Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis, biological, and biophysical studies of DAG-indololactones

designed as selective activators of RasGRP

Lia C. Garcia ^a, Lucia Gandolﬁ Donadío ^a, Ella Mann ^c, Soﬁya Kolusheva ^c, Noemi Kedei ^b, Nancy E. Lewin ^b,

Colin S. Hill ^b, Jessica S. Kelsey ^b, Jing Yang ^b, Timothy E. Esch ^b, Marina Santos ^a, Megan L. Peach ^d,

James A. Kelley ^e, Peter M. Blumberg ^b, Raz Jelinek ^c, Victor E. Marquez ^e, Maria J. Comin ^a,

⇑

^aLaboratory of Organic Synthesis, Center of Research and Development in Chemistry, National Institute of Industrial Technology, Buenos Aires, Argentina

^bLaboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA

^cDepartment of Chemistry, Ben Gurion University, Beer Sheva 84105, Israel

^dBasic Science Program, Leidos Biomedical, Inc., Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, MD

21702, USA

^eChemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD

21702, USA

a r t i c l e i n f o

a b s t r a c t

Article history:

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms

and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important chal-

lenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP)

isoforms play both in immune responses as well as in the development of prostate cancer and melanoma,

suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the

N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity

for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical

analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2–5) that

differ in the position of the linkage between the indole ring and the lactone moiety. These structural

variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular

membranes, which is believed to be an important factor for selectivity in the activation of DAG-respon-

sive C1 domain containing signaling proteins. All compounds were potent and selective activators of Ras-

Received 23 December 2013

Revised 5 April 2014

Accepted 14 April 2014

Available online xxxx

Keywords:

Indolo-lactones

C1 domain

RasGRP

Cancer

GRP when compared to PKCa with selectivities ranging from 6 to 65 fold. However, the parent compound

1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the

patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles

as model membranes did show substantial differences in terms of molecular interactions impacting lipid

organization, dynamics and membrane insertion. However, these differences did not yield correspond-

ingly large changes in patterns of biological response, at least for the parameters examined.

1. Introduction

Abbreviations: DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMAP, 4-dimethyl-

aminopyridine; DMPC/DMPG, 1,2-dimyristoyl-sn-glycero-3-phospocholine/1,2-

dimyristoyl-sn-glycero-3-phosphoglycerol; Erk1/2, extracellular signal-regulated

kinases 1 and 2; LiHMDS, lithium hexamethyldisilazide; MsCl, methanesulfonyl

chloride; PCC, pyridinium chlorochromate; NBD-PE, [1,2-dipalmitoyl-sn-glycero-3-

phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)] (ammonium salt);

PDBu, [20-^cH]phorbol 12,13-dibutyrate; PKC, protein kinase C; PC, phosphatidyl

choline; PG, 1-(3-sn-phosphatidyl)-rac-glycerol sodium salt; PKD, protein kinase D;

PMA, phorbol 12-myristate 13-acetate; PPTS, pyridinium p-toluene sulfonate;

RasGRP, Ras guanine nucleotide-releasing protein; TBDMSCl, tert-butyldimethylsi-

lyl chloride; TBDPSCl, tert-butyldiphenylsilyl chloride.

The lipid diacylglycerol (DAG) represents one of the central sec-

ond messengers in cell signaling. Increased levels of DAG in the

plasma membrane occur in response to either tyrosine-kinase

receptor or G-protein coupled receptor stimulation, primarily

through the activation of phospholipase C. This enzyme catalyzes

the hydrolysis of phosphatidylinositol 4,5-bisphosphate into a pair

of second messengers: inositol triphosphate and DAG. Increased

levels of DAG are transduced into cellular signals via various PKC

isoforms and six other families of proteins through the interaction

with structurally similar C1 domains.^1–3

⇑

Corresponding author. Tel./fax: +54 11 4724 6289.

E-mail address: jcomin@inti.gob.ar (M.J. Comin).

http://dx.doi.org/10.1016/j.bmc.2014.04.024

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2

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

C1 domains are zinc ﬁnger structures of approximately 50

characterization revealed that the orientation of the 1-methyl-

1H-indole ring on the DAG-lactone had substantial effects on the

nature of the interaction of the ligand with the membrane. Further-

more, it was found that the removal of the 1-methyl group pro-

duced only an insigniﬁcant change in binding afﬁnity, suggesting

that the free indole NH was not involved in any critical interac-

tion.²³Since the presence of the 1-methyl group greatly facilitates

the syntheses, we chose to prepare the complete family of regioiso-

meric DAG-lactones with an invariant 1-methyl-1H-indole ring to

study their PKC and RasGRP binding afﬁnities, biological activity,

and translocation characteristics (Fig. 1). These isomers differ from

one another in terms of the position of the linkage between the

benzene ring of the 1-methyl-1H-indole and the DAG-lactone,

which changes the orientation of the heteroaromatic ring relative

to the lactone template. In addition, we examined the inﬂuence

of the various 1-methyl-1H-indole ring orientations with model

membrane vesicle systems using a range of biophysical techniques.

Given that the proposed binding mode for DAG-lactones is with the

amino acids in length that were originally discovered as lipid bind-

ing modules in PKCs, a family of related kinases that regulate pro-

liferation, differentiation and malignant transformation.⁴C1

domains can be sub-classiﬁed into two families, those that are

responsive to DAG and those that are not. The classic and novel

PKC isoforms represent the ﬁrst recognized and most extensively

studied family of effectors for DAG.^5,6The C1 domains of the atyp-

ical PKCs provide one paradigm for the so-called ‘atypical’ C1

domains that do not respond to DAG.

There is a large body of evidence supporting the potential of

PKC isoforms as therapeutic targets in cancer, immunological, car-

diovascular and neurological diseases. Examples of DAG mimetics

in clinical trials that bind the C1 domain are the natural products

bryostatin 1 and ingenol 3-angelate (PEP005).⁷Indeed, the use of

ingenol-3-angelate for the topical treatment of actinic keratosis

has been approved recently.^8,9After the identiﬁcation of the PKCs,

six other families of proteins with homologous, DAG-responsive C1

domains have been identiﬁed. The protein kinase D family is

involved in Golgi function, proliferation, metastasis and apopto-

sis.¹⁰The chimaerins act as inhibitors (GAPs, GTPase activating

proteins) for the small GTPase Rac and are candidate tumor sup-

pressors.¹¹The Unc-13 family members are responsible for pro-

moting vesicle priming.¹²The DAG kinases terminate DAG

signaling by phosphorylating DAG.¹³MRCK is a downstream effec-

tor of cdc42 involved in ﬁlopodia formation, contributing to tumor

invasion.¹⁴Finally, the RasGRP family members function as activa-

tors (GEFs, GTP Exchange factors) for Ras¹⁵and are prominently

expressed in blood cells. RasGRP malfunction likely contributes

to autoimmunity and may contribute to blood malignancies.¹⁶In

addition, the role of RasGRP3 in prostate cancer¹⁷and melanoma¹⁸

has been demonstrated and, very recently, it was shown that Ras-

GRPs are targets of the anticancer drug ingenol-3-angelate.¹⁹Given

the important biological roles of RasGRP family members, the dis-

covery of selective agents capable of speciﬁcally interacting with

their C1 domain could provide exciting lead structures for drug

development.

When DAG or phorbol esters bind to the C1 domain, they com-

plete a hydrophobic surface on the top face of it. Likewise, the

hydrophobic substituents on the ligand contribute an additional

hydrophobic element. Together, these factors drive membrane

association of the C1 domain and the accompanying stabilization

of the active, membrane-associated conformation of the protein.

Because the membrane environment constitutes the third element

of the ternary binding complex, the membrane microdomain com-

position, as well as its variation as a function of localization within

the cell, provides a potentially important basis for establishing

selectivity of regulation.²⁰

DAG-lactones were developed as a synthetically more tractable

alternative to complex natural products such as bryostatin 1 or

phorbol esters that are ultrapotent analogues of DAG. DAG-

lactones combine a rigid template, responsible for enhanced

afﬁnity, with relative chemical simplicity, allowing the synthetic

exploration of a wide chemical space through the combined action

of a myriad of substituents at the sn-1 and sn-2 positions that we

have deﬁned as ’chemical zip codes’.²¹As reported previously,

nanomolar binding afﬁnities for PKC approaching those of natural

products have been achieved.²⁰Furthermore, compounds with

marked binding selectivity for RasGRP, as compared to PKC, were

developed with the most selective compound incorporating a

1-methyl-1H-indole ring at the sn-2 position of the DAG-lactone

(compound 1, Fig. 1).²²Following up on these ﬁndings, two other

DAG-indololactones were prepared to evaluate the effect on bind-

ing afﬁnity and selectivity as a function of the point of attachment

on the pyrrole ring of the 1-methyl-1H-indole, as well as the role of

the 1-methyl group. While selectivity did not improve, our

a-arylidene moiety oriented toward the surface of the C1 domain,

adjacent to the lipid interface,²⁴we postulated that the orientation

of the 1-methyl-1H-indole group at this position could inﬂuence

membrane binding selectivity. Our results show that indeed the

orientation of the 1-methyl-1H-indole group plays a critical role

in selectivity with the most selective compound being the one

linked through position 3 (1). Moreover, the biophysical data sug-

gest a differential mode of membrane interaction depending on the

point of attachment. Finally, the successful outcome of this study

was critically dependent on the development of a new synthetic

strategy for the synthesis of the novel analogues 2–5 that took

into account the high reactivity of the 2- and 3-unsubstituted

1-methyl-1H-indole ring.

2. Results

2.1. Chemistry

The synthesis of compound 5 was initially attempted following

the same strategy reported for lead compound 1 using bis-silylated

lactone 6a as starting material.²²Unfortunately, the selective ﬁnal

monoacylation was not reproducible, giving low yields and varying

amounts of diacylated product (Scheme 1). Alternatively, we tried

to prepare compound 5 starting from the known lactone 6b, pro-

tected with a benzyl and a TBDPS groups as already reported for

related derivatives of compound 1.²³In the present case, total

decomposition was observed when treating the precursor 7b under

typical conditions employed for benzyl group cleavage (BCl₃,

ꢀ78 °C, CH₂Cl₂) for this type of compounds. In addition, several

attempts to selectively remove the benzyl group in the presence

of the conjugated double bond by transfer hydrogenation were

made. Unfortunately, in our hands, no selectivity could be achieved

employing different hydrogen sources and catalysts.^25,26In all

cases, we obtained complex mixtures of the desired product

together with considerable amounts of the corresponding satu-

rated derivatives.

In order to overcome these difﬁculties, we decided to prepare a

new starting lactone 11 protected with TBDPS and TBDMS groups.

This strategy was based on the possible selective removal, under

very mild conditions (PPTS, EtOH),²⁷of the TBDMS protecting

group in the presence of the TBDPS group (Scheme 2). We envi-

sioned that these conditions would be compatible with the pres-

ence of highly reactive 2- and 3-unsubstituted indole rings.

Therefore, starting from commercially available 1,3-dihydroxyace-

tone dimer, the monoprotected 1,3-dihydroxyketone was

obtained.²⁸Introduction of the second silyl protective group was

performed under standard conditions to yield the fully protected

ketone 9. Then, treatment with allylmagnesium chloride produced

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L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

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Figure 1. Structures of DAG-indololactones.

Scheme 1. Monoacylation approach towards DAG-indololactones. Reagents and conditions: (a) (i) LiHMDS, indole-7-carboxyaldehyde, THF, ꢀ78 °C, (ii) Et₃N, MsCl, DBU,

CH₂Cl₂, 0 °C ? rt, 78% (7a), 61% (7b); (b) HFꢁEt₃N, THF, 70 °C, 74%.

and ﬁnal deprotection of the second primary hydroxyl group. In

all cases, oleﬁnation of the aldol products resulting from the reac-

tion with the corresponding 1-methylindole-carboxaldehydes

afforded almost exclusively the E-isomers. The E/Z geometry

around the double bond was assigned by ¹H NMR; the vinyl proton

for the E-isomers displayed a characteristic multiplet that was fur-

ther downﬁeld from that of the corresponding Z-isomers.²⁹Selec-

tive deprotection was achieved by treatment of alkylated

products 12a–d in ethanolic solution with PPTS at 60 °C. Under

these conditions, the desired alcohols 13a–d were obtained in very

good yields. Acylation followed by deprotection afforded the

Scheme 2. Synthesis of starting lactone 11. Reagents and conditions: (a) (i)

expected DAG-indololactones 2, 3, 4 and 5.

TBDPSCl, imidazole, DMF, 0 °C, 51%, (ii) TBDMSCl, imidazole, CH₂Cl₂, 0 °C, 87%; (b)

allylmagnesium chloride, THF, 0 °C ? rt, 89%; (c) (i) BH₃ꢁSMe₂in THF, CH₂Cl₂,

2.2. Biological results

ꢀ78 °C ? rt, (ii) PCC, CH₂Cl₂, rt, 74% (two steps).

2.2.1. Binding of ligands to PKC

To examine possible binding selectivity, the binding afﬁnities of

compounds 1–5 to PKC , to PKC , to the C1 domain of RasGRP1,

and to RasGRP3 were measured by competition for [³H]PDBu bind-

ing in the presence of 100 g/mL phosphatidylserine (Table 1). All

compounds were appreciably more selective for the RasGRP iso-

forms than for PKC or PKC , with selectivities ranging from 5 to

a, PKCe and to RasGRP1/3

the homoallylic alcohol 10 in excellent yield and purity. Finally,

tandem hydroboration and PCC oxidation yielded the protected

lactone 11 with 74% yield.

With key intermediate 11 at hand, the desired positional iso-

mers of lead compound 1 were prepared as depicted in Scheme 3.

Thus, target compounds 2, 3, 4 and 5 were synthesized by sequen-

tial alkylation–elimination followed by selective deprotection of

the TBDMS group, acylation of the free primary hydroxyl group,

a

e

l

a

e

84 fold. Relative to both PKC isoforms, the parent compound 1

was the most selective. Compound 3 approached compound 1 in

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L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

Scheme 3. Synthesis of regioisomeric DAG-indololactones. Reagents and conditions: (a) (i) LiHMDS, indole-aldehyde, THF, ꢀ78 °C, (ii) Et₃N, MsCl, DBU, CH₂Cl₂, 0 °C ? rt; (b)

PPTS, EtOH, 60 °C; (c) Et₃N, DMAP, (CH₂CH₂CH₃)₂CHCOCl, CH₂Cl₂; (d) HFꢁEt₃N, THF, 70 °C.

