.08; N, 4.55.

₂₆H₂₃NO₂[M^þ] 381.1729, found 381.1726. Anal. Calcd for.

₂₆H₂₃NO₂: C, 81.86; H, 6.08; N, 3.67. Found: C, 81.67; H, 5.97; N, 3.95.

Compound (8aS)-6f was isolated as a colorless oil; [

]

_Dꢁ227 (c

0.024, CHCl₃); IR

(500 MHz):

¼ 3692, 3608, 2929, 1640, 1441 cm^ꢁ1

;

¹H NMR

4.1.4.5. (2R,8aR)-2-Hydroxy-6-phenyl-7-(naphthalen-2-yl)-2,3,8,8a-

tetrahydroindolizin-5(1H)-one (8aR)-6e and (2R,8aS)-2-hydroxy-6-

phenyl-7-(naphthalen-2-yl)-2,3,8,8a-tetrahydroindolizin-5(1H)-one

(8aS)-6e. Substrate cis-9e (104 mg, 0.25 mmol) underwent the

General Cyclization Procedure, and after work-up and puriﬁcation

of the residue by column chromatography (EtOAc/MeOH 95:5),

compounds (8aR)-6e (44 mg, 50%) and (8aS)-6e (26 mg, 29%) were

isolated (79% overall yield, 70 mg, dr 6:4).

¼ 1.87 (ddd, J ¼ 6.9, 8.2,12.7 Hz,1H), 2.50 (ddd, J ¼ 6.3,

6.3, 12.7 Hz, 1H), 2.70 (dd, J ¼ 13.9, 16.4 Hz, 1H), 3.27 (dd, J ¼ 4.4,

17.0 Hz, 1H), 3.63 (dd, J ¼ 5.0, 12.6 Hz, 1H), 3.71 (dd, J ¼ 6.3, 12.6 Hz,

1H), 3.96 (m, 1H), 4.47 (dddd, J ¼ 5.8, 5.8, 6.3, 6.3 Hz, 1H), 5.44 (d,

J ¼ 3.8 Hz, 1H), 6.20 (dd, J ¼ 1.9, 3.7 Hz, 1H), 7.19 (d, J ¼ 7.6 Hz, 2H),

7.30e7.40 (m, 4H); ¹³C NMR (125.7 MHz):

¼ 32.2 (CH₂), 41.1 (CH₂),

52.6 (CH₂), 54.1 (CH), 69.3 (CH), 112.2 (CH), 113.5 (CH), 127.6 (CH),

128.6 (2ꢃ CH), 129.4 (C), 129.7 (2ꢃ CH), 134.6 (C), 136.6 (C), 142.8

(CH), 151.0 (C), 164.0 (C); MS (EI) m/z 295 (M^þ, 100), 210 ([COe

C(Ph)]C(furyl)eCH₂]^þ, 86); HRMS calcd for C₁₈H₁₇NO₃[M^þ]

295.1208, found 295.1212. Anal. Calcd for. C₁₈H₁₇NO₃: C, 73.20; H,

5.80; N, 4.74; Found: C, 73.46; H, 6.12; N, 4.50.

Compound (8aR)-6e was isolated as a colorless oil; [

]

_Dþ72 (c

0.14, CHCl₃); IR

(400 MHz):

¼ 3383, 3015, 1638, 1444 cm^ꢁ1

;

¹H NMR

¼ 1.84 (ddd, J ¼ 4.1, 11.3, 12.8 Hz, 1H), 2.32 (dd, J ¼ 5.2,

12.9 Hz, 1H), 2.85 (dd, J ¼ 14.0, 16.2 Hz, 1H), 2.97 (dd, J ¼ 4.7,

16.3 Hz, 1H), 3.72 (d, J ¼ 13.4 Hz, 1H), 3.84 (dd, J ¼ 4.3, 13.5 Hz, 1H),

4.35 (m, 1H), 4.63 (m, 1H), 7.02 (br d, J ¼ 8.6 Hz, 1H), 7.05e7.20 (m,

5H), 7.39e7.45 (m, 2H), 7.54 (d, J ¼ 8.5 Hz, 1H), 7.57 (br s, 1H), 7.65e

4.1.4.7. (2R,8aR)-2-Hydroxy-6-phenyl-7-(N-methyl-1H-pyrrol-2-yl)-

2,3,8,8a-tetrahydroindolizin-5(1H)-one (8aR)-6g and (2R,8aS)-2-

552

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

hydroxy-6-phenyl-7-(N-methyl-1H-pyrrol-2-yl)-2,3,8,8a-tetrahy-dro

indolizin-5(1H)-one (8aS)-6g. Substrate cis-9g (92 mg, 0.25 mmol)

underwent the General Cyclization Procedure, and after work-up

and puriﬁcation of the residue by column chromatography

(EtOAc/MeOH, 95:5), compounds (8aR)-6g (51 mg, 66%) and (8aS)-

6g (15 mg, 20%) were isolated (86% overall yield, 66 mg, dr 3:1).

