Conformationally constrained analogues of diacylglycerol. 29. Cells sort diacylglycerol-lactone chemical zip codes to produce diverse and selective biological activities

5198

J. Med. Chem. 2008, 51, 5198–5220

Conformationally Constrained Analogues of Diacylglycerol. 29. Cells Sort

Diacylglycerol-Lactone Chemical Zip Codes to Produce Diverse and Selective Biological

Activities

Dehui Duan,^†,+Dina M. Sigano,^†James A. Kelley,^†Christopher C. Lai,^†Nancy E. Lewin,^‡Noemi Kedei,^‡Megan L. Peach,^§

,X

Jeewoo Lee,^|Thushara P. Abeyweera,

Susan A. Rotenberg, Hee Kim,^#Young Ho Kim,^#Saïd El Kazzouli,^†

Jae-Uk Chung,^|,∞Howard A. Young, Matthew R. Young,^OAlyson Baker,^ONancy H. Colburn,^O

Adriana Haimovitz-Friedman,^[Jean-Philip Truman,^[Damon A. Parrish,^¶Jeffrey R. Deschamps,^¶Nicholas A. Perry,^‡

Robert J. Surawski,^‡Peter M. Blumberg,^‡and Victor E. Marquez*^,†

Laboratory of Medicinal Chemistry, National Cancer Institute at Frederick, National Institutes of Health, 376 Boyles Street,

Frederick, Maryland 21702, Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health,

Bethesda, Maryland 20892, Basic Research Program SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702, Laboratory of

Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National UniVersity,

Seoul 151-742, South Korea, Department of Chemistry and Biochemistry, Queens College, City UniVersity of New York, Flushing,

New York 11367, Digital Biotech, 1227 Sin Gil Dong, Danwon-Ku, Ansa-Si, Kyonggi-Do 425-839, South Korea, Laboratory of Experimental

Immunology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, Laboratory of Cancer

PreVention, National Cancer Institute at Frederick, National Institutes of Health, Frederick Maryland 21702, Department of Radiation

Oncology, Memorial Sloan-Kettering Cancer Center, 1275 York AVe, New York, New York 10021, NaVal Research Laboratory,

Washington, D.C. 20375

ReceiVed February 22, 2008

Diacylglycerol-lactone (DAG-lactone) libraries generated by a solid-phase approach using IRORI technology

produced a variety of unique biological activities. Subtle differences in chemical diversity in two areas of

the molecule, the combination of which generates what we have termed “chemical zip codes”, are able to

transform a relatively small chemical space into a larger universe of biological activities, as membrane-

containing organelles within the cell appear to be able to decode these “chemical zip codes”. It is postulated

that after binding to protein kinase C (PKC) isozymes or other nonkinase target proteins that contain

diacylglycerol responsive, membrane interacting domains (C1 domains), the resulting complexes are directed

to diverse intracellular sites where different sets of substrates are accessed. Multiple cellular bioassays show

that DAG-lactones, which bind in vitro to PKCR to varying degrees, expand their biological repertoire into

a larger domain, eliciting distinct cellular responses.

Introduction

(nPKCs: δ, ε, η, θ), as well as other proteins containing similar

C1 membrane targeting domains, typically requires recruitment

to membranes and allosteric activation by DAG, which results

from the speciﬁc molecular interactions of DAG with the C1

domains and the modulation of the physical properties of

membranes by the DAG alkyl chains. The combination of these

two factors contributes to the localization of an isozyme to

speciﬁc intracellular sites where different substrates are accessed.

Molecular interactions revealed by the crystal structure of the

PKCδ C1b domain bound to phorbol-13-acetate¹showed the

existence of a network of hydrogen bonds between phorbol ester

and conserved amino acids Thr242, Leu251, and Gly253, which

were reproduced precisely by computer modeling when DAG

was inserted into the same C1 domain.²Other highly conserved

hydrophobic amino acids of the δ-C1b domain, such as Pro231,

Phe243, Leu250, and Trp252, which form a contiguous hydro-

phobic ring around the top of the C1 domain, are likely to

interact with the membrane by sensing changes in the physical

properties of the lipid bilayer induced by DAG, including

ﬂuidity, curvature, hydrocarbon volume, and headgroup separa-

tion, all of which may facilitate the insertion of the enzyme

into membranes.^3-6It has become increasingly clear that these

type of physical perturbations of lipid membranes contribute

to the modulation of PKC activity.⁷

Protein kinase C isozymes (PKCs) are key signaling mol-

ecules, responding to the second messenger diacylglycerol

(DAG), which is generated from phosphoinositides upon activa-

tion of many cellular receptors. The activation of conventional

protein kinases (cPKCs: R, ꢀI, ꢀII, γ) and novel protein kinases

* To whom correspondence should be addressed. Phone: 301-846-5954.

Fax: 301-846-6033; E-mail: marquezv@mail.nih.gov.

†

Laboratory of Medicinal Chemistry, National Cancer Institute at

Frederick, National Institutes of Health.

‡

Laboratory of Cancer Biology and Genetics, National Cancer Institute,

National Institutes of Health.

§

Basic Research Program SAIC-Frederick, Inc., NCI-Frederick.

Laboratory of Medicinal Chemistry, Research Institute of Pharmaceuti-

|

cal Sciences, College of Pharmacy, Seoul National University.

Department of Chemistry and Biochemistry, Queens College, City

University of New York.

#

Digital Biotech.

Laboratory of Experimental Immunology, National Cancer Institute

at Frederick, National Institutes of Health.

O

Laboratory of Cancer Prevention, National Cancer Institute at Frederick,

National Institutes of Health.

[

Department of Radiation Oncology, Memorial Sloan-Kettering Cancer

Center.

¶

Naval Research Laboratory.

Current address: Sequoia Pharmaceuticals, 401 Professional Drive,

+

Gaithersburg, MD 20879.

X

Current address: Memorial Sloan-Kettering Cancer Center, Program

The cellular distribution of activated PKC isozymes is

additionally controlled by interactions with other signaling or

scaffolding proteins through the formation of multiprotein

in Immunology, 1275 York Avenue, New York, NY 10021.

∞

Current address: Choongwae Pharma Corporation, 146-141, Annyung-

dong, Hwaseong-si, Gyeonggi-do 445-380, South Korea.

10.1021/jm8001907 CCC: $40.75

2008 American Chemical Society

Published on Web 08/13/2008

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5199

Figure 1. “Chemical zip codes” for the DAG-lactones libraries 1 and 2. For Library 1 (top), the rows correspond to a set of R groups adjacent to

the sn-2 carbonyl, which are derived from a collection of aldehydes. The columns correspond to R′ groups, which are derived from a set of acid

chlorides and are connected to the sn-1 carbonyl. For Library 2 (bottom), the orientation is reversed and the rows display the R′ groups at sn-1,

whereas the columns display the R groups at sn-2. The red numbers in each library correspond to measured K_ivalues (nM); the blue numbers

correspond to calculated log P values, and the green numbers in parentheses correspond to the observed m/z for MH⁺(or M^•+).

complexes that facilitate signal transduction and confer speciﬁc-

ity for individual PKC isoforms by regulating their activity and

cellular localization, allowing the modulation of biological

functions within the cell.^8-10Furthermore, these signaling

complexes are not conﬁned to the plasma membrane alone but

are found on various cellular membranes, including Golgi,

mitochondrial, and nuclear membranes.^11,12Recent studies have

also shown that lipid-protein interactions can similarly provide

the necessary speciﬁcity and afﬁnity to achieve a particular

subcellular localization of signaling proteins.¹³Thus, membrane

5200 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Scheme 1

lipids may help compartmentalize signaling complexes and

regulate the spatiotemporal dynamics of PKC activation through

interacting membrane microdomains, some of which can be

formed transiently during signaling after the release of DAG.^14-16

An additional level of control where lipids play a critical role

in enzyme activation is that of disrupting intramolecular,

interdomain interactions between C1 and C2 domains, as

demonstrated with PKCR,^17,18and between the C1 domain and

at least four different regions of the protein in ꢀ2-chimaerin

(the N-terminus, the SH2 and RacGAP domains, and the linker

between C1 and SH2) as elegantly described by Hurley et al.¹⁹

Combinatorial libraries built on our potent DAG-lactone

template²⁸using carefully chosen R and R′ groups (Figure 1)

that function as “chemical zip codes” were expected to create

a unique microenvironment surrounding the binding site. These

“chemical zip codes” result from the exclusive combination of

two diversity elements on the DAG-lactone at the sn-1 (R′) and

sn-2 (R) positions (Figure 1).

The chosen elements of our “chemical zip codes” were

selected to exploit membrane-ligand interactions and protein-

ligand interactions that operate outside the C1 domain in the

ternary complex (C1-domain-DAG-membrane) through vari-

ous mechanisms for which the side chains would be expected

to be critical, such as Coulombic interactions between cationic

residues and anionic lipids, interactions between aromatic

residues and zwitterionic lipids, and hydrophobic interactions

between conserved amino acids along the rim of the C1 domain

and membrane lipids. By applying this approach, we have been

able to identify combinations of R and R′ groups that produce

DAG-lactones capable of eliciting distinct, speciﬁc cellular

responses, thus providing strong proof-of-principle for the use

of “chemical zip codes” on DAG-lactones to develop novel

therapeutic strategies for PKC agonists with unique biology.

The most useful pharmacological probes for C1 domains, the

family of phorbol esters (e.g., TPA), cannot distinguish between

PKC isoforms, and PKC is only one of multiple families of

proteins that have appropriate C1 domains to act as DAG/

phorbol receptors.^20,21There are other proteins with typical C1

domains that are subject to DAG modulation including: the PKD

family, a distinct class of serine/threonine kinases,²²the

chimaerin family, inhibitors of p21Rac,²³the munc-13 family,

proteins involved in synaptic vesicle priming,²⁴the RasGRP

family, guanyl nucleotide exchange factors for Ras and Rap1,^25,26

and the DAG kinase family, which function to abrogate DAG

signaling, thus providing negative feedback on DAG signaling

pathways.²⁷Of these C1 domain-containing families, the PKC

family is the best-studied mediator in the DAG signaling

pathways and an important target for drug development if one

could learn how to selectively impact PKC regulation in cells

subject to proliferating or differentiating stimuli.

Chemistry

In a previous publication, we reported the development of a

solid-phase method using a PL-DHP (3,4-dihydro-2H-pyran)

resin for the synthesis of DAG-lactones with an exploratory

set of R and R′ groups.²⁹This method was tested with the

synthesis of a small, nine-member array with the idea of

forecasting the reliability of the biological data that could be

obtained when using larger chemical libraries synthesized under

the same conditions. From that study, we concluded that the

biological data acquired using crude samples directly obtained

from the resin provided valuable information as the difference

in binding afﬁnities (K_i) for PKCR between pure and crude

materials varied only by a factor between 1.5 and 3.7.

The synthetic approach utilized here was essentially the same

(Scheme 1), except that we chose two starting lactones (1b and

1d) with different protecting groups (Scheme 2), which were

designed to overcome incompatibilities between some of the

chemical groups selected for the library and the conditions for

deprotection. Using this method, two exploratory libraries were

investigated (Figure 1).

Because the actual DAG binding site is a complex of lipid

bilayer and C1 domain, for which the C1 domain is only a half-

site, the interactions with the membrane and other important

protein components are difﬁcult to study. We surmise that the

speciﬁc cellular localization of the activated PKC isozyme, or

C1 domain containing protein, ought to be determined in part

by the different lipid composition of the membranes, the

targeting information intrinsic to the individual C1 domain

bound to DAG, and the binding to scaffolding or signaling

proteins. This multifaceted control of diversity suggests that,

in order to improve our chance of developing DAG-like

molecules capable of translocating these proteins to speciﬁc

cellular targets, we need to gain access to the chemical space

surrounding the DAG binding site. Because structure-activity

analyses that incorporate the lipid bilayer and other proteins

into the model are still rudimentary, we explored this aspect

indirectly by combinatorial chemistry.

The basic concept behind the design of Library 1 was to

explore the effect of swapping similar groups, containing simple

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5201

Scheme 2

contains very active ligands, including the most potent com-

pound of the entire Library (L1-G2). Because of this ﬁnding,

the triﬂuoromethylphenyl group was more thoroughly studied

in Library 2, which included positional isomers and multiple

CF substitutions (vide infra).

An interesting observation that supports the binding mode

described before is the effect of exchanging the positions of

polar and nonpolar groups. For example, a comparison between

compounds L1-B5, L1-B6, L1-B7, and L1-B8, with polar

dimethylamino, methoxy, triﬂuoromethyl, and nitro groups as

part of R′ (sn-1), vis-a`-vis compounds L1-E2, L1-F2, L1-G2,

and L1-H2, where the same polar groups become part of R

(sn-2), showed that the latter arrangement produced better

ligands. Comparing the K_ivalues for L1-B8 and L1-H2 between

crude and pure samples (E-isomers) corroborated the proposed

binding mode, as the 10-fold difference in binding afﬁnity

observed between the crude samples was magniﬁed to a 100-

fold difference for the pure compounds (Table 1, rows 3 and

12).

For the compounds that were synthesized in pure form (Table

1), the differences in afﬁnity between crude and pure samples

were variable. Pure compounds L1-A12, L1-B5, and L1-B8

were obtained as Z-isomers, whereas the rest of the compounds

with R-arylidene moieties were obtained almost exclusively as

E-isomers (vide infra). The majority of the compounds showed

K_icrude/K_ipure ratios of e10-fold, except L1-A12, L1-F11,

L1-F12, and L1-H2. Obviously, factors such as purity of the

crude samples (see Analysis of Chemical Libraries in the

Experimental Section), solubility, and operational variation can

explain these differences. Nevertheless, for the most part, the

results with the crude samples mimicked well the real trends

of the pure samples and aided in the selection of compounds

for additional studies.

polar substituents that are either electron-withdrawing or

electron-donating and nonpolar alkyl or aryl moieties, between

the two available positions on the DAG-lactone scaffold. Hence,

in Library 1, the R groups chosen for the rows of the matrix

(A-H) were the same as the R′ groups of the ﬁrst eight columns

(1-8). Four additional acid chlorides (columns 9-12) were

added to explore the effect of branching on the aromatic moiety.

The reason for selecting branched chains was based on the

excellent activity achieved earlier with DAG-lactones containing

aliphatic, branched acyl chains.²⁸In addition, the chosen groups

allowed us to explore a range of log P values, which is also an

important parameter for activity and selectivity.

On the basis of a previous study where we found differences

between DAG-lactones bearing identical functionalized phenyl

groups at either the sn-2 (R) or the sn-1 (R′) positions, we

concluded that DAG-lactones prefer to bind to the C1 domain,

with the R′ acyl chain oriented toward the interior of the

membrane and the R alkylidene or arylidene group directed to

the surface of the C1 domain, adjacent to the lipid interface.³⁰

Keeping that concept in mind, we decided to expand the

chemical R space by designing Library 2 with a larger aldehyde-

derived set (columns 1-12) consisting exclusively of aromatic

moieties. The smaller, acid chloride-derived rows were com-

prised mostly of alkyl groups (A, B, C, F, G, H) and aryl-alkyl

groups (D and E), all of which are nonpolar and able to interact

effectively with the membrane.

The selection of these two libraries was intended to showcase

the potential of this approach as every member of the library

was expected to bind to the C1 domain but where differences

between the various bioassays and cellular activities would

reﬂect the manner in which the ternary complex, C1-domain-

DAG-lactone-membrane, could sense the different composition

of the “chemical zip codes”. All of the crude compounds

synthesized in libraries 1 and 2 were racemates and consisted

of variable mixtures of geometrical E- and Z-isomers. The

identity of individual DAG-lactone library members and the

overall library quality were initially assessed by FAB/MS

analysis employing a modiﬁed combine-and-analyze strategy³¹

(see Experimental Section).

Library 1, RasGRP. The high conservation of the C1

domains of PKC has been a challenge for the development of

selective ligands. Furthermore, the identiﬁcation of other families

of signaling molecules containing DAG-responsive C1 domains

makes achieving speciﬁcity an even more daunting task. The

PKCR binding assay of the puriﬁed members of Library 1 (Table

1) showed that several compounds produced unusually shaped

dose-response curves. These curves were shallow and could

be modeled as two binding components with different afﬁnities

and with each representing half of the total binding sites,

suggesting that the compounds might show selectivity between

the C1a and C1b domains. To test that model, we assayed these

compounds for RasGRP1/3 binding because it only has a single

C1 domain. This led to the discovery of two compounds, L1-

H5 and L1-H6, which displayed much higher binding afﬁnities

for RasGRP1/3 than for PKCR and other PKC isozymes.³⁴This

ﬁnding provided strong support for the hypothesis that the

complexes formed between the ligand, the C1 domain, mem-

brane phospholipids, and intervening proteins can decipher the

“chemical zip codes” and show speciﬁcity. Compound L1-H5,

also known as 130C037,³⁴served as the basis for the develop-

ment of a small library composed of all of its possible isomers

(Table 2).

Biological Assays

Library 1, PKCr. PKCR binding afﬁnities were ascertained

with partially puriﬁed mouse enzyme. The K_ivalues were

determined by competition with [³H]PDBU as described previ-

ously, except that values were calculated from a single ligand

concentration.³²The groups chosen for Library 1 covered a

useful range of log P values between 2.42 and 7.30 calculated

according to the atom-based program MOE SLogP.³³There

appears to be a parabolic dependence between K_iand log P,

which peaks near a log P value of 4.8, as observed for compound

L1-G2. Column 12, which contains the most hydrophobic

compounds with the highest log P values (between 6 and 7),

did not produce very potent compounds, except for L1-G12.

