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#-Arylidene Diacylglycerol-Lactones (DAG-Lactones) as Selective Ras Guanine Releasing Protein 3 (RasGRP3) Ligands Jihyae Ann, Agnes Czikora, Amandeep Saini, Xiaoling Zhou, Gary A. Mitchell, Nancy E. Lewin, Megan L Peach, Peter M. Blumberg, and Jeewoo Lee J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00661 • Publication Date (Web): 03 Jun 2018 Downloaded from http://pubs.acs.org on June 3, 2018

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Journal of Medicinal Chemistry

α-Arylidene Diacylglycerol-Lactones (DAG-Lactones) as Selective Ras Guanine Releasing Protein 3 (RasGRP3) Ligands

Jihyae Ann,† Agnes Czikora,‡ Amandeep S. Saini,‡ Xiaoling Zhou,‡ Gary A. Mitchell,‡ Nancy E. Lewin,‡ Megan L. Peach,§ Peter M. Blumberg,‡ Jeewoo Lee*,†

†

Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul

08826, Republic of Korea ‡

Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer

Institute, NIH, Bethesda, MD 20892, USA §

Basic Science Program, Leidos Biomedical Research Inc., Chemical Biology Laboratory,

Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, MD 21702, USA

* To whom correspondence should be addressed. Phone: 82-2-880-7846 Fax: 82-2-888-0649 E-mail: [email protected]

ACS Paragon Plus Environment

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Key Words: RasGRPs, Protein Kinase C, Diacylglycerol-Lactone.

ABSTRACT Diacylglycerol-lactones have proven to be a powerful template for the design of potent ligands targeting C1 domains, the recognition motif for the cellular second messenger diacylglycerol. A major objective has been to better understand the structure activity relations distinguishing the seven families of signaling proteins that contain such domains, of which the protein kinase C (PKC) and RasGRP families are of particular interest. Here, we synthesize a series of aryl/alkyl substituted diacylglycerol-lactones and probe their relative selectivities for RasGRP3 versus PKC. Compound 96 showed 73-fold selectivity relative to PKC alpha and 45-fold selectivity relative to PKC epsilon for in vitro binding activity. Likewise, in intact cells compound 96 induced Ras activation, a downstream response to RasGRP stimulation, with 8-29 fold selectivity relative to PKC delta S299 phosphorylation, a measure of PKC delta stimulation.


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INTRODUCTION Diacylglycerol (DAG) is a lipid second messenger that is produced through phosphoinositol 4,5-bisphosphate (PIP2) hydrolysis following the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D.1 Increased levels of DAG are transduced into cellular signals by binding to the C1 domains of the various PKC isoforms and six other families of proteins.2 The binding of DAG translocates the target proteins to membranes and may further lead to conformational change of the protein, as is the case with PKC, leading to enzyme activation. These proteins represent promising therapeutic targets for cancer, dementia, HIV/AIDS, and multiple other disorders, with multiple ligands in various points in the developmental pipeline.3 C1 domains are the major recognition motif for DAG and were first identified as lipidbinding modules in protein kinase C (PKC) isoforms. C1 domains are zinc-finger structures of approximately 50 amino acid residues containing a highly conserved structural motif.4 They have been classified based on their ligand binding properties into two classes: typical and atypical. Typical C1 domains bind and respond to DAG/phorbol ester and these are found in the conventional PKCs (PKCα, β, and γ) and novel PKCs (PKCδ, ε, η, and θ), as well as in the Ras Guanine Releasing Protein (RasGRPs), chimaerin, and Munc13 families, among others. The atypical C1 domains do not respond to DAG and these are so named for their presence in the atypical PKCs (PKCι and ξ) and many of the DAG kinase isoforms, among other examples.4 Reflecting the different patterns of expression and the complexities of signaling downstream from the multiple DAG-responsive proteins, ligands with selectivity between these various families have great potential for differential regulation of these downstream signaling pathways.