(m, 3H), 3.94 (dd, J ¼ 3.3, 3.3 Hz, 1H), 4.53 (m, 1H), 4.60 (m, 1H),

7.24e7.28 (m, 3H), 7.33 (dd, J ¼ 7.3, 7.8 Hz, 2H), 7.58 (d, J ¼ 8.5 Hz,

2H), 7.91 (d, J ¼ 8.6 Hz, 2H); ¹³C NMR (125.7 MHz):

¼ 38.1 (CH₂),

42.5 (CH₂), 42.6 (CH₂), 54.3 (CH), 55.8 (CH₂), 71.3 (CH), 127.0 (CH),

128.5 (C), 128.8 (2ꢃ CH), 129.1 (2ꢃ CH), 130.0 (2ꢃ CH), 132.0 (2ꢃ

CH), 134.3 (C), 135.4 (C), 170.1 (C), 198.4 (C); MS (EI) m/z 403/401

(M^þ, 6/7), 284/282 (M^þꢁ COCH₂Ph, 27/32), 185/183 ([CO-C₆H₄-p-

Br]^þ, 59/65), 91 ([CH₂Ph]^þ, 100); HRMS calcd for C₂₀H₂₀NO⁸₃¹Br

[M^þ] 403.0606; found, 403.0591; calcd for C₂₀H₂₀NO⁷₃⁹Br [M^þ]

401.0627, found 401.0621. Anal. Calcd for C₂₀H₂₀NO₃Br: C, 59.71; H,

5.01; N, 3.48; Found: C, 60.09; H, 5.12; N, 3.34.

Compound (8aR)-6g was isolated as a colorless oil; [

]

þ95 (c

0.06, CHCl₃); IR

(500 MHz):

¼ 3611, 3013, 1636, 1438 cm^ꢁ1

;

¹H NMR

¼ 1.76 (ddd, J ¼ 4.4, 12.0, 13.2 Hz, 1H), 2.25 (br dd,

J ¼ 5.1, 13.2 Hz, 1H), 2.64 (dd, J ¼ 13.9, 16.4 Hz, 1H), 2.79 (dd, J ¼ 4.4,

16.4 Hz, 1H), 2.87 (s, 3H), 3.67 (d, J ¼ 13.3 Hz, 1H), 3.79 (dd, J ¼ 4.4,

13.2 Hz,1H), 4.16 (m,1H), 4.57 (dd, J ¼ 3.8, 4.4 Hz,1H), 6.10e6.12 (m,

2H), 6.40 (dd, J ¼ 2.2, 2.2 Hz, 1H), 7.10e7.24 (m, 5H); ¹³C NMR

4.1.5.4. Preparation of (2S,4R)-4-(hydroxy)-2-(2-[4-bromophenyl]-2-

oxoethyl)-1-(2-phenylacetyl) pyrrolidine (trans-16c). The acetate

trans-9c (111 mg, 0.25 mmol) underwent the General Saponiﬁca-

tion Procedure. After puriﬁcation by column chromatography

(hexanes:EtOAc 40:60), compound trans-16c was isolated as a

(125.7 MHz):

¼ 34.7 (CH₃), 38.3 (CH₂), 42.3 (CH₂), 53.3 (CH), 54.3

(CH₂), 69.2 (CH), 108.5 (CH), 110.5 (CH), 123.8 (CH), 126.9 (CH), 127.5

(2ꢃ CH), 130.8 (2ꢃ CH), 131.6 (C), 132.1 (C), 136.2 (C), 137.4 (C), 164.0

(C); MS (EI) m/z 308 (M^þ, 100), 223 ([COeC(Ph)]C(N-methyl-1H-

pyrrol-2-yl)eCH₂]^þ, 62); HRMS calcd for C₁₉H₂₀N₂O₂[M^þ]

308.1525, found 308.1533. Anal. Calcd for. C₁₉H₂₀N₂O₂: C, 74.00; H,

6.54; N, 9.08; Found: C, 74.15; H, 6.80; N, 9.52.

syrup (100 mg, 99%): IR

(500 MHz): A 15:1 mixture of rotamers was observed, only the

¼ 3388, 1682, 1636, 1586 cm^ꢁ1; ¹H NMR

major rotamer is described:

¼ 1.96 (m, 1H), 2.16 (m, 1H), 2.84 (dd,

Compound (8aS)-6g was isolated as a colorless oil; [

]