Table 1

Binding selectivity of DAG-indololactones for PKC

a and PKCe versus RasGRP

Compound

PKCa^aK_i(nM)

PKCe

K_i(nM)

RasGRP3 K_i(nM)

PKC

a:RasGRP3

PKCe

:RasGRP3

RasGRP1-C1 K_i(nM)

PKCa:RasGRP1

PKCe:RasGRP1

1

2

3

4

16.2 1.0

17.8 2.0

8.25 0.88

7.5 1.0

12.9 1.4

0.17 0.02

21.1 1.9

14.6 2.7

2.17 0.36

8.08 0.31

11.7 0.30

0.22 0.05

0.33 0.06

1.63 0.18

0.34 0.01

1.22 0.21

1.12 0.05

0.42 0.03

49

11

24

6

10

—

64

9.0

6.4

6.6

11

—

0.25 0.10

1.55 0.10

0.41 0.10

0.87 0.04

0.69 0.14

0.12 0.01

65

10

24

9

19

—

84

9.4

5.3

9.3

17

—

5

[³H]PDBu^b

a

Values represent mean SEM of triplicate independent experiments.

[K_d(nM)].

b

selectivity with respect to PKCa, being about half as selective, and

experiments. As illustrated for compound 1 (Fig. 2A), Erk1/2 phos-

compound 4 was the least selective. Differences in selectivity were

somewhat more pronounced when compared to PKC (up to 64 to

84-fold) than they were for PKC (up to 49 to 65-fold). In terms of

phorylation was greatly enhanced in the HEK cells overexpressing

e

RasGRP3. Phosphorylation began to be seen at 1

lM and was

a

intense by 10 M. RasGRP3 T133 phosphorylation increased appre-

l

absolute potencies for RasGRP1/3, the compounds ranged from

slightly more potent than the phorbol ester PDBu to 13-fold less

potent. For both PKCa and PKCe, the compounds were all apprecia-

bly less potent than PDBu, reﬂecting their selectivity. Compounds 1

and 3 were the most potent for RasGRP1/3. For RasGRP1/3 and for

PKCa, compound 2 was the least potent. For PKCe, compound 1

was a little less potent. RasGRP3 and the C1 domain of RasGRP1

behaved very similarly to one another.

ciably less with increasing ligand concentration than did Erk1/2

phosphorylation, consistent with the important role of C1 domain

occupancy by the ligand for RasGRP activity. The dose response

curve for PKCd S299 phosphorylation in response to the com-

pounds was shifted to somewhat higher concentrations relative

to that of RasGRP3 T133 phosphorylation, suggesting that other

PKC isoforms may be more important than PKCd for RasGRP3 phos-

phorylation. A generally similar pattern of responses was observed

with PMA, although the potency of PMA was considerably greater

(Fig. 2B). Within the resolution of the experiments, results for com-

pounds 2–5 were similar (Supplementary Fig. 1).

Biological activity was also examined in Ramos cells, which

endogenously express RasGRP3 to a high level. Once again all ﬁve

compounds showed approximately similar potencies for Erk1/2

activation and for PKCd S299 phosphorylation, as well as for phos-

phorylation of PKD, a downstream substrate of PKC (Supplemen-

tary Fig. 2).

2.2.2. Biological activity of ligands

The biological activities and relative potencies of compounds 1–

5 were examined in HEK293 cells and compared with that in

HEK293 cells transfected with RasGRP3. PMA was used as a phor-

bol ester control. Erk1/2 phosphorylation is a reporter for activa-

tion of the Ras pathway and thus should reﬂect RasGRP3

activation. Phosphorylation of PKCd at S299 provides a measure

of PKCd activation,³⁰while phosphorylation of RasGRP3 at T133

is thought to be needed for activation,^31,32along with occupancy

of its C1 domain by DAG, phorbol ester, or related ligands,³³and

is brought about by various PKC isoforms.^34,35All ﬁve compounds

behaved similarly, within the range of resolution of the

2.2.3. Translocation of PKCs and RasGRP3 in response to ligands

Translocation of PKC isoforms in response to phorbol esters

or other DAG analogs is

a

sensitive reporter of the

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5

Figure 2. Response of HEK293 and HEK293 cells overexpressing RasGRP3 to treatment with compound 1 (A) or with PMA (B). Cells were treated for 30 min with the indicated

concentrations of compound, after which the cells were lysed and the lysates subjected to electrophoresis and immunoblotting with the antibodies as indicated. Results are

representative of the triplicate experiments performed.

PKC–ligand–membrane interactions.^36,37We visualized cellular

translocation of exogenously expressed GFP-PKC YFP-PKC

GFP-PKCd, and GFP-RasGRP3 as a function of time of treatment

with 10 M ligand. The GFP-PKC , GFP-PKCd and GFP-RasGRP3

Quantitation of images indicated that, for PKCa, compounds 3, 4,

a

,

e,

and 5 induced more translocation that did 1 and 2. It was also

noteworthy that the rates of translocation varied, with response

to 4 reaching a maximum after 2–5 min; those for 1 and PMA were

l

e

were expressed in LNCaP cells, which we have extensively used

appreciably slower. For PKCe, compounds 2 and 3 induced more

for visualization of PKC translocation, as well as for biological anal-

rapid translocation than did 1, 4, and 5. For PKCd, little difference

was seen among compounds, all of which induced less transloca-

tion than did PMA. For RasGRP3, all of the compounds induced

greater clustering at internal membranes, as evidenced by

increased patchiness of its distribution, but without increased

association with the plasma membrane. For further comparison,

we also examined translocation in response to PDBu (Supplemen-

tary Fig. 3). PDBu, being less lipophilic than PMA, induces localiza-

tion of PKCd predominantly to internal membranes rather than the

ysis of PKC ligands.^38–40GFP-PKC

a

was expressed in CHO cells,

since PKCa shows only limited translocation in response to phor-

bol esters in LNCaP cells, presumably reﬂecting a low basal level

of internal calcium, which functions as a co-activator of classic

PKC isoforms.³⁸Representative images are presented for PKC

a,

for PKC

e

, for PKCd, and for RasGRP3 (Figs. 3A, C, E, and 2F, respec-

tively). For PKC

a

and PKC , which showed substantial differences

e

among ligands, the results were also quantitated as the ratio of

membrane/cytoplasmic signal (Fig. 3B, D, respectively).

plasma membrane. Its effects on PKCa, PKCe, and RasGRP3 are

Figure 3. Translocation of different PKC isoforms in living cells. CHO-K1 cells were transiently transfected with GFP-PKC

with YFP-PKC (C, D), GFP-PKCd (E), or GFP-RasGRP3 (F). The translocation pattern was examined by confocal microscopy as a function of time after treatment with the

indicated drugs (10,000 nM) or PMA (1000 nM). Each panel represents images typical of the experiments performed (n = 3–6). Bars are 10 m. Quantitative analysis of PKC

(B) and PKC (D) translocation. Average SEM of calculated values are presented.

a (A, B) and LNCaP cells were transiently transfected

e

l

a

e

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L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

similar to those of PMA but somewhat less complete, reﬂecting its

lower absolute afﬁnity.

2.3. Biophysical studies

To assess the modes of membrane interactions of DAG-indolo-

lactones 1–5 and to determine both the effects of the compounds

upon lipid organization and dynamics and their relative insertion

into membrane bilayers, we applied several ﬂuorescence spectros-

copy techniques. Figure 4 depicts the results of ﬂuorescence

anisotropy measurements utilizing giant PC/PG vesicles, which

also contained the ﬂuorescent dye diphenylhexatriene-trimethy-

lammonium (DPH-TMA). DPH-TMA is embedded in the hydropho-

bic environment of the lipid bilayer, and changes to its

ﬂuorescence anisotropy generally provide a mean for evaluating

modulation of bilayer ﬂuidity induced by membrane-active

compounds.^41,42

The ﬂuorescence anisotropy data in Figure 4 point to distinct

bilayer modulation effects induced by the DAG-indololactones

studied. All ﬁve compounds appear to modulate the ﬂuorescence

anisotropy, albeit to different degrees. Speciﬁcally, the data pre-

sented in Figure 4 point to roughly two main groupings. Com-

pounds 2 and 3 exhibited only minor changes in anisotropy,

indicating minimal effect upon bilayer ﬂuidity. Compounds 1, 4,

and 5, however, gave rise to a much more signiﬁcant increase in

ﬂuorescence anisotropy, corresponding to lowered bilayer ﬂuidity

upon binding of these indololactones.

While Figure 4 highlights the impact of the DAG-indololactones

upon the bilayer properties, we carried out ﬂuorescence spectros-

copy experiments utilizing the intrinsic 1-methyl-1H-indole

ﬂuorescence emission⁴³to probe the effect of vesicle interactions

upon the DAG-indololactones’ environment (Fig. 5). Figure 5A indi-

cates that the intrinsic ﬂuorescence emission of 1, 3 and 5 is low.

However, the ﬂuorescence signals of 2 and 4 undergo dramatic

modulations ascribed to bilayer internalization (Fig. 5B and C).

Speciﬁcally, the ﬂuorescence emission peaks of both 2 and 4 clearly

increase in intensity and shift to lower wavelengths following

increasing vesicle concentrations in the aqueous solutions. These

effects are indicative of more hydrophobic environments of the

1-methyl-1H-indole moieties,⁴⁴most likely brought about by

insertion of these two DAG-indololactones into the bilayer. Impor-

tantly, the ﬂuorescence shift appears most signiﬁcant in the case of

DAG-indololactone 4, which is consistent with the anisotropy data

in Figure 4 pointing to pronounced interaction of this compound

Figure 5. Fluorescence emission spectra of DAG-indololactones 1–5 in the absence

and in the presence of increasing concentration of giant vesicles. (A) Compounds 1

(- - -), 3 (ꢁꢁꢁꢁ) and 5 (solid line); samples (6.05

egg-PC giant vesicle solutions (30 L) in the scale of compound 4. Excitation

wavelength = 358 nm, 343 nm, 364 nm respectively; (B) Compound 2 (6.05 M),

control sample (- - -) titrated with an increasing volume of DMPG/egg-PC giant

vesicle solutions (5, 10, 20, 30 L). Excitation wavelength = 373 nm; (C) Compound

4 (6.05 M), control sample (- - -) titrated with an increasing volume of DMPG/egg-

PC giant vesicle solutions (5, 10, 20, 30 L). Excitation wavelength = 349 nm.

lM) in buffer in the presence DMPG/

l

Figure 4. DAG-indololactones modulation of ﬂuorescence anisotropy of DHP-TMA

embedded in giant PC/PG vesicles. DHP-TMA was present in 1.25 lM concentration.

Anisotropy was recorded in control vesicles without compound addition (left

column), and in the presence of two concentrations of DAG-indololactones 1–5:

2.42 lM (middle column) and 6.05 lM (right column).

with the lipid bilayer. Additionally, the ﬂuorescence excitation

and emission spectra of indololactones 1 to 5 in solvents of varying

polarities were measured (Supplementary Fig. 4). In accordance

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L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

7

with the effect of lipid addition, the results showed a strong depen-

dence of ﬂuorescence emission with solvent polarity. A shift

towards lower wavelengths was observed with decreasing solvent

polarity.

should be emphasized that an insigniﬁcant FRET effect was

recorded in the case of 1 (Fig. 6D), supporting the proposal that 1

experiences a different mode of bilayer insertion. By addition of

Triton X-100, the emission spectra of each DAG-indololactone were

obtained after irradiation at its corresponding intrinsic excitation

wavelength, as a consequence of vesicle disruption and FRET elim-

ination. [No FRET could be measured in the case of 2 since there is a

signiﬁcant mismatch between its emission wavelength and the

excitation wavelength of NBD-PE].

To further probe membrane interactions of the DAG-

indololactones we carried out ﬂuorescence energy transfer experi-

ments utilizing giant egg-PC/PG vesicles containing the ﬂuorescent

dye NBD-PE. Figure 6 depicts the results of ﬂuorescence resonance

energy transfer (FRET) experiments in which the ﬂuorescence

emission spectra of NBD-PE were recorded following excitation

at 330–370 nm (the intrinsic excitation wavelength of the

DAG-indololactones). In addition, one measurement for each

sample was done also in the presence of 20% Triton X-100 that

disrupts vesicles and, consequently, eliminates the FRET.

The FRET data in Figure 6 provide further insight into mem-

brane interactions of the DAG-indololactones and the differences

among the compounds. Consistent with the ﬂuorescence anisot-

ropy data (Fig. 4) and ﬂuorescence emission analysis (Fig. 5), 4

appears to enmesh most efﬁciently within the bilayer, giving rise

to signiﬁcant FRET to the bilayer-embedded NBD-PE (Fig. 6A).

The FRET results also underscore highly effective FRET in the case

of 5 (Fig. 6B), likewise consistent with the anisotropy results in Fig-

ure 3. Intriguingly, DAG-indololactone 3 also appears to facilitate

FRET, giving rise to the notable increase in the ﬂuorescence

emission of the co-encapsulated NBD-PE (Fig. 6C). This result is

somewhat surprising in light of the relatively smaller effect of 3

upon the DPH-TMA anisotropy (Fig. 4) and suggests that this

DAG-indololactone undergoes distinct bilayer interactions in

comparison with the other compounds studied. In this context, it

2.4. Modeling studies

In order to explain and reconcile the biological and biophysical

results, we decided to investigate the interaction of the DAG-indo-

lolactone compounds through molecular dynamics simulations

with a modeled bilayer that approximates the solvation effects of

a lipid membrane, including the heterogeneous dielectric environ-

ment of the membrane interfacial region where water interacts

with the lipid headgroups and glycerol backbones.⁴⁵The results

show that all the compounds reach equilibrium at approximately

the same depth in the bilayer (Fig. 7A), with both the indole ring

and the branched alkyl sidechain located in the interfacial region.