In this paper, we seek to further our understanding of the structural features


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conferring selectivity between representatives of the RasGRP and PKC families of DAGsignaling proteins, starting from a previously identified selective, lead DAG-lactone structure. It should be appreciated that the regulation of the C1 domain containing proteins is highly complex. Typical C1 domains have a hydrophobic binding site that inserts into the membrane to bind DAG or its analogs. Below the binding site there is a ring of positively charged residues that controls the intrinsic affinity of these C1 domains for anionic lipids. As illustrated in Figure 1 comparing the C1 domains of RasGRP3, conventional PKCα, and novel PKCε, subtle differences in binding site sequence and surface hydrophobicity and shape control the affinities in the ternary complex between receptor, ligand, and phospholipids, as well as cellular membrane targeting and translocation. Variable membrane compositions in the cell along with their microheterogeneity provide a powerful generator of diversity.5

Figure 1. Binding site surfaces of homology models of the C1 domains of RasGRP3 (A), PKCα C1a (B), and PKCε C1b (C). Surfaces were rendered in Chimera6, and colored according to electrostatic potential: neutral, hydrophobic regions are in grey, positively-charged regions are blue, and negatively-charged regions are red.

Additionally, extensive phosphorylation, protein adapters, and conformation dependent proteolysis play a further role.7 The present study focuses on assessing structural features


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contributing to differential selectivity for RasGRP versus PKC. While development of drug candidates requires attention to many additional features such as metabolic stability and bioavailability, understanding the basis for selectivity is an essential first step.

Figure 2. The structure of DAG-lactones

Previously, DAG-lactones were developed as ultrapotent DAG surrogates, overcoming the weak binding affinities of DAG to the C1 domain by exploiting conformational restriction (Figure 2).8 On the high affinity template of 5-acyloxymethyl-5’-hydroxymethyl γ–lactone (1), the two side chains at the sn-1 and sn-2 positions function as “chemical zip codes”, which provide specific interactions with the membrane in the chemical space outside the C1 domain of PKC and other C1-domain containing proteins. The DAG-responsive proteins vary in their patterns of tissue distribution, subcellular localization, substrate specificity, and biological function.5 Owing to the different lipid compositions of plasma membranes, nuclear membranes and membranes of cellular organelles, along with subdomains within these membranes, the interactions between the “chemical zip codes” and the characteristic lipid-water-protein microenvironment outside the C1-domain are thought to direct the DAG-lactone-C1-domain complexes to functionally distinct membrane locations producing unique biological responses.9


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Accordingly, we had explored a series of DAG-lactones containing homo/hetero aromatic moieties as R1 (next to the sn-1 carbonyl) and/or R2 (next to the sn-2 carbonyl) groups.10-12 Among the compounds, 5-(hydroxymethyl)-5’-(4-methoxybenzoyloxy)-3E-(4-nitrobenzylidene) γ–lactone

(130C032,

2)

and

5-(hydroxymethyl)-5’-(4-dimethylaminobenzoyloxy)-3E-(4-

nitrobenzylidene) γ–lactone (130C037, 3) displayed significant degrees of specificity for RasGRP3 comparing with PKCα (20 fold for 2 and 90 fold for 3).12 On the basis of this finding, we decided to investigate systematically the structure activity relationship (SAR) of α-arylidene DAG-lactones employing representative homo/hetero aryl and alkyl groups as their sn-1 and sn-2 substituents to evaluate PKC/RasGRP3 selectivity. Then, for further optimization, the two series of DAG-lactones, in which N-methylindolidene was fixed as the sn-2 substituent or the pivaloyl group was fixed as the sn-1 substituent, were examined. Finally, we characterized the most selective DAG-lactone (96) for its activity on RasGRP3 in intact cells.

RESULTS AND DISCUSSION Chemistry. In this study, we prepared a series of DAG-lactones incorporating a variety of heteroaryl substituents which were obtained from commercial sources or chemical synthesis. The synthesis of heteroaryl substituents was shown in Scheme 1. Indole 3-aldehyde derivatives (11-18) were synthesized by simple N-alkylation with sodium hydride and appropriate alkyl halides. Benzofuran-3-carbaldehyde (10) was prepared by the methyl oxidation with selenium (IV) oxide. Among the acyclic sn-1 substituents, 3-isobutyl-5-methylhexanoyl chloride (32) was prepared according to previous methods.9 Knoevenagel condensation of methylindole carbaldehyde (11)


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with malonate provided the α,β-unsaturated carboxylic acid (35).13 Commercial sources of benzo[b]thiophene-3-carbaldehyde (19), picolinaldehyde (20), p-phenyl derivatives (21-26), linear, cyclo- or branched- alkyl derivatives (27-31) and aryl acrylic acids (33-34) were directly used for R1, R2 substituents.