ꢁ29 (c

J ¼ 9.4, 15.5 Hz, 1H), 3.51 (br d, J ¼ 11.1 Hz, 1H), 3.57 (dd, J ¼ 4.4,

11.1 Hz, 1H), 3.62 (d, J ¼ 15.1 Hz, 1H), 3.66 (d, J ¼ 15.1 Hz, 1H), 3.89

(dd, J ¼ 3.2, 15.5 Hz, 1H), 4.42 (m, 1H), 4.60 (m, 1H), 7.20e7.25 (m,

3H), 7.27e7.33 (m, 2H), 7.57 (d, J ¼ 8.6 Hz, 2H), 7.99 (d, J ¼ 8.6 Hz,

0.10, CHCl₃); IR

(500 MHz):

6.7, 12.6 Hz, 1H), 2.75 (dd, J ¼ 4.4, 16.4 Hz, 1H), 2.87 (dd, J ¼ 13.9,

16.4 Hz, 1H), 2.88 (s, 3H), 3.70 (dd, J ¼ 5.4, 12.6 Hz, 1H), 3.74 (dd,

J ¼ 6.0, 12.6 Hz, 1H), 4.04 (m, 1H), 4.54 (dddd, J ¼ 5.7, 6.0, 6.0, 6.1 Hz,

1H), 6.10 (d, J ¼ 2.2 Hz, 2H), 6.40 (dd, J ¼ 1.9, 2.2 Hz, 1H), 7.10e7.20

¼ 3610, 3011, 1636, 1438 cm^ꢁ1

;

¹H NMR

¼ 1.85 (ddd, J ¼ 7.0, 7.3, 12.6 Hz, 1H), 2.51 (ddd, J ¼ 6.0,

2H); ¹³C NMR (125.7 MHz):

¼ 39.4 (CH₂), 42.3 (CH₂), 42.6 (CH₂),

53.4 (CH), 55.4 (CH₂), 69.8 (CH),126.9 (CH),128.5 (C),128.7 (2ꢃ CH),

129.0 (2ꢃ CH), 129.9 (2ꢃ CH), 131.9 (2ꢃ CH), 134.4 (C), 135.2 (C),

170.5 (C), 197.7 (C); MS (EI) m/z 403/401 (M^þ, 4/4), 284/282

(M^þꢁ COCH₂Ph, 24/24), 185/183 ([CO-C₆H₄-p-Br]^þ, 47/50), 91

([CH₂Ph]^þ, 100); HRMS calcd for C₂₀H₂₀NO₃⁸¹Br [M^þ] 403.0606;

(m, 5H); ¹³C NMR (125.7 MHz):

¼ 34.7 (CH₃), 38.8 (CH₂), 41.2

(CH₂), 52.7 (CH₂), 54.1 (CH), 69.6 (CH), 108.5 (CH), 110.6 (CH), 123.9

(CH), 126.9 (CH), 127.5 (2ꢃ CH), 128.6 (C), 129.3 (C), 130.8 (2ꢃ CH),

131.4 (C), 132.0 (C), 136.2 (C), 137.6 (C), 164.0 (C); MS (EI) m/z 308

(M^þ, 100), 223 ([COeC(Ph)]C(N-methyl-1H-pyrrol-2-yl)eCH₂]^þ,

62); HRMS calcd for C₁₉H₂₀N₂O₂[M^þ] 308.1525, found 308.1525.

Anal. Calcd for. C₁₉H₂₀N₂O₂: C, 74.00; H, 6.54; N, 9.08; Found: C,

74.25; H, 6.75; N, 9.12.

found, 403.0613; calcd for

₂₀H₂₀NO₃⁷⁹Br 401.0627, found

401.0642. Anal. Calcd for C₂₀H₂₀NO₃Br: C, 59.71; H, 5.01; N, 3.48;

Found: C, 59.73; H, 5.10; N, 3.25.

4.1.5.5. Preparation of (2R,4R)-4-(hydroxy)-2-(2-[p-biphenyl]-2-oxo-

ethyl)-1-(2-phenylacetyl)pyrrolidine (cis-16d). The acetate cis-9d

(110 mg, 0.25 mmol) underwent the General Saponiﬁcation Pro-

cedure, and after usual puriﬁcation by column chromatography

(hexanes:EtOAc 40:60), compound cis-16d was isolated as a white

4.1.5. General procedure for the saponiﬁcation step

4.1.5.1. Preparation of (2R,4R)-4-(hydroxy)-2-(2-[4-methoxyphenyl]-

2-oxoethyl)-1-(2-phenylacetyl)pyrrolidine (cis-16b). To a solution of

product cis-9b (100 mg, 0.25 mmol) in methanol (8 mL) was added

K₂CO₃(800 mg, 5.8 mmol), and the mixture was stirred at 26 ^ꢀC for

18 h. Then it was partially evaporated under vacuum, poured into

saturated aqueous NH₄Cl solution and extracted three times with

CH₂Cl₂. The combined organic layers were dried, ﬁltered and

concentrated as usual, and the residue was puriﬁed by column

chromatography (hexanes:EtOAc 40:60), affording alcohol (cis-16b)