This is consistent with experimental NMR^46–48and all-atom

molecular dynamics results^49,50for the bilayer localization of

indole and tryptophan. Shown in this ﬁgure also are the approxi-

mate experimentally determined locations of DPH-TMA (the

central double bond)⁵¹and NBD-PE.⁵²

The measured changes in DPH-TMA ﬂuorescence anisotropy

sense the perturbation induced by the insertion of the

Figure 6. Fluorescence energy transfer induced by DAG-indololactones in giant egg-PC/PG vesicles containing NDB-PE. (A) Compound 4, 6.05

wavelength = 349 nm). NBD-PE (ꢁꢁꢁꢁ), NBD-PE+4 (solid line), and NBD-PE+4+Triton (- - -); (B) compound 5, 6.05 M (excitation wavelength = 364 nm). NBD-PE (ꢁꢁꢁꢁ), NBD-

PE+5 (solid line) and NBD-PE+5+Triton (- - -); (C) compound 3, 6.05

M (excitation wavelength = 343 nm). NBD-PE (ꢁꢁꢁꢁ), NBD-PE+3 (solid line) and NBD-PE+3+Triton (- - -);

(D) compound 1, 6.05

M (excitation wavelength = 358 nm). NBD-PE (ꢁꢁꢁꢁ), NBD-PE+1 (solid line) and NBD-PE+1+Triton (- - -).

lM (excitation

l

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8

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

A

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

DPH

NBD

1 indole

2 indole

3 indole

4 indole

5 indole

1 alkyl

2 alkyl

3 alkyl

4 alkyl

5 alkyl

0

5

10

15

20

25

30

35

Distance from center of bilayer (A)

B

0.12

0.1

0.08

0.06

0.04

0.02

0

1

2

3

4

5

-45

-30

-15

0

15

30

45

60

75

90

105

120

135

150

165

180

Angle between indole dipole and bilayer normal

Figure 7. (A) Histogram of the density of indole ring atoms (solid lines) and branched alkyl sidechain atoms (dashed lines) as a function of distance from the bilayer center.

(B) Histogram of the occupancy of the angle between the indole emission dipole moment and the bilayer normal.

indololactones in the bilayer and, as such, they depend on several

factors that inﬂuence the ﬂuidity and ordering in the acyl chain

region of the membrane. This is not directly observable in our

simulations, since we are not explicitly including any lipid atoms,

and since the DAG-indololactones do not penetrate deeply into this

region of the bilayer (Fig. 7A), but may be related to changes in

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L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

9

lipid headgroup spacing and hydration⁵³induced to different

degrees by the different DAG-indololactones.

are summarized for our simulations of each DAG-indololactone

in Table 2.

On the other hand, FRET measurements are directly related to

the distance and the angle between the dye (NBD) and the indole

ring of the DAG-indololactones. Although all the indole rings

occupy approximately the same depth in the bilayer, the magni-

tude and efﬁciency of energy transfer in FRET is also a function

of the relative angles of the donor emission dipole of the indole

and the acceptor absorption dipole of NBD. Indole has two excited

states, L_aand L_b, whose energies and dipole moment directions

depend both on the polarity of the solvent environment and on

the nature and position of any ring substituents.^54,55Here for sim-

plicity we assumed that in the bilayer environment the emitting

state will be L_b, and deﬁned an approximate dipole moment as a

vector from the 3-position to the 7-position on the ring. In Fig-

ure 7B the occupancy of the angle between this vector and the

bilayer normal is calculated for each compound. It can be observed

that compound 1 is an outlier with a signiﬁcantly more restricted

range of motion, differing from the other compounds that exhibit a

greater freedom of rotation.

The ﬂuorescence resonance energy transfer (FRET) depicted in

Figure 6 shows that for compounds 3, 4, and 5 there is a signiﬁcant

FRET to the bilayer-embedded NBD agreeing with the proximity

observed in our simulations between it and the indole moieties

as well as the orientational heterogeneity of the indole ring. In

the case of compound 1, however, the simulations suggest that

the insigniﬁcant FRET observed may be a function of the restricted

rotation of the indole ring in the bilayer environment. This may

also be an explanation for the low intrinsic ﬂuorescence emission

of this compound, as shown in Figure 5.

The conformation that all DAG-lactones must adopt in order to

bind the C1 domain has been well-established by our previous

modeling studies.^56–60Figure 8 shows a representation of com-

pound 1 relative to the binding site loops of the C1 domain and

the bilayer dielectric environment. The carbon atoms of the glyc-

erol backbone (or what would be the glycerol backbone if these

compounds were standard open-chain diacylglycerols) are num-

bered.⁶¹For effective C1 domain binding, the h₄dihedral angle

(around the bond between carbon atoms 1 and 2) must be the

+sc rotamer (ꢂ60°), and the plane of the glycerol backbone should

be approximately 45° relative to the plane of the bilayer. The

occupancy of these angular conformations, as well as the average

depth of the chiral carbon in the lactone ring (atom 2 of the glyc-

erol backbone), which falls near the center of the binding site,

All of the DAG-indololactones reach equilibrium at essentially

identical membrane depths for the lactone ring, and also adopt

an average angle relative to the bilayer plane that is close to 45°.

This suggests that the depth and angle of membrane penetration

by the C1 domain for ligand binding will be nearly the same for

all compounds. Additionally, the binding conformation of the h₄

dihedral angle, which controls the position of the sn-1 branched

alkyl sidechain, is energetically accessible and occupied a signiﬁ-

cant fraction of the time in our simulations for each compound.

These results shed light on the relatively minor differences in bind-

ing afﬁnity, for a given C1 domain, between the DAG-indololac-

tones (Table 1).

3. Discussion

The synthesis of DAG-indololactones with an unsubstituted

1-methyl-1H-indole ring at the 3- and/or 2-positions required

the preparation of the lactone precursor 11 where the two

hydroxyl moieties were distinctively protected either as TBDPS

or TBDMS ethers. This material was prepared from economical,

commercially available 1,3-dihydroxyacetone dimer in ﬁve steps

with an overall yield of 29%. This result constitutes a valuable

synthetic achievement when compared to known synthetic

routes towards DAG-lactones that employ expensive glycidyl

4-methoxyphenyl ether as starting material.⁶²With 11 at hand,

the development of a synthetic approach based on the selective

removal of the TBDMS group under mild conditions allowed the

preparation of the desired indololactones 2–5 that we have

failed to synthesize employing already developed conditions. In

the case of the monoacylation approach reported for the synthe-

sis of compound 1, no selectivity could be achieved and low and

variable yields of the desired indololactone 5 were obtained.

When trying the use of 6b, differentially protected as benzyl

and a TBDPS ethers, the benzyl group could not be removed

either because of poor stability of the molecule in the presence

of a Lewis acid (position 2 and 3 of the indole ring are very reac-

tive) or because of lack of selectivity in transfer hydrogenation

conditions.^22,23

It has become increasingly clear that physical perturbations of

lipid membranes such as ﬂuidity, curvature, hydrocarbon volume

and headgroup separation contribute to the modulation of PKC

activity.⁶³It is also accepted that lipid–protein interactions can

provide the necessary speciﬁcity and afﬁnity to achieve a particular

subcellular localization of signaling proteins.⁶⁴Thus, membrane

lipids may help compartmentalize signaling complexes and regu-

late the spatiotemporal dynamics of PKC activations through inter-

acting membrane microdomains, some of which can be formed

transiently during signaling after release of DAG. Accordingly, we

surmise that the speciﬁc cellular localization of the C1 domain

containing protein ought to be determined in part by the different

lipid composition of the membranes, the targeting information

Table 2

Binding conformation of DAG-indololactones

Compound

Ring depth^a

Backbone angle^b

+sc rotamer^c

1

2

3

4

5

17.38

17.34

17.54

17.58

17.34

44.31

43.10

48.31

59.04

49.18

0.63

0.51

0.50

0.44

Figure 8. Cartoon representation of the C1 domain binding conformation of a DAG-

indololactone. The carbon atoms in the glycerol backbone are numbered. The

colored background represents the varying dielectric environments in the modeled

bilayer: blue = lipid headgroups and water; green = ester groups and glycerol

backbone; yellow = hydrophobic acyl chains.

a

b

c

Average distance of ring chiral carbon from bilayer center.

Average value of angle between plane of glycerol backbone and plane of bilayer.

Occupancy of 60 30° range for h₄dihedral angle.

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10

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

intrinsic to the individual C1 domain bound to DAG, and the

binding to scaffolding or signaling proteins.

the experiments. A similar pattern of activity was also evidenced

in Ramos cells, which endogenously express high levels of Ras-

GRP3. The response with PMA was analogous although in all cases

the potency of PMA was considerably greater. Here again the con-

ditions of the assay are widely different from the binding afﬁnity

experiments, as the ligand–protein complexes have to be translo-

cated to the cellular membrane. These interactions therefore occur

in a different lipid milieu.

The most useful pharmacological probes for C1 domains—the

families of phorbol esters—are not known to distinguish between

DAG-responsive C1 domain containing proteins. Therefore, our

objective has been to design DAG-lactones that could selectively

direct the ﬂow of information of DAG signaling pathways through

their multiple signal transducers in order to achieve a speciﬁc cel-

lular response with a potential therapeutic value. Previously, a

combinatorial approach allowed the identiﬁcation of DAG-lactones

containing substituted aromatic rings at sn-1 and sn-2 positions

that showed important selectivity for RasGRP3.^21,65The discovery

of these compounds provided additional support for the feasibility

of designing compounds selective for speciﬁc members of the fam-

ilies of signaling proteins with DAG-responsive C1 domains,

through the combination of a ‘core’ DAG-lactone with an array of

sn-1 and sn-2 substituents acting as a ‘chemical zip codes’ as we

have described before.²¹

With this in mind, further variation of the aromatic moieties

identiﬁed an DAG-indololactone 1 that was the most selective

and potent compound known for the activation of RasGRP3.²²On

the basis of this ﬁnding we have synthesized compounds that dif-

fer with respect to the point of attachment of the indole ring to the

DAG-lactone template. This variation inﬂuences the orientation of

the indole relative to the lactone ring and, consequently, could

affect membrane interactions and selectivity.

Another way of examining protein–ligand–membrane interac-

tions for compounds 1–5 was to observe the extent of transloca-

tion of various PKC isoforms and RasGRP3. These were visualized

with exogenously expressed GFP-PKC

GFP-RasGRP3 as a function of time with 10

cells for GFP-PKC and LNCaP cells for the rest of the enzymes.

In these assays substantial differences among ligands were

observed, for example between PKC and PKC , but little differ-

ences if any were detected for GFP-PKCd and GFP-RasGRP3. For

PKC , compounds 3, 4, and 5 induced more translocation than

did 1 and 2, whereas for PKC the difference was not with the

a, YFP-PKCe, GFP-PKCd, and

l

M ligand using CHO

a

e

a

e

extent of translocation but rather with its rate, where compounds

2 and 3 induced a more rapid translocation than did 1, 4, and 5.

These differences obviously reﬂect different modes of PKC–

ligand–membrane interactions, which are probably a function of

the different lipid composition of the membranes.

Finally, the biophysical studies were conducted employing

giant egg-PC/PG vesicles, which obviously do not correspond to a

typical cellular membrane composition but which are useful to

explore more speciﬁc molecular interactions impacting lipid orga-

nization and dynamics and the extent of insertion into membrane

bilayers. Overall, the biophysical experiments conﬁrmed that all

ﬁve DAG-indololactones experience signiﬁcant interactions with

membranes and all associate with the membrane bilayer to a var-

ied degree. The experiments pointed, however, to distinct mecha-

nisms of bilayer binding and insertion. In particular, 4 appears to

exhibit the most pronounced bilayer interactions and insertion,

thereby both affecting most signiﬁcantly the lipid organization

(Fig. 4) and, conversely, experiencing the most hydrophobic envi-

ronment following bilayer association (Figs. 5 and 6). Interestingly,

compound 1 showed signiﬁcantly reduced ﬂuorescent energy

transfer to the NBD dye and restricted rotation of the indole ring

in the interfacial region of the bilayer. This could be related per-

haps with its higher selectivity towards RasGRP, but this may be

conﬁrmed with further experimentation.

All isomers tested were selective and potent activators when

comparing binding afﬁnities towards RasGRP1/3 versus PKCa.

However, at the cellular level, RasGRP3 activation differences

amongst the compounds in HEK293 cells were modest, suggesting

that at this level of analysis these structural changes were not

sensed by the lipid environment of the cell membrane. On the

other hand, translocation experiments in living cells did show

measurable differences between PKC isoforms although not with

RasGRP1/3. Signiﬁcantly, at the molecular level, biophysical exper-

iments were able to detect important differences in the way these

molecules interact with model membranes.

A global structure–activity relationship (SAR) for these com-

pounds based on the results from all the biological assays used

in this study is difﬁcult to achieve. Each individual assay provides

strong evidence that the compounds behave differently, showing

unique properties depending on the speciﬁc assay and the condi-

tions of the test. For example, individual afﬁnities for PKC

and RasGRP1/3, which were determined in a milieu with a high

concentration of phosphatidyl serine (100 g/mL), show modest

a, PKCe

Our modeling results are consistent with physicochemical stud-

ies with membrane proteins that demonstrate that tryptophan has

a penchant for membrane surfaces due to distinct interfacial inter-

actions.⁴⁶Such interactions may involve the formation of hydrogen

bonds between the indole’s NH and lipid acyl carbonyls⁶⁶as well as

l

variations relative to the point of attachment of the indole ring.

This result was supported by the modeling, which showed little

difference in average membrane depth and orientation across the

DAG-indololactone compound series, suggesting that differences

in the indole attachment point, and consequent strong differences

in indole–bilayer interactions, may not necessarily be reﬂected in

differences in C1 domain binding patterns.

Previously, it was determined that between DAG-lactones linked

at the 2- and 3-positions of the indole, the one linked through the 3-

position (compound 1) had greater discriminating capacity between

PKC and RasGRP.²³In the present study, the point of attachment

through the four remaining options available on the benzene ring

(compounds 2–5) produced somewhat smaller changes in binding

afﬁnity with greater discriminating selectivity (24-fold) for the

compound linked through the 5-position (compound 3). Overall,

amongst all of the indole-substituted DAG-lactones, compound 1

still shows the greatest selectivity between PKC and RasGRP with

ratios ranging from 49-fold to 84-fold (Table 1).

tryptophan–lipid cation–p

interactions.⁴⁷In the speciﬁc case of the

DAG-indololactones we found no differences between compound 1

and the analogue with the free indole NH in terms of PKC binding

afﬁnity²³suggesting that the primary force responsible for trypto-

phan’s interfacial preference in these compounds is the aromaticity

of the indole ring, its pi electron cloud, and the associated quadru-

pole moment. This is conﬁrmed by our molecular dynamics simu-

lations where the bilayer solvent dielectric ﬁeld (in the absence of

any hydrogen bonds or other speciﬁc atomic interactions) is

enough to localize and orient the indole group.