Scheme 1. Synthesis of indole derivatives and benzofuran-3-carbaldehydea

a

Reagents and conditions: (a) NaH, DMF, R-Br or R-I, 0 °C, 2-5 h; (b) Malonic acid, piperidine, pyridine, reflux, 2 h, (ii) 2N HCl, r.t., 5 h; (c) SeO2, 1,4-dioxane, sealed, 105 °C, 24 h.

The general synthesis of α-arylidine DAG-lactones is described in Scheme 2. The synthesis started from either racemic lactone 36 or 37, the choice of which was determined depending on lability during deprotection steps. In particular, for the synthesis of αheterobiarylidene DAG-lactones including 1-methylindolidene, the lactone 36 with the two TBDPS protecting groups was exclusively used due to its lability under the deprotection conditions with boron trichloride (BCl3) and ceric ammonium nitrate (CAN). Aldol reaction of


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the selected lactone with the aldehydes followed by β-hydroxy elimination provided the corresponding α-arylidenes, where the more stable E-isomer was generated as the major isomer. The geometry of E/Z-isomers was assigned based on 1H-NMR spectra in which the vinyl proton for the E-isomers displayed a characteristic multiplet that was farther downfield than that of corresponding Z-isomers.14 After separation of E/Z isomers at this stage, bis-TBDPS protected lactones (52-61) were deprotected with tetrabutylammonium fluoride (TBAF) and then selectively monoacylated with the corresponding acyl chloride to afford the final compounds 9699 and 116-132. On the other hand, the p-methoxyphenyl (PMP) group of 62-65 was deprotected with CAN to afford 76-79, which were acylated with the appropriate R1 acid or acyl chloride. The final deprotection of 80-95 using BCl3 provided the desired DAG-lactones 100-115.

Scheme 2. General synthesis of α-heteroarylidene DAG-lactones.a

a

Reagents and conditions: (a) LiHMDS, THF, -78 °C, 4 h; (b) MsCl, DBU, CH2Cl2, 0 °C, 2-15 h; (c) TBAF, THF, 0 °C, 2 h for 66-75 or CAN, CH3CN/H2O, 0 °C, 1 h. for 76-79; (d) i) EDC, DMAP, CH2Cl2, r.t., 2-15 h for R1CO2H or TEA, DMAP, CH2Cl2, r.t., 2-3 h for R1COCl; (e) BCl3, CH2Cl2, -78 °C, 1.5 h.


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Biological Evaluation. Binding of ligands to PKCα, PKCε and RasGRP3. For the biological studies, we selected PKCα and PKCε as representative of classical PKCs and novel PKCs, respectively, following previous studies assessing the selectivity of DAG-lactones between PKC and RasGRP isoforms.11 In most cases, the E-isomer of the αarylidene was investigated because of the reported higher binding affinities of E-isomers compared to that of the corresponding Z-isomers.12 The binding affinities of compounds for PKCα, PKCε and RasGRP3 were measured by competition with [20-3H]phorbol 12,13dibutyrate ([3H]PDBu) in the presence of 100 µg/mL phosphatidylserine to determine the inhibition constants (Ki) of the DAG-lactones. The dissociation constants (Kd) of the isolated proteins for [3H]PDBu were measured in the presence of 100 µg/mL PS and previously determined by our laboratory.15 Initially, we selected the five representatives of the α-arylidene group (referred to as the sn-2 moiety), namely N-methylindole, pyridine, 4-dimethylaminophenyl, 4-methoxyphenyl, 4chlorophenyl, and four representatives of the O-acyl group (referred to as the sn-1 moiety), namely pivaloyl, 4-chlorobenzoyl, 4-methoxybenzoyl and 4-dimethylaminobenzoyl. The combination provided a series of twenty DAG-lactones (96-115) (listed in Table 1) to be investigated. The lipophilicity (ClogP) of the compounds, calculated by PerkinElmer ChemDraw Ultra v. 16.0., ranged from 1.95 to 4.11, In order to quantify the selectivity for RasGRP3, the ratios for PKC isoforms/RasGRP3 were calculated and listed in Table 1. Generally, all DAG-lactones in Table 1 were appreciably more selective for RasGRP3 than for PKCα and PKCε, with the range of selectivity from 1 to 72 (mean = 16), contrasting