(88 mg, 98%) as a syrup: Compound cis-16b has been previously

reported [9].

foam (96 mg, 96%): IR

¼ 3409, 1677, 1636, 1605 cm^ꢁ1

;

¹H NMR

(500 MHz, 26 ^ꢀC). A 6:1 mixture of rotamers was observed, only the

major rotamer is described:

¼ 1.98 (br d, J ¼ 14.2 Hz, 1H), 2.20

(ddd, J ¼ 5.1, 8.8, 14.1 Hz, 1H), 3.34 (dd, J ¼ 10.3, 15.6 Hz, 1H), 3.56e

3.65 (m, 4H), 4.00 (dd, J ¼ 3.3, 15.6 Hz, 1H), 4.49 (m, 1H), 4.64

(brddd, J ¼ 2.9, 9.5, 9.9 Hz, 1H), 7.23e7.28 (m, 3H), 7.32 (dd, J ¼ 7.2,

7.5 Hz, 2H), 7.38 (dd, J ¼ 7.4, 7.5 Hz, 1H), 7.45 (dd, J ¼ 7.3, 7.8 Hz, 2H),

7.60 (d, J ¼ 8.5 Hz, 2H), 7.65 (d, J ¼ 8.4 Hz, 2H), 8.10 (d, J ¼ 8.5 Hz,

2H); MS (EI) m/z 399 (M^þ, 4),181 ([CO-C₆H₄-Ph]^þ,100); HRMS calcd

for C₂₆H₂₅NO₃[M^þ] 399.1834, found 399.1829. Anal. Calcd for

4.1.5.2. Preparation of (2S,4R)-4-(hydroxy)-2-(2-[4-methoxyphenyl]-2-

oxoethyl)-1-(2-phenylacetyl)pyrrolidine (trans-16b). Obtained from

acetate trans-9b (100 mg, 0.25 mmol) as reported for cis-16b. After

puriﬁcation by column chromatography (hexanes:EtOAc 40:60), the

alcohol trans-16b was obtained as a white solid (86 mg, 96%). Com-

pound trans-16b has been previously reported [9].

C₂₆H₂₅NO₃: C, 78.17; H, 6.31; N, 3.51; Found: C, 77.93; H, 6.38; N,

3.85.

4.1.5.6. Preparation of (2S,4R)-4-(hydroxy)-2-(2-[p-biphenyl]-2-oxo-

ethyl)-1-(2-phenylacetyl) pyrrolidine (trans-16d). The acetate trans-

9d (110 mg, 0.25 mmol) underwent the General Saponiﬁcation

Procedure, and after usual puriﬁcation by column chromatography

(hexanes:EtOAc 40:60), compound trans-16d was isolated as a

4.1.5.3. Preparation of (2R,4R)-4-(hydroxy)-2-(2-[4-bromophenyl]-2-

oxoethyl)-1-(2-phenylacetyl) pyrrolidine (cis-16c). The acetate cis-

9c (111 mg, 0.25 mmol) underwent the General Saponiﬁcation

Procedure, and after usual puriﬁcation by column chromatography

(hexanes:EtOAc 40:60), compound cis-16c was isolated as a syrup

white foam (95 mg, 95%): IR

¼ 3405, 1681, 1638, 1607, 1427 cm^ꢁ1

;

¹H NMR (500 MHz, 26 ^ꢀC). A 9:1 mixture of rotamers was observed,

only the major rotamer is described:

¼ 2.00 (ddd, J ¼ 5.1, 6.9,

13.8 Hz, 1H) 2.17 (m, 1H), 2.89 (dd, J ¼ 9.5, 15.3 Hz, 1H), 3.49 (br d,

J ¼ 11.1 Hz, 1H), 3.58 (dd, J ¼ 4.5, 11.1 Hz, 1H), 3.63 (d, J ¼ 15.2 Hz,

1H), 3.67 (d, J ¼ 15.3 Hz, 1H), 3.97 (dd, J ¼ 3.2, 15.3 Hz, 1H), 4.45 (m,

1H), 4.65 (m, 1H), 7.21e7.25 (m, 3H), 7.29 (dd, J ¼ 7.3, 7.5 Hz, 2H),

7.36 (dd, J ¼ 7.3, 7.8 Hz, 1H), 7.45 (dd, J ¼ 7.3, 7.9 Hz, 2H), 7.59 (d,

(99 mg, 99%): IR

(500 MHz): A 5:1 mixture of rotamers was observed, only the major

¼ 3408, 1682, 1635, 1586 cm^ꢁ1

;