Remarkably, the entire G row, which contains the triﬂuo-

romethylphenyl R group, is rather unique and for the most part

A remarkable point about this mini-library was the relative

small variance in K_ivalues as a function of the substitution

pattern on either aromatic moiety. Furthermore, K_i(PKCR)/

K_i(RasGRP3) ratios varied only from 4-fold to 33-fold with the

original p,p-isomer (L1-H5) showing the greatest selectivity for

RasGRP3. In addition, as previously reported, the inverted p,p-

isomer, known as 130C045 (not shown),³⁴where the dimethyl-

aminophenyl group and the nitrophenyl group were switched,

5202 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Table 1. Comparison of K_iValues for PKCR between Crude Library Samples and Pure Compounds in Library 1

row^a

compd

library sample K_i(nM)

pure sample K_i(nM)

ratio^cK_icrude/K_ipure

1

2

3

4

5

6

7

8

L1-A12

L1-B5

L1-B8

L1-E2

L1-E5

126 ( 38

466 ( 55

460 ( 46

96 ( 12

2.5 ( 0.1^b

133.8 ( 8.7^b

334 ( 29^b

12.9 ( 0.6

102 ( 14

102.8 ( 5.5

213 ( 20

48.0 ( 7.6

129 ( 19

50.4

3.4

1.4

7.4

10.6

8.7

5.8

7.5

2.6

1080 ( 190

890 ( 170

1230 ( 230

359 ( 25

332 ( 40

118.5 ( 9.5

197 ( 37

41 ( 2.8

a

L1-E6

L1-E8

a

L1-F5

L1-F6

L1-F11

L1-F12

L1-H2

9

10

11

12

13

14

15

16

17

3.7 ( 0.6

8.0 ( 1.6

32.0

24.6

13.2

3.10 ( 0.40

73.5 ( 7.3^b

127 ( 30

41 ( 2.8

0.56

a

L1-H5

362 ( 37

281 ( 35

660 ( 220

2.9

0.34

2.7

5.2

L1-H5

1076 ( 89^b

103 ( 17

a

L1-H6

L1-H8

127 ( 16

^aSamples dissolved in DMSO for testing. ^bZ-isomer. ^cRatio calculated relative to the unresolved library mixture of E- and Z-isomers.

Table 2. K_i(nM) Values for Binding to RasGRP3 (Blue) and PKCR (Red) Competing with [³H]PDBu (K_iValues Correspond to Pure Samples)

showed a similar selectivity to that of L1-H5 with a K_iof 7.8

( 0.94 nM for RasGRP3 and a K_iof 215 ( 14 nM for PKCR.³⁴

Not surprisingly, in the present study, we also found that pure

Library 1 compounds L1-E5 and L1-H8 with identical p-

dimethylaminophenyl or p-nitrophenyl groups at both sn-1 and

sn-2 positions displayed similar afﬁnities for RasGRP3 with K_i

values of 4.68 ( 0.39 and 2.22 ( 0.08 nM, respectively.

Furthermore, the bis-p-nitro compound (L1-H8) now appears

to be the ligand with the highest selectivity ratio [K_i(PKCR)/

K_i(RasGRP3) ) 57], suggesting that these types of compounds,

irrespective of the orientation of the polar moieties, are able to

form productive electrostatic interactions at the lipid interface

when bound to the C1 domain of RasGRP. The different

decoding of the “chemical zip codes” for these compounds was

also demonstrated in living cells where L1-H5 effectively

translocated RasGRP3 while failing to translocate PKCR.³⁴

Because RasGRP1/3 are enzymes that contain a solitary C1

domain, L1-H5 was also assayed with individual C1 domains

for PKC isozymes R and δ. The results showed that only the

δC1b domain bound effectively to L1-H5 in the same nano-

molar range as with intact RasGRP1/3, and that it was

exclusively translocated in living cells.³⁴Figure 2 shows the

close structural similarity between (A) the empty δC1b domain

(obtained from the crystal structure in complex with phorbol

13-acetate),¹(B) the RasGRP1 and (C) RasGRP3 C1 domains

(built from the coordinates of the δC1b by homology modeling)

in contrast to the subtle structural differences in the C1 domains

of (D) δC1a, (E) RC1a, and (F) RC1b, which do not translocate

in response to L1-H5.³⁴This illustrates that, while the binding

sites of all six C1 domains are highly hydrophobic, even small

differences in sequence and surface shape can affect binding

afﬁnity to DAG-lactones and the targeting of the ternary

complex (C1-domain-DAG-membrane), resulting in large

differences in biological effects.

One advantage of performing the syntheses of pure samples

of DAG-lactones with R-arylidene moieties was the isolation

of small amounts of the minor Z-isomers in cases where they

were formed. When that was possible, the distinct chemical

1

shifts for the vinyl protons in the H NMR spectra conﬁrmed

the identity of both isomers. In the case of the Z-isomers of

L1-H5 and L1-H2, the compounds displayed characteristic

multiplets at δ 6.93-7.04, while the corresponding signal for

the E-isomers appeared more downﬁeld at δ 7.58-7.59. The

relative afﬁnities of the L1-H5 geometric isomers for PKCR

and RasGRP3 revealed that the Z-isomer was a poor ligand for

both enzymes. The K_ivalue for PKCR was 1076 ( 89 nM,

which corresponds to a 8-fold drop in binding afﬁnity relative

to the E-isomer (see Table 1), while the afﬁnity for RasGRP3

decreased even more, 21-fold from 3.8 ( 0.1 nM (Table 2) to

79 ( 17 nM. The situation for the Z-isomer of L1-H2 was

similar, displaying a 24-fold drop in binding afﬁnity for PKCR

(K_i) 73.5 ( 7.2 nM, Table 1) relative to the E-isomer (K_i)

3.10 ( 0.40 nM, Table 1). This preference for the E-isomer is

exactly opposite to what we observed with R-alkylidene moieties

where the Z-isomers generally showed greater binding afﬁni-

ties.²⁸

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5203

Figure 2. Binding site surface of the crystal structure of the C1b domain of PKCδ¹(A) compared to homology models of RasGRP1 (B), RasGRP3

(C), δC1a (D), RC1a (E), and RC1b (F). Residues are colored according to hydrophobicity³⁵from highly hydrophobic residues in red to hydrophilic

residues in blue. C1 domains in the top row (A, B, C) are capable of binding and translocating L1-H5, whereas those in the bottom row (D, E, F)

are not.

Library 1, Activation of r-Secretase. It is known that

activation of R-secretase upon PKC stimulation by various

ligands causes increased degradation of the amyloid precursor

protein (APP), resulting in enhanced secretion of sAPPR and

reduced deposition of ꢀ-amyloid peptide (Aꢀ). Formation of

this ꢀ-amyloid peptide is a key factor implicated in the

pathogenesis of Alzheimer’s disease.^36,37The release of sAPPR

and a second fragment (C83), which is further processed to the

N-terminally truncated Aꢀ variant (p3), are not involved in the

pathogenesis of the disease and instead show neuroprotective

effects.

One of our previously synthesized, branched DAG-lactones

(8, Figure 3) was earlier found to efﬁciently increase R-secretase

activity in a dose-dependent manner.³⁸Hence, the compounds

in Library 1 offered the opportunity to optimize the scaffold of

this DAG-lactone with more drug-like substituents with reduced

lipophilicities. R-Secretase activity was measured in W4 cells

(a human APP695 transfected rat neuroblastoma cell line).^39,40

The amount of secreted sAPPR, the hydrolysis product of APP

upon cleavage by R-secretase, was measured by gel electro-

phoresis and immunoblot analysis with monoclonal antibody

6E10 that recognizes the N-terminus of the Aꢀ peptide.⁴¹The

augmented amount of sAPPR reﬂects an increase in R-secretase

activity. The intensity of the sAPPR band in the experimental

groups was analyzed by densitometry and compared to that of

untreated cells, which were normalized to 100%. A preliminary

screening of Library 1 revealed 14 active compounds (L1-A9,

L1-A10, L1-B2, L1-B10, L1-B11, L1-C2, L1-D2, L1-D10, L1-

D11, L1-D12, L1-E2, L1-E6, L1-F2, and L1-F6) that increased

basal enzymatic activity to a level equal to or above that induced

by control DAG-lactone (8). These compounds were then

assayed in triplicate (Figure 3). As compounds L1-A10 and L1-

C2 were chemically more similar to the parent DAG-lactone

8, compounds belonging to rows E and F, particularly L1-E6

and L1-F6, were of special interest despite their weak PKCR

binding afﬁnity (Table 1) because of their polar “chemical zip

codes”. As a crude sample, L1-E6 equaled the activity of control

DAG-lactone 8 (177%) relative to untreated cells (100%). Under

more rigorous conditions, pure samples of L1-E6 and L1-F6

increased enzymatic activity to 154% and 162% above untreated

cells (data not shown). Such potent activities were achieved

despite a >2 orders of magnitude reduction in log P values and

weaker PKCR binding afﬁnities compared to DAG-lactone 8.²⁸

These compounds, as well as the compounds previously

identiﬁed as selective ligands for RasGRP3, demonstrate that

the DAG-lactone scaffold accepts a variety of novel substitution

patterns and that good PKCR binding afﬁnity is not the exclusive

predictor of R-secretase activity.

Library 2, PKCr and Isozyme Selectivity. The groups

chosen for Library 2 also cover a range of log P values between

2.36 and 7.12, similar to Library 1. For this library, the acid

chlorides in the R′ rows were limited to nonpolar alkyl groups

(A, B, C, F, G, H) and aryl-alkyl groups (D and E), all of which

were expected to interact effectively with membranes. For the

aldehyde columns (R), we expanded the important triﬂuorom-

ethylphenyl group discovered in Library 1 to include positional

isomers as well as multiple CF₃substitutions (columns 1-6).

The rest of the groups included mostly common halogens such

as Cl, Br, and F in various substitution patterns. The afﬁnity

for the most potent ligand from Library 1, compound L1-G2,

which in Library 2 corresponded to compound L2-C4, gave an

almost identical K_ivalue (15.5 nM versus 13.1 nM), reﬂecting

the reproducibility of our assay. Because we wanted to limit

the number of isomers to just the racemic DAG-lactone, plus

5204 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Figure 3. Densitometric analysis of Western blots of secreted sAPPR over basal activity (100%) for the 14 active compounds (average of three

experiments). All compounds were assayed at 1 µM and the control was the cell culture medium.

Table 3. Comparison of K_iValues for PKCR between Crude Library Samples and Pure Compounds in Library 2

row no.

compd

library sample K_i(nM)

pure sample K_i(nM)

ratio^bK_icrude/K_ipure

1

2

3

4

5

6

7

8

L2-A1

L2-B4

L2-B7

157.6 ( 7.3

16.6 ( 0.4

17.1 ( 1.4

9.3 ( 1.2

21.5 ( 1.9

8.1 ( 1.2

310 ( 38^a

2.96 ( 0.34

5.53 ( 0.59

2.45 ( 0.65

3.38 ( 0.15

2.66 ( 0.44

2.90 ( 0.15

4.89 ( 0.69

10.1 ( 1.3^a

2.07 ( 0.9

18.3 ( 3.8

10.9 ( 0.4

7.9 ( 3.8

0.51

5.6

3.0

3.8

6.3

3.0

4.3

2.5

1.2

2.9

5.8

1.8

3.6

0.77

L2-B10

L2-B11

L2-B12

L2-C8

L2-C11

L2-C12

L2-D6

12.7 ( 0.3

12.4 ( 0.2

6.1 ( 0.5

9

10

11

12

13

14

108 ( 12

L2-F10

L2-F11

20.5 ( 1.2

28.8 ( 1.8

36.73 ( 0.71^a

^aZ-isomer. ^bRatio calculated relative to the unresolved library mixture of E- and Z-isomers.

the inevitable mixture of E- and Z- isomers, we selected the

similar compound L2-B4 (K_i) 16.6 nM) with a symmetric

acyl moiety for further synthesis. Although intuitively this

selection made sense, we found later that even these two very

similar branched acyl chains could be discriminated by the cell

in some speciﬁc bioassays (see next section). The selected

compound, L2-B4, and all the members of column 4, had good

binding afﬁnities and potencies that varied by less than 5-fold.

The o-isomers (column 1) were less effective, while the

m-isomers (column 2) were very similar to the p-isomers

(column 4). The presence of an extra triﬂuoromethyl group on

the aromatic ring does not seem to improve binding afﬁnity

despite an increase in log P (column 3). In terms of PKCR

binding afﬁnity, the most potent compounds from this library

are clustered in the upper right-hand corner in rows B and C.

Again, for the same reason stated above, the symmetric acyl

chain of row B was preferred for further synthesis.

library was better (see Analysis of Chemical Libraries in the

Experimental Section), and this was reﬂected in the improved

match of the K_ivalues between crude and pure samples (Table

3). For these compounds, the differences in K_icrude/K_ipure

ratios averaged 3.6. As with Library 1, we were also able to

obtain signiﬁcant amounts of Z-isomers in two cases (L2-C11

and L2-F11) and, surprisingly, in the case of L2-A1, only the

Z-isomer was obtained. As before, the ¹H NMR spectra

conﬁrmed the chemical structures, and, in the case of the

E-isomer of L2-F11, a crystal structure further corroborated the

assignment (Figure 4). Again, as opposed to the DAG-lactones

with an R-alkylidene side chain where the Z-isomer outperforms

the E-isomer, R-arylidene DAG-lactones showed that binding

afﬁnity increases in favor of the more abundant E-isomer.

Indeed, for compounds L2-C11 and L2-F11, the differences in

potency favoring the E-isomer were 2.1 and 4.6-fold, respec-

tively (Table 3).

Because the compounds in Library 2 are more homogeneous

in terms of their physicochemical properties, the quality of the

The discovery of the highly potent PKCR ligand, DAG-

lactone L2-C12 (K_i) 2 nM) prompted the study of this

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5205

Figure 4. Crystal structure of L2-F11 represented as one enantiomer (displacement ellipsoid plot drawn at the 50% probability level).

Figure 5. Translocation of PKCR, ꢀ, δ and ε in living CHO cells by L2-C12 (300 nM). Resuts for PKCR and δ are representative of three

independent experiments; those for PKCꢀ and ε are representative of two independent experiments.

compound in more detail, particularly in terms of cellular

translocation of various PKC isozymes. Translocation of PKCs

from cytosol to membrane compartments provides one measure

of their activation. This process can be detected in real time by

overexpressing different green ﬂuorescent protein-tagged (GFP-

tagged) PKC isoforms in Chinese hamster ovary (CHO) cells

and following their localization before and after ligand treatment

using confocal microscopy. All of the isozymes tested translo-

cated very quickly at low doses of L2-C12 (300 nM), and there

was no evidence of selectivity between the conventional

isozymes R and ꢀ and the novel calcium-independent isozymes

δ (Figure 5). The behavior of this ligand is akin to that of the

potent phorbol esters, which are unable to distinguish between

PKC isoforms.^20,21

Library 2, PKCr Activation and Cellular Motility. Some

of the critical issues in developing PKC agonists, which are

evident with the use of the prototypic activator 12-O-tetrade-

canoyl-phorbol-13-O-acetate (TPA), are the appearance of

phenotypic changes associated with malignancy, including an

increase in metastatic behavior. In light of observations that

correlate overexpression and activation of PKCR with metastatic

behavior, we screened Library 2 in a cell motility assay using

MCF-10A cells.⁴²These cells are immortalized, nontransformed,

and nontumorigenic human breast epithelial cells that show

enhanced cell motility after treatment with TPA. Such enhanced

motility is believed to be the consequence of PKCR activation.

To examine the effects of the DAG-lactones in this system, we

screened the entire library using a semihigh throughput method

for quantifying rates of cell movement. The assay measures the

movement of cells that have been plated in a concentrated

circular area on a slide, enabled by a manifold that accom-

modates 10 cell samples. After cell attachment, the manifold is

removed and time-resolved radial movement of the cells can

be captured via photomicroscopy.⁴³From the image data, the

extent of movement can be calculated by the time-dependent

increase in area or radius after a 20 h exposure. The concentra-

tion of each member of the library was adjusted to 10 µM, and

the same DAG-lactone (8) used in the R-secretase assay was

employed as a positive control, while the negative control

was the vehicle DMSO. When the results were analyzed, there

was clear discordance between PKCR binding afﬁnity and cell

motility (Figure 6).

Because of the lack of correlation that showed either very

potent PKCR activators (large log 1/K_i) with little or no effect

on cell motility, as well as weak PKCR activators with

signiﬁcant effects on cell motility, we selected four compounds

at each extreme of the ﬁgure for testing as pure, single agents

(L2-A1, L2-B12, L2-C12, and L2-D6). A dose-response

experiment where the total area rather than the radius measure-

5206 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Figure 6. Plot of radius vs log (1/K_i) for Library 2. K_ivalues were taken from Figure 1. Each motility assay was done in triplicate at 10 µM

DAG-lactone and compared with a positive control (compound 8) and a negative control (1% DMSO v/v). Each measurement was done after 20 h

of exposure to achieve the greatest radius traveled by the cells (in micrometers). The y-axis is the ratio of the radius induced by the sample to that

radius produced by the positive control. For highly motile cells, including those receiving the positive control, the radius was g4 µm and the error

was typically within 20%. Red circles indicate selected compounds for further studies.

ment was used gave much better statistics without qualitatively

changing the results (Figure 7).

as well as enhanced AP-1 dependent transcription. Expectedly,

TPA induces AP-1 activation but at the same time it is a

transforming agent that induces tumor formation.⁴⁶Thus, our

library strategy provided an opportunity to search for DAG-

lactones that could activate AP-1 without the tumor-promoting

activity characteristic of TPA.