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with a selectivity of 0.2 for phorbol 12,13-dibutyrate (PDBu). The analysis of structure activity relationship indicated that the selectivity for RasGRP3 was more susceptible to the nature of the sn-1 substituents than the sn-2 substituents. Among the set of DAG-lactones containing each αarylidene group, the compounds with the pivaloyl group (R1= t-butyl) at the sn-1 position exhibited the highest selectivity for RasGRP3. For examples, 96, 104, 108 showed high ratios of PKCα/RasGRP3 with values above 60 and 96, 100 exhibited ratios of PKCε/RasGRP3 above 45. Overall, 96 with R1 = t-butyl (sn-1 moiety) and R2 = N-methylindole (sn-2 moiety) displayed the highest selectivity with PKCα/RasGRP3 = 72.6 and PKCε/RasGRP3 = 45.1. In terms of absolute affinities, 96 exhibited a similar binding affinity to RasGRP3 as did PDBu but displayed 450- and 360-fold lower affinities to PKCα and PKCε, respectively, than did PDBu.

Table 1. Binding affinities of α-arylidene DAG-lactones for PKCs and RasGRPs.

Ki(nM)a R1

R2

ClogP

[3H]PDBub

ratio

hPKCα

hPKCε

RasGRP3

0.28

0.22

1.17

PKCα/ GRP3 0.24

PKCε/ GRP3 0.19

96

2.80

127 (±3.2)

78.9 (±5.5)

1.75 (±0.34)

72.6

45.1

97

3.32

146 (±38)

208 (±40)

11.2 (±1.1)

13

18.5

98

2.64

446 (±62)

380 (±17)

26.5 (±2.3)

16.8

14.3

99

3.05

186 (±23)

220 (±44)

18.2 (±2.1)

10.2

12.1


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100

2.36

3640 (±490)

1124 (±57)

192 (±3)

19

59.2

101

2.11

383 (±32)

92.3 (±4.5)

29.0 (±4.9)

13.2

3.18

102

2.63

1188 (±55)

199 (±24)

89.7 (±9.5)

13.2

2.22

103

1.95

1480 (±190)

328 (±41)

271 (±19)

5.4

1.21

104

3.15

65.9 (±1.9)

7.87 (±0.21)

1.07 (±0.20)

61.6

7.36

105

3.56

25.4 (±4.6)

6.39 (±0.92)

1.81 (±0.18)

14

3.53

106

3.30

128 (±1.1)

18.2 (±4.2)

7.48 (±1.25)

17.2

2.43

107

3.85

252 (±37)

39.4 (±7.4)

26.8 (±3.7)

9.4

1.47

108

3.42

72 (±11)

10.1 (±0.11)

1.09 (±0.13)

65.6

9.3

109

2.74

30.1 (±6.0)

6.80 (±0.64)

1.54 (±0.04)

19.5

4.42

110

3.16

84.7 (±4.1)

13.4 (±1.1)

4.17 (±0.77)

20.3

3.2

111

2.89

82.2 (±4.8)

18.3 (±0.8)

7.0 (±1.1)

11.7

2.61

112

3.58

13.6 (±1.7)

2.57 (±0.20)

0.53 (±0.06)

25.7

4.85

113

4.11

25.2 (±6.4)

6.51 (±0.68)

2.69 (±0.42)

9.4

2.42

114

3.42

27.6 (±1.7)

7.1 (±1.3)

2.25 (±0.21)

12.3

3.16

115

3.85

30.4 (±2.1)

9.5 (±1.5)

2.73 (±0.13)

11.1

3.48

Values represent mean ± SEM of triplicate or more independent experiments (where indicated by italics, ± range of duplicate experiments). b Kd (nM) a

Proceeding from the notable result with 96, we next sought to optimize the discrimination between PKCs and RasGRPs by modifying the sn-1 and sn-2 substituents of 96 with various