¹H NMR

rotamer is described:

3.27 (dd, J ¼ 10.3, 15.7 Hz, 1H), 3.59 (d, J ¼ 11.3 Hz, 1H), 3.62e3.69

¼ 1.95 (br d, J ¼ 14.2 Hz, 1H), 2.21 (m, 1H),

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

553

J ¼ 7.0 Hz, 2H), 7.65 (d, J ¼ 8.0 Hz, 2H), 8.07 (d, J ¼ 8.2 Hz, 2H); MS

(EI) m/z 399 (M^þ, 4), 181 ([CO-C₆H₄-Ph]^þ, 100); HRMS calcd for

(m, 1H), 4.57 (m, 1H), 6.10 (dd, J ¼ 2.5, 4.1 Hz, 1H), 6.78 (dd, J ¼ 1.8,

1.8 Hz, 1H), 7.22e7.32 (m, 6H); For this product, the spectrum at

70 ^ꢀC (one rotamer observed) presented better signal resolution,

₂₆H₂₅NO₃[M^þ] 399.1834, found 399.1823.

and it is also described:

¼ 1.98 (d, J ¼ 14.1 Hz, 1H), 2.16 (m, 1H),

4.4.5.7. (2R,4R)-4-(Hydroxy)-2-(2-[furan-2-yl]-2-oxoethyl)-1-(2-

phenylacetyl)pyrrolidine (cis-16f). The acetate cis-9f (89 mg,

0.25 mmol) underwent the General Saponiﬁcation Procedure, and

after usual puriﬁcation by column chromatography (hexanes:EtOAc

40:60), compound cis-16f was isolated as a white foam (75 mg,

3.13 (m, 1H), 3.48 (d, J ¼ 11.7 Hz, 1H), 3.54e3.70 (m, 4H), 3.88 (s,

3H), 4.54 (m, 1H), 4.56 (br dd, J ¼ 8.9, 9.4 Hz, 1H), 6.07 (dd, J ¼ 2.6,

3.9 Hz, 1H), 6.74 (brs, 1H), 7.12 (br b, 1H), 7.17e7.30 (m, 5H); ¹³C

NMR (125.7 MHz):

¼ 37.6 (CH₃), 37.9 (CH₂), 42.3 (CH₂), 43.1 (CH₂),

55.3 (CH), 55.8 (CH₂), 71.1 (CH), 108.3 (CH), 126.1 (CH), 126.8 (CH),

128.6 (2ꢃ CH), 129.0 (2ꢃ CH), 130.7 (C), 131.4 (CH), 134.4 (C), 169.9

(C), 190.1 (C); MS (EI) m/z 326 (M^þ, 6), 308 (M^þꢁ H₂O, 10), 217

96%): IR

¼ 3416, 1639, 1468, 1424 cm^ꢁ1; ¹H NMR (500 MHz): A 5:1

mixture of rotamers was observed, only the major rotamer is

described:

¼ 2.01 (br d, J ¼ 14.2 Hz, 1H), 2.20 (ddd, J ¼ 5.1, 8.9,

(M^þ

ꢁ

CO-(N-methylpyrrolyl) eH, 21), 108 ([CO-(N-methyl-

14.2 Hz, 1H), 3.21 (dd, J ¼ 10.1, 15.2 Hz, 1H), 3.58 (brd, J ¼ 11.3 Hz,

1H), 3.61e3.68 (m, 3H), 3.75 (dd, J ¼ 3.3, 15.2 Hz, 1H), 4.52 (m, 1H),

4.57 (m, 1H), 6.51 (dd, J ¼ 1.7, 3.6 Hz, 1H), 7.23e7.30 (m, 3H), 7.33

(dd, J ¼ 7.2, 7.6 Hz, 2H), 7.42 (d, J ¼ 3.6 Hz, 1H), 7.58 (d, J ¼ 1.2 Hz,

1H); For this product, the spectrum at 70 ^ꢀC (one rotamer observed)

pyrrolyl)]^þ, 100), 91 ([CH₂Ph]^þ, 77); HRMS calcd for C₁₉H₂₂N₂O₃

[M^þ] 326.1630, found 326.1629. Anal. Calcd for C₁₉H₂₂N₂O₃: C,

69.92; H, 6.79; N, 8.58; Found: C, 70.04; H, 7.05; N, 8.24.