The results for the pure compounds were consistent with the

raw data obtained from the crude library and conﬁrmed that

while compounds L2-B12 and L2-C12 are potent PKCR ligands

(Table 3), only L2-C12, which possesses an unsymmetrical acyl

chain, induced cell motility. On the other hand, L2-D6, which

behaves as a weaker PKCR ligand (Table 3) mimics L2-C12

in terms of inducing cell motility. Compound L2-A1 represents

the case where both PKCR binding afﬁnity and induction of

cell motility are low; interestingly, this was the only compound

for which the pure isolated product was obtained exclusively

as the Z-isomer.

These striking results for DAG-lactones L2-B12 and L2-C12

also argue in favor of mechanisms through which very minor

structural changes in the “chemical zip codes” can be decoded

differently in the cellular milieu despite their similar potencies

in binding to PKCR in vitro. This ﬁnding appears to be

consistent with other contrasting cellular responses observed

previously with DAG-lactones bearing slightly different branched

patterns, which probably have an impact on lipid organization

in membranes.⁴⁴Analysis of the dose-response curves on cell

motility (Figure 7) also supports this concept as the correspond-

ing pairs of active and less potent DAG-lactones engendered

similar curves in terms of cell motility. The similar sharp rise

in the curves for L2-C12 and L2-D6 at 10 µM is very clear

and suggests perhaps the existence of off-target effects beyond

this concentration.

The Balb/C JB6 model is a well characterized model of

genetic variants for a neoplastic transformation response to

tumor promoters and is suited for studying tumor promotion

and promotion-relevant molecular events.⁴⁷AP-1 activity

measured in JB6 cells transfected with the AP-1-driven lu-

ciferase reporter gene is strongly induced by TPA, a response

that can be used as a measure of AP-1 dependent transcription.⁴⁸

Concomitantly, a transformation assay that measures anchorage

independent colony induction can give an estimation of the

tumor promoting activity of TPA.⁴⁸After a preliminary screen-

ing of the entire Library 2, several compounds were identiﬁed

as capable of inducing AP-1 activation without transforming

JB6 cells (Figure 8). Responses in each experiment were

normalized to those of TPA. In these assays, JB6 cells

transfected with an AP-1 luciferase reporter⁴⁸were treated with

DMSO, TPA (10 ng/mL), or pure synthesized DAG-lactone

(100 ng/mL) and AP-1 dependent luciferase activity was

determined 18 h later. For the transformation assays in soft agar,

10⁴cells were suspended in top agar containing DMSO, TPA

(10 ng/mL), or DAG-lactone (100 ng/mL) and layered over

bottom agar also containing the compound of interest. Trans-

formed colonies that grow anchorage independently were

counted 14 days later using automated image analysis.⁴⁸

Library 2, AP-1 Activation. The transcription factor AP-1

is a heterodimer composed of a Jun and a Fos family member,⁴⁵

and the association between the Jun and Fos proteins is required

for binding to DNA. AP-1 binds to DNA sequences (TPA

response elements) in the promoter region of many genes, which

are involved in regulating cell proliferation and oncogenesis.

Several signaling pathways and factors, including PKC, may

regulate AP-1-mediated gene expression as the activation of

PKC results in an increase in nuclear AP-1 DNA binding activity

It is interesting that compounds L2-B4 and L2-B7, with

similar K_ivalues for PKCR (Table 3), acted correspondingly in

the AP-1 and anchorage independent transformation assays

showing very strong AP-1 transcription factor activation and

minimal transforming properties in dramatic contrast to TPA.

The other three compounds, L2-C8 and L2-C11 (E- and

Z-isomers), showed that the addition of ﬂuorine ortho to the

chlorine is a critical factor in differentiating AP-1 activation

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5207

Figure 7. Migration results as % of positive control and DAG-lactone concentration. Each DAG-lactone concentration was tested in triplicate, and

the increase in area occupied by the cells was measured in cm². The error of these measurements was within 10%. The results are representative

of two or more independent experiments.

Figure 8. DAG-lactones that retain AP-1 activation but lack potential for inducing transformation. Values were normalized to TPA induced

activity. For soft agar transformation, an average of four individual assays is shown. For AP-1 induced luciferase, an average of six individual

assays is shown. Methods employed for TPA induced transformation and AP-1 luciferase activities have been previously described.⁴⁸

from oncogenic transformation. Compound L2-C8, although less

potent than TPA, showed signiﬁcant transforming activity, while

both geometric isomers of L2-C11 were comparable to L2-B4

and L2-B7. The apparent equal potency of both geometric

isomers of L2-C11 in this assay contrasts with their 2-fold

difference in PKCR binding afﬁnities (Table 3). The conclusion

5208 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Figure 9. Effect of DAG-lactones L2-B4 and L2-B7 on PKCR-mediated apoptosis. LNCaP cells were pretreated for 16 h with 10 µM of DAG-

lactones L2-B4 and L2-B7 followed by 20 Gy irradiation. Cells were ﬁxed 24 h post radiation, stained with bis-benzimide, and scored for apoptosis.

The results are the averages of two separate experiments with standard deviation shown.

from these results is that some DAG-lactones are indeed capable

of inducing AP-1 luciferase activity to similar levels as TPA

but without its transforming activity. Although a detailed

interpretation could be complicated by the rather different

conditions of the two assays, it is clear that rather subtle

chemical changes in the nature of the “chemical zip codes” have

marked effects on outcome.

An initial comparison between DAG-lactones L2-B4 and L2-

B7 in terms of their capacity to induce apoptosis in LNCaP

cells at two different doses (10 and 20 µM) 24 h post radiation

(20 Gy) showed induction of apoptosis by both compounds,

with the more potent PKCR activator of the two (L2-B4, Table

3) being more effective (Figure 9).

To explore the potential of L2-B4 for tumor radiosensitiza-

tion, the orthotopic LNCaP model was employed using Swiss

nude mice. LNCaP tumors reaching a size of ∼100 mm³were

injected ip with L2-B4 (12 mg/kg). Two injections were

delivered at 24 h intervals with prostate resection at 48 h after

the ﬁrst injection. ATM levels in tumor extracts were signiﬁ-

cantly decreased following L2-B4 treatment, dropping to a level

between 7% and 3% of vehicle control. These results compare

favorably with TPA treatment that reduced ATM levels to

12-27% of baseline (data not shown).

We next tested whether the reduction in ATM correlated with

radiation response using a fractionated radiation scheme em-

ploying 1 Gy delivered daily ﬁve times per week (Monday-

Friday) to a total dose of 10 Gy. Mice received daily ip

injections of L2-B4 (12 mg/kg) 16 h prior to each radiation

treatment during the ﬁrst week, whereas during the second week,

only two additional injections were delivered at a 48 h interval.

Tumor response was assessed indirectly by measuring serum

PSA levels, shown to correlate with tumor weight.⁵⁶PSA

doubling time in our orthotopically implanted tumors was

approximately 10 days (data not shown). Figure 10 shows that

fractionated radiotherapy alone resulted in an initial tumor

growth response. However, by week 4, PSA started to rise, and

by week 8, when mice were sacriﬁced, the tumor had increased

to 1 cm³. In contrast, the combination of L2-B4 and fractionated

radiation completely inhibited tumor growth for the duration

of the experiment, and no elevation of serum PSA was detected

during this period. These data showed that L2-B4 signiﬁcantly

enhances the LNCaP tumor response to radiotherapy, indicating

that it might serve as a potent radiosensitizer in vivo.

Library 2, Apoptosis and Radiosensitization. As discussed

in the previous section, TPA-mediated activation of PKC has

been shown to be associated with cellular transformation and

proliferation. TPA-induced PKC activation protects many cell

types from apoptosis induced by TNFa,⁴⁹chemotherapy,⁵⁰

growth factor withdrawal,⁵¹and radiation.⁵²However, in other

cells, it has also been shown that TPA can induce apoptosis

via PKC activation. Because the subcellular distribution or

translocation of PKC isozymes to various membranes of cellular

organelles is a critical determinant, we evaluated as apoptosis

inducers the two nontransforming DAG-lactones (L2-B4 and

L2-B7) that were obtained as single geometrical isomers and

which also behaved as potent AP-1 activators. Garcia-Bermejo

et al. reported that both PKCR and PKCδ mediate TPA-induced

apoptosis in LNCaP cells and showed that DAG-lactone 8

(Figure 4) was selective in inducing only PKCR-mediated

apoptosis in these cells.⁵³More recently, Haimovitz-Friedman

et al. have shown that treatment with TPA and the same DAG-

lactone (8) leads to increased ceramide generation via ceramide

synthetase (CS).⁵⁴The proposed pathway is that PKCR activa-

tion leads to down-regulation of ATM (ataxia telengiectasia

mutated gene), a protein that functions to constrain CS activa-

tion.⁵⁵A key ﬁnding of that work was that PKCδ, an isoform

known to be involved in TPA-induced apoptosis in LNCaP cells,

had no effect on ATM levels.⁵⁴Because reduced ATM levels

enhance radiation-induced CS activation, it was not surprising

that PKCR activation alone could sensitize these cells to ionizing

radiation. Indeed, while prostate-tumor (LNCaP) bearing nude

mice treated with DAG-lactone 8 signiﬁcantly decreased ATM

levels and delayed tumor growth, the combination of radiation

and DAG-lactone treatment greatly potentiated tumor growth

delay.⁵⁴

Library 2, Immunostimulatory Activity. PKC activators

can not only induce apoptosis but also regulate the host immune

response.

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5209

Figure 10. Effect of DAG-lactone L2-B4 (12 mg/kg) in the orthotopic prostate cancer model. Control nonirradiated mice (n ) 14, red); L2-B4

treated mice (n ) 13, blue); radiation only (XRT) treated mice (n ) 14, black); combination L2-B4 plus radiation treated mice (n ) 12, green).

Values represent the mean.

One measure of the effects of these compounds on their ability

to modulate the host immune response is their effect on the

production of a key immunoregulatory cytokine, interferon-

gamma (IFN-γ), produced by T cells and NK cells.^57,58Thus,

a human cell line, NK92, was utilized to analyze if the different

DAG-lactone derivatives were able to alter IFN-γ gene expres-

sion. As it has been previously shown that IL-12 synergizes

strongly with TPA to induce IFN-γ,⁵⁹we tested the induction

of IFN-γ by the DAG-lactone library members both alone and

in combination with IL-12. As shown in Figure 11, some

selected compounds induced IFN-γ independently and also

strongly synergized with IL-12 to induce IFN-γ. Thus, these

compounds may also have an important immunostimulatory

activity in addition to their pro-apoptotic activity.

C1 domains and ﬁnding which structural elements inﬂuence the

translocation of the ligand-protein complexes to speciﬁc cellular

compartments represent important goals of our work. The

ultimate objective is to design DAG-lactones that could

selectively direct the ﬂow of information from the DAG

signaling pathways through their multiple signal transducers,

the PKC isoforms and other signaling proteins that along with

the PKCs possess DAG-responsive C1 domains, in order to

achieve a speciﬁc cellular response with a potential therapeutic

value.

The disadvantages of the weak afﬁnity of endogenous DAGs

and the complicated structures and restricted supply of exog-

enous, natural product ligands for the C1 domains, the diterpenes

(phorbol, ingenol, and daphnane esters), the macrocyclic lactones

(bryostatins), the indole alkaloids (teleocidin, lynbyatoxin), and

the polyacetates (aplysiatoxin), prompted us to devise a strategy

to enhance the afﬁnity of the endogenous ligand through

appropriate constraint of the DAG by means of an optimized

DAG-lactone. Because of the rudimentary nature of current

structure-activity analyses that incorporate the lipid bilayer into

the model, we decided to explore this binding site combinato-

rially by generating DAG-lactone libraries where arrangements

of R and R′ groups produced a series of “chemical zip codes”

capable of creating a different PKC-lipid microenvironment that

would direct the activated complex to different subcellular sites.

Discussion

The preliminary data obtained with only two 96-member

DAG-lactone libraries, which are characterized by a modest

array of “chemical zip codes”, demonstrate that the compounds

obtained are indeed a source of exciting biological activities.

A primary focus of our work has been to understand the basic

mechanisms by which 1,2-diacylglycerol (DAG) and the newly

developed DAG-lactones activate speciﬁc downstream subsets

of cellular responses that occur through the activation of PKC

isozymes, RasGrRP, and the other families of C1 domain target

proteins. The multiplicity of cellular responses, often antago-

nistic, which result from PKC activation, or DAG binding to

other C1 domain target proteins, can be explained by two still

inadequately understood events: (1) the diversity of substrates

for PKC and C1 domain proteins, which have different roles in

downstream events, and (2) the localization of these ligand-

activated complexes into speciﬁc cellular compartments, which

aside from the plasma membrane include the endoplasmic

reticulum, the Golgi, and the nucleus, thus allowing access to

unique substrates. Therefore, gaining knowledge about how the

regulation of PKC isozymes and the nonkinase protein targets

can be controlled by the speciﬁc binding of DAG-lactones to

Our preliminary results provide a strong proof-of-principle

for this concept that could form the basis for therapeutic

strategies targeting speciﬁc pathways. One key difference

between a conventional combinatorial library approach and our

targeted DAG-lactone libraries is that every member of the

library was expected to bind, to a certain degree, to C1 domain

containing proteins. This was conﬁrmed by the K_ivalues

obtained for the PKCR isozyme with Libraries 1 and 2. On the

other hand, in the more speciﬁc bioassays, the cellular activities

that were measured reﬂected the manner in which the activated

complex “C1-domain-DAG-lactone-membrane” was able to

5210 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Figure 11. NK92 IFN-γ production in response to TPA and selected DAG-lactone derivatives plus IL12. The human NK cell line, NK92, (10⁶

cells/mL) was incubated at 37 °C for 6 h, in the presence or absence of the derivatives (1 µg/mL) in 10% RPMI plus 10% FCS, glutamine and

Pen/Strep. Duplicate wells were set up with derivatives + IL12 (100u/10⁶cells). Cell supernatants were analyzed for IFN-γ by Elisa (R & D

Systems, Minneapolis, MN). Data are representative of multiple experiments. Standard deviation within the Elisa ranged from 0.001-0.49.

induce a unique biological response according to the nature of

the “chemical zip codes”.

L2-C12 failed to show any speciﬁcity in the cellular translo-

cation of PKC isozymes R, ꢀ, δ, and ε (Figure 5), and while

the compound was able to stimulate cell migration effectively

as expected for a PKCR activator, this activity was matched by

a much weaker PKCR ligand, L2-D6 (Figures 6 and 7). Another

dramatic result was the speciﬁc and potent activity associated

with DAG-lactones bearing polar substituents simultaneously

at sn-1 and sn-2 positions for non-PKC targets like RasGRP

and the effect of some of these compounds as inducers of

R-secretase activity. This clearly implies that membrane as-

sociations with these ligand-C1 domain complexes can occur

by different mechanisms, most likely involving Coulombic

interactions with phospholipid headgroups.

The triad of compounds L2-B4, L2-B7, L2-C8 represents

another interesting example of the lack of a correlation between

PKCR binding afﬁnity and cellular activity. These compounds

have K_ivalues of 2.96, 5.53, and 2.90 nM, respectively, as

resynthesized pure samples (Table 3). All were able to induce

AP-1 activation, but only L2-C8 with a K_ivalue identical to

L2-B4 was able to induce cellular transformation. The L2-C11

isomers (both E and Z), which had up to a 3-fold difference in

binding afﬁnity for PKCR relative to L2-B4, matched the

activity of L2-B4 in AP-1 activation, as well as in their lack of

induction of cellular transformation (Figure 8). The small

variation in chemical structure from L2-C8 to L2-C11 involved

the simple addition of a ﬂuorine atom. Surprisingly, this change

was sufﬁcient to abrogate the effect of L2-C8 on cellular

transformation. Another important minor structural change

associated with a dramatic difference in biological behavior is

represented by compounds L2-B12 and L2-C12, which differ

only in the level of branching of the R acyl chain. The DAG-

lactone L2-B12 with a symmetrical branched chain had a very

weak effect on cell motility, while L2-C12 with the asymmetric

branched chain was very effective despite both compounds

The design of Library 2 evolved from the initial results

obtained with Library 1. The more polar DAG-lactones in

Library 1 showed good activity and selectivity for RasGRP and

R-secretase, despite having low PKCR binding afﬁnity. Then,

from Library 1, we selected the most potent PKCR binding

ligand (L1-G2) and explored its chemical space with less polar

groups to generate Library 2. Therefore, the selection of

bioassays for each of the libraries was different and depended

on the intrinsic afﬁnities of the compounds for PKCR. The K_i

values that were obtained for PKCR in both libraries simply

established a benchmark against a known C1 domain containing

protein in our standard assay. Aside from the general observation

that nonpolar groups correlate better with activity when present

as R′ acyl groups (sn-1) and polar groups are better when present

as R groups at sn-2, as exempliﬁed by compounds L1-B8 and

L1-H2, we found it difﬁcult to establish a clear structure-activity

relationship for this enzyme. Some differences were also noted

for the same chemical functionality depending on its location

on the molecule. For example, when the triﬂuoromethylphenyl

group was present as an sn-2, R-arylidene R group (Library 2,

column 4), it correlated consistently with more potent ligands

than when the same functional group appeared as an sn-1, R′

group (Library 1, column 7). An interesting observation that

was gathered from all of the various biological and cellular

assays was the lack of clear correlation between potency as a

PKCR ligand and activity in other biological assays, bearing in

mind that all the compounds were designed to have activity on

PKCR. This is entirely consistent with the expectation that other

isozymes, as well as other C1 domain containing targets, are

likely involved in directing speciﬁc biological responses. This

fact was exempliﬁed for compound L2-C12, which was

identiﬁed as the most potent ligand for PKCR in both libraries.