4. Conclusions

In summary, we believe that the structural differences between

these DAG-lactones reﬂect changes limited to the membrane envi-

ronment. Contrary to the contacts of the critical pharmacophores

on the DAG-lactone with the protein environment, which involve

precise hydrogen bonding interactions with the C1 domain,

The response to compounds 1–5 in HEK293 and HEK293 cells

overexpressing RasGRP3 showed that in this type of assay all ﬁve

compounds behaved similarly within the range of resolution of

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L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

11

interactions with the lipid environment are less sensitive to

5.2. Chemistry

relatively small structural changes amongst isosteres. The biophys-

ical differences detected are real but perhaps of lesser magnitude

in the protein–ligand–membrane interactions equation.

5.2.1. (E)-5,5-Bis((tert-butyldiphenylsilyloxy)methyl)-3-((1-

methyl-1H-indol-7-yl)methylene)dihydrofuran-2(3H)-one (7a)

A solution of 6a (549 mg, 0.88 mmol) in anhydrous tetrahydro-

furan (8 mL) at ꢀ78 °C was treated dropwise with LiHMDS (1.3 mL,

1.30 mmol) and stirred at the same temperature for 0.3 h. A solu-

tion of indole-7-carboxyaldehyde (293 mg, 1.83 mmol) in anhy-

drous tetrahydrofuran (1 mL) was added dropwise, and the

mixture was stirred at ꢀ78 °C for 2 h. A saturated aqueous solution

of NH₄Cl (15 mL) was added at room temperature and the aqueous

phase was extracted with ethyl ether (3 ꢃ 20 mL). The organic

phase was dried (Na₂SO₄) and concentrated to give a mixture of

diastereomers, which was used directly in the next step. Thus,

the residue was dissolved in anhydrous dichloromethane (6 mL)

and treated with triethylamine (0.54 mL, 3.52 mmol) and MsCl

(0.14 mL, 1.76 mmol) at 0 °C for 2 h. DBU (0.66 mL, 4.4 mmol)

was added and the mixture was allowed to reach room tempera-

ture for 2 h. The reaction was quenched by the addition of NH₄Cl

(ss, 10 mL) and the aqueous phase was extracted with CH₂Cl₂

(3 ꢃ 20 mL). The organic phase was dried (Na₂SO₄) and concen-

trated. The residue was puriﬁed by silica gel ﬂash column chroma-

tography (gradient 0–20% EtOAc/hexanes) to give 528 mg (78%

yield) of 7a as a colorless syrup. ¹H NMR (400 MHz, CDCl₃) d 8.25

(m, 1H, –CH@C–), 7.65 (d, J = 7.6 Hz, 1H, H-4⁰), 7.57 (d, J = 7.8 Hz,

8H, Ph), 7.38–7.16 (m, 13H, Ph, H-6⁰), 7.10 (t, J = 7.6 Hz, 1H, H-5⁰),

6.98 (d, J = 3.0 Hz, 1H, H-2⁰), 6.52 (d, J = 3.0 Hz, 1H, H-3⁰), 3.89 (s,

3H, CH₃N–), 3.82 (d, J = 10.6 Hz, 2H, –CH_aHOTBDPS), 3.74 (d,

J = 10.6 Hz, 2H, –CHH_bOTBDPS), 3.07 (mAB, 2H, H-4), 0.99 (s, 18H,

–SiC(CH₃)₃); ¹³C NMR (50 MHz, CDCl₃) d 171.44, 135.58, 135.51,

134.42, 132.83, 132.59, 132.38, 131.26, 130.38, 129.78, 127.73,

126.61, 122.85, 122.50, 119.84, 119.21, 101.38, 85.55, 66.15,

53.39, 37.43, 32.00, 26.71, 19.23; IR (neat) 1751 cm^ꢀ1; ESI-MS m/

z 781 [M+NH₄]⁺, 764 [M+H⁺]; HRMS (ESI) m/z [M+H]⁺calcd for C_48-

H₅₃NO₄Si₂: 764.3586, found: 764.3553.

5. Experimental section

5.1. General procedures

All chemical reagents were commercially available.

[20-³H]phorbol 12,13-dibutyrate (PDBu) was obtained from Per-

kin-Elmer, Waltham, MA. PMA was from LC Laboratories, Woburn,

MA, USA. Melting points were determined on an Electrothermal

IA9000series digital melting point apparatus and are uncorrected.

Column chromatography was performed on a Teledyne Isco Com-

biFlash Companion instrument under gradient elution conditions

with RediSep disposable ﬂash columns. Analytical TLC was per-

formed on Merck silica gel 254F plates. ¹H and ¹³C NMR spectra

were recorded on a Bruker Avance DPX 400 instrument at 400

and 100 MHz, respectively. Spectra are referenced to the solvent

in which they were run (7.26 ppm for CDCl₃). Both low resolution

and high resolution positive ion electrospray ionization (ESI) mass

spectra were obtained for all compounds. Low resolution ESI anal-

ysis was carried out on an Agilent LC/MSD single quadrupole sys-

tem, equipped with an in-line diode-array UV detector, to assess

compound identity and homogeneity. Initial analyses were carried

out in ﬂow-injection analysis (FIA) mode with the sample injected

directly into the LC/MSD using 1:1 CH₃OH/H₂O containing 0.1%

CH₃COOH at a ﬂow rate of 300

were additionally analyzed by LC/MS using

m), Zorbax Rapid-Resolution

lL/min. Final DAG-lactone products

a

narrow-bore

(100 ꢃ 2.1 mm), small-particle (3.5-

l

reversed-phase C₁₈column coupled with a C₁₈guard column

(12.5 ꢃ 2.1 mm) eluted with a 5–90% gradient of CH₃OH/H₂O

containing 0.1% CH₃COOH at a ﬂow rate of 300 lL/min for

separations. Both the total-ion chromatogram (TIC) and the

UV-chromatogram were used to conﬁrm compound purity. The full

scan (210–400 nm) diode-array UV spectra for both FIA and LC/MS

analyses of the ﬁnal DAG-lactone products were also generated

and compared to assess similarity. UV spectra in all cases were

equivalent and are reported for the individual products. High

resolution MS analysis was conducted on a Thermo-Fisher LTQ-XL

Orbitrap hybrid mass spectrometer operated at a resolution of

300,000 (FWHM) using either FIA or LC/MS sample introduction,

depending on which mode was the most suitable based on previous

low resolution analyses. For LC/MS analyses on the Orbitrap, a nar-

row-bore (50 ꢃ 2.1 mm) Zorbax Rapid-Resolution reversed-phase

C₁₈column coupled with a C₁₈guard column was eluted with a

5.2.2. (E)-5-(Benzyloxymethyl)-5-(tert-

butyldiphenylsilyloxy)methyl)-3-((1-methyl-1H-indol-7-

yl)methylene)dihydrofuran-2(3H)-one (7b)

A solution of 6b (302 mg, 0.63 mmol) in anhydrous tetrahydro-

furan (6 mL) at ꢀ78 °C was treated dropwise with LiHMDS

(0.95 mL, 0.945 mmol) and stirred at the same temperature for

0.3 h. A solution of indole-7-carboxyaldehyde (200 mg, 1.26 mmol)

in anhydrous tetrahydrofuran (1 mL) was added dropwise, and the

mixture was stirred at ꢀ78 °C for 2 h. A saturated aqueous solution

of NH₄Cl (15 mL) was added at room temperature and the aqueous

phase was extracted with ethyl ether (3 ꢃ 20 mL). The organic

phase was dried (Na₂SO₄) and concentrated to give a mixture of

diastereomers, which was used directly in the next step. Thus,

the residue was dissolved in anhydrous dichloromethane (5 mL)

and treated with triethylamine (0.39 mL, 2.58 mmol) and MsCl

(0.10 mL, 1.29 mmol) at 0 °C for 2 h. DBU (0.49 mL, 3.23 mmol)

was added and the mixture was allowed to reach room tempera-

ture for 2 h. The reaction was quenched by the addition of NH₄Cl

(ss, 10 mL) and the aqueous phase was extracted with CH₂Cl₂

(3 ꢃ 20 mL). The organic phase was dried (Na₂SO₄) and concen-

trated. The residue was puriﬁed by silica gel ﬂash column chroma-

tography (gradient 0–20% EtOAc/hexanes) to give 240 mg (61%

yield) of 7b as a colorless syrup. ¹H NMR (400 MHz, CDCl₃) d 8.25

(m, 1H, –CH@C–), 7.64 (d, J = 7.6 Hz, 1H, H-4⁰), 7.59 (d, J = 7.8 Hz,

4H, Ph), 7.38–7.18 (m, 12H, Ph, H-6⁰), 7.11 (t, J = 7.6 Hz, 1H, H-5⁰),

7.03 (d, J = 3.0 Hz, 1H, H-2⁰), 6.50 (d, J = 3.0 Hz, 1H, H-3⁰), 4.53

(mAB, 2H, –OCH₂Ph), 3.87 (s, 3H, CH₃N–), 3.82 (d, J = 10.6 Hz, 1H,

–CH_aHOTBDPS), 3.75 (d, J = 10.6 Hz, 1H, –CHH_bOTBDPS), 3.62

5–90% gradient of CH₃CN/H₂O containing 0.1% HCOOH at 250 lL/

min. The resulting accurate mass measurement of a molecular

species ([M+H]⁺, [M+Na]⁺or M+NH₄]⁺) was then used to determine

a unique elemental composition for each particular compound.

Where appropriate, ¹H and ¹³C NMR data were used to set elemental

constraints for this calculation. Elemental analyses were performed

by Atlantic Microlab, Inc., Norcross, GA and by UMYMFOR-CONICET,

Argentina. 1,2-Dimyristoyl-sn-glycerophosphocholine (DMPC), 1-(3

sn-phosphatidyl)-rac-glycerol sodium salt (PG), phosphatidylserine,

and the ﬂuorescent dye 1,2-dipalmitoyl-sn-glycero-3-phosphoeth-

anolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl])(ammonium salt)

(NBD-PE) were purchased from Avanti (Alabaster, AL, USA). Sodium

dithionite (Na₂O₄S₂) and Tris(hydroxymethyl)aminomethane (TRIZMA

base buffer, C₄H₁₁NO₃) KCl, and sucrose were purchased from

Sigma–Aldrich. 1-(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-

hexatriene (TMA-DPH) was obtained from Molecular Probes, Inc.

(Eugene, Oregon, USA).

Please cite this article in press as: Garcia, L. C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.024

12

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

(mAB, 2H, –CH₂OCH₂Ph), 3.10 (mAB, 2H, H-4), 1.00 (s, 9H, –

SiC(CH₃)₃); ¹³C NMR (50 MHz, CDCl₃) d 171.33, 137.58, 135.56,

134.36, 132.82, 132.73, 132.57, 131.27, 130.32, 129.76, 128.36,

127.68, 126.37, 122.91, 122.84, 122.58, 122.51, 119.78, 119.23,

101.41, 84.79, 73.70, 71.91, 66.37, 37.45, 32.40, 26.72, 19.34; IR

(neat) 1749 cm^ꢀ1; ESI-MS m/z 616 [M+H⁺]; HRMS (ESI) m/z

[M+H]⁺calcd for C₃₉H₄₁NO₄Si: 616.2883, found: 616.2892.

(50 mL) was added and the aqueous phase was extracted with

EtOAc (3 ꢃ 100 mL). The organic phase was dried (Na₂SO₄) and

concentrated. The residue was puriﬁed by silica gel ﬂash column

chromatography (gradient 0–5% EtOAc/hexanes) to give 7.18 g

(89% yield) of 10 as a colorless oil: ¹H NMR (400 MHz, CDCl₃) d

7.65 (d, J = 6.7 Hz, 4H, Ph), 7.44–7.35 (m, 6H, Ph), 5.92–5.81 (m,

1H, –CH₂CH@CH₂), 5.07 (m, 2H, –CH₂CH@CH₂), 3.60 (d, 1H,

J = 9.0 Hz, –CH_aHOTBDPS)^⁄, 3.58 (d, 1H, J = 9.0 Hz, –CH_aHOTBMPS)^⁄,

3.52 (d, 2H, J = 9.0 Hz, –CHH_bOTBDPS, –CHH_bOTBDMS), 2.53 (s, 1H,

–OH), 2.30 (d, J = 7.2 Hz, –CH₂CH@CH₂), 1.07 (s, 9H, –SiC(CH₃)₃),

0.87 (s, 9H, –SiC(CH₃)₃), 0.05 (s, 6H, –Si(t-Bu)(CH₃)₂); ¹³C NMR

5.2.3. (E)-5,5-Bis(hydroxymethyl)-3-((1-methyl-1H-indol-7-

yl)methylene)dihydrofuran-2(3H)-one (8)

To a solution of 7 (164 mg, 0.21 mmol) in anhydrous THF (8 mL)

was added triethylamine trihydroﬂuoride (0.34 mL, 2.13 mmol).