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aryl/alkyl groups, respectively. For this study, we investigated the two series of DAG-lactones with R2 = N-methylindole (Table 2) fixed as the sn-2 substituent or with R1 = t-butyl (Table 3) fixed as the sn-1 substituent, respectively. The series of α-N-methylindolidene DAG-lactones having the fixed sn-2 substituent of 96 are shown in Table 2. Our previous findings indicated that a highly branched acyl group increased the stability of the DAG lactones and their membrane affinity. Therefore, we replaced the t-butyl moiety of 96 with representatives of straight, branched and cyclo alkyl groups and aryl acrylates which mimic sn-2 arylidene moieties. The SAR analysis indicated that the branched analogues (117-120) showed higher binding affinities for PKC isoforms, while retaining their binding affinities for RasGRP3, resulting in lower selectivity to RasGRP3. In particular, 119 showed excellent binding affinity to RasGRP3 with Ki = 0.84 nM and a little higher PKCε/GRP3 ratio than that of 96. However, the more lipophilic and branched side chain in 120 reduced the binding affinity for RasGRP3, while displaying similar binding affinities for PKCs. The aryl acrylate substituted analogues (121-123) showed significant differences from the others. They all exhibited much reduced binding affinities for PKCs and RasGRP3 but retained as high a ratio of PKCε/GRP3 (48.9 in 122, 50.0 in 123) as did 96. Another characteristic observed in the series of α-N-methylindolidene DAG-lactones was reversal in the selectivity among PKCs relative to RasGRP3. Generally, whereas the DAGlactones described in Table 1 generally exhibited a higher selectivity ratio for PKCα/RasGRP3 than for PKCε/RasGRP3, the α-N-methylindolidene DAG-lactones in Table 2 displayed higher ratios for PKCε/RasGRP3. Nevertheless, none of the compounds were appreciably more selective than was the parent compound 96.


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Table 2. Binding affinities of α-methylindolelidene DAG-lactones

R1

a b

ClogP

Ki(nM)a

ratio

hPKCα

hPKCε

RasGRP3

PKCα/GRP3

PKCε/GRP3

96

2.80

127 (±3.2)

78.9 (±5.5)

1.75 (±0.34)

72.6

45.1

116

3.19

135 (±11)

122 (±9)

5.3 (±1.3)

25.7

23.2

117

4.39

31.3 (±1.5)

42.0 (±7.5)

2.15 (±0.47)

14.6

19.6

118

2.84

112 (±29)

98.2 (±9.8)

2.86 (±0.43)

39.0

23.9

119

3.76

31.7 (±1.3)

42.8 (±2.2)

0.84 (±0.07)

37.6

50.8

120

4.42

39.0 (±2.1)

41.5 (±6.9)

5.07 (±0.35)

7.69

8.17

121

3.11

439 (±32)

672 (±73)

25.0 (±3.2)

17.6

26.9

122

2.19

2308 (±64)

3408 (±520)

149 (±79)

33.1

48.9

123

2.88

375 (±73)

780 (±130)

15.7 (±3.7)

24.0

50.0

Values represent mean ± SEM of triplicate independent experiments [Kd (nM)]

The series of O-pivaloyl DAG-lactones having the fixed sn-1 substituent of 96 are shown in Table 3. Previously, it had been found that the orientation of the N-methylindolidene group at the sn-2 position was critical for the selectivity of PKCs/RasGRP3 and its N-methyl group was also important for the interaction with the lipid environment.16,17 Therefore, we further modified the N-methylindole group of 96 with N-alkylindoles (124-126) and 5-substituted-N-


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methylindoles (127-129), along with its bioisosteric heterocycles including benzofuran (130), benzothiophene (131), and indazole (132). In this series, we investigated the two geometric E/Z isomers, respectively, except when only one isomer was obtained in the synthesis (124-127 and 132). In the more lipophilic N-alkylindoles (124-126), whereas their binding affinities to the PKC isoforms increased, those to RasGRP3 decreased, resulting in a reduced selectivity ratio to RasGRP3. In the 5-substituted N-methylindole analogues (127-129), whereas the lipophilic 5chloro substituent (128) enhanced binding affinity for both PKCs and RasGRP3 while keeping high selectivity for RasGRP3, the more hydrophilic 5-nitro substituent (129) almost abolished the activity. For the bioisosteric heterocyclic analogues (130-132), their binding affinities for PKCs and RasGRP3 improved as their lipophilicities increased (ClogP: 131 (3.83) > 130 (3.36) > 96 (2.80) > 132 (2.30)). Nevertheless, the selectivity for RasGRP3 was lower than that of 96. The analysis of geometric isomers in this series indicated that both binding affinity for PKCs and RasGRP3 and selectivity for RasGRP3 were more favorable with the E-isomer than the Zisomer, as was previously found for diaryl DAG-lactones.5