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5211

prevent the binding site loops from closing during the minimization.

Harmonic positional restraints on the backbone atoms were gradu-

ally relaxed over the course of the minimization to eliminate steric

clashes in the sidechains without inducing deformations in the

backbone.

Activation of r-Secretase Activity. The protocol for the

R-secretase activation assay was followed as previously described.³⁸

Translocation of GFP-Tagged PKC Isoforms in CHO

Cells. Translocation of GFP-tagged PKCR, ꢀ, δ, and ε was followed

in Chinese hamster ovary (CHO) cells as described previously.⁶¹

The GFP-tagged PKC isoforms were constructed by subcloning

the PKC isoforms into a modiﬁed pEGFP-N1 plasmid (Clontech,

Palo Alto, CA) as described by Wang et al.⁶²

Cellular Motility Assay. Cell culture serum, growth factors, and

media were purchased from Invitrogen, Inc. (Carlsbad, CA).

Midpassage MCF-10A human breast epithelial cells were obtained

from the Barbara Ann Karmanos Cancer Institute (Detroit, MI).

MCF-10A cells were cultured as previously described.⁴²

Motility of MCF-10A cells was analyzed by monitoring the

extent of cell movement using a computer-assisted digital camera

(Moticam 2000) attached to an inverted Nikon Diaphot microscope.

The cells were applied to a 10-well slide through a 10-hole manifold

(CSM, Inc., Phoenix, AZ) that restricts sedimentation of cells to a

small, circumscribed area. Upon removal of the manifold (t ) 0),

the DAG-lactone was added to the cells to a ﬁnal concentration of

10 µM. Cells receiving no reagent were treated with an equivalent

volume of DMSO to a ﬁnal concentration of 1% (v/v). Following

addition of the DAG-lactone, cells moved radially over a 20 h period

while incubated at 37 °C, 5% CO₂. The extent of movement was

determined by measuring either a change in radius (in micrometers)

or a change in total area (in cm²) occupied by the cells using Motic

Images Plus 2.0 software. Each reported value is the average of

triplicate measurements for which the error was typically within

10%.

having nearly identical K_ivalues of ca. 2 nM as PKCR ligands

(Table 3). This is possibly due to changes in membrane

organization induced by both compounds, which affect in a

different fashion membrane curvature and other parameters

associated with cell motility. This ﬁnding is in agreement with

studies on DAGs of different acyl chain lengths and degrees of

unsaturation that are capable of differentially perturbing mem-

branes.⁵

Finally, the radiosensitization properties of PKC ligands

associated with transforming activity, as in the case of the

phorbol ester TPA, were dissociated in the nontransforming

DAG-lactones L2-B4 and L2-B7. These two compounds were

strong apoptosis inducers whose activities were augmented by

ionizing radiation as demonstrated in vivo in an orthotopic

prostate cancer model that measured PSA levels associated with

tumor growth. The more potent compound, L2-B4, is an

interesting drug candidate and represents a prototypic DAG-

lactone that has speciﬁc targeting properties without the side

effects that are typically associated with the phorbol esters. This

compound and others described in this article provide a strong

proof-of-principle for the concept of “chemical zip codes” and

strongly support the continued search for additional sets of

DAG-lactones with more speciﬁc targeting properties.

Experimental Section

General Techniques. All reagents and solvents purchased were

of the highest commercial quality and used without further

puriﬁcation unless otherwise stated. MicroKan (IRORI) reactors

were purchased from Discovery Partners International, San Diego,

CA (Now Nexus Biosystems, Poway, CA). DHP (3,4-Dihydro-2H-

pyran) resin (1.7 mmol/g, 150-300 µM) was purchased from

Polymer Laboratories, Ltd., Amherst, MA. All solid-phase reactions

were performed in MicroKan reactors ﬁlled with 14.7 mg (0.025

mmol) of DHP resin per Kan. Final crude products were obtained

after cleavage from the resin and evaporation of the solvent.

Selected, individual samples of pure materials were synthesized

through conventional solution-phase methods as described pre-

viously^30,44and were fully characterized by FT-IR, ¹H NMR, FAB-

MS, and elemental analysis or HRMS.

AP-1 Luciferase Assay. On day 1, 10⁴JB6 P+ cells were

seeded. Cells were transfected with a 3 to 1 ratio of Fugene (Roche

Applied Science) to 4× AP-1 luciferase reporter DNA (0.2 µg).

On day 3, DMSO, TPA (10 ng/mL) or DAG-lactone (100 ng/mL)

were added to cells and 18 h later cells were harvested and luciferase

activity determined.⁴⁸Six independent luciferase assays are reported.

Anchorage Independent Transformation. First, 10⁴cells were

suspended in 0.33% top agar with DMSO, TPA 10 ng/mL, or DAG-

lactone at 100 ng/mL and were layered over 0.5% bottom agar

also containing the compound of interest. After 14 days, anchorage

independent colonies were counted. Colonies with more than eight

cells were counted as transformed.⁴⁸Four independent plates were

seeded and six independent assays were scored. The average number

of colonies is reported with TPA used as the 100% value.

Apoptosis and Radiosensitization. Cell culture products were

obtained through Mediatech (800-Cellgro, VA), except for fetal

calf serum, which was purchased from Gemini Bioproducts (CA).

The DNA-binding ﬂuorochrome bis-benzimide trihydrochloride was

obtained from Sigma-Aldrich (St. Louis, MO).

The human prostate cancer cell line LNCaP was obtained from

ATCC. Cells were cultured at 37 °C in a humidiﬁed 5% CO₂

atmosphere in RPMI-1640 supplemented with 10% fetal calf serum,

2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL strepto-

mycin, and 10 mM HEPES (pH 7.2). For experiments, cells were

cultured in the same medium, except 0.2% human albumin (HA)

was substituted for FCS 24 h before the beginning of the

experiment. LNCaP cells were treated with L2-B4 and L2-B7 (10

or 20 µM) for 16 h prior to irradiation.

Analysis of Inhibition of [³H]PDBU Binding by Nonradioac-

tive Ligands. Enzyme-ligand interactions were analyzed by

competition with [³H]PDBU binding for the single isozyme PKCR

essentially as described previously.³²The ID₅₀values were

determined by ﬁtting a theoretical sigmoidal competition curve to

the binding data. The K_iwas calculated from the ID₅₀values

according to the relationship:

K_i) ID₅₀⁄ (1 + L ⁄ K_d)

(1)

where L is the concentration of free [³H]PDBU at the ID₅₀and K_d

is the dissociation constant for [³H]PDBU under the assay condi-

tions. Values represent the mean ( SEM. All values represent a

minimum of three independent experiments. For the puriﬁed

compounds, complete dose-response curves with 6-7 concentra-

tions of ligand were performed. For the unpuriﬁed library com-

pounds, a single ligand concentration (1 µM) was used. In either

case, triplicate determinations were made at each ligand concentra-

tion in each experiment.

Molecular Modeling. Homology models of the C1 domains of

RasGRP1 and RasGRP3, the C1a and C1b domains of PKCR, and

the C1a domain of PKCδ, shown in Figure 2, were built on the

backbone coordinates of the crystal structure of the C1b domain

of PKCδ.¹Side chains for the models were constructed using the

program SCWRL,⁶⁰which uses a backbone-dependent rotamer

library to place residues in their most likely conformation given

the backbone φ-ψ angles at that position. Homologous residues

were left unchanged from their crystallographic positions. The

models were reﬁned with a small energy minimization, with

phorbol-13-O-acetate left in position from the crystal structure to

Morphologic changes in nuclear chromatin of cells undergoing

apoptosis were detected by staining with the DNA-binding ﬂuo-

rochrome bis-benzimide trihydrochloride, as described.⁶³Cells were

visually inspected using an Olympus BH2 ﬂuorescence microscope

equipped with a Dich mirror cube ﬁlter (BH2-DMU2UV). Cells

displaying at least three apoptotic bodies were scored as apoptotic.

Orthotopic Prostate Cancer Model. Eight to ten week old male

Swiss nude (nu/nu) mice were implanted orthotopically with 3.0

× 10⁶LNCaP cells. Immediately before tumor implantation,

5212 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Table 4. Mass Spectrometrically Determined Combinatorial Purity

cultured LNCaP cells were trypsinized and resuspended in RPMI

1640 with 10% FBS and viability determined by trypan blue

exclusion. Only single cell suspensions with >90% viability were

used for in vivo injection. Mice were anesthetized with 12.5 mg

Ketamine + 1.25 mg Xylazine (ip, intraperitoneal), and orthotopic

tumor implantation was performed as described.⁶⁴Brieﬂy, a low

midline abdominal incision was made with a no. 15 blade (Bard

Parker). The peritoneal cavity was entered by sharply incising the

linea alba. The bladder and seminal vesicles were identiﬁed and

gently raised, thus exposing the dorsal lobes of the mouse prostate.

LNCaP cells were injected in 0.1 mL of medium using a 26 gauge

needle. Proper implantation of cell suspension was indicated by

blebbing under the prostatic capsule. Visceral contents were then

replaced into the abdominal cavity and the wound closed with

surgical autoclips (Becton Dickinson). Mice were monitored during

the postoperative period according to animal care facility guidelines.

Injected mice were housed (5 mice/cage) in a pathogen-free

environment, using ﬁltered, laminar airﬂow hoods in standard vinyl

cages with air ﬁlter tops. Cages, bedding, and water were autoclaved

before use.

Orthotopically transplanted LNCaP tumors in nude mice secrete

PSA that can be detected histochemically in tumor cells and by

radioimmunassay in mouse serum.⁵⁶To assess LNCaP tumor take

and tumor volume after intraprostatic transplantation, mice were

anesthetized with 12.5 mg Ketamine + 1.25 mg Xylazine (ip).

Phlebotomy was performed by accessing the retro-orbital venous

plexus with a microcapillary pipet (Fisher). Serum PSA determina-

tions were performed by radioimmunoassay (Hybritech) according

to the recommendations of the manufacturer. Using serum PSA

determinations as an indicator of tumor size, mice were only utilized

in experiments when PSA levels ranged from 5.0-15.0 ng/mL

representing tumors with a mass in the range of 70-150 mg. Mice

were divided into four treatment groups: (a) control, DMSO ip;

(b) DAG-Lactone L2-B4 at 12 mg/kg/day in DMSO (MTD dose)

ip; (c) DMSO 16 h before 10 × 1 Gy; (d) DAG-Lactone L2-B4 at

12 mg/kg/day 16 h before 10 × 1 Gy. Mice were locally irradiated

by exposure to a small pelvic RT ﬁeld using a specially designed

Lucite jig. All procedures and postoperative care with animals were

in accordance with NIH guidelines and received prior approval by

the Institutional Animal Care and Use Committee at Memorial

Sloan-Kettering Cancer Center.

(Library 2)^a

library

component

library quality

rating

library purity

(%)

measurements

(n)

L2-A1

L2-B4

L2-B7

+++

++

+++

++

+++

+

++

25 ( 1.2

47 ( 1.2

40 ( 1.8

43 ( 1.3

50 ( 2.0

19 ( 3.4

62 ( 2.1

37 ( 2.3

56 ( 1.3

6

3

4

3

1

L2-B10

L2-B11

L2-B12

L2-C8

L2-C11

L2-D6

L2-F10

L2-F11

35

^aComparison of the semi-quantitative compound quality rating derived

from an individual mass spectrum of the crude solid-phase combinatorial

product and the quantitatively determined synthetic purity. Library quality

rating: +++ ) MH⁺g 1 V (spectrum similar to that of a pure or standard

compound); ++ ) MH⁺g 250 mV (spectrum contains diagnostic fragment

ions and may indicate presence of minor amounts of synthetic by-products);

+ ) MH⁺g 25 mV (overall spectrum is weak and/or contains evidence

of substantial by-product formation); 0 - (no mass spectral evidence for

the desired compound). Library components are mixtures of E/Z isomers.

product.⁶⁷As shown for Library 2 (Table 4), the average purity

of the DAG-lactones was 38%.

Library 1. For Library 1, column 6 and row H were

individually analyzed (Figure 1S in Supporting Information) to

see whether the desired DAG-lactone products were present.

The compounds in column 6 all have the same p-methoxyphenyl

(PMP) acyl moiety but different alkylidene groups, so their mass

spectra exhibited the same characteristic acylium ion

(MeOC₆H₄-CtO⁺, m/z 135) but different masses for MH⁺.

The compounds of row H all have the same p-nitrophenyl

alkylidene moiety, but each has a different acyl group, so the

masses observed for both the acylium ion and MH⁺varied in

concert. The initial analytical evaluation of Library 1 gave clear

evidence for the presence of 94 out of the 96 desired DAG-

lactone analogues. For the mixture analysis, mixtures were made

using compounds of the same row so that the acylium ions

representing the different acyl groups (R′) would not overlap.

For example, the three, 4-component mixtures representing row

A of Library 1 were composed of L1-A1, L1-A4, L1-A7, and

L1-A10, L1-A2, L1-A5, L1-A8, and L1-A11, and L1-A3, L1-

A6, L1-A9, and L1-A12. Those compounds that were not

observed during mixture analysis were subsequently analyzed

individually (e.g., L1-B11 and L1-B12). One compound, L1-

H10, was questionable because its MH⁺peak was present but

below the absolute intensity threshold required for a minimal

rating. There was no mass spectral evidence for the presence

of DAG-lactone L1-H8.

Analysis of Immunostimulatory Activity. The human NK cell

line NK92 (10⁶cells/mL) was incubated in 10% RPMI plus 10%

FCS, glutamine and Pen/Strep at 37 °C for 6 h in the presence or

absence of derivatives (1 µg/mL) alone or in the presence of

derivatives plus IL12 (100 u/million cells). Cell supernatants were

analyzed for IFN-γ expression by Elisa (R & D Systems, Min-

neapolis, MN).

Analysis of Chemical Libraries

For a typical DAG-lactone derivative, the FAB mass spectrum

consisted of either MH⁺or M^•+to designate the molecular

weight plus several structure-indicating fragment ions derived

from both the acyl and the alkylidene portions of the molecule.⁶⁵

Use of a 3-nitrobenzyl alcohol (NBA) matrix was found to offer

signiﬁcant advantages in terms of speed, simplicity, and

information content for the analysis of these lipophilic deriva-

tives. Furthermore, because these DAG-lactones are all structur-

ally alike, their surface activity in the FAB matrix, and hence

FAB ionization efﬁciency, were expected to be similar.⁶⁶

The issue of library quality and individual library component

purity was investigated further with pure, individually synthe-

sized DAG-lactone standards. Thus we constructed standard

curves over the range of linear mass spectral response using

the absolute intensity of diagnostic ions (e.g., R′-CtO⁺, MH⁺)

from the analysis of measured amounts of individually synthe-

sized standards and used these to measure the amount of DAG-

lactone in similar amounts of crude combinatorial library

Library 2. A similar analysis strategy was followed for

Library 2. The components of column 4 and row H were chosen

for individual mass spectral analysis to assess the suitability of

this library for the combine-and-analyze strategy (Figure 2S in

Supporting Information). Thus, the anticipated acylium ion (R′-

CtO⁺) varied in concert with MH⁺for each member of column

4, all of which have the same p-triﬂuoromethyl-phenyl alky-

lidene (R) moiety; while the expected acylium ion

(C₈H₁₇-CtO⁺, m/z 141) remained constant for all the com-

pounds of row H, which possess the same acyl group. For the

mixture analysis, mixtures were made using compounds of the

same column so that the acylium ions representing the different

acyl groups (R′) would not overlap. For example, for the analysis

of column 4, two mixtures were generated consisting of

components L2-A4, L2-C4, L2-E4, and L2-G4 (Mix 4A) and

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5213

compounds L2-B4, L2-D4, L2-F4, and L2-H4 (Mix 4B),

respectively.

Library 1: “PMP Approach”. Procedure for Loading 5-(hy-

droxymethyl)-5-[(4-methoxyphenoxy)methyl]-3,4,5-trihydrofu-

ran-2-one (1b) onto the DHP Resin in 96 MicroKan Reactors.

First, 96 MicroKan reactors, each containing DHP resin (0.0147

g, 0.025 mmol) and a radio frequency tag capsule, were placed

in a 500 mL round-bottom ﬂask equipped with a stir bar. DAG-

lactone 1b (6.1 g, 24 mmol, 10 equiv), pyridinium p-toluene-

sulfonate (6.0 g, 24 mmol, 10 equiv), and 1,2-dichloroethane

(240 mL) were introduced into the ﬂask. The reactors were

degassed at -30 °C in vacuo for 2 min. The temperature was

then raised to 60 °C, and the solution was vigorously stirred

for 24 h. After cooling to room temperature, the solution was

decanted into another ﬂask to recover the excess of unreacted

DAG-lactone (1b). The reactors containing polymer-bound

lactone 2b were washed sequentially with dichloromethane (1),

1:1 DMF/water (3×), DMF (3×), methanol (2×), dichlo-

romethane (3×), and ﬁnally dried in vacuo.