The reaction mixture was stirred at 70 °C for 6 h. The crude solu-

tion was then concentrated in vacuo and puriﬁed by silica gel ﬂash

column chromatography (gradient 50–100% EtOAc/hexanes) to

(100 MHz, CDCl₃)

d 136.30, 136.26, 134.09, 133.87, 133.82,

130.39, 128.36, 118.53, 74.73, 66.53, 65.80, 39.03, 27.57, 26.54,

19.96, 18.86, ꢀ4.80, ꢀ4.84; IR (neat) 3072, 2954, 2929, 2857,

1471, 1428, 1252, 1080, 835, 776, 739, 700 cm^ꢀ1(OH); ESI-MS

m/z 507 [M+Na]⁺. Elemental Analysis Calcd for C₂₈H₄₄O₃Si₂: C,

69.37; H, 9.15. Found: C, 69.61; H, 9.29.

give 46 mg (75% yield) of

8 as a

yellow syrup: ¹H NMR

(400 MHz, CDCl₃, CD₃OD) d 8.32 (m, 1H, –CH@C–), 7.65 (d,

J = 7.6 Hz, 1H, H-4⁰), 7.29 (d, J = 7.3 Hz, 1H, H-6⁰), 7.11 (t,

J = 7.6 Hz, 1H, H-5⁰), 7.03 (d, J = 3.0 Hz, 1H, H-2⁰), 6.51 (d,

J = 3.0 Hz, 1H, H-3⁰), 4.04 (s, 3H, CH₃N–), 3.75 (d, J = 12.1 Hz, 2H,

–CH_aHOH), 3.68 (d, J = 12.1 Hz, 2H, –CHH_bOH), 3.10 (d, J = 2.8 Hz,

2H, H-4); ¹³C NMR (100 MHz, CDCl₃) d 172.47, 134.25, 133.96,

131.29, 130.29, 125.56, 123.13, 122.61, 119.31, 119.12, 101.31,

86.32, 64.14, 37.28, 31.43; IR (neat) 3493, 2925, 1690, 1627,

722 cm^ꢀ1, ESI-MS m/z 310 [M+Na]⁺, 288 [M+H⁺]; HRMS (ESI) m/z

[M+H]⁺calcd for C₁₆H₁₇NO₄: 288.1230, found: 288.1229.

5.2.6. 5-((tert-Butyldimethylsilyloxy)methyl)-5-((tert-butyldi-

phenylsilyloxy)methyl)dihydrofuran-2(3H)-one (11)

A THF solution of BH₃.SMe₂(2.0 M, 15.5 mL, 30.95 mmol) was

added slowly to a stirred solution of 10 (6.0 g, 12.37 mmol) in

anhydrous THF (50 mL) at ꢀ78 °C. The reaction mixture was stirred

at ꢀ78 °C for 0.25 h and then at room temperature for 4 h. Metha-

nol (30 mL) was added slowly and the resulting solution was con-

centrated to yield a colorless oil that was used as such in the next

step. The obtained oil (6.92 g) was dissolved in anhydrous dichlo-

romethane (160 mL) and PCC (20.02 g, 92.99 mmol) and 4 Å

molecular sieves (7.02 g) were added. The reaction mixture was

stirred at room temperature for 72 h. Ethyl ether (150 mL) was

added and the resulting solution was stirred at room temperature

for 1 h. The reaction mixture was then ﬁltrated through a column

of silica gel and concentrated. The residue was puriﬁed by silica

gel ﬂash column chromatography (gradient 0–10% EtOAc/hexanes)

to give 4.55 g (74% yield) of 11 as a colorless oil: ¹H NMR (400 MHz,

CDCl₃) d 7.65 (d, J = 6.8 Hz, 4H, Ph), 7.44–7.37 (m, 6H, Ph), 3.72 (d,

1H, J = 10.9 Hz, –CH_aHOTBDPS)^⁄, 3.71 (d, 1H, J = 10.6 Hz, –CH_aHOT-

BMPS)^⁄, 3.65 (d, 1H, J = 9.0 Hz, –CHH_bOTBDMS)^–, 3.64 (d, 1H,

J = 9.0 Hz, –CHH_bOTBDPS)^–, 2.60 (t, J = 8.5 Hz, 2H, H-3), 2.16 (t,

J = 8.2 Hz, 2H, H-4), 1.06 (s, 9H, –C(CH₃)₃), 0.85 (s, 9H, –C(CH₃)₃),

0.03 (s, 6H, –Si(CH₃)₂); ¹³C NMR (50 MHz, CDCl₃) d 177.18,

135.61, 135.57, 132.83, 132.62, 129.88, 127.82, 88.19, 66.47,

65.92, 29.45, 26.78, 25.75, 25.54, 19.22, 18.17, ꢀ5.55, ꢀ5.61; IR

5.2.4. 1-(tert-Butyl-dimethyl-silanyloxy)-3-(tert-butyl-

diphenyl-silanyloxy)-propan-2-one (9)

A solution of 1,3-dihydroxyacetone dimer (11.0 g, 60.79 mmol)

and imidazole (1.84 g, 26.88 mmol) in anhydrous DMF (40 mL) at

0 °C was treated dropwise with a solution of TBDPSCl (9.6 mL,

37.68 mmol) in anhydrous DMF (9 mL). The reaction mixture was

stirred at this temperature for 4 h. Water was added (40 mL) and

the mixture was extracted with dichloromethane (3 ꢃ 40 mL).

The combined organic phases were dried (Na₂SO₄) and concen-

trated. The residue was puriﬁed by silica gel ﬂash column chroma-

tography (gradient 0–20% EtOAc)/hexanes) to give 6.31 g of

slightly impure monoprotected product 1-(tert-butyldiphenylsilyl-

oxy)-3-hydroxypropan-2-one²⁸that was used as such for the next

step. Thus, to

a solution of 1-(tert-butyldiphenylsilyloxy)-3-

hydroxypropan-2-one (6.31 g, 19.20 mmol) in anhydrous dichloro-

methane (80 mL) were added imidazole (3.0 g, 44.00 mmol) and a

solution of TBDMSCl (3.43 g, 22.66 mmol) in anhydrous dichloro-

methane (40 mL). The reaction mixture was stirred at 0 °C for

2 h. Water (80 mL) was added and the aqueous phase was

extracted with dichloromethane (3 ꢃ 80 mL). The organic phase

was dried (Na₂SO₄) and concentrated. The residue was puriﬁed

by silica gel ﬂash column chromatography (gradient 0–5% EtOAc/

hexanes) to give 7.42 g (44% two steps yield) of 9 as a white solid:

mp 56 °C; ¹H NMR (400 MHz, CDCl₃) d 7.64 (d, J = 6.7 Hz, 4H, Ph),

7.46–7.37 (m, 6H, Ph), 4.42 (s, 2H, –CH₂OTBDMS)^⁄, 4.40 (s, 2H, –

CH₂OTBDPS)^⁄, 1.10 (s, 9H, –SiC(CH₃)₃)(CH₃)₂)^⁄, 0.85 (s, 9H, –SiPh_2-

C(CH₃)₃)^⁄, 0.02 (s, 6H, –Si(t-Bu)(CH₃)₂); ¹³C NMR (100 MHz, CDCl₃)

d 208.58, 136.17, 133.29, 130.66, 128.54, 69.14, 68.68, 27.43, 26.41,

19.92, 18.98, ꢀ4.91; IR (neat) 1740, 1097, 837, 825, 777, 741,

702 cm^ꢀ1; ESI-MS m/z 465 [M+Na]⁺. Elemental Analysis Calcd for

(neat) 2954, 2929, 2857, 1778, 1112, 836, 777, 738, 700 cm^ꢀ1

;

ESI-MS m/z 521 [M+Na]⁺, 499 [M+H]⁺, 421 [M+H-C₆H₆]⁺; HRMS

(ESI) m/z [M+H]⁺calcd for C₂₈H₄₂O₄Si₂: 499.2694, found:

499.2693. Elemental Analysis Calcd For C₂₈H₄₂O₄Si₂: C, 67.42; H,

8.49. Found: C, 67.48; H, 8.30.

^⁄,–Signals could be interchanged.

5.2.7. General procedure A. Aldol condensation followed by

oleﬁnation^67,62

Under argon, a solution of 11 (1 equiv) in THF (5 mL/mmol) at

ꢀ78 °C was treated dropwise with [(CH₃)₃Si]₂N-Li (LiHMDS, 1 M

in THF, 1.5 equiv) and stirred at the same temperature for 0.5 h. A

solution of indole aldehyde (1.5 equiv dissolved in THF) was then

added dropwise, and the reaction was stirred at ꢀ78 °C for 2 h.

The reaction mixture was then quenched with a saturated aqueous

solution of NH₄Cl and allowed to warm to room temperature. The

layers were separated, and the aqueous layer was extracted with

Et₂O (3ꢃ). The combined organics were washed with H₂O (2ꢃ)

and brine (1ꢃ), dried (Na₂SO₄), and concentrated in vacuo. The

obtained residue was used directly without puriﬁcation. Thus, it

was dissolved in CH₂Cl₂(10 mL/mmol) and Et₃N (4 equiv) was

added. The resulting solution was cooled to 0 °C, treated dropwise

with CH₃SO₂Cl (MsCl, 2 equiv), and then stirred at room

C

₂₅H₃₈O₃Si₂: C, 67.82; H, 8.65. Found: C, 67.91; H, 8.71.

^⁄Signals could be interchanged.

5.2.5. 2-(tert-Butyl-dimethyl-silanyloxymethyl)-1-(tert-butyl-

diphenyl-silanyloxy)-pent-4-en-2-ol (10)

To a solution of 9 (7.38 g, 16,67 mmol) in anhydrous tetrahy-

drofuran (30 mL) was added allylmagnesium chloride (2 M,

20 mL, 40.00 mmol). The reaction mixture was stirred at room

temperature for 2 h. A saturated aqueous solution of NH₄Cl

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L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

13

temperature for 1–2 h. The reaction mixture was then cooled again

5.2.11. (E)-5-((tert-Butyldimethylsilyloxy)methyl)-5-((tert-

butyldiphenylsilyloxy)methyl)-3-((1-methyl-1H-indol-7-

yl)methylene)dihydrofuran-2(3H)-one (12d)

to 0 °C and treated dropwise with 1,8-diazabicyclo[5.4.0]non-5-ene

(DBU, 5 equiv). When the addition of DBU was completed, the reac-

tion mixture was allowed to reach room temperature overnight.

The residue was treated with EtOAc (10 mL/mmol) and saturated

solution of NH₄Cl (10 mL/mmol). The layers were separated, and

the aqueous layer was extracted with EtOAc (1ꢃ). The combined

organics were washed with H₂O (2ꢃ) and brine (1ꢃ), dried (Na_2-

SO₄), and concentrated in vacuo. Puriﬁcation by silica gel ﬂash col-

umn chromatography (gradient EtOAc/hexanes) gave 12 (a–d).

Starting from 11 (710 mg, 1.42 mmol) and following general

procedure A, 12d was obtained as a yellowish oil (674 mg, 74%

yield); ¹H NMR (400 MHz, CDCl₃) d 8.25 (m, 1H, –CH@C–), 7.65

(d, J = 7.8 Hz, 1H, H-4⁰), 7.60 (d, J = 7.1 Hz, 4H, Ph), 7.38–7.22 (m,

7H, Ph, H-6⁰), 7.11 (t, J = 7.6 Hz, 1H, H-5⁰), 6.99 (d, J = 2.5 Hz, 1H,

H-2⁰), 6.51 (d, J = 2.5 Hz, 1H, H-3⁰), 3.96 (s, 3H, CH₃N–), 3.79 (d,

1H, J = 10.6 Hz, –CH_aHOTBDPS)^⁄, 3.77 (d, 1H, J = 10.6 Hz, –CH_aHOT-

BMPS)^⁄, 3.71 (d, 2H, J = 10.6 Hz, –CHH_bOTBDMS, –CHH_bOTBDPS),

3.08 (mAB, 2H, J = 7.1 Hz, H-4), 1.01 (s, 9H, –C(CH₃)₃), 0.83 (s, 9H,

–C(CH₃)₃), 0.03 (s, 6H, –Si(CH₃)₂); ¹³C NMR (50 MHz, CDCl₃) d

171.43, 135.60, 135.51, 134.40, 132.89, 132.63, 132.31, 131.26,

130.36, 129.77, 127.71, 126.81, 122.79, 122.47, 119.88, 119.21,

101.36, 85.53, 66.17, 65.39, 37.44, 31.68, 26.70, 25.71, 19.22,

18.14, ꢀ5.52; IR (neat) 2952, 2928, 2856, 1750, 1111, 836, 778,

700 cm^ꢀ1; ESI-MS m/z 662 [M+Na]⁺, 640 [M+H]⁺, 562 [M+H-

C₆H₆]⁺; HRMS (ESI) m/z [M+H]⁺calcd for C₃₈H₄₉NO₄Si₂: 640.3273,

found: 640.3276.

5.2.8. (E)-5-((tert-Butyldimethylsilyloxy)methyl)-5-((tert-butyl-

diphenylsilyloxy)methyl)-3-((1-methyl-1H-indol-4-

yl)methylene)dihydrofuran-2(3H)-one (12a)

Starting from 11 (1.30 g, 2.60 mmol)and following general pro-

cedure A, 12a was isolated as a greenish oil (997 mg, 60% yield); ¹H

NMR (400 MHz, CDCl₃) d 8.01 (m, 1H, –CH@C–), 7.62 (t, J = 6.6 Hz,

4H, Ph), 7.40–7.28 (m, 9H, Ph, H-5, H-6, H-7), 7.15 (d, J = 2.8 Hz, 1H,

H-2⁰), 6.73 (d, J = 2.8 Hz, 1H, H-3⁰), 3.84 (s, 3H, CH₃N), 3.77 (m, 4H,

–CH₂OTBDPS, –CH₂OTBDMS), 3.14 (mAB, 2H, H-4), 0.99 (s, 9H,

–C(CH₃)₃), 0.81 (s, 9H, –C(CH₃)₃), 0.02 (d, J = 3.5 Hz, 6H, –Si(CH₃)₂):

¹³C NMR (50 MHz, CDCl₃) d 172.05, 136.85, 135.63, 135.60, 132.92,

132.81, 132.76, 129.76, 129.73, 129.49, 127.74, 127.02, 125.50,

121.37, 119.38, 110.68, 99.60, 85.34, 66.19, 65.39, 32.99, 32.55,

26.66, 25.71, 19.20, 18.14, ꢀ5.54; IR (neat) 2928, 2856, 1749,

1110, 836, 777, 700 cm^ꢀ1; ESI-MS m/z 662 [M+Na]⁺. Elem. Anal.

Calcd for C₃₈H₄₉NO₄Si₂: C, 71.32; H, 7.72; N, 2.19. Found: C,

70.87; H, 7.84; N, 1.91.

^⁄Signals could be interchanged.

5.2.12. General procedure B. TBDMS deprotection

Pyridinium p-toluene sulfonate (0.80 equiv) was added to a

solution of fully protected DAG-indololactone (12a–d) in absolute

ethanol (10 mL/mmol). The reaction mixture was stirred at 60 °C

till disappearance of starting material by TLC. Brine solution was

added and the mixture was extracted with AcOEt (3ꢃ). The com-

bined organic layers were dried (Na₂SO₄) and the volatiles were

removed. The residue was puriﬁed by silica gel ﬂash column chro-

matography (gradient EtOAc/hexanes) to yield compounds 13a–d.