Table 3. Binding affinities of O-pivaloyl DAG-lactones

R2

96

ClogP

2.80

Ki(nM)a

ratio

hPKCα

hPKCε

RasGRP3

PKCα/GRP3

PKCε/GRP3

127 (±3.2)

78.9 (±5.5)

1.75 (±0.34)

72.6

45.1


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124E

4.85

25.65 (±0.94)

19.18 (±0.65)

1.87 (±0.16)

13.70

10.24

125E

4.72

15.6 (±1.5)

19.36 (±0.16)

3.03 (±0.11)

5.16

6.38

126E

4.23

29.0 (±1.6)

60.6 (±7.4)

3.58 (±0.23)

8.10

16.93

127E

3.32

249 (±10)

208 (±7.4)

4.76 (±0.13)

52.18

43.71

128E

4.02

42.3 (±1.4)

39.3 (±2.7)

1.01 (±0.04)

41.82

38.81

128Z

4.02

79.1 (±3.4)

103.6 (±7.0)

4.32 (±0.38)

18.31

23.99

129E

3.09

>30,000

>30,000

3490 (±160)

NA

NA

129Z

3.09

>30,000

>30,000

1390 (±160)

NA

NA

130E

3.36

114 (±2.3)

35.3 (±1.8)

2.8 (±0.29)

40.70

12.62

130Z

3.36

62.5 (±1.7)

73.3 (±3.0)

11.67 (±0.47)

5.35

6.28

131E

3.83

28.77 (±0.82)

8.17 (±0.43)

1.67 (±0.13)

17.22

4.89

131Z

3.83

37.0 (±2.9)

12.5 (±0.12)

4.26 (±0.16)

8.68

2.93

132E

2.30

183.8 (±7.6)

130.9 (±3.9)

7.22 (±0.69)

25.45

18.00

a b

Values represent mean ± SEM of triplicate independent experiments [Kd (nM)]


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Further characterization of the activity of 96. The binding analysis revealed multiple compounds with substantial in vitro selectivity for binding to RasGRP3 versus PKCα and PKCε as well as good binding potency. Among these, we chose the lead structure, 96, for further characterization. A model of the ternary RasGRP3 + compound 96 + membrane bilayer complex is shown in the Supporting Information.

First, we examined the potency of 96 for RasGRP1 for comparison with that for RasGRP3 (Figure 3). Compound 96 showed similar binding affinity for both. We likewise had previously observed lack of selectivity between RasGRP1 and RasGRP3 for a pair of diaryl DAG lactones12 and for a limited series of positional isomers of indololactones17 that again showed good selectivity relative to PKC.

Figure 3. Dose response curves of 96 for binding to RasGRP and PKC isoforms. Binding of [3H]PDBu to PKCα, PKCε, RasGRP1, and RasGRP3 was measured in the presence of 100 µg/ml PS and half-log increasing concentrations of 96. Results are from single, representative experiments. Values, mean ± SE. Where error bars are not visible, they are smaller than the symbols and thus not visible. All experiments were performed at least 3 times.