X-ray Crystal Structure of L2-F11. Single-crystal X-ray

diffraction data on compound L2-F11 were collected at 103 K

using Mo KR radiation and a Bruker APEX 2 CCD area

detector. A 0.35 × 0.31 × 0.04 mm³crystal was prepared for

data collection by coating with high viscosity microscope oil

(Paratone-N, Hampton Research). The oil-coated crystal was

mounted on a MicroMesh mount (MiTeGen, Ithaca, NY) and

transferred immediately to the cold stream (-170 °C) on the

diffractometer. The crystal was monoclinic in space group P2₁/c

with unit cell dimensions a ) 13.986(7) Å, b ) 15.392(8) Å,

c ) 8.137(4) Å, and ꢀ ) 103.17(1)°. Corrections were applied

for Lorentz, polarization, and absorption effects using the

program SADABS (Bruker, SADABS v2.10, 2000a, Bruker

AXS Inc., Madison, WI). Data were 98.6% complete to 28.29°

θ (approximately 0.75 Å) with an average redundancy of 3.03.

The structure was solved by direct methods and reﬁned by full-

matrix least-squares on F²values using the programs found in

the SHELXTL suite (Bruker, SHELXTL v6.14, 2000b, Bruker

AXS Inc., Madison, WI). Parameters reﬁned included atomic

coordinates and anisotropic thermal parameters for all non-

hydrogen atoms. Hydrogen atoms on carbons were included

using a riding model (coordinate shifts of C applied to H atoms)

with C-H distance set at 0.96 Å. The asymmetric unit contains

a single molecule. Atomic coordinates for compound L2-F11

are included in the Supporting Information available from this

journal and have also been deposited with the Cambridge

Crystallographic Data Centre (CCDC 680078). Copies of the

data can be obtained, free of charge, on application to CCDC,

12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44(0)-

1223-336033; E-mail: deposit@ccdc.cam.ac.uk).

Procedure for the Aldol Reaction of Polymer-Bound Lac-

tone 2b with Aldehydes. The 96 MicroKan reactors were

sorted into 8 vessels to react with eight different aldehydes.

Each vessel contained 12 reactors. The eight parallel reactions

were performed following an identical, general procedure:

MicroKan reactors containing polymer-bound lactone 2b (12

reactors, 0.3 mmol, 1 equiv) were suspended in THF (30 mL)

in a Schlenk vessel equipped with a stir bar. After degassing

at -78 °C in vacuo for 2 min, LDA (1.5 mL, 2.0 M in

heptane/THF/ethylbenzene, 3.0 mmol, 10 equiv) was added

while the temperature was maintained at -78 °C. After

stirring 6 h at -78 °C, the solution was treated with ZnCl₂

(7.2 mL, 0.5 M in THF, 3.6 mmol, 12 equiv) and the reaction

was allowed to warm to -45 °C for 30 min. A solution of

the corresponding aldehyde (3.6 mmol, 12.0 equiv) in THF

(2 mL) was then added to each of the vessels and stirring

continued at -45 °C for 3 h. The reactions were quenched

with a saturated solution of ammonium chloride and allowed

to reach room temperature. All MicroKan reactors containing

polymer-bound aldol product 3b were pooled together and

washed sequentially with 1:1 DMF/water (3×), DMF (3×),

methanol (2×), and dichloromethane (3×) and dried in vacuo.

Preparation of Starting Materials

5-(Hydroxymethyl)-5-[(4-methoxyphenoxy)methyl]-3,4,5-tri-

hydrofuran-2-one (1b). This compound was prepared by the

treatmentof5-[(4-methoxyphenoxy)methyl]-5-[(phenylmethoxy)m-

ethyl]-3,4,5-trihydrofuran-2-one (1a)⁶⁸with boron trichloride

as described.³⁰

5-(Hydroxymethyl)-5-[(tert-butyldiphenylsilyloxy)methyl]-

3,4,5-trihydrofuran-2-one (1d). A stirred solution of 1b (4.0 g,

15.9 mmol, 1.0 equiv) in dichloromethane (160 mL) was treated

with tert-butyldipenylsilyl (TBDPS) chloride (8.2 mL, 31.7

mmol, 2.0 equiv) in the presence of imidazole (2.2 g, 31.7 mmol,

2.0 equiv) at room temperature for 1.5 h. The reaction was

quenched with water, and the separated organic phase was

washed once with water, dried (Na₂SO₄), and concentrated to

dryness. The crude product (15.9 mmol, 1.0 equiv) was

dissolved in acetonitrile:water (4:1, 125 mL) and treated with

ammonium cerium(IV) nitrate (CAN, 26.2 g, 47.7 mmol, 3.0

equiv) at 0 °C. After stirring for 30 min at this temperature, the

reaction was quenched with a 10% aqueous sodium thiosulfate

solution. After most of the acetonitrile was removed under

vacuum, the ﬁnal solution was extracted with ethyl acetate (3×),

dried (Na₂SO₄), and concentrated to dryness. The residue was

puriﬁed by silica gel ﬂash column chromatography with ethyl

acetate:hexane (1:2) as eluant to afford a light yellow oil (5.24

g, 86% after two steps) that solidiﬁed upon standing; mp 82-83

°C (hexane). IR (neat) 3451 (OH), 1774 (CdO) cm^-1. ¹H NMR

(400 MHz, CDCl₃) δ 7.61-7.64 (m, 4 H, ArH) 7.35-7.46 (m,

6 H, ArH), 3.72 (d, J ) 12.3 Hz, 2H, HOCH₂), 3.63 (ABq, J

) 11.6 Hz, 2 H, SiOCH₂), 2.52-2.72 (m, 2 H, H_3a,b), 2.05-2.22

(m, 3 H, H_4a,band OH), 1.04 (s, 9 H, C(CH₃)₃). FAB-MS (m/z,

relative intensity) 385 (MH⁺, 14), 135 (100). Anal. (C₁₃H₁₆O₅)

C, H.

Procedure for the Triﬂation of Polymer-Bound Aldol Prod-

ucts 3b Followed by DBU-Assisted Elimination. MicroKan

reactors containing polymer-bound aldol product 3b (96 Mi-

croKan reactors, 2.4 mmol, 1 equiv) were placed in a round-

bottom ﬂask equipped with a stir bar and suspended in

dichloromethane (240 mL). After degassing at -78 °C in vacuo

for 2 min, pyridine (3.9 mL, 48 mmol, 20 equiv), 4-(dimethy-

lamino)pyridine (4-DMAP, 0.29 g, 2.4 mmol, 1 equiv), and

triﬂuoromethylsulfonic anhydride (4.0 mL, 24 mmol, 10 equiv)

were subsequently introduced into the vessel at the same

temperature. The resulting mixture was stirred at -20 °C for

20 h, after which time the reaction was quenched with a

saturated solution of ammonium chloride and allowed to reach

room temperature. The MicroKan reactors were washed se-

quentially with 1:1 DMF/water (3×), DMF (3×), methanol

(2×), and dichloromethane (3×) and dried in vacuo. All reactors

were then suspended in toluene (240 mL), and after degassing

at -78 °C in vacuo for 2 min, DBU (7.2 mL, 48 mmol, 20

equiv) was added at 0 °C. The MicroKan reactors were stirred

at the same temperature for 6 h, after which time the reaction

was quenched with a saturated solution of ammonium chloride.

The MicroKan reactors containing polymer-bound R-alkylidene-

lactone 4b were washed with 1:1 DMF/water (3×), DMF (3×),

methanol (2×), and dichloromethane (3×) and dried in vacuo.

5214 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

Procedure for Removal of the p-Methoxyphenyl (PMP)

Group from Polymer-Bound r-Alkyl/Arylidene-Lactones 4b.

MicroKan reactors containing polymer-bound R-alkyl/arylidene-

lactone 4b (96 MicroKan reactors, 2.4 mmol, 1.0 equiv) were

suspended in THF (192 mL) in a round-bottom ﬂask equipped

with a stir bar. After degassing at -78 °C in vacuo for 2 min,

the solution was warmed to 0 °C. Water (48 mL) and

ammonium cerium(IV) nitrate (CAN, 26.3 g, 48 mmol, 20

equiv) were subsequently introduced into the vessel and stirring

continued at the same temperature for 6 h, after which time the

reaction was quenched with a 5% aqueous solution of sodium

thiosulfate. Finally, the MicroKan reactors containing polymer-

bound diol-R-alkyl/arylidene-lactone 5 were washed with a 5%

aqueous solution of sodium thiosulfate (2×), 1:1 DMF/water

(2×), DMF (3×), methanol (2×), and dichloromethane (3×)

and dried in vacuo.

Procedure for Removal of the t-Butyldiphenylsilyl (TB-

DPS) Group from Polymer-Bound r-Alkyl/Arylidene-Lac-

tones 4d. MicroKan reactors containing polymer-bound R-alkyl/

arylidene-lactone 4d (96 MicroKan reactors, 2.4 mmol, 1 equiv)

were suspended in THF (200 mL) in a round-bottom ﬂask

equipped with a stir bar. After degassing at -78 °C in vacuo

for 2 min, the solution was warmed to 0 °C and slowly treated

with a solution of tetra-n-butylammonium ﬂuoride (TBAF) in

THF (1.0 M in THF, 48 mL, 20 equiv). Stirring continued at 0

°C for 6 h, after which time the reaction was quenched with a

saturated aqueous solution of ammonium chloride. Finally, the

MicroKan reactors containing polymer-bound diol-R-alkyl/

arylidene-lactone 5 were washed with 1:1 DMF/water (3×),

DMF (3×), methanol (2×), and dichloromethane (3×) and dried

in vacuo.

Library 1: Hybrid “PMP” and “TBDPS” Approach. Library

components in Rows E and F were prepared through the

“TBDPS” approach, whereas the others were synthesized

through the “PMP” approach. The procedure was identical

as described previously, except for the number of MicroKan

reactors loaded with starting DAG-lactones 1b and 1d and

the sorting of the polymer-bound reactors containing the

different starting materials.

Procedure for the Acylation of Polymer-Bound Diol Lac-

tones 5 with Acid Chlorides. The 96 MicroKan reactors were

sorted into 12 vessels to react with 12 different acid chlorides.

Each vessel contained eight reactors. The 12 parallel reactions

were performed following an identical, general procedure:

MicroKan reactors containing polymer-bound diol-R-alkyl/

arylidene-lactone 5 (8 reactors, 0.2 mmol, 1 equiv) were

suspended in CH₂Cl₂(20 mL) in a Schlenk vessel equipped

with a stir bar. After degassing at -78 °C in vacuo for 2 min,

triethylamine (0.56 mL, 4.0 mmol, 20 equiv), 4-DMAP (0.024

g, 0.2 mmol, 1 equiv), and the corresponding acid chloride (2.0

mmol, 10 equiv) were subsequently introduced into the vessel.

The mixture was stirred at 0 °C for 6 h, after which time the

reactions were quenched with a saturated solution of ammonium

chloride. All reactors were pooled together and washed with

1:1 DMF/water (3×), DMF (3×), methanol (2×), and dichlo-

romethane (3×) and dried in vacuo.

Procedure for TFA-Assisted Parallel Cleavage. The 96

MicroKan reactors containing polymer-bound DAG-lactone 6

were sorted into 96 individual wells using the IRORI 96-

cleavage station. To each well was added 1.5 mL of 1:10 TFA/

dichloromethane. After shaking (15 min), the solution was

ﬁltered into 96 individual preweighed vials. An additional 1.5

mL of dichloromethane was introduced into each well for

washing the MicroKan reactors. The combined organic solution

in each vial was directly concentrated in vacuo and weighed.

Procedure for Sorting Polymer-Bound Lactone 2b and 2d.

MicroKan reactors containing polymer-bound lactone 2b were

sorted into vessels 1, 2, 3, 4, 7, and 8, to respectively react

with aldehydes A, B, C, D, G, and H. MicroKan reactors

containing polymer-bound lactone 2d were sorted into vessels

5 and 6 to react with aldehydes E and F, respectively. Each

vessel contained 12 MicroKan reactors.

Procedure for Scanning Polymer-Bound r-Alkyl/Arylidene-

Lactones 4b and 4d. The 96 MicroKan reactors were grouped

into two vessels using the “decode tag” button in the

UTILITIES menu. The 72 reactors containing polymer-bound

R-alkyl/arylidene-lactone 4b, which were identiﬁed by al-

dehydes A, B, C, D, G, and H, were placed into one vessel,

whereas the 24 reactors containing polymer-bound R-alkyl/

arylidene-lactone 4d, which were identiﬁed by aldehydes E

and F, were placed into another vessel. The deprotection of

the 72 MicroKan reactors containing R-alkyl/arylidene-

lactones 4b (protected with the PMP group) and the 24

MicroKan reactors containing R-alkyl/arylidene-lactones 4d

(protected with the TBDMS group) was separately performed

as described previously.

Library 1: “TBDPS Approach”. With the exception of the

loading step and the removal of the TBDPS group, all the other

steps were performed in the same fashion as in Library 1 (PMP

approach).

Library 2: “PMP” Approach. The loading of 5-(hydroxy-

methyl)-5-[(4-methoxyphenoxy)methyl]-3,4,5-trihydrofuran-2-

one (1b) onto the DHP resin was performed as described for

Library 1. The execution of this library was identical to the

procedure previously described for Library 1 except that the

matrix of this library consisted of 12 aldehydes and 8 acid

chlorides.

Procedure for Loading 5-(Hydroxymethyl)-5-[(tert-butyl-

diphenylsilyloxy)methyl]-3,4,5-trihydrofuran-2-one (1d) onto

the DHP Resin. First, 100 MicroKan reactors, each containing

DHP-resin (0.0147 g, 0.025 mmol) and a radiofrequency tag

capsule, were placed in a 500 mL round-bottom ﬂask equipped

with a stir bar. Then DAG-lactone 1d (4.8 g, 12.5 mmol, 5

equiv), pyridinium p-toluenesulfonate (2.5 g, 10 mmol, 4 equiv),

and 1,2-dichloroethane (150 mL) were introduced into the ﬂask.

These reactors were degassed at -30 °C in vacuo for 2 min.

The temperature was then raised to 60 °C, and the solution was

stirred for 24 h. After reaching the room temperature, the

solution was decanted into another vessel to recover the

unreacted lactone 1d. The reactors containing polymer-bound

lactone 2d were washed sequentially with dichloromethane (1×),

1:1 DMF/water (3×), DMF (3×), methanol (2×), and dichlo-

romethane (3×) and ﬁnally dried in vacuo.

(Z)-[4-Heptylidene-2-(hydroxymethyl)-5-oxo-2-2,3-dihydro-

furyl]methyl 2,4,6-tris(methylethyl)benzoate (L1-A12, Library

1). Oil; IR (neat) 3440 (OH), 2960 (CH), 2929 (CH), 2871 (CH),

1761 (CdO), 1734 (CdO), 1680 (CdC) cm^-1. ¹H NMR (400

MHz, CDCl₃) δ 6.98 (s, 2 H, ArH), 6.72 (tt, J ) 7.5, 2.8 Hz,

1 H, CdCH), 4.42 (AB q, J ) 11.8 Hz, 2 H, CCH₂OC), 3.71

(AB q, J ) 12.1 Hz, 2 H, CCH₂OH), 2.79 (m, 5 H, H_3aband

C(O)C₆H₂(CH(CH₃)₂)₃), 2.32 (v br s, 1 H, CCH₂OH), 2.11 (m,

2 H, CdCHCH₂CH₂CH₂CH₂CH₂CH₃), 1.41 (m, 2 H, Cd-

CHCH₂CH₂CH₂CH₂CH₂CH₃), 1.25 (m, 24 H, CdCHCH₂-

CH₂CH₂CH₂CH₂CH₃and C(O)C₆H₂(CH(CH₃)₂)₃), 0.85 (t, J )

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5215

6.9 Hz, 3 H, CdCHCH₂CH₂CH₂CH₂CH₂CH₃). FAB-MS (m/z,

relative intensity) 473 (MH⁺, 20), 231 (100). Anal. (C₂₉H₄₄O₅)

C, H.

z, relative intensity) 425 (MH⁺, 77), 424 (M^•+, 84), 148 (100).

Anal. (C₂₄H₂₈N₂O₅·0.3H₂O) C, H, N.

(E)-(4-{[4-(Dimethylamino)phenyl]methylene}-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl)methyl 4-Methoxybenzoate

(L1-E6, Library 1). Light yellow solid, mp ) 190-192 °C; IR

(Z)- [4-(2-Ethylhexylidene)-2-(hydroxymethyl)-5-oxo-2-2,3-

dihydrofuryl]methyl 4-(dimethylamino)benzoate (L1-B5, Li-

brary 1). White solid, mp ) 108-112 °C; IR (neat) 3417 (OH),

2962 (CH), 2924 (CH), 1728 (CdO), 1698 (CdO), 1609 (CdC)

1

(neat) 3370 (OH), 1705 (CdO), 1593 (CdC) cm^-1. H NMR

(400 MHz, CDCl₃) δ 7.92 (d, J ) 8.8 Hz, 2 H, ArH), 7.51 (t,

J ) 2.7 Hz, 1 H, CdCH), 7.40 (d, J ) 8.9 Hz, 2 H, ArH), 6.87

(d, J ) 8.9 Hz, 2 H, ArH), 6.76 (d, J ) 8.7 Hz, 2 H, ArH),

4.48 (AB q, J ) 11.9 Hz, 2 H, CCH₂OC), 3.83 (s, 3 H,

C(O)C₆H₄OCH₃), 3.78 (app t, 2 H, CCH₂OH), 3.21 (dd, J )

cm^-^{1 1}H NMR (400 MHz, CDCl₃) δ 7.85 and 7.84 [2 m

.