5.2.9. (E)-5-((tert-Butyldimethylsilyloxy)methyl)-5-((tert-butyl-

diphenylsilyloxy)methyl)-3-((1-methyl-1H-indol-5-

yl)methylene)dihydrofuran-2(3H)-one (12b)

Starting from 11 (382 mg, 0.76 mmol) and following general

procedure A, 12b was isolated as a colorless oil (250 mg, 51%

5.2.13. (E)-5-((tert-Butyldiphenylsilyloxy)methyl)-5-

(hydroxymethyl)-3-((1-methyl-1H-indol-4-

yl)methylene)dihydrofuran-2(3H)-one (13a)

1

yield); H NMR (400 MHz, CDCl₃) d 7.78 (sa, 1H, H–4⁰), 7.66–7.62

(m, 5H, CH@C, Ph), 7.40–7.30 (m, 8H, Ph, H–6⁰, H–7⁰), 7.10 (d,

J = 3.0 Hz, 1H, H–2⁰), 6.55 (d, J = 3.0 Hz, 1H, H–3⁰), 3.82 (s, 3H,

CH₃N), 3.77 (m, 4H, –CH₂OTBDPS, –CH₂OTBDMS), 3.13 (mAB, 2H,

H–4), 1.01 (s, 9H, –C(CH₃)₃), 0.82 (s, 9H, –C(CH₃)₃), 0.03 (s, 3H, –

Si(CH₃)₂), 0.02 (s, 3H, –Si(CH₃)₂); ¹³C NMR (50 MHz, CDCl₃) d

172.45, 137.52, 137.14, 135.63, 132.92, 132.77, 130.07, 129.75,

128.70, 127.74, 126.57, 123.95, 123.55, 122.06, 109.54, 102.00,

85.15, 66.17, 65.39, 32.96, 32.26, 26.64, 25.71, 19.19, 18.14,

Starting from 12a (997 mg, 1.55 mmol) and following general

procedure B, 13a was obtained as a yellow oil (550 mg, 68% yield);

¹H NMR (400 MHz, CDCl₃) d 8.05 (m, 1H, CH@C), 7.60 (t, J = 6.6 Hz,

4H, Ph), 7.41- 7.28 (m, 9H, Ph, H-5⁰, H-6⁰, H-7⁰), 7.16 (d, J = 2.8 Hz,

1H, H-2⁰), 6.73 (d, J = 2.5 Hz, 1H, H-3⁰), 3.84 (s, 3H, CH₃N–), 3.74 (m,

4H, –CH₂OTBDPS, –CH₂OH), 3.21 (mAB, 2H, 17.6 Hz, H-4_a,b), 1.89 (t,

J = 6.8 Hz, 1H, OH), 0.99 (s, 9H, –C(CH₃)₃), ¹³C NMR (50 MHz, CDCl₃)

d 171.92, 136.85, 135.63, 135.57, 133.85, 132.69, 132.48, 129.89,

129.57, 127.80, 126.66, 124.56, 121.39, 119.59, 111.00, 99.50,

85.27, 66.20, 65.24, 33.02, 32.49, 26.64, 19.16; IR (neat) 3401,

2929, 2856, 1732, 1111, 730, 700 cm^ꢀ1; ESI-MS m/z 548 [M+Na]⁺,

526 [M+H]⁺, 448 [M+H-C₆H₆]⁺; HRMS (ESI) m/z [M+H]⁺calcd for

ꢀ5.52; IR (neat) 2928, 2856, 1749, 1110, 836, 777, 700 cm^ꢀ1

;

ESI-MS m/z 662 [M+Na]⁺, 640 [M+H]⁺, 562 [M+H-C₆H₆]⁺; HRMS

(ESI) m/z [M+H]⁺calcd for C₃₈H₄₉NO₄Si₂: 640.3273, found:

640.3271.

C₃₂H₃₅NO₄Si: 526.2408, found: 526.2400.

5.2.10. (E)-5-((tert-Butyldimethylsilyloxy)methyl)-5-((tert-butyl-

diphenylsilyloxy)methyl)-3-((1-methyl-1H-indol-6-yl)methylene)

dihydrofuran-2(3H)-one (12c)

5.2.14. (E)-5-((tert-Butyldiphenylsilyloxy)methyl)-5-(hydroxy-

methyl)-3-((1-methyl-1H-indol-5-yl)methylene)dihydrofuran-

2(3H)-one (13b)

Starting from 11 (565 mg, 1.13 mmol) and following general

procedure A, 12c was obtained as a yellowish oil (457 mg, 63%

yield); ¹H NMR (400 MHz, CDCl₃): d 7.68–7.61 (m, 6H, CH@C, Ph,

H-4⁰), 7.43–7.28 (m, 8H, Ph, H-5⁰, H-7⁰), 7.17 (d, J = 3.0 Hz, 1H, H-

2⁰), 6.52 (d, J = 2.8 Hz, 1H, H-3⁰), 3.82 (s, 3H, CH₃N–), 3.76 (m, 4H,

–CH₂OTBDPS, –CH₂OTBDMS), 3.14 (m, 2H, H-4), 1.01 (s, 9H, –

C(CH₃)₃), 0.82 (s, 9H, –C(CH₃)₃), 0.03 (s, 3H, –Si(CH₃)₂), 0.02 (s,

3H, –Si(CH₃)₂); ¹³C NMR (50 MHz, CDCl₃): d 172.27, 137.43,

136.70, 135.60, 132.77, 131.20, 129.77, 129.63, 128.61, 127.74,

122.96, 121.13, 121.07, 111.79, 101.36, 85.18, 66.20, 65.36, 32.87,

32.35, 26.67, 25.71, 19.22, 18.14, ꢀ5.50; IR (neat): 2928, 2856,

1747, 1111, 836, 777, 700 cm^ꢀ1(CO); ESI-MS m/z 662 [M+Na]⁺,

640 [M+H]⁺; HRMS (EI) m/z [M+H]⁺calcd for C₃₈H₄₉NO₄Si₂:

640.3273, found: 640.3271.

Starting from 12b (250 mg, 0.39 mmol) and following general

procedure B, 13b was obtained as a white solid (158 mg, 77% yield);

mp 185–186 °C; ¹H NMR (400 MHz, CDCl₃) d 7.79 (s, 1H, H-4⁰), 7.69

(t, J = 2.5 Hz, 1H, CH@C), 7.61 (m, 4H, Ph), 7.41–7.30 (m, 8H, Ph, H-6⁰,

H-7⁰), 7.11 (d, J = 3.0 Hz, 1H, H-2⁰), 6.55 (d, J = 3.0 Hz, 1H, H-3⁰), 3.83

(s, 3H, CH₃N), 3.79 (m, 4H, –CH₂OTBDPS, –CH₂OH), 3.18 (mAB, 2H, H-

4), 1.00 (s, 9H, (–C(CH₃)₃); ¹³C NMR (50 MHz, CDCl₃) d 172.22,

138.62, 137.32, 135.62, 135.60, 132.75, 132.58, 130.19, 129.88,

128.78, 127.79, 126.33, 124.13, 123.78, 121.21, 109.60, 102.13,

85.00, 66.27, 65.37, 33.00, 32.27, 26.69, 19.20; IR (neat) 3372,

2923, 2858, 1708, 1113, 805, 699 cm^ꢀ1; ESI-MS m/z 548 [M+Na]⁺,

526 [M+H]⁺, 448 [M+H-C₆H₆]⁺; HRMS (ESI) m/z [M+H]⁺calcd for

C₃₂H₃₅NO₄Si: 526.2408, found: 526.2401.

Please cite this article in press as: Garcia, L. C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.024

14

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

5.2.15. (E)-5-((tert-Butyldiphenylsilyloxy)methyl)-5-(hydroxy-

methyl)-3-((1-methyl-1H-indol-6-yl)methylene)dihydrofuran-

2(3H)-one (13c)

5.2.19. (E)-(2-((tert-butyldiphenylsilyloxy)methyl)-4-((1-

methyl-1H-indol-6-yl)methylene)-5-oxotetrahydrofuran-2-

yl)methyl 2-propylpentanoate (14c)

Starting from 12c (457 mg, 0.71 mmol) and following general

procedure B, 13c was obtained as a white solid (167 mg, 45%

yield); mp 171 °C, ¹H NMR (400 MHz, CDCl₃) d 7.70 (t, J = 2.6 Hz,

1H, CH@C), 7.66 (d, J = 8.3 Hz, 1H, H-4⁰), 7.60 (m, 4H, Ph), 7.42–

7.25 (m, 8H, Ph, H-5⁰, H-7⁰), 7.17 (d, J = 3.0 Hz, 1H, H-2⁰), 6.52 (d,

J = 3.0 Hz, 1H, H-3⁰), 3.81 (s, 3H, –NCH₃), 3.79 (m, 4H, –CH₂OTBDPS,

–CH₂OH), 3.19 (mAB, 2H, H-4), 1.00 (s, 9H, (–C(CH₃)₃); ¹³C NMR

(50 MHz, CDCl₃) d 172.24, 138.42, 136.64, 135.60, 132.69, 132.48,

131.40, 129.86, 128.26, 127.77, 122.15, 121.22, 121.10, 111.96,

101.39, 85.24, 66.20, 65.15, 32.90, 32.17, 26.64, 19.19; IR (neat)

3424, 2929, 2857, 1708, 1111, 1056, 802, 698 cm^ꢀ1; ESI-MS m/z

548 [M+Na]⁺, 526 [M+H]⁺, 448 [M+H-C₆H₆]⁺; HRMS (ESI) m/z

[M+H]⁺calcd for C₃₂H₃₅NO₄Si: 526.2408, found: 526.2409.

Starting from 13c (167 mg, 0.32 mmol) and following general

procedure C, 14c was obtained as an amber oil (198 mg, 96% yield);

¹H NMR (400 MHz, CDCl₃) d 7.71 (m, 1H, CH@C), 7.66 (d, J = 8.3 Hz,

1H, H-4⁰), 7.61 (m, 4H, Ph), 7.41–7.24 (m, 8H, Ph, H-5⁰, H-7⁰), 7.18

(d, J = 2.8 Hz, 1H, H-2⁰), 6.53 (d, J = 3.0 Hz, 1H, H-3⁰), 4.33 (mAB,

2H, –CH₂OCO–), 3.82 (s, 3H, –NCH₃), 3.81 (d, 1H, J = 10.6 Hz,

–CH_aHOTBDPS), 3.76 (d, 1H, J = 10.6 Hz, –CHH_bOTBDPS), 3.23 (dd,

1H, J = 17.2, 2.5 Hz, C-4a), 3.06 (dd, 1H, J = 17.2, 2.5 Hz, C-4b),

2.35 (m, 1H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.48 (m, 2H, (CH₃CH₂

CH₂)₂CHCO₂CH₂–), 1.35 (m, 2H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.23

(m, 4H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.01 (s, 9H, –C(CH₃)₃), 0.79 (t,

3H, J = 7.3 Hz, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 0.75 (t, 3H, J = 7.3 Hz,

(CH₃CH₂CH₂)₂CHCO₂CH₂–); ¹³C NMR (50 MHz, CDCl₃) d 176.00,

171.67, 138.29, 136.72 , 135.62, 135,57, 132.61, 132.42, 131.43,

129.91, 128.23, 127.81, 121.59, 121.16, 120.95, 112.09, 103.74,

101.47, 83.00, 66.35, 65.37, 45.17, 34.41, 32.92, 32.84, 26.68,

20.59, 19.23, 13.87; IR (neat) 2956, 2931, 2858, 1737, 1105, 810,

701 cm^ꢀ1; ESI-MS m/z 674 [M+Na]⁺, 652 [M+H]⁺; HRMS (ESI) m/z

[M+H]⁺calcd for C₄₀H₄₉NO₅Si: 652.3453, found: 652.3449.

5.2.16. (E)-5-((tert-Butyldiphenylsilyloxy)methyl)-5-(hydroxy-

methyl)-3-((1-methyl-1H-indol-7-yl)methylene)dihydrofuran-

2(3H)-one (13d)

Starting from 12d (316 mg, 0.63 mmol) and following general

procedure B, 13d was obtained as a white solid (180 mg, 54%

yield); mp 165–167 °C; ¹H NMR (400 MHz, CDCl₃) d 8.30 (m, 1H,

CH@C), 7.67 (d, J = 7.6 Hz, 1H, H-4⁰), 7.57 (d, J = 7.6 Hz, 4H, Ph),

7.36 (m, 2H, Ph), 7.26 (m, 5H, H-6⁰, Ph), 7.12 (t, J = 7.6 Hz, 1H, H-

5⁰), 7.00 (d, J = 3.0 Hz, 1H, H-2⁰), 6.52 (d, J = 3.0 Hz, 1H, H-3⁰), 3.94

(s, 3H, –NCH₃), 3.85- 3.69 (m, 4H, –CH₂OTBDPS, –CH₂OH), 3.12

(mAB, 2H, H-4), 1.88 (t, J = 6.8 Hz, 1H, OH), 1.00 (s, 9H, –C(CH₃)₃);

¹³C NMR (50 MHz, CDCl₃) d 171.49, 135.57, 135.48, 134.37,

133.27, 132.66, 132.37, 131.35, 130.39, 129.86, 127.74, 125.87,

123.14, 122.64, 119.56, 119.24, 101.42, 85.59, 66.11, 65.12, 37.50,

31.74, 26.67, 19.22; IR (neat) 3391, 2929, 2857, 1708, 1296,

1112, 796, 700 cm^ꢀ1; ESI-MS m/z 548 [M+Na]⁺, 526 [M+H]⁺, 448

[M+H-C₆H₆]⁺; HRMS (ESI) m/z [M+H]⁺calcd for C₃₂H₃₅NO₄Si:

526.2408, found: 526.2407.