4.4.5.10. (2S,4R)-4-(Hydroxy)-2-(2-[1-methyl-1H-pyrrol-2-yl]-2-

oxoethyl)-1-(2-phenylacetyl) pyrrolidine (trans-16g). The acetate

trans-9g (92 mg, 0.25 mmol) underwent the General Saponiﬁcation

Procedure, and after usual puriﬁcation by column chromatography

(hexanes:EtOAc 40:60), compound trans-16g was isolated as a

white foam (80 mg, 98%): IR

(500 MHz): A 12:1 mixture of rotamers was observed, only the

major rotamer is described:

(m, 1H), 3.47e3.60 (m, 2H), 3.60e3.80 (m, 3H), 3.88 (s, 3H), 4.47 (m,

1H), 4.63 (m,1H), 6.11 (dd, J ¼ 2.4, 4.1 Hz,1H), 6.79 (br s,1H), 7.18 (br

s, 1H), 7.20e7.27 (m, 3H), 7.27e7.33 (m, 2H); For this product, the

spectrum at 70 ^ꢀC (one rotamer observed) presented better signal

resolution, and it is also described: 2.12 (m, 2H), 2.71 (m, 1H), 3.49

(m, 1H), 3.58 (dd, J ¼ 4.9, 11.4 Hz, 1H), 3.64 (br b, 1H), 3.66 (br s, 2H),

3.91 (s, 3H), 4.47 (m, 1H), 4.65 (m, 1H), 6.11 (dd, J ¼ 2.5, 4.1 Hz, 1H),

6.77 (br s, 1H), 7.15 (brb, 1H), 7.20e7.34 (m, 5H); ¹³C NMR

presented better signal resolution, and it is also described:

¼ 2.00

(br d, J ¼ 13.9 Hz, 1H), 2.25 (m, 1H), 3.24 (m, 1H), 3.54 (br d,

J ¼ 11.7 Hz, 1H), 3.61e3.75 (m, 4H), 4.49 (m, 1H), 4.60 (m, 1H), 6.51

(br s, 1H), 7.21e7.38 (m, 6H), 7.56 (br s, 1H); ¹³C NMR (125.7 MHz):

¼ 38.1 (CH₂), 42.5 (2ꢃ CH₂), 54.4 (CH), 55.8 (CH₂), 71.3 (CH), 112.3

¼ 3402, 1640, 1528 cm^ꢁ1

;

¹H NMR

(CH), 118.9 (CH), 126.9 (CH), 128.7 (2ꢃ CH), 129.0 (2ꢃ CH), 134.3 (C),

146.8 (CH), 152.3 (C), 170.0 (C), 188.0 (C); MS (EI) m/z 313 (M^þ, 5),

295 (M^þꢁ H₂O, 8), 194 (M^þꢁ COCH₂Ph, 28), 95 (([CO-furyl)]^þ, 68),

91 ([CH₂Ph]^þ,100); HRMS calcd for C₁₈H₁₉NO₄[M^þ] 313.1314, found

313.1303. Anal. Calcd for C₁₈H₁₉NO₄: C, 68.99; H, 6.11; N, 4.47;

Found: C, 69.04; H, 6.41; N, 4.67.

¼ 2.10 (m, 2H), 2.44 (br b, 1H), 2.69

4.4.5.8. (2S,4R)-4-(Hydroxy)-2-(2-[furan-2-yl]-2-oxoethyl)-1-(2-

phenylacetyl)pyrrolidine (trans-16f). The acetate trans-9f (89 mg,

0.25 mmol) underwent the General Saponiﬁcation Procedure, and

after usual puriﬁcation by column chromatography (hexanes:EtOAc

40:60), compound trans-16f was isolated as a white foam (77 mg,

(125.7 MHz):

¼ 37.6 (CH₃), 39.2 (CH₂), 42.4 (CH₂), 42.8 (CH₂), 54.6

(CH), 55.4 (CH₂), 69.9 (CH), 108.3 (CH), 120.7 (CH), 126.9 (CH), 128.7

(2ꢃ CH), 129.0 (2ꢃ CH), 130.6 (C), 131.4 (CH), 134.4 (C), 170.7 (C),

188.8 (C); MS (EI) m/z 326 (M^þ, 20), 217 (M^þꢁ CO-(N-methyl-

pyrrolyl)eH, 8), 207 (M^þꢁ COCH₂Ph, 55), 108 ([CO-(N-methyl-

pyrrolyl)]^þ, 100), 91 ([CH₂Ph]^þ, 66); HRMS calcd for C₁₉H₂₂N₂O₃

[M^þ] 326.1630, found 326.1632. Anal. Calcd for C₁₉H₂₂N₂O₃: C,

69.92; H, 6.79; N, 8.58; Found: C, 70.18; H, 6.88; N, 8.47.