(diastereomers), 2 H, ArH], 6.61 and 6.59 (2 m, 2 H, ArH),

6.56 and 6.53 [2 t, J ) 2.8 Hz (diastereomers), 1 H, CdCH],

4.41 [2 AB q, J ) 12.0 Hz (diastereomers), 2 H, CCH₂OC],

3.71 (m, 2 H, CCH₂OH), 3.02 (2 s, 6 H, N(CH₃)₂), 2.86 (app

dt, J ) 17.0 Hz, 1 H, H_3a), 2.72 (app dt, J ) 17.0 Hz, 1 H,

H_3b), 2.38 (m, 1 H, CCH₂OH), 2.09 (m, 1 H, CdCHCH

(CH₂CH₃)CH₂CH₂CH₂CH₃), 1.48 (m, 2 H, CdCHCH(CH₂-

CH₃)CH₂CH₂CH₂CH₃), 1.21 (m, 6 H, CdCHCH(CH₂CH₃)-

CH₂CH₂CH₂CH₃),0.85(m,3H,CdCHCH(CH₂CH₃)CH₂CH₂CH₂-

CH₃),0.76(m,3H,CdCHCH(CH₂CH₃)CH₂CH₂CH₂CH₃).FAB-MS

(m/z, relative intensity) 404 (MH⁺, 49), 403 (M^•+, 49), 148

(100). Anal. (C₂₃H₃₃NO₅) C, H, N.

17.3, 2.8 Hz,

1 H, H_3a), 3.06 (m, 7 H, H_3band

CdCHC₆H₄N(CH₃)₂), 2.33 (t, J ) 6.7 Hz, 1 H, CCH₂OH).

FAB-MS (m/z, relative intensity) 412 (MH⁺, 75), 411 (M^•+

,

75), 135 (100). Anal. (C₂₃H₂₅NO₆) C, H, N.

(E)-(4-{[4-(Dimethylamino)phenyl]methylene}-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl)methyl 4-Nitrobenzoate (L1-

E8, Library 1). Brown solid, mp ) 191-193 °C; IR (neat)

3324(OH), 1721 (CdO), 1707 (CdO), 1594 (CdC) cm^-1. ¹H

NMR (400 MHz, CDCl₃) δ 8.22 (d, J ) 9.0 Hz, 2 H, ArH),

8.10 (d, J ) 9.0 Hz, 2 H, ArH), 7.52 (t, J ) 2.7 Hz, 1 H,

CdCH), 7.41 (d, J ) 8.9 Hz, 2 H, ArH), 6.83 (br d, J ≈ 7.4

Hz, 2 H, ArH), 4.56 (AB q, J ) 11.9 Hz, 2 H, CCH₂OC), 3.84

(br AB q, J ) 11.7 Hz, 2 H, CCH₂OH), 3.23 (dd, J ) 17.2, 2.8

Hz, 1 H, H_3a), 3.08 (dd, J ) 17.3, 2.5 Hz, 1 H, H_3b), 3.04 (v br

s, 6 H, CdCHC₆H₄N(CH₃)₂), 2.15 (s, 1 H, CCH₂OH). FAB-

MS (m/z, relative intensity) 427 (MH⁺, 100), 426 (M^•+, 93).

Anal. (C₂₂H₂₂N₂O₇) C, H, N.

(Z)-[4-(2-Ethylhexylidene)-2-(hydroxymethyl)-5-oxo-2-2,3-di-

hydrofuryl]methyl 4-nitrobenzoate (L1-B8, Library 1). White

solid, mp ) 112-115 °C; IR (neat) 3392 (OH), 1716 (CdO)

cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (m, 2 H, ArH), 8.13

(m, 2 H, ArH), 6.59 and 6.56 [m (diastereomers), 1 H, CdCH],

4.52 [2 AB q, J ) 11.9 Hz (diastereomers), 2 H, CCH₂OC],

3.78 [2 AB q, J ) 12.1 Hz (diastereomers), 2 H, CCH₂OH],

2.90 [2 dd, J ) 17.1, 3.0 Hz (diastereomers], 1 H, H_3a), 2.74 [2

dd, J ) 17.0, 2.7 Hz (diastereomers), 1 H, H_3b], 2.20 (m, 1 H,

CCH₂OH), 2.08 (m, 1 H, CdCHCH(CH₂CH₃)CH₂CH₂CH₂-

CH₃), 1.47 (m, 2 H, CdCHCH(CH₂CH₃)CH₂CH₂CH₂CH₃), 1.27

(m, 4 H, CdCHCH(CH₂CH₃)CH₂CH₂CH₂CH₃), 1.10 (m, 2 H,

CdCHCH(CH₂CH₃)CH₂CH₂CH₂CH₃), 0.84 (m, 3 H, Cd

CHCH(CH₂CH₃)CH₂CH₂CH₂CH₃), 0.72 (m, 3 H, CdCHCH

(CH₂CH₃)CH₂CH₂CH₂CH₃). FAB-MS (m/z, relative intensity)

406 (MH⁺, 100). Anal. (C₂₁H₂₇NO₇) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(4-methoxyphenyl)methylene]-5-

oxo-1-2,3-dihydrofuryl}methyl 4-(dimethylamino)benzoate (L1-

F5, Library 1). White solid, mp ) 180 °C. ¹H NMR (300 MHz,

CDCl₃) δ 7.85 (d, J ) 9.0 Hz, 2 H, ArH), 7.57 (m, 1 H, CdCH),

7.46 (d, J ) 9.0 Hz, 2 H, ArH), 6.95 (d, J ) 9.0 Hz, 2 H,

ArH), 6.61 (d, J ) 9.0 Hz, 2 H, ArH), 4.57 (d, J ) 12.0 Hz, 1

H, CCH₂OC), 4.38 (d, J ) 12.0 Hz, 1 H, CCH₂OC), 3.85 (s, 3

H, CdCHC₆H₄OCH₃), 3.73-3.85 (m, 2 H, CCH₂OH), 3.25 (dd,

J ) 17.4, 2.7 Hz, 1 H, H_3a), 3.07 (dd, J ) 17.4, 2.7 Hz, 1 H,

H_3b), 3.04 (s, 6 H, C(O)C₆H₄N(CH₃)₂). FAB-MS (m/z, relative

intensity) 412 (MH⁺, 100). Anal. (C₂₃H₂₅NO₆) C, H, N.

(E)-(4-{[4-(Dimethylamino)phenyl]methylene}-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl)methyl 2-ethylhexanoate (L1-

E2, Library 1). Light yellow solid, mp ) 89-91 °C; IR (neat)

3371(OH), 2963 (CH), 2932 (CH), 2859 (CH), 1734 (CdO),

1712 (CdO), 1636 (CdC) cm^-1. ¹H NMR (400 MHz, CDCl₃)

δ 7.49 (t, J ) 2.7, 1 H, CdCH), 7.41 (d, J ) 8.9 Hz, 2 H,

ArH), 6.85 (d, J ) 8.2 Hz, 2 H, ArH), 4.28 [2 AB q, J ) 11.9

Hz (diastereomers), 1 H, CCH₂OC], 4.28 [2 AB q, J ) 11.9

Hz (diastereomers), 1 H, CCH₂OC], 3.71 (m, 2 H, CCH₂OH),

3.14 (dd, J ) 17.3, 2.8 Hz, 1 H, H_3a), 3.04 (s, 6 H,

CdCHC₆H₄N(CH₃)₂), 2.96 (dd, J ) 17.3, 2.8 Hz, 1 H, H_3b),

2.27 (m, 1 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃), 2.15 (v br

s, 1 H, CCH₂OH), 1.50 (m, 4 H, C(O)CH(CH₂CH₃)CH₂-

CH₂CH₂CH₃),1.19(m,4H,C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃),

0.82 (m, 6 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃). FAB-MS

(m/z, relative intensity) 404 (MH⁺, 74), 403 (M^•+, 100). Anal.

(C₂₃H₃₃NO) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(4-methoxyphenyl)methylene]-5-

oxo-1-2,3-dihydrofuryl}methyl 4-methoxybenzoate (L1-F6, Li-

1

brary 1). White solid, mp ) 169 °C. H NMR (300 MHz,

CDCl₃) δ 7.92 (d, J ) 9.0 Hz, 2 H, ArH), 7.55-7.59 (m, 1 H,

CdCH), 7.46 (d, J ) 9.0 Hz, 2 H, ArH), 6.95 (d, J ) 9.0 Hz,

2 H, ArH), 6.88 (d, J ) 9.0 Hz, 2 H, ArH), 4.58 (d, J ) 12.0

Hz, 1 H, CCH₂OC), 4.42 (d, J ) 12.0 Hz, 1 H, CCH₂OC),

3.85 (s, 6 H, CdCHC₆H₄OCH₃and C(O)C₆H₄OCH₃), 3.80-3.90

(m, 2 H, CCH₂OH), 3.26 (dd, J ) 17.4, 2.7, Hz, 1 H, H_3a),

3.08 (dd, J ) 17.4, 2.7 Hz, 1 H, H_3b), 2.35-2.45 (m, 1 H,

CCH₂OH). FAB-MS (m/z, relative intensity) 399 (MH⁺, 100).

Anal. (C₂₂H₂₂O₇) C, H.

(E)-{2-(Hydroxymethyl)-4-[(4-methoxyphenyl)methylene]-5-

oxo-2-2,3-dihydrofuryl}methyl 2,4,6-trimethylbenzoate (L1-

F11, Library 1). White solid, mp ) 56-58 °C; IR (neat) 3300

(E)-(4-{[4-(Dimethylamino)phenyl]methylene}-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl)methyl 4-(Dimethylamino)ben-

zoate (L1-E5, Library 1). Yellow solid, mp ) 230-232 °C; IR

(neat) 3386 (OH), 2898 (CH), 1713 (CdO), 1697 (CdO, 1606

(CdC) cm^-1. ¹H NMR (400 MHz, CD₂Cl₂) δ 7.82 (d, J ) 9.1

Hz, 2 H, ArH), 7.45 (m, 3 H, ArH and CdCH), 6.76 (d, J )

8.9 Hz, 2 H, ArH), 6.63 (d, J ) 9.1 Hz, 2 H, ArH), 4.42 (AB

q, J ) 11.8 Hz, 2 H, CCH₂OC), 3.77 (irregular AB q, 2 H,

CCH₂OH), 3.19 (dd, J ) 17.3, 2.8 Hz, 1 H, H_3a), 3.09 (dd, J )

17.3, 2.6 Hz, 1 H, H_3b), 3.03 (s, 12 H, N(CH₃)₂). FAB-MS (m/

1

(OH), 1728 (CdO), 1649 (CdC) cm^-1. H NMR (400 MHz,

CDCl₃) δ 7.47 (t, J ) 2.7 Hz, 1 H, CdCH), 7.41 (d, J ) 8.8

Hz, 2 H, ArH), 6.92 (d, J ) 8.8 Hz, 2 H, ArH), 6.80 (s, 2 H,

ArH), 4.49 (AB q, J ) 11.9 Hz, 2 H, CCH₂OC), 3.82 (s, 3 H,

CdCHC₆H₄OCH₃), 3.77 (AB q, J ≈ 12.2 Hz, 2 H, CCH₂OH),

3.18 (dd, J ) 17.5, 2.8 Hz, 1 H, H_3a), 3.05 (dd, J ) 17.5, 2.8

Hz, 1 H, H_3b), 2.24 (s, 3 H, C(O)C₆H₂(CH₃)₃), 2.23 (s, 6 H,

C(O)C₆H₂(CH₃)₃). FAB-MS (m/z, relative intensity) 411 (MH⁺,

51), 147 (100). Anal. (C₂₄H₂₆O₆·0.5H₂O) C, H.

5216 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

(E)-{2-(Hydroxymethyl)-4-[(4-methoxyphenyl)methylene]-5-

oxo-2-2,3-dihydrofuryl}methyl 2,4,6-tris(methylethyl)benzoate

(L1-F12, Library 1). White solid, mp ) 62-64 °C; IR (neat)

3687 (OH), 2966 (CH), 1731 (CdO), 1715 (CdO), 1647 (CdC)

cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 7.49 (t, J ) 2.7 Hz, 1 H,

CdCH), 7.41 (d, J ) 8.8 Hz, 2 H, ArH), 6.97 (s, 2 H, ArH),

6.92 (d, J ) 8.8 Hz, 2 H, ArH), 4.49 (AB q, J ) 11.8 Hz, 2 H,

CCH₂OC), 3.83 (s, 3 H, CdCHC₆H₄OCH₃), 3.78 (AB q, J ≈

11.9 Hz, 2 H, CCH₂OH), 3.14 (dd, J ) 17.5, 2.8 Hz, 1 H, H_3a),

3.08 (dd, J ) 17.6, 2.8 Hz, 1 H, H_3b), 2.86 (sept., J ) 6.9 Hz,

1 H, C(O)C₆H₂(CH(CH₃)₂)₃), 2.76 (sept, J ) 6.7 Hz, 2 H,

C(O)C₆H₂(CH(CH₃)₂)₃), 1.22 (d, J ) 6.9 Hz, 6 H, C(O)C₆-

H₂(CH(CH₃)₂)₃), 1.19 (d, J ) 6.8 Hz, 6 H, C(O)C₆H₂(CH-

(CH₃)₂)₃), 1.16 (d, J ) 6.8 Hz, 6 H, C(O)C₆H₂(CH(CH₃)₂)₃).

FAB-MS (m/z, relative intensity) 495 (MH⁺, 25), 231 (100).

HRMS (FAB) calcd for C₃₀H₃₈O₆(M+K⁺): 533.231; found:

533.230. HRMS (FAB) calcd for C₃₀H₃₈O₆(MH⁺): 495.2747;

found: 495.2766.

CCH₂OC), 3.83 (br s, 2 H, CCH₂OH), 3.64 (app AB q, 2 H, H_3ab),

3.04 (s, 6 H, C(O)C₆H₄N(CH₃)₂); FAB-MS (m/z, relative intensity)

427 (MH⁺, 100), 426 (M^•+, 73); Anal. (C₂₂H₂₂N₂O₇·0.3H₂O) C,

H, N.

(E)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 4-Methoxybenzoate (L1-H6, Library

1). White solid, mp ) 202-204 °C; IR (neat) 3388 (OH), 1711

1

(CdO) cm^-1. H NMR (400 MHz, CDCl₃) δ 8.26 (d, J ) 8.9

Hz, 2 H, ArH), 7.88 (d, J ) 8.9 Hz, 2 H, ArH), 7.59 (m, 3 H,

CdCH and ArH), 6.87 (d, J ) 8.9 Hz, 2 H, ArH), 4.50 (AB q, J

) 12.0 Hz, 2 H, CCH₂OC), 3.87 (dd, J ) 12.2, 6.7 Hz, 1 H,

CCH₂OH), 3.84 (s, 3 H, CC₆H₄OCH₃), 3.79 (dd, J ) 12.2, 6.8

Hz, 1 H, CCH₂OH), 3.33 (dd, J ) 18.0, 3.2 Hz, 1 H, H_3a), 3.11

(dd, J ) 18.0, 2.8 Hz, 1 H, H_3b), 2.20 (t, J ) 6.8 Hz, 1 H,

CCH₂OH). FAB-MS (m/z, relative intensity) 414 (MH⁺, 56), 135

(100). Anal. (C₂₁H₁₉NO₈) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 4-Nitrobenzoate (L1-H8, Library

1). White solid, mp ) 205-208 °C; IR (neat) 3427 (OH), 1720

(CdO), 1606 (CdC) cm^-1. ¹H NMR (400 MHz, CD₂Cl₂) δ 8.26

(m, 3 H, ArH), 8.11 (m, 1 H, ArH), 7.67 (d, J ) 8.8 Hz, 2 H,

ArH), 7.61 (t, J ) 3.0 Hz, 1 H, CdCH), 4.59 (AB q, J ) 12.0 Hz,

2 H, CCH₂OC), 3.90 (AB q, J ) 12.0 Hz, 2 H, CCH₂OH), 3.37

(dd, J ) 18.1, 3.2 Hz, 1 H, H_3a), 3.18 (dd, J ) 18.1, 2.8 Hz, 1 H,

H_3b). FAB-MS (m/z, relative intensity) 429 (MH⁺, 63), 89 (100).

Anal. calcd for (C₂₀H₁₆N₂O₉) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 2-Ethylhexanoate (L1-H2, Library

1). White solid, mp ) 98-100 °C; IR (neat) 3392 (OH), 1716

1

(CdO) cm^-1. H NMR (400 MHz, CDCl₃) δ 8.28 (d, J ) 8.9

Hz, 2 H, ArH), 7.63 (d, J ) 8.8 Hz, 2 H, ArH), 7.59 (t, J ) 3.0

Hz, 1 H, CdCH), 4.28 (d AB q, J ) 12.0 Hz, 2 H, CCH₂OC),

3.77 (d AB q, J ) 12.1, Hz, 2 H, CCH₂OH), 3.28 (dd, J ) 18.0,

3.1 Hz, 1 H, H_3a), 3.03 (dd, J ) 18.0, 2.6 Hz, 1 H, H_3b), 2.24 (m,

2 H, CCH₂OH and C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃), 1.47 (m,

4H,C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃),1.18(m,4H,C(O)CH(CH₂-

CH₃)CH₂CH₂CH₂CH₃), 0.80 (m, 6 H, C(O)CH(CH₂CH₃)

CH₂CH₂CH₂CH₃). FAB-MS (m/z, relative intensity) 406 (MH⁺,

86), 57 (100). Anal. (C₂₁H₂₇NO₇) C, H, N.