5.2.20. (E)-(2-((tert-butyldiphenylsilyloxy)methyl)-4-((1-

methyl-1H-indol-7-yl)methylene)-5-oxotetrahydrofuran-2-yl)

methyl 2-propylpentanoate (14d)

Starting from 13d (180 mg, 0.34 mmol) and following general

procedure C, 14d was obtained as an amber oil (191 mg, 86% yield);

¹H NMR (400 MHz, CDCl₃) d 8.31 (m, 1H, CH@C), 7.67 (d, J = 7.6 Hz,

1H, H-4⁰), 7.58 (d, J = 6.9 Hz, 4H, Ph), 7.40–7.17 (m, 7H, Ph, H-6⁰),

7.11 (t, J = 7.6 Hz, 1H, H-5⁰), 7.00 (d, J = 2.8 Hz, 1H, H-2⁰), 6.52 (d,

J = 3.0 Hz, 1H, H-3⁰), 4.29 (qAB, 2H, J = 11.9 Hz, –CH₂OCO–), 3.96

(s, 3H, –NCH₃), 3.76 (qAB, 2H, J = 10.6 Hz, –CH₂OTBDPS), 3.16 (dd,

1H, J = 17.6, 2.1 Hz, H-4a), 3.01 (dd, 1H, J = 17.6, 2.1 Hz, H-4b),

2.36 (m, 1H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.52 (m, 2H, (CH₃CH_2-

CH₂)₂CHCO₂CH₂–), 1.37 (m, 2H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.24

(m, 4H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.01 (s, 9H, –C(CH₃)₃), 0.81 (t,

3H, J = 7.1 Hz, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 0.79 (t, 3H, J = 7.1 Hz,

(CH₃CH₂CH₂)₂CHCO₂CH₂–); ¹³C NMR (50 MHz, CDCl₃) d 175.88,

170.91, 135.57, 135.48, 134.43, 133.27, 132.60, 132.31, 131.38,

130.50, 129.92, 127.80, 125.23, 123.23, 122.50, 119.50, 119.27,

101.51, 83.29, 66.17 , 65.18, 45.15, 37.53, 34.41, 32.32, 26.70,

20.62, 20.56, 19.22, 13.86; IR (neat) 2957, 2931, 2858, 1736,

5.2.17. General procedure C. Acylation

A solution of 13 (a–d) (1 equiv) in dichloromethane (12 mL/

mmol) was treated with Et₃N (3 equiv), valproic acid chloride

(1.3 equiv) and a catalytic amount of DMAP (0.1 equiv). The reac-

tion was stirred at room temperature and monitored by TLC, and

upon completion it was concentrated in vacuo. Puriﬁcation by sil-

ica gel ﬂash column chromatography gave 14 (a–d).

1111, 701 cm^ꢀ1

;

ESI-MS m/z 674 [M+Na]⁺; HRMS (ESI) m/z

5.2.18. (E)-(2-((tert-butyldiphenylsilyloxy)methyl)-4-((1-

methyl-1H-indol-4-yl)methylene)-5-oxotetrahydrofuran-2-yl)

methyl 2-propylpentanoate (14a)

[M+H]⁺calcd for C₄₀H₄₉NO₅Si: 652.3453, found: 652.3456.

5.2.21. General procedure D. TBDPS deprotection

Starting from 13a (549 mg, 1.04 mmol) and following general

procedure C, 14a was obtained as an amber oil (623 mg, 92% yield);

¹H NMR (400 MHz, CDCl₃) d 8.06 (m, 1H, CH@C), 7.61 (m, 4H, Ph),

7.42- 7.22 (m, 9H, Ph, H-5⁰, H-6⁰, H-7⁰), 7.16 (d, J = 3.0 Hz, 1H, H-2⁰),

6.72 (d, J = 2.5 Hz, 1H, H-3⁰), 4.30 (mAB, 2H, –CH₂CO₂–), 3.85 (s, 3H,

–NCH₃), 3.81 (d, 1H, J = 10.6 Hz, –CH_aHOTBDPS), 3.73 (d, 1H,

J = 10.6 Hz, –CHH_bOTBDPS), 3.22 (dd, 1H, J = 17.7, 2.5 Hz, C-H_4a),

3.06 (dd, 1H, J = 17.7, 2.8 Hz, C-H_4b), 2.34 (m, 1H, (CH₃CH₂CH₂)_2-

CHCO₂CH₂–), 1.15–1.56 (m, 8H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.00

(s, 9H, –C(CH₃)₃), 0.78 (t, J = 7.32 Hz, 3H, (CH₃CH₂CH₂)₂CHCO₂CH₂),

0.74 (t, J = 7.07 Hz, 3H, (CH₃CH₂CH₂)₂CHCO₂CH₂); ¹³C NMR

(50 MHz, CDCl₃) d 175.97, 171.49, 136.85, 135.60, 135.54, 133.65,

132.54, 132.37, 129.89, 129.57, 127.80, 126.54, 124.04, 121.36,

119.44, 111.03, 99.44, 83.17, 66.32, 65.33, 45.13, 34.38, 33.02,

26.61, 20.56, 19.16, 13.86; IR (neat): 2956, 2931, 2858, 1751,

To a solution of compounds 14a–d in anhydrous THF (20 mL/

mmol) was added triethylamine hydroﬂuoride (10 equiv). The

reaction mixture was stirred at 70 °C until the starting material

disappeared by TLC analysis. Volatiles were removed and the resi-

due was puriﬁed by silica gel ﬂash column chromatography to

yield pure ﬁnal DAG-indololactones 2, 3, 4 and 5.

5.2.22. (E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-4-

yl)methylene)-5-oxotetrahydrofuran-2-yl)methyl 2-propyl-

pentanoate (2)

Starting from 14a (547 mg, 0.84 mmol) and following general

procedure D,

2 was obtained as a yellow crystalline solid

(287 mg, 83% yield); mp 100–102 °C; ¹H NMR (400 MHz, CDCl₃) d

8.10 (t, J = 2.8 Hz, 1H, CH@C), 7.40 (dd, J = 6.4, 2.0 Hz, 1H, H-7⁰),

7.27 (m, 2H, H-5⁰, H-6⁰), 7.17 (d, J = 3.0 Hz, 1H, H-2⁰), 6.73 (d,

J = 3.0 Hz, 1H, H-3⁰), 4.31 (qAB, 2H, J = 12.1 Hz, –CH₂OCO–), 3.84

(s, 3H, –NCH₃), 3.76 (qAB, J = 12.1 Hz, 2H, –CH₂OH), 3.26 (dd, 1H,

J = 17.7, 2.8 Hz, H-4a), 3.09 (dd, 1H, J = 17.7, 2.8 Hz, H-4b), 2.38

1736, 1105, 742, 701 cm^ꢀ1

;

ESI-MS m/z 674 [M+Na]⁺, 652

[M+H]⁺; HRMS (ESI) m/z [M+H]⁺calcd for C₄₀H₄₉NO₅Si: 652.3453,

found: 652.3453.

Please cite this article in press as: Garcia, L. C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.024

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

15

(m, 1H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 2.15 (m, 1H, OH), 1.53 (m, 2H,

5.2.25. (E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-7-

yl)methylene)-5-oxotetrahydrofuran-2-yl)methyl 2-

propylpentanoate (5)

(CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.37 (m, 2H, (CH₃CH₂CH₂)₂CHCO₂

CH₂–), 1.21 (m, 4H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 0.81 (t, 3H,

J = 7.3 Hz, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 0.79 (t, 3H, J = 7.3 Hz, (CH_3-

Starting from 14d (94 mg, 0.14 mmol) and following general

procedure D, 5 was obtained as a pale yellow oil (40 mg, 67% yield);

¹H NMR (400 MHz, CDCl₃) d 8.38 (m, 1H, CH@C), 7.66 (d, J = 7.6 Hz,

1H, H-4⁰), 7.24 (d, J = 7.6 Hz, 1H, H-6⁰), 7.11 (t, J = 7.6 Hz, 1H, H-5⁰),

7.01 (d, J = 3.1 Hz, 1H, H-2⁰), 6.51 (d, J = 3.1 Hz, 1H, H-3⁰), 4.34

(qAB, 2H, J = 11.9 Hz, –CH₂OCO–), 4.05 (s, 3H, –NCH₃), 3.79 (dd,

J = 6.6, 12.1 Hz, –CH_aOH), 3.70 (dd, J = 6.6, 12.1 Hz, –CHH_bOH),

3.21 (dd, 1H, J = 17.7, 2.8 Hz, H-4a), 3.03 (dd, 1H, J = 17.7, 2.8 Hz,

H-4b), 2.39 (m, 1H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 2.13 (t, 1H,

J = 6.8 Hz, –OH), 1.55 (m, 2H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.40 (m,

2H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.24 (m, 4H, (CH₃CH₂CH₂)₂CHCO_2-

CH₂–), 0.83 (t, J = 7.3 Hz, 3H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 0.81 (t,

J = 7.3 Hz, 3H, (CH₃CH₂CH₂)₂CHCO₂CH₂–); ¹³C NMR (50 MHz,

CDCl₃) d 176.25, 170.84, 134.41, 131.48, 130.57 , 124.51, 123.53,

122.65, 119.31, 101.62, 83.34, 65.09, 64.88, 45.21, 37.63, 34.43,

32.14, 20.61, 13.86; IR (neat) 3430, 2957, 2931, 2872, 1732, 1292,

1207, 1015, 719 cm^ꢀ1;); UV (CH₃OH/H₂O [1:1] with 0.1% CH₃CO₂H)

k_max@ 261 nm, 355 nm (conjugated 1-methyl indole); HPLC (gradi-

ent elution) pure (>98%); ESI-MS m/z 436 [M+Na]⁺, 414 [M+H]⁺;

HRMS (ESI) m/z [M+H]⁺calcd for C₂₄H₃₁NO₅: 414.2275, found:

414.2275; Elem. Anal. Calcd For: C₂₄H₃₁NO₅: C, 69.71; H, 7.56; N,

3.39. Found: C, 69.44; H, 7.51, N, 3.43.

CH₂CH₂)₂CHCO₂CH₂–); ¹³C NMR (50 MHz, CDCl₃)

d 176.37,

171.36, 136.91, 134.72, 130.08, 129.65, 126.32, 123.17, 121.41,

119.64, 111.33, 99.36, 83.16, 65.22, 64.95, 45.19, 34.38, 33.02,

32.75, 20.57, 13.86, 13.83; IR (neat) 3465, 2932, 1724, 1225,

1042, 731, 746 cm^ꢀ1; UV (CH₃OH/H₂O [1:1] with 0.1% CH₃CO₂H)

k_max@ 256 nm, 363 nm (conjugated 1-methyl indole); HPLC

(gradient elution) pure (>98%); ESI-MS m/z 436 [M+Na]⁺, 414

[M+H]⁺; HRMS (ESI) m/z [M+H]⁺calcd for C₂₄H₃₁NO₅: 414.2275,

found: 414.2269.

5.2.23. (E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-5-

yl)methylene)-5-oxotetrahydrofuran-2-yl)methyl 2-

propylpentanoate (3)

Starting from 13b (137 mg, 0.26 mmol) and following general

procedures C and D, 3 was obtained as a white solid (48 mg, 44%

yield); mp 107–109 °C; ¹H NMR (400 MHz, CDCl₃) d 7.80 (s, 1H,

H-4⁰), 7.73 (sa, 1H, CH@C), 7.37 (m, 2H, H-6⁰, H-7⁰), 7.10 (d,

J = 3.0 Hz, 1H, H-2⁰), 6.54 (d, J = 3.0 Hz, 1H, H-3⁰), 4.35 (qAB, 2H,

J = 11.8 Hz, –CH₂OCO–), 3.82 (s, 3H, –NCH₃), 3.80 (dd, 1H,

J = 12.2, 6.6 Hz, –CH_aHOH), 3.73 (dd, 1H, J = 12.2, 5.8 Hz, –CHH_b-

OH), 3.26 (dd, 1H, J = 17.4, 2.8 Hz, H-4a), 3.08 (dd, 1H, J = 17.4,

2.8 Hz, H-4b), 2.45 (t, 1H, J = 6.3 Hz, OH), 2.39 (m, 1H, (CH₃CH_2-

CH₂)₂CHCO₂CH₂–), 1.54 (m, 2H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.37

(m, 2H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.23 (m, 4H, (CH₃CH₂CH₂)_2-

CHCO₂CH₂–), 0.82 (t, 3H, J = 7.3 Hz, (CH₃CH₂CH₂)₂CHCO₂CH₂–),

0.78 (t, 3H, J = 7.3 Hz, (CH₃CH₂CH₂)₂CHCO₂CH₂–); ¹³C NMR

5.3. Biological studies

5.3.1. Cell culture

LNCaP cells (ATCC, Manassas, VA) were cultured in RPMI-1640

(50 MHz, CDCl₃),

d

176.42, 171.79, 139.45, 137.40, 130.30,

medium (ATCC) containing 4 mM L-glutamine supplemented with

128.78, 125.95, 124.09, 123.93, 119.78, 109.66, 102.14, 83.01,

65.28, 64.97, 45.17, 34.39, 34.38, 32.98, 32.49, 20.58, 20.57,

13.88, 13.84; IR (neat) 3363, 2952, 2932, 2872, 1741, 1717,

1147, 811, 714 cm^ꢀ1; UV (CH₃OH/H₂O [1:1] with 0.1% CH₃CO₂H)

k_max@ 280 nm, 329 nm (conjugated 1-methyl indole); HPLC

(gradient elution) pure (>98%); ESI-MS m/z 849 [M₂+Na]⁺, 436

[M+Na]⁺, 414 [M+H]⁺; HRMS (ESI) m/z [M+H]⁺calcd for

10% FBS (ATCC). CHO-K1 cells (ATCC) were grown in F-12 medium

(Invitrogen) supplemented with 10% FBS (ATCC). HEK-293-RasGRP3

cells were grown in DMEM supplemented with 10% FBS (ATCC).

5.3.2. Molecular constructs

The constructs used for confocal imaging (human GFP-PKC

a,

human YFP-PKC

e

, and human GFP-PKCd) were described earlier.³⁸

C

₂₄H₃₁NO₅: 414.2275, found: 414.2259.

Human RasGRP3 was PCR ampliﬁed and cloned into the pQBI25fN1

vector using NheI restriction enzyme. Sequence analysis of the

construct was conducted by the DNA Minicore (Center for Cancer

Research, NCI, National Institutes of Health).