98%): IR

¼ 3406, 1666, 1638, 1468 cm^ꢁ1

;

¹H NMR (500 MHz): A

16:1 mixture of rotamers was observed, only the major rotamer is

described:

¼ 2.07 (m, 1H), 2.16 (m, 1H), 2.88 (dd, J ¼ 9.0, 15.2 Hz,

1H), 3.51 (br d, J ¼ 11.1 Hz,1H), 3.55e3.75 (m, 4H), 4.47 (m,1H), 4.61

(m, 1H), 6.51 (dd, J ¼ 1.7, 3.6 Hz, 1H), 7.20e7.27 (m, 3H), 7.31 (dd,

J ¼ 7.1 Hz, 2H), 7.37 (d, J ¼ 3.4 Hz, 1H), 7.57 (br d, J ¼ 1.6 Hz, 1H); For

this product, the spectrum at 70 ^ꢀC (one rotamer observed) pre-

sented better signal resolution, and it is also described:

¼ 2.11

4.2. Evaluation of the biological activity

(ddd, J ¼ 5.7, 6.0, 13.6 Hz, 1H), 2.17 (ddd, J ¼ 3.5, 7.3, 13.8 Hz, 1H),

2.95 (dd, J ¼ 8.8, 15.1 Hz, 1H), 3.52 (m, 1H), 3.60 (brdd, J ¼ 4.0,

10.5 Hz, 1H), 3.65 (m, 1H), 3.66 (brs, 2H), 4.48 (m, 1H), 4.64 (m, 1H),

6.50 (dd, J ¼ 1.6, 3.5 Hz, 1H), 7.21e7.32 (m, 6H), 7.56 (dd, J ¼ 0.6,

4.2.1. Cell culture and cytotoxicity assays for the indolizidinone

series

SHSY-5Y and MCF-7 cells were grown in DMEM/F12 Ham’s

medium supplied with 10% fetal bovine serum (FBS) and DMEM

phenol red supplemented with 10% FBS, respectively. Cultures were

maintained at 37 ^ꢀC under a 5% CO₂atmosphere in T75 ﬂasks until

reaching 80% conﬂuence. Once trypsinized, approximately 10 ꢃ 10⁶

cells were seeded in P25 Petri dishes, allowed to grow for 24 h, and

then exposed for additional 24 or 48 h to either vehicle (0.05%

DMSO) or the different compounds at increasing concentrations,

always dissolved in DMSO (1%). Cell counting was performed by

trypan blue staining, using Vi-Cell-XR analyzer (Beckman). Statis-

tical analyses of cell viability/mortality were performed by one-way

analysis of variance followed by Student’s t-test. Signiﬁcance levels

were computed at probability (p) values 0.05, 0.01 and 0.005, and

indicated in the ﬁgures.

1.6 Hz, 1H); ¹³C NMR (125.7 MHz):

¼ 39.3 (CH₂), 42.0 (CH₂), 42.5

(CH₂), 53.6 (CH), 55.4 (CH₂), 70.0 (CH), 112.3 (CH), 118.4 (CH), 126.9

(CH), 128.7 (2ꢃ CH), 129.0 (2ꢃ CH), 134.5 (C), 146.7 (CH), 152.5 (C),

170.4 (C), 187.1 (C); MS (EI) m/z 313 (M^þ, 4), 295 (M^þꢁ H₂O, 6), 194

(M^þꢁ COCH₂Ph, 26), 95 ([(CO-furyl)]^þ, 64), 91 ([CH₂Ph]^þ, 100);

HRMS calcd for C₁₈H₁₉NO₄[M^þ] 313.1314, found 313.1311. Anal.

Calcd for C₁₈H₁₉NO₄: C, 68.99; H, 6.11; N, 4.47; Found: C, 65.17; H,

6.27; N, 4.29.

4.4.5.9. (2R,4R)-4-(Hydroxy)-2-(2-[1-methyl-1H-pyrrol-2-yl]-2-

oxoethyl)-1-(2-phenylacetyl)pyrrolidine (cis-16g). The acetate cis-

9g (92 mg, 0.25 mmol) underwent the General Saponiﬁcation

Procedure, and after usual puriﬁcation by column chromatography

(hexanes:EtOAc 40:60), compound cis-16g was isolated as a white

foam (79 mg, 97%): IR

(500 MHz): A 7:1 mixture of rotamers was observed, only the major

¼ 3389, 1636, 1528, 1408 cm^ꢁ1

;

¹H NMR

4.2.2. Cell culture and cytotoxicity assays for the pyrrolidine series

Cell lines were cultured in DMEM (MCF-7) and DMEM/F12

(SHSY-5Y) media supplemented with 10% Fetal Bovine Serum

(FBS) up to 70% conﬂuence. Compounds were dissolved in DMSO

rotamer is described:

¼ 2.02 (br d, J ¼ 14.1 Hz, 1H), 2.14 (ddd,

J ¼ 5.3, 8.8, 14 Hz, 1H), 3.06 (dd, J ¼ 10.1, 14.1 Hz, 1H), 3.56 (d,

J ¼ 11.7 Hz, 1H), 3.60e3.67 (m, 3H), 3.72 (dd, J ¼ 3.6, 14 Hz, 1H), 4.48

(0.05%) and added to the media at 10 mM for 24 h or 30 mM for

554

J. Miguélez et al. / European Journal of Medicinal Chemistry 66 (2013) 540e554

V. Chandra Sekhar, A. Venkateswarlu, S. Rajagopal, R. Ajaykumar, D.S. Deevi,

N.V.S. Rao Mamidi, R. Rajagopalan Bioorg. Med. Chem. Lett. 13 (2003) 1679e

1682.

48 h. Cells were quantiﬁed by the trypan blue exclusion method

in an automated Vi-CELL XR analyzer (Beckman). Control

vehicle-treated cells were incubated with DMSO (0.05%) for the

same period. Total cells, viable cells, viability, mortality and

proliferation rates were calculated for each compound and dose.

Data were submitted to one way ANOVA followed by Students’s t

test. Results are expressed as mean ꢄ SEM of 5e6 different

assays.

[9] F.S. Kimball, B.J. Turunen, K.C. Ellis, R.H. Himes, G.I. Georg, Bioorg. Med. Chem.

16 (2008) 4367e4377.

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2238;

(b) M.J. Niphakis, G.I. Georg, J. Org. Chem. 75 (2010) 6019e6022.

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J. Nat. Prod. 65 (2002) 1299e1302.

Acknowledgments

[13] (a) For a preliminary communication, see: A. Boto, J. Miguélez, R. Marín,

M. Díaz, Bioorg. Med. Chem. Lett. 22 (2012) 3402e3407;

(2005) 3461e3468;

[14] (a) C.J. Saavedra, A. Boto, R. Hernández, J.I. Miranda, J.M. Aizpurua, J. Org.

Chem. 77 (2012) 5907e5913;

This work was supported by the Research Projects CTQ2009-

07109 (AB) and SAF2010-22114-C02-01 (MD)/02 (RM) (Plan

Nacional de I þ D, Ministerio de Ciencia e Innovación, currently

Economia y Competitividad, Spain), and Proyectos Intramurales

2004-8-0E211 and 2004-80E642 (CSIC, Spanish Research Council,

Spain). We also acknowledge ﬁnancial support from FEDER (Euro-

pean Funds for Regional Development). J. M. thanks CSIC for a JAE

predoctoral fellowship. We also thank technician Irene Guerra-

Palma for laboratory support. This article is dedicated to the

memory of D. Juan Marín, who inspired his daughter (RM) the love

for science.

(b) C.J. Saavedra, A. Boto, R. Hernández, Org. Lett. 14 (2012) 3542e3545;

(d) A. Boto, D. Hernández, R. Hernández, Tetrahedron Lett. 50 (2009) 3974e

3977;

(e) C. Saavedra, R. Hernández, A. Boto, E. Alvarez, J. Org. Chem. 74 (2009)

4655e4665;

(f) A. Boto, D. Hernández, R. Hernández, Tetrahedron Lett. 49 (2008) 455e458

(and references cited therein).

[15] S.G. Hansen, T. Skrydstrup, Top. Curr. Chem. 264 (2006) 135e162.

[16] (a) K.M. Bonger, T. Wennekes, D.V. Filippov, G. Lodder, G.A. van der Marel,

H.S. Overkleeft, Eur. J. Org. Chem. (2008) 3678e3688;

Appendix A. Supplementary data

(b) D. Smith, K.A. Woerpel, Org. Biomol. Chem. 4 (2006) 1195e1201;

Soc. 127 (2005) 10879e10884 (and references cited therein).

[17] In the ¹H NMR of the trans -compounds trans -9aeg , the signals of 3-H_aand 3-

H_bappeared as multiplets; in the case of their isomers cis -9aeg the signal of

3-H_aand 3-H_bwere usually a broad doublet (J_2,3az J_3a,4z 0 Hz) and a

multiplet, respectively. Besides, in the NOESY experiments of cis -9aeg , 2-H/4-

H spatial interactions were observed.

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2013.06.009.

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ketone 13 displayed similar NMR data (J_2,3a

J_3a,4

0.5 Hz, while

J_2,3a¼ 8.6 Hz and J_3a,4¼ 5.8 Hz; NOESY spatial interactions between 1⁰-H_aand

3-H_a).

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(2 R ,8a S ) the theoretical coupling constants were J_8a,8¼ 3.8, 11.7 Hz; J_8a,1¼ 6.1,

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