(Z)-(2-(Hydroxymethyl)-5-oxo-4-{[2-(triﬂuoromethyl)phenyl]-

methylene}-2-2,3-dihydrofuryl)methyl 2-Methylpropanoate (L2-

A1, Library 2). Oil; IR (neat) 3522 (OH), 2980 (CH), 2880

(CH), 1769 (CdO), 1745 (CdO), 1609 (CdC) cm^-1. ¹H NMR

(400 MHz, CDCl₃) δ 7.64 (d, J ) 7.7 Hz, 1 H, ArH), 7.49 (t,

J ) 7.5 Hz, 1 H, ArH), 7.36 (m, 2 H, ArH), 6.68 (t, J ) 1.6

Hz, 1 H, CdCH), 4.28 (AB q, J ) 11.9 Hz, 2 H, CCH₂OC),

3.77 (br s, 2 H, CCH₂OH), 3.69 (br s, 2 H, H_3ab), 2.52 (br s, 1

H, CCH₂OH), 2.46 (sept, J ) 7.0 Hz, 1 H, C(O)CH(CH₃)₂),

1.06 (t, J ) 6.8 Hz, 6 H, C(O)CH(CH₃)₂). FAB-MS (m/z,

relative intensity) 373 (MH⁺, 100). Anal. (C₁₈H₁₉F₃O₅) C, H.

(Z)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 2-Ethylhexanoate (L1-H2, Library

1). Colorless oil; IR (neat) 3467 (OH), 2961 (CH), 2935 (CH),

1

2873 (CH), 1764 (CdO), 1738 (CdO), 1605 (CdC) cm^-1. H

NMR (400 MHz, CDCl₃) δ 8.16 (d, J ) 8.8 Hz, 2 H, ArH), 7.40

(d, J ) 8.7 Hz, 2 H, ArH), 6.93 (app q, J ) 1.4 Hz, 1 H, CdCH),

4.31 (2 AB q, J ) 11.9 Hz, 2 H, CCH₂OC), 3.75 (br s, 2 H,

CCH₂OH), 3.71 (br s, 2 H, H_3ab), 2.22 (m, 1 H, C(O)CH-

(CH₂CH₃)CH₂CH₂CH₂CH₃), 1.46 (m, 4 H, C(O)CH(CH₂CH₃)-

CH₂CH₂CH₂CH₃), 1.21 (m, 4 H, C(O)CH(CH₂CH₃)CH₂CH₂-

CH₂CH₃), 0.83 (m, 6 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃).

FAB-MS (m/z, relative intensity) 406 (MH⁺, 83), 57 (100). Anal.

(C₂₁H₂₇NO₇) C, H, N.

(E)-(2-(Hydroxymethyl)-5-oxo-4-{[4-(triﬂuoromethyl)phenyl-

]methylene}-2-2,3-dihydrofuryl)methyl 2-Propylpentanoate (L2-

B4, Library 2). White solid, mp ) 70-72 °C; IR (neat) 3381

1

(OH), 2935 (CH), 1727 (CdO), 1655 (CdC) cm^-1. H NMR

(400 MHz, DMSO-d₆) δ 7.67 (d, J ) 8.2 Hz, 2 H, ArH), 7.57

(d, J ) 8.4 Hz, 2 H, ArH), 7.55 (t, J ) 3.0 Hz, 1 H, CdCH),

4.26 (AB q, J ) 12.0 Hz, 2 H, CCH₂OC), 3.75 (AB q, J )

12.2 Hz, 2 H, CCH₂OH), 3.25 (dd, J ) 17.9, 3.1 Hz, 1 H, H_3a),

3.01 (dd, J ) 17.9, 2.8 Hz, 1 H, H_3b), 2.33 (m, 2 H, CCH₂OH

and C(O)CH(CH₂CH₂CH₃)₂), 1.48 (m, 2 H, C(O)CH(CH₂-

CH₂CH₃)₂), 1.34 (m, 2 H, C(O)CH(CH₂CH₂CH₃)₂), 1.17

(m, 4 H, C(O)CH(CH₂CH₂CH₃)₂), 0.79 (t, J ) 7.3 Hz, 3 H,

C(O)CH(CH₂CH₂CH₃)₂), 0.75 (t, J ) 7.3 Hz, 3 H, C(O)-

CH(CH₂CH₂CH₃)₂). FAB-MS (m/z, relative intensity) 429

(MH⁺, 100). Anal. (C₂₂H₂₇F₃O₅) C, H.

(E)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 4-(Dimethylamino)benzoate (L1-

H5, Library 1). Yellow solid, mp ) 212-215 °C; IR (neat) 3383

(OH), 1703 (CdO) cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 8.25 (d,

J ) 8.8 Hz, 2 H, ArH), 7.78 (d, J ) 8.8 Hz, 2 H, ArH), 7.58 (m,

3 H, CdCH and ArH), 6.58 (d, J ) 9.0 Hz, 2 H, ArH), 4.47 (AB

q, J ) 12.1 Hz, 2 H, CCH₂OC), 3.83 (dd, J ) 12.2, 6.8 Hz, 1 H,

CCH₂OH), 3.77 (dd, J ) 12.3, 6.7 Hz, 1 H, CCH₂OH), 3.31 (dd,

J ) 18.0, 3.1 Hz, 1 H, H_3a), 3.11 (dd, J ) 18.0, 2.8 Hz, 1 H, H_3b),

3.03 (s, 6 H, CC₆H₄N(CH₃)₂), 2.34 (t, J ) 6.9 Hz, 1 H, CCH₂OH).

FAB-MS (m/z, relative intensity) 427 (MH⁺, 69), 426 (M^•+, 51),

148 (100). Anal. (C₂₂H₂₂N₂O₇) C, H, N.

(E)-{4-[(4-Fluorophenyl)methylene]-2-(hydroxymethyl)-5-

oxo-2-2,3-dihydrofuryl}methyl 2-Propylpentanoate (L2-B7, Li-

brary 2). Oil; IR (neat) 2965 (CH), 1742 (CdO) cm^-1. ¹H NMR

(400 MHz, DMSO-d₆) δ 7.50 (t, J ) 2.9 Hz, 1 H, CdCH),

7.46 (m, 2 H, ArH), 7.11 (m, 2 H, ArH), 4.26 (AB q, J ) 12.0

Hz, 2 H, CCH₂OC), 3.73 (AB q, J ) 12.1 Hz, 2 H, CCH₂OH),

3.19 (dd, J ) 17.6, 2.9 Hz, 1 H, H_3a), 2.96 (dd, J ) 17.6, 2.7

Hz, 1 H, H_3b), 2.33 (m, 2 H, CCH₂OH and C(O)CH-

(CH₂CH₂CH₃)₂), 1.49 (m, 2 H, C(O)CH(CH₂CH₂CH₃)₂), 1.34

(m, 2 H, C(O)CH(CH₂CH₂CH₃)₂), 1.17 (m, 4 H, C(O)CH-

(CH₂CH₂CH₃)₂), 0.77 (overlap t, J ) 7.3 Hz, 6 H, C(O)-

(Z)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 4-(dimethylamino)benzoate (L1-H5,

Library 1). Yellow solid, mp ) 188-191 °C; IR (neat) 3466 (OH),

1

2923 (CH), 1746 (CdO), 1736 (CdO), 1606 (CdC) cm^-1. H

NMR (400 MHz, CDCl₃) δ 7.89 (d, J ) 8.8 Hz, 2 H, ArH), 7.68

(d, J ) 9.1 Hz, 2 H, ArH), 7.23 (d, J ) 10.0 Hz, 2 H, ArH), 7.04

(t, J ) 1.3 Hz, 1 H, CdCH), 6.56 (d, J ) 9.1 Hz, 2 H, ArH), 4.76

(d, J ) 12.0 Hz, 1 H, CCH₂OC), 4.40 (d, J ) 12.0 Hz, 1 H,

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5217

1

CH(CH₂CH₂CH₃)₂). FAB-MS (m/z, relative intensity) 379

(CH), 2874 (CH), 1735 (CdO), 1657 (CdC) cm^-1. H NMR

(MH⁺, 100). Anal. (C₂₁H₂₇FO₅) C, H.

(400 MHz, CDCl₃) δ 7.42 (m, 2 H, CdCH and ArH), 7.22 (m,

2 H, ArH), 4.25 (2 AB q, J ≈ 11.9, 2 H, CCH₂OC), 3.75 (AB

q, J ≈ 12.2 Hz, 2 H, CCH₂OH), 3.20 (dd, J ) 17.8, 3.0 Hz, 1

H, H_3a), 2.95 (dd, J ) 17.8, 2.6 Hz, 1 H, H_3b), 2.66 (v br s, 1

H, CCH₂OH), 2.23 (m, 1 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃),

1.44 (m, 4 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃), 1.15 (m, 4

H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃), 0.78 (m, 6 H, C(O)-

CH(CH₂CH₃)CH₂CH₂CH₂CH₃). FAB-MS (m/z, relative inten-

sity) 413 (MH⁺, 100). Anal. (C₂₁H₂₆ClFO₅) C, H, Cl.

(E)-{4-[(3-Chloro-4-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2-Propylpentanoate

(L2-B10, Library 2). Oil; IR (neat) 3508 (OH), 2959 (CH), 2872

1

(CH), 1741 (CdO), 1663 (CdC) cm^-1. H NMR (400 MHz,

CDCl₃) δ 7.24 (m, 1 H, CdCH), 7.02 (m, 3 H, ArH), 4.06 (AB

q, J ) 12.0 Hz, 2 H, CCH₂OC), 3.54 (AB q, J ) 12.1 Hz, 2 H,

CCH₂OH), 2.99 (dd, J ) 17.8, 3.0 Hz, 1 H, H_3a), 2.76 (dd, J )

17.8, 2.8 Hz, 1 H, H_3b), 2.23 (v br s, 1 H, CCH₂OH), 2.14 (m, 1

H, C(O)CH(CH₂CH₂CH₃)₂), 1.29 (m, 2 H, C(O)CH(CH₂CH₂-

CH₃)₂), 1.15 (m, 2 H, C(O)CH(CH₂CH₂CH₃)₂), 0.97 (m, 4 H,

C(O)CH(CH₂CH₂CH₃)₂), 0.60 (t, J ) 6.5 Hz, 3 H, C(O)CH-

(CH₂CH₂CH₃)₂), 0.57 (t, J ) 6.5 Hz, 3 H, C(O)CH(CH₂CH₂-

CH₃)₂). FAB-MS (m/z, relative intensity) 413 (MH⁺, 100). Anal.

(C₂₁H₂₆ClFO₅) C, H.

(Z)-{4-[(4-Chloro-3-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2-Ethylhexanoate (L2-

C11, Library 2). Oil; IR (neat) 3461 (OH), 2960 (CH), 2934

(CH), 2874 (CH), 1766 (CdO), 1735 (CdO), 1582 (CdC)

cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 7.31 (t, J ) 7.9 Hz, 1 H,

ArH), 7.01 (dd, J ) 9.7, 1.9 Hz, 1 H, ArH), 6.95 (dd, J ) 8.2,

1.4 Hz, 1 H, ArH), 6.90 (d, J ) 1.3 Hz, 1 H, CdCH), 4.36 (dd,

J ) 11.9, 5.4 Hz, 1 H, CCH₂OC), 4.25 (dd, J ) 11.9, 7.9 Hz,

1 H, CCH₂OC), 3.74 (app s, 2 H, CCH₂OH), 3.56 (s, 2 H, H_3ab),

2.21 (m, 2 H, CCH₂OH and C(O)CH(CH₂CH₃)CH₂CH₂-

CH₂CH₃), 1.46 (m, 4 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃),

1.21 (m, 4 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃), 0.83 (m, 6 H,

C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃). FAB-MS (m/z, relative in-

tensity) 413 (MH⁺, 21), 57 (100). Anal. (C₂₁H₂₆ClFO₅) C, H, Cl.

(E)-{4-[(3-Chloro-4-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2-Propylpentanoate

(L2-B11, Library 2). Oil; IR (neat) 3459(OH), 2958 (CH), 2872

1

(CH), 1735 (CdO) cm^-1. H NMR (400 MHz, CDCl₃) δ 7.50

(dd, J ) 6.9, 2.0 Hz, 1 H, ArH), 7.42 (t, J ) 2.8 Hz, 1 H, CdCH),

7.35 (m, 1 H, ArH), 7.19 (t, J ) 8.6 Hz, 1 H, ArH), 4.26 (AB q,

J ) 12.0 Hz, 2 H, CCH₂OC), 3.75 (AB q, J ) 12.2 Hz, 2 H,

CCH₂OH), 3.19 (dd, J ) 17.7, 2.7 Hz, 1 H, H_3a), 2.95 (dd, J )

17.7, 2.5 Hz, 1 H, H_3b), 2.57 (v br s, 1 H, CCH₂OH), 2.34 (m, 1

H, C(O)CH(CH₂CH₂CH₃)₂), 1.49 (m, 2 H, C(O)CH(CH₂-

CH₂CH₃)₂), 1.34 (m, 2 H, C(O)CH(CH₂CH₂CH₃)₂), 1.18 (m, 4

H, C(O)CH(CH₂CH₂CH₃)₂), 0.80 (t, J ) 6.3 Hz, 3 H, C(O)CH-

(CH₂CH₂CH₃)₂), 0.77 (t, J ) 6.3 Hz, 3 H, C(O)CH(CH₂CH₂-

CH₃)₂). FAB-MS (m/z, relative intensity) 413 (MH⁺, 100). Anal.

(C₂₁H₂₆ClFO₅) C, H.

(E)-{4-[(3-Bromo-4-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2-Propylpentanoate

(L2-B12, Library 2). Oil; IR (neat) 3244 (OH), 1757 (CdO),

1658 (CdC) cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 7.65 (dd, J

) 6.5, 2.1 Hz, 1 H, ArH), 7.40 (t, J ) 2.7 Hz, 1 H, CdCH),

7.38 (dd, J ) 4.6, 2.2 Hz, 1 H, ArH), 7.16 (t, J ) 8.3 Hz, 1 H,

ArH), 4.25 (AB q, J ) 12.0 Hz, 2 H, CCH₂OC), 3.75 (AB q,

J ) 12.2 Hz, 2 H, CCH₂OH), 3.20 (dd, J ) 17.7, 2.9 Hz, 1 H,

H_3a), 2.95 (dd, J ) 17.7, 2.7 Hz, 1 H, H_3b), 2.73 (v br s, 1 H,

CCH₂OH), 2.32 (m, 1 H, C(O)CH(CH₂CH₂CH₃)₂), 1.48 (m, 2 H,

C(O)CH(CH₂CH₂CH₃)₂), 1.33 (m, 2 H, C(O)CH(CH₂CH₂CH₃)₂),

1.16 (m, 4 H, C(O)CH(CH₂CH₂CH₃)₂), 0.79 (t, J ) 6.4 Hz, 3 H,

C(O)CH(CH₂CH₂CH₃)₂), 0.75 (t, J ) 6.4 Hz, 3 H, C(O)CH(CH₂-

CH₂CH₃)₂). FAB-MS (m/z, relative intensity) 457 (MH⁺, 47), 57

(100). Anal. (C₂₁H₂₆BrFO₅) C, H, Br.

(E)-{4-[(4-Chlorophenyl)methylene]-2-(hydroxymethyl)-5-

oxo-2-2,3-dihydrofuryl}methyl 2-Ethylhexanoate (L2-C8, Li-

brary 2). Light yellow solid, mp ) 41-45 °C; IR (neat) 3384

(OH), 2959 (CH), 2932 (CH), 2873 (CH), 1733 (CdO), 1715

(CdO), 1650 (CdC) cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 7.48

(t, J ) 2.9 Hz, 1 H, CdCH), 7.39 (m, 4 H, ArH), 4.26 [2 AB

q, J ) 12.0 Hz (diastereomers), 2 H, CCH₂OC], 3.74 [2 AB q,

J ) 12.1 Hz (diastereomers), 2 H, CCH₂OH], 3.19 (dd, J )

17.7, 3.0 Hz, 1 H, H_3a), 2.96 (dd, J ) 17.6, 2.6 Hz, 1 H, H_3b),

2.40 (v br s, 1 H, CCH₂OH), 2.24 (m, 1 H, C(O)CH(CH₂-

CH₃)CH₂CH₂CH₂CH₃), 1.46 (m, 4 H, C(O)CH(CH₂CH₃)CH₂-

CH₂CH₂CH₃), 1.15 (m, 4 H, C(O)CH(CH₂CH₃)CH₂CH₂-

CH₂CH₃), 0.79 (m, 6 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂CH₃).

FAB-MS (m/z, relative intensity) 395 (MH⁺, 57), 57 (100).

Anal. (C₂₁H₂₇ClO₅) C, H, Cl.

(E)-{4-[(3-Bromo-4-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2-Ethylhexanoate (L2-

C12, Library 2). Oil; IR (neat) 3533 (OH), 2963 (CH), 2874

(CH), 1758 (CdO), 1739 (CdO), 1657 (CdC) cm^-1. ¹H NMR

(400 MHz, CDCl₃) δ 7.66 (dd, J ) 6.5, 2.2 Hz, 1 H, ArH),

7.44 (t, J ) 2.9 Hz, 1 H, CdCH), 7.40 (m, 1 H, ArH), 7.17 (t,

J ) 8.3 Hz, 1 H, ArH), 4.27 (2 AB q, J ) 12.0 Hz, 2 H,

CCH₂OC), 3.75 (AB q, J ) 11.9 Hz, 2 H CCH₂OH), 3.20 (dd,

J ) 17.7, 3.0 Hz, 1 H, H_3a), 2.95 (dd, J ) 17.7, 2.7 Hz, 1 H,

H_3b), 2.33 (br s, 1 H, CCH₂OH), 2.25 (m, 1 H, C(O)CH-

(CH₂CH₃)CH₂CH₂CH₂CH₃), 1.48 (m, 4 H, C(O)CH(CH₂-

CH₃)CH₂CH₂CH₂CH₃), 1.17 (m, 4 H, C(O)CH(CH₂CH₃)

CH₂CH₂CH₂CH₃), 0.80 (m, 6 H, C(O)CH(CH₂CH₃)CH₂CH₂CH₂-

CH₃). FAB-MS (m/z, relative intensity) 457 (MH⁺, 50), 57 (100).