5.2.24. (E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-6-

yl)methylene)-5-oxotetrahydrofuran-2-yl)methyl 2-

propylpentanoate (4)

Starting from 14c (146 mg, 0.22 mmol) and following general

procedure D, 4 was obtained as a pale yellow oil (59 mg, 63%

yield); ¹H NMR (400 MHz, CDCl₃) d 7.73 (m, 1H, CH@C), 7.65 (d,

J = 8.3 Hz, 1H, H-4⁰), 7.43 (s, 1H, H-7⁰), 7.27 (dd, J = 9.1, 2.8 Hz,

1H, H-5⁰), 7.18 (d, J = 3.0 Hz, 1H, H-2⁰), 6.52 (d, J = 2.8 Hz, 1H, H-

3⁰), 4.31 (qAB, 2H, J = 11.9 Hz, –CH₂OCO–), 3.83 (s, 3H, –NCH₃),

3.81 (d, J = 12.1 Hz, 1H, –CH_aHOH), 3.73 (d, J = 12.1 Hz, 1H, –CHH_b-

OH), 3.28 (dd, 1H, J = 17.3, 2.5 Hz, H-4a), 3.08 (dd, 1H, J = 17.3,

2.5 Hz, C-4b), 2.37 (m, 1H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.54 (m,

2H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 1.37 (m, 2H, (CH₃CH₂CH₂)₂CHCO_2-

CH₂–), 1.23 (m, 4H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 0.82 (t, J = 7.3 Hz,

3H, (CH₃CH₂CH₂)₂CHCO₂CH₂–), 0.77 (t, J = 7.3 Hz, 3H, (CH₃CH_2-

CH₂)₂CHCO₂CH₂–); ¹³C NMR (100 MHz, CDCl₃) d 176.34, 171.67,

139.31, 136.66, 131.59, 130.07, 127.91, 121.19, 121.00, 120.71,

112.28, 101.47, 83.11, 65.29, 64.91, 45.17, 34.39, 32.92, 32.44,

20.56, 13.87, 13.81; IR (neat) 3435, 2956, 2932, 2872, 1732,

1644, 1164, 1043, 731, 625 cm^ꢀ1; UV (CH₃OH/H₂O [1:1] with

0.1% CH₃CO₂H) k_max@ 271 nm, 325 nm (conjugated 1-methyl

indole); HPLC (gradient elution) pure (>98%); ESI-MS m/z 436

[M+Na]⁺, 414 [M+H]⁺; HRMS (ESI) m/z [M+H]⁺calcd for

5.3.3. Ligand binding

Binding of [³H]PDBu to PKC

a

, to RasGRP3, and to RasGRP1-C1

and competition for binding by DAG-indololactones were mea-

sured in the presence of 100 g/mL phosphatidylserine as

described previously.²³To optimize stability, binding to PKC

and RasGRP1-C1 was performed at 37 °C; binding to RasGRP3

was performed at 18 °C.

l

a

5.3.4. Biological activity of ligands

HEK-293 cells and HEK-293 with overexpressed RasGRP3 (LEG-

FP-HEK-N-RasGRP3) cells were seeded at 2 ꢃ 10⁶/dish, cultured for

24 h, then treated with different concentrations of PMA or indolo-

lactones 1–5 for 30 min. The cells were then collected, lysed, and

Western blotting was performed. Brieﬂy, equal amounts of protein

were separated by SDS gel electrophoresis and then transferred to

immobilon-P membranes (Millipore Corp.). The membranes were

blocked and labeled with primary antibodies and secondary anti-

bodies. The signals were developed by the ECL western blotting

detection system and imaged on BioMax XAR ﬁlm. The following

antibodies were used: anti-RasGRP3 (1:1000), ERK (1:2000) and

p-ERK (1:1000) antibodies (Cell Signaling Technology Inc.); anti-

p-RasGRP3 (pT133) (1:2000), p-PKCd(pS299) (1:3000) antibodies

(Epitomics Company); anti-PKCd (1:2000) and b-actin (1:2000)

C

₂₄H₃₁NO₅: 414.2275, found: 414.2274; Elem. Anal. Calcd for

₂₄H₃₁NO₅: C, 69.71; H, 7.56; N, 3.39. Found: C, 69.70; H, 7.49, N,

3.40.

Please cite this article in press as: Garcia, L. C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.024

16

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

antibodies (Santa Cruz Biotechnology Inc). Results represent tripli-

cate independent experiments for all compounds.

giant vesicles. Fluorescence emission spectra were acquired at

27 °C on a FL920 spectroﬂuorimeter (Edinburgh, Co., Edinburgh,

UK) by using the compound’s excitation wavelength. Total sample

volumes were 1 mL, and the solutions were placed in a quartz cell

with a 0.5 cm optical path-length. Light scattering from the vesi-

cles was conﬁrmed to account for less than 5% of the emission

intensity.

Ramos cells grown in RPMI-1640 (ATCC) containing 10% fetal

bovine serum and 1%

concentration of 300,000 cells/mL. After 24 h cells were exposed

to compounds at 0.01, 0.1, 1, and 10 M concentrations for

L-glutamine were plated in 6 cm dishes at a

l

30 min. Cells were then pelleted and cell lysates were prepared

in PBS containing 0.5% Triton X-100 with protease and phospha-

tase inhibitors (Roche). Cells were sonicated 3 times for 6 s each

5.4.3. Fluorescence anisotropy

and 7.5

l

g of protein lysate (concentration calculated using BIO-

The ﬂuorescence probe DHP-TMA was incorporated into the

GVs, that were prepared according to the procedure described

above, by adding the dye dissolved in THF (1 mM) to the vesicles

RAD DC protein assay) in SDS containing beta-mercaptoethanol

was separated on 10% Tris-glycine gels (Novex) and transferred

to nitrocellulose membranes (Whatman). The membranes were

blocked with 5% nonfat dry milk (BIO-RAD) and incubated over-

night in the primary antibody, washed with PBS containing 0.05%

Triton X-100 and incubated 1 h in the secondary antibody and

washed in PBS containing 0.05% Triton X-100. The blots were

developed by ECL (Amersham) and signals detected on chemilumi-

nescence ﬁlm (Amersham). The primary antibodies used were:

RasGRP3 (C33A3, Cell Signaling), pRasGRP3 (pT133, Epitomics),

PKCd (Epitomics), pPKCd (S299, Epitomics), pPKD1 (S744/748, Cell

Signaling), ERK (Cell Signaling), pERK (T202/Y204, Cell Signaling),

up to a ﬁnal concentration of 1.25 lM and incubating at room

temperature for 30 min in order to allow the incorporation of

the probe into the vesicles. The required amount of solutions of

compounds 1–5 (DMSO, 1 mg/mL) were added to the vesicles con-

taining TMA-DPH probe (30 lL) and the volume was adjusted to

1 mL with buffer (0.1 M sucrose, 0.1 M KCl, 2 mM Tris solution,

pH 7.4). The samples were placed in a quartz cell with a 0.5 cm

optical path-length. TMA-DPH ﬂuorescence anisotropy was

measured at 430 nm (excitation 355 nm) on

a

FL920

spectroﬂuorimeter. Anisotropy values were automatically calcu-

lated by the spectroﬂuorimeter software by using conventional

PKCa (c-terminus, Epitomics), and PKCe (C-15, Santa Cruz). The

secondary antibody was a horseradish peroxidase conjugated

anti-rabbit (BIO-RAD) antibody.

methodology. The concentrations of compounds were 2.42

and 6.05 M.

lM

l

5.3.5. Visualization of translocation of PKC isoforms and

RasGRP3 in living cells in response to ligands

5.4.4. Förster resonance energy transfer (FRET) measurements

Vesicles prepared according to the procedure described above

CHO-K1 cells (40,000 cells/mL) or LNCaP cells (80,000 cells/mL)

were plated onto ibidi dishes (ibidi LLC, Verona, WI). After 24 or

48 h in culture, respectively, cells were transfected with recombi-

nant constructs using Lipofectamine reagent and Plus reagent

(both from Invitrogen) according to the manufacturer’s protocol.

Cellular expression and translocation of ﬂuorescent fusion proteins

after drug treatment were examined 24 h after transfection using a

Zeiss LSM 510 NLO confocal microscopy system (Carl Zeiss, Inc.,

Thornwood, NY) with excitation from a 30-milliwatt argon laser

tuned to 488 nm and emission collected with a BP 500–530 ﬁlter.

Images were acquired every 30 s for 30 min at varying zoom set-

tings (1.4-2) using Zeiss AIM software and a 63 ꢃ 1.4 NA Zeiss

Plan-Apochromat oil immersion objective. Quantitation of confocal

images was performed as described earlier.⁵⁷In each cell, regions

were additionally supplemented with NBD-PE at a 100:1

(phospholipid:ﬂuorophore) molar ratio. Indololactones 1 to 5 were

dissolved in DMSO (1 mg/mL) and the required amount of each

solution was placed in a quartz cell with a 0.5 cm optical path-

length containing 30 lL of vesicles and the volume was adjusted

to 1 mL with buffer (0.1 M sucrose, 0.1 M KCl, 2 mM Tris solution,

pH 7.4). Emission spectra were acquired at 530 nm (NBD-PE) with

an excitation wavelength of the indololactone at 25 °C on a FL920

spectroﬂuorimeter. Energy transfer was determined by three types

of measurement: (1) only vesicles, (2) vesicles with addition of

indololactone and (3) vesicles with addition of indololactone and

addition of 20% Triton X-100.

5.5. Molecular modeling

of 4 l

m²each were selected in the cytoplasm and the cell mem-

brane. Mean intensities in the selected regions were calculated

using the Zeiss AIM software at the indicated time points. The ratio

of the intensities for membrane and cytoplasm was then calculated

and normalized to the time 0 values. The increase in the mem-

brane/cytoplasm ratio indicates translocation.

Molecular dynamics simulations with an implicit solvent

bilayer were run in CHARMM⁶⁹version c35b5. Forceﬁeld parame-

ters for the lactone ring and alkyl sidechain of the DAG-indololac-

tones were developed by analogy with existing parameters for

lipids⁷⁰and furanose sugars, and checked against ab-initio geome-

try-optimized structures calculated at the RHF/6-31G^⁄theory level.

Parameters for the N-methylindole sidechain were based on tryp-

tophan, and the indole-lactone linker was derived from styrene

in the CHARMM General Force Field.⁷¹

5.4. Biophysical studies

5.4.1. Giant vesicle (GV) preparation

GVs were prepared through the rapid evaporation method.⁶⁸GVs

comprising combinations of lipids were prepared in the molar ratio

of 9:1, DMPC/DMPG. Thelipid constituents were dissolved in chloro-

form/ethanol (1:1, v/v) and subsequently added to a round-bottom

ﬂask (250 mL) containing chloroform (1 mL). The aqueous phase

(5 mL of 0.1 M sucrose, 0.1 M KCl, 2 mM Tris solution, pH 7.4) was

then carefully added along the ﬂask walls. The organic solvent was

removed in a rotary evaporator under reduced pressure (ﬁnal pres-

sure 40 mbar) at 25 °C and 40 rpm. After evaporation for 4–5 min, an

opalescent ﬂuid was obtained with a volume of approximately 5 mL.

The implicit solvent bilayer was simulated using the HDGB

(heterogeneous dielectric generalized Born) method [Ref. 44] in

CHARMM. In this method the bilayer is modeled as a series of slabs,

set up perpendicular to the z-axis, with varying dielectric con-

stants. The inner slab, centered at z = 0 with a width of 20 Å, has

a dielectric constant of 2.0 as a representation of the hydrophobic

core of the bilayer, a middle slab with a thickness of 5 Å has a

dielectric constant of 7.0 as a representation of the glycerol back-

bone region, and the outer slab has a dielectric constant of 80.0

for bulk water. An apparent dielectric constant as a function of

membrane depth z was calculated by solving the Poisson equation

for a probe ion moving across the membrane. This apparent dielec-

tric is then used to calculate the electrostatic component of the sol-

vation energy for each atom by solving the Born equation. The

5.4.2. Fluorescence measurements

Changes in the compound’s intrinsic emission were measured

for 2.42 lM and 6.05 lM solutions titrated with DMPC/DMPG

Please cite this article in press as: Garcia, L. C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.024

L. C. Garcia et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx

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surface tension coefﬁcient for the nonpolar portion of the solvation

energy was 0.015 kcal/mol Å.²

Four molecular dynamics simulations were run for each indolo-

lactone, each starting from a different orientation relative to the

bilayer, with two inside the membrane core at z = 0 and two out-

side the membrane entirely at z = 25. Each simulation underwent

200 steps of adopted-basis Newton Raphson miminization, was

heated in 10-degree increments from a starting temperature of

50 K to 300 K over the course of 10 ps, and run at constant temper-

ature for 100 ns, with coordinate snapshots saved every 5 ps. Any

rotational and translational movement of the molecules was

stopped after every picosecond. After 20 ns of simulation time,

each of the indololactones had reached its equilibrium position

in the membrane, and the remaining 80 ns were used for analysis.

The absolute value of the z-position of each heavy atom in the

indole ring and branched alkyl group was extracted from each

saved coordinate snapshot, binned in 0.25 Å increments from

z = 0 to z = 30, and averaged over the four simulations, to give

the density of each type of atom at each z-position. The transition

dipole moment of the indole ring was deﬁned as a vector from

atom CG to atom CZ2. This vector was then normalized, and its

angle to the bilayer normal (which is the z-axis) was calculated

by taking the arccosine of its z-component. This angle was

extracted from each saved coordinate snapshot, binned in 5 degree

increments from ꢀ180 to 180, and averaged over the four simula-

tions, to give the occupancy of each angle.

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The research was supported in part by The National Institute of

Industrial Technology (INTI) and Consejo Nacional de Investigación

Cientíﬁca y Tecnológica (CONICET); in part by the Intramural

Research Program of the National Institutes of Health, Center for

Cancer Research, National Cancer Institute (Z1A BC 005720) and

in part with Federal funds from the Frederick National Laboratory

for Cancer Research, National Institutes of Health, under contract

HHSN261200800001E. The content of this publication does not

necessarily reﬂect the views or policies of the Department of

Health and Human Services, nor does mention of trade names,

commercial products or organizations imply endorsement by the

US Government.

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Please cite this article in press as: Garcia, L. C.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.024

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Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP

DOI: 10.1016/j.bmc.2014.04.024

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