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Cellular activity of 96. The in vitro analysis on the binding potencies of 96 for RasGRP1/3 and for PKCα/ε was carried out in the presence of 100 µg/mL phosphatidylserine. In the intact cell, potency will be influenced both by the different phospholipid compositions of the various membranes together with the complex influences of the intracellular environment, including phosphorylation and interaction with other proteins. We therefore examined the response to 96 in three different cell lines: LNCaP, HEK293, and Ramos. HEK293 and LNCaP cells were chosen because of a difference in their membranes. Unlike HEK293 cells, LNCaP cells are mutant in PTEN, leading to highly elevated levels of phosphatidylinositol 3,4,5-triphosphate, which has been reported to interact with the PT region of RasGRP1 to enhance its membrane binding.18 RasGRP3 lacks this PT domain and might therefore act differently from RasGRP1 in cells with a PTEN mutation. Since HEK293 and LNCaP cell lines have low endogenous RasGRP expression, we expressed exogenous GFP-tagged RasGRP1/3 DNA in these cell lines. In contrast, Ramos cells express RasGRP3 endogenously, where it plays a central role in cell signaling,19 so we were able to use these cells to measure the RasGRP3 response in the absence of overexpression. While there is not a convenient readout of in vivo activation for most PKC isoforms, phosphorylation of PKCδ at S299 provides a measure of activation for this isoform.20 We therefore monitored the response of endogenous PKCδ using an antibody specific for phosphorylation of PKCδ at this site. RasGTP activation, the immediate downstream response to RasGRP1/3, was detected using a pulldown assay with anti-Pan-Ras monoclonal antibody. Phosphorylation of Erk1/2 provided a measure of response further downstream from RasGRP1/3 but is less informative because it is also subject to activation through PKC mediated pathways independent of RasGRP1/3. To


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ensure uniform overexpression of the GFP-tagged RasGRP1/3, we detected GFP levels using anti-GFP antibody.

Figure 4. Activation of Ras upon treatment with 96 of (A) HEK293 cells over-expressing RasGRP1 or RasGRP3, (B) LNCaP cells over-expressing RasGRP1 or RasGRP3, and C) Ramos cells which endogenously express RasGRP3. After 48 h, cells were treated with 96 at the indicated doses, PMA (1000 nM), or DMSO (vehicle control) at 37 °C for 30 min in a 5% CO2 atmosphere. The cells were then lysed, and the levels of activation of endogenous Ras protein were evaluated by pull-down of the activated Ras and detection by immunoblotting with antiPan-Ras antibody. Representative immunoblots from three independent experiments under each set of conditions are illustrated.

Figure 5. Activation of signaling pathways downstream of RasGRPs. HEK293 (A) and LNCaP (B) cells overexpressing RasGRP3/1 and Ramos cells (C), which endogenously express RasGRP3, were incubated with the indicated concentrations of 96, DMSO (negative control) and 1000 nM PMA (positive control) at 37 °C for 30 min in a 5% CO2 atmosphere. Cell lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels and subjected to immunoblotting


18

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with anti-phospho-PKCδS299 and anti-phospho-ERK antibodies. Equal loading of all lanes was confirmed by blotting of the RasGRP3/1-overexpressing cells with antibody directed against the GFP tag incorporated into the RasGRP3/1 constructs and for Ramos cells with anti-actin antibody. Representative images from three independently performed experiments are shown. Representative results for activation of Ras by 96 are illustrated in Figure 4. Representative results for the activation by 96 of PKCδ, as detected by PKCδS299 phosphorylation, and for activation of ERK1/2, as detected by its phosphorylation, are illustrated in Figure 5. Response was compared to that by a maximally effective dose of PMA (1000 nM). The gels were quantitated and the EC50 values for the multiple experimental replicates were determined (Table 4).

Table 4. Quantitative comparison of the EC50 values for response to 96 for different overexpressed cell lines EC50 (µM) Selectivity PKCδ(S299) pERK RasGTP:PKCδ 12.6 (±2.6) 14.7 4.88 (±2.61)

RasGTP HEK293

LNCaP Ramos

Selectivity pERK:PKCδ 2.6

RasGRP1

0.86 (±0.26)

RasGRP3

1.83 (±0.38)

16.9 (±7.3)

9.2

4.66 (±0.81)

3.6

RasGRP1

0.97 (±0.38)

8.03 (±2.52)

8.3

3.73 (±1.66)

2.2

RasGRP3

0.64 (±0.31)

8.28 (±1.07)

12.9

2.98 (±1.21)

2.8

RasGRP3

0.86 (±0.22)

24.6 (±5.4)

28.6

6.09 (±1.92)

4.0

0.082 (±0.033)

0.29 (±0.02)

3.5

N/A

N/A

HEK RasGRP3 (PMA)

The EC50 (µM) values for HEK293 and LNCaP cells overexpressing RasGRP1 or RasGRP3 or for the Ramos cells were determined. Levels of RasGTP, pPKCδS299, and pERK were determined from scanning of western blots and analysis using ImageJ and GraphPad Prism 6 software. Image intensities were normalized to those