Anal. (C₂₁H₂₆BrFO₅) C, H.

(E)-[2-(Hydroxymethyl)-5-oxo-4-({4-[4-(triﬂuoromethyl)phe-

nyl]phenyl}methylene)-2-2,3-dihydrofuryl]methyl 2-Phenylbu-

tanoate (L2-D6, Library 2). White solid, mp ) 113-115 °C;

IR (neat) 3729 (OH), 1745 (CdO), 1714 (CdO), 1648 (CdC)

cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (m, 2 H, ArH), 7.65

(m, 2 H, ArH), 7.51 (m, 5 H, CdCH and ArH), 7.12 (m, 4 H,

ArH), 4.27 (2 AB q, J ) 11.9 Hz (diastereomers), 2 H, CH₂OC),

3.62 (2 AB q, J ) 12.2 Hz (diastereomers), 2 H, CH₂OH), 3.43

(m, 1 H, C(O)CHPhCH₂CH₃), 3.08 (m (diastereomers), 1 H,

H_3a), 2.84 (m (diastereomers), 1 H, H_3b), 2.05 and 1.76 (m

(diastereomers), 2H, C(O)CHPhCH₂CH₃), 0.82(m, 3H, C(O)CH-

PhCH₂CH₃). FAB-MS (m/z, relative intensity) 525 (MH⁺, 100). Anal.

(C₃₀H₂₇F₃O₅) C, H.

(E)-{4-[(3-Chloro-4-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2,2-Dimethylpro-

panoate (L2-F10, Library 2). White solid, mp ) 138-140 °C;

IR (neat) 3495(OH), 2977 (CH), 1728 (CdO), 1717 (CdO),

1656 (CdC) cm^-1. H NMR (400 MHz, CDCl₃) δ 7.46 (m, 2

1

H, ArH), 7.22 (m, 2 H, CdCH and ArH), 4.25 (AB q, J )

12.0 Hz, 2 H, CCH₂OC), 3.75 (AB q, J ) 12.0 Hz, 2 H,

CCH₂OH), 3.21 (dd, J ) 17.6, 2.4 Hz, 1 H, H_3a), 2.96 (dd, J )

17.3, 1.8 Hz, 1 H, H_3b), 1.13 (s, 9 H, C(O)C(CH₃)₃). FAB-MS

(m/z, relative intensity) 371 (MH⁺, 74), 57 (100). Anal.

(C₁₈H₂₀ClFO₅) C, H.

(E)-{4-[(4-Chloro-3-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2-Ethylhexanoate (L2-

C11, Library 2). Oil; IR (neat) 3464 (OH), 2960 (CH), 2934

5218 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17

Duan et al.

1

(E)-{4-[(4-Chloro-3-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2,2-Dimethylpro-

panoate (L2-F11, Library 2). White solid, mp ) 188-190 °C;

IR (neat) 3377(OH), 2977 (CH), 2875 (CH), 1714 (CdO), 1654

1602 (CdC) cm^-1. H NMR (400 MHz, CDCl₃) δ 8.22 (s, 2

H, ArH), 7.71 (d, J ) 7.8 Hz, 1 H, ArH), 7.58 (m, 3 H, ArH

and CdCH), 7.22 (m, 3 H, ArH), 6.89 (s, 1 H, ArH), 4.51 (AB

m, 2 H, CCH₂OC), 3.86 (AB q, J ) 11.2 Hz, 2 H, CCH₂OH),

3.35 (dd, J ) 17.9, 3.1 Hz, 1 H, H_3a), 3.15 (dd, J ) 18.0, 2.4

Hz, 1 H, H_3b), 2.93 (s, 6 H, C(O)C₆H₄N(CH₃)₂), 2.35 (s, 1 H,

CCH₂OH). FAB-MS (m/z, relative intensity) 427 (MH⁺, 100),

426 (M^•+, 79). HRMS (FAB) calcd for C₂₂H₂₂N₂O₇(M⁺):

426.1427; found: 426.1439.

1

(CdC) cm^-1. H NMR (400 MHz, CDCl₃) δ 7.51 (dd, J )

6.9, 2.2 Hz, 1 H, ArH), 7.43 (t, J ) 2.9 Hz, 1 H, CdCH), 7.35

(m, 1 H, ArH), 7.19 (t, J ) 8.6 Hz, 1 H, ArH), 4.25 (AB q, J

) 12.0 Hz, 2 H, CCH₂OC), 3.75 (AB q, J ) 12.2 Hz, 2 H,

CCH₂OH), 3.21 (dd, J ) 17.6, 2.9 Hz, 1 H, H_3a), 2.95 (dd, J )

17.7, 2.6 Hz, 1 H, H_3b), 1.12 (s, 9 H, C(O)C(CH₃)₃). FAB-MS

(m/z, relative intensity) 371 (MH⁺, 69), 57 (100). Anal. (C₁₈H₂₀-

ClFO₅) C, H, Cl.

(Z)-{4-[(4-Chloro-3-ﬂuorophenyl)methylene]-2-(hydroxy-

methyl)-5-oxo-2-2,3-dihydrofuryl}methyl 2,2-Dimethylpro-

panoate (L2-F11, Library 2). White solid, mp ) 97-99 °C;

IR (neat) 3410 (OH), 1726 (CdO) cm^-1. ¹H NMR (400 MHz,

CDCl₃) δ 7.31 (t, J ) 7.9 Hz, 1 H, ArH), 7.01 (dd, J ) 9.7, 1.9

Hz, 1 H, ArH), 6.95 (dd, J ) 8.2, 1.3 Hz, 1 H, ArH), 6.87 (t,

J ) 1.5 Hz, 1 H, CdCH), 4.30 (AB q, J ) 11.9 Hz, 2 H,

CCH₂OC), 3.73 (AB q, J ) 12.1 Hz, 2 H, CCH₂OH), 3.56 (d,

J ) 1.4 Hz, 2 H, H_3ab), 1.10 (s, 9 H, C(O)C(CH₃)₃). FAB-MS

(m/z, relative intensity) 371 (MH⁺, 24), 57 (100). Anal.

(C₁₈H₂₀ClFO₅)

(E)-{2-(Hydroxymethyl)-4-[(3-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 2-(Dimethylamino)benzoate (Table

2). Yellow solid, mp ) 54-56 °C; IR (neat) 3480 (OH), 2885

(CH), 2833 (CH), 2801 (CH), 1757 (CdO), 1715 (CdO), 1597

(CdC) cm^-1. ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1 H, ArH),

8.22 (d, J ) 8.0 Hz, 1 H, ArH), 7.75 (d, J ) 7.8 Hz, 1 H,

ArH), 7.58 (m, 3 H, CdCH and ArH), 7.35 (m, 1 H, ArH),

6.94 (d, J ) 8.2 Hz, 1 H, ArH), 6.80 (t, J ) 7.4 Hz, 1 H, ArH),

4.49 (AB q, J ) 12.0 Hz, 2 H, CCH₂OC), 3.84 (br AB q, J )

12.2 Hz, 2 H, CCH₂OH), 3.35 (dd, J ) 17.9, 3.0 Hz, 1 H, H_3a),

3.15 (dd, J ) 17.9, 2.8 Hz, 1 H, H_3b), 2.79 (s, 7 H,

C(O)C₆H₄N(CH₃)₂and CCH₂OH). FAB-MS (m/z, relative

intensity) 427 (MH⁺, 100), 426 (M^•+, 18). Anal. (C₂₂H₂₂-

N₂O₇·H₂O) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(2-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 4-(Dimethylamino)benzoate (Table

2). Yellow solid, mp ) 173-175 °C; IR (neat) 3409(OH), 2926

(CH), 1756 (CdO), 1699 (CdO), 1603 (CdC) cm^-1. ¹H NMR

(400 MHz, CDCl₃) δ 8.09 (dd, J ) 8.2, 1.3 Hz, 1 H, ArH),

7.95 (t, J ) 2.9 Hz, 1 H, CdCH), 7.82 (d, J ) 9.1 Hz, 2 H,

ArH), 7.64 (td, J ) 7.6, 1.2 Hz, 1 H, ArH), 7.51 (m, 2 H, ArH),

6.69 (d, J ) 9.0 Hz, 2 H, ArH), 4.42 (AB q, J ) 12.1 Hz, 2 H,

CCH₂OC), 3.76 (AB q, J ) 12.2 Hz, 2 H, CCH₂OH), 3.16 (dd,

J ) 17.7, 3.2 Hz, 1 H, H_3a), 3.04 (s, 6 H, C(O)C₆H₄N(CH₃)₂),

2.92 (dd, J ) 17.7, 2.8 Hz, 1 H, H_3b). FAB-MS (m/z, relative

intensity) 427 (MH⁺, 53), 426 (M^•+, 40), 148 (100). Anal.

(C₂₂H₂₂N₂O₇) C, H, N.

C, H.

(E)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 3-(dimethylamino)benzoate (Table

2). Yellow solid, mp ) 173-175 °C; IR (neat) 3421 (OH), 1735

(CdO) cm^-1. H NMR (400 MHz, CDCl₃) δ 8.24 (d, J ) 8.9

1

Hz, 2 H, ArH), 7.56 (m, 3 H, CdCH and ArH), 7.25 (m, 3 H,

ArH), 6.93 (br s, 1 H, ArH), 4.51 (br s, 2 H, CCH₂OC), 3.85

(AB q, J ) 12.2 Hz, 2 H, CCH₂OH), 3.33 (dd, J ) 18.0, 3.2

Hz, 1 H, H_3a), 3.13 (dd, J ) 18.0, 2.8 Hz, 1 H, H_3b), 2.95 (s, 6

H, C(O)C₆H₄N(CH₃)₂), 2.27 (v br s, 1 H, CCH₂OH). FAB-MS

(m/z, relative intensity) 427 (MH⁺, 100), 426 (M^•+, 84). Anal.

(C₂₂H₂₂N₂O₇·0.3H₂O) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(4-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 2-(Dimethylamino)benzoate (Table

2). Yellow solid, mp ) 57-60 °C; IR (neat) 3486(OH), 2880

(CH), 1755 (CdO), 1714 (CdO), 1597 (CdC) cm^-1. ¹H NMR

(400 MHz, CDCl₃) δ 8.24 (d, J ) 8.8 Hz, 2 H, ArH), 7.59 (d,

J ) 8.8 Hz, 3 H, ArH), 7.56 (t, J ) 2.8 Hz, 1 H, CdCH), 7.35

(m, 1 H, ArH), 6.95 (d, J ) 8.2 Hz, 1 H, ArH), 6.80 (t, J ) 7.4

Hz, 1 H, ArH), 4.48 (AB q, J ) 12.0 Hz, 2 H, CCH₂OC), 3.83

(m, 2 H, CCH₂OH), 3.32 (dd, J ) 18.0, 3.0 Hz, 1 H, H_3a), 3.13

(dd, J ) 18.0, 2.8 Hz, 1 H, H_3b), 2.78 (s, 7 H, C(O)C₆H₄N(CH₃)₂

and CCH₂OH). FAB-MS (m/z, relative intensity) 427 (MH⁺,

100), 426 (M^•+, 28). HRMS (FAB) calcd for C₂₂H₂₂N₂O₇

(MH⁺): 427.1505; found: 427.1483.

(E)-{2-(Hydroxymethyl)-4-[(2-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 3-(Dimethylamino)benzoate (Table

2). Yellow solid, mp ) 62-64 °C; IR (neat) 3461(OH), 2880

(CH), 2813 (CH), 1756 (CdO), 1718 (CdO), 1604 (CdC)

1

cm^-1. H NMR (400 MHz, CDCl₃) δ 8.07 (dd, J ) 8.2, 1.2

Hz, 1 H, ArH), 7.91 (t, J ) 2.9 Hz, 1 H, CdCH), 7.63 (td, J )

7.7, 1.3 Hz, 1 H, ArH), 7.52 (m, 2 H, ArH), 7.25 (m, 3 H,

ArH), 6.91 (v br s, 1 H, ArH), 4.46 (AB q, J ) 12.0 Hz, 2 H,

CCH₂OC), 3.80 (AB q, J ) 12.2 Hz, 2 H, CCH₂OH), 3.15 (dd,

J ) 17.8, 3.2 Hz, 1 H, H_3a), 2.94 (m, 7 H, H_3band

C(O)C₆H₄N(CH₃)₂), 2.45 (v br s, 1 H, CCH₂OH). FAB-MS (m/

z, relative intensity) 427 (MH⁺, 100), 426 (M^•+, 69). Anal.

(C₂₂H₂₂N₂O₇·0.9H₂O) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(3-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 4-(dimethylamino)benzoate (Table

2). Yellow solid, mp ) 231-233 °C; IR (neat) 3379(OH), 1707

(CdO), 1608 (CdC) cm^-1. ¹H NMR (400 MHz, DMSO-d₆) δ

8.41 (s, 1 H, ArH), 8.25 (d, J ) 8.5 Hz, 1 H, ArH), 8.07 (d, J

) 7.6 Hz, 1 H, ArH), 7.76 (t, J ) 8.0 Hz, 1 H, ArH), 7.60 (m,

3 H, CHdCH, ArH), 6.65 (d, J ) 8.8 Hz, 2 H, ArH), 5.74 (s,

2 H, CCH₂OC), 5.40 (v br s, 1 H, CCH₂OH), 4.40 (s, 2 H,

CCH₂OC), 3.70 (AB q, J ) 11.9 Hz, 2 H, H_3ab), 2.98 (s, 6 H,

C(O)C₆H₄N(CH₃)₂). FAB-MS (m/z, relative intensity) 427

(MH⁺, 51), 426 (M^•+, 38), 148 (100). Anal. (C₂₂H₂₂N₂O₇·

0.25H₂O) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(2-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 2-(Dimethylamino)benzoate (Table

2). Yellow solid, mp ) 47-49 °C; IR (neat) 3509 (OH), 2795

1

(CH), 1757 (CdO), 1597 (CdC) cm^-1. H NMR (400 MHz,

CDCl₃) δ 8.07 (dd, J ) 8.2, 1.3 Hz, 1 H, ArH), 7.88 (t, J ) 2.9

Hz, 1 H, CdCH), 7.63 (m, 2 H, ArH), 7.52 (m, 1 H, ArH),

7.46 (d, J ) 7.7 Hz, 1 H, ArH), 7.36 (m, 1 H, ArH), 6.96 (d,

J ) 8.3 Hz, 1 H, ArH), 6.85 (t, J ) 7.4 Hz, 1 H, ArH), 4.44

(AB q, J ) 12.0 Hz, 2 H, CCH₂OC), 3.78 (AB m, 2 H,

CCH₂OH), 3.13 (dd, J ) 17.8, 3.1 Hz, 1 H, H_3a), 2.92 (dd, J )

17.7, 2.9 Hz, 1 H, H_3b), 2.80 (s, 7 H, C(O)C₆H₄N(CH₃)₂and

CCH₂OH). FAB-MS (m/z, relative intensity) 427 (MH⁺, 100),

426 (M^•+, 17). Anal. (C₂₂H₂₂N₂O₇·H₂O) C, H, N.

(E)-{2-(Hydroxymethyl)-4-[(3-nitrophenyl)methylene]-5-oxo-

2-2,3-dihydrofuryl}methyl 3-(Dimethylamino)benzoate (Table

2). Yellow solid, mp ) 160-162 °C; IR (neat) 3462(OH), 1717

(CdO), 2883 (CH), 2804 (CH), 1755 (CdO), 1718 (CdO),

Acknowledgment. This paper is dedicated to Dr. John S.

Driscoll, former Chief of the Laboratory of Medicinal Chemistry

Constrained Analogues of Diacylglycerol. 29.

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5219

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at NCI. This research was supported in part by the Intramural

Research Program of the NIH, National Cancer Institute, Center

for Cancer Research, and in part with federal funds from the

National Cancer Institute, National Institutes of Health, under

contract N01-CO-12400. 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 U.S. Government. This work was also supported in part by

the Korea Health 21 R&D Grant (A020601) from the Ministry

of Health and Welfare, by NIH Grant RO1 no. CA105125-02

to A.H.-F. and by NIH CA125632 to S.A.R. X-ray crystal-

lographic studies were supported in part under contract Y1-

DA6002 from the National Institute on Drug Abuse (NIDA).

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Supporting Information Available: Crystal data and structural

reﬁnement, atomic coordinates, bond lengths, anisotropic displace-

ment parameters, hydrogen coordinates and isotropic displacement

1

parameters, torsion angles and hydrogen bonds for L2-F11; H

NMR spectra, ¹³C NMR spectra, and combustion analysis results.

This material is available free of charge via the Internet at http://

pubs.acs.org.

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JM8001907

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Conformationally constrained analogues of diacylglycerol. 29. Cells sort diacylglycerol-lactone chemical zip codes to produce diverse and selective biological activities

DOI: 10.1021/jm8001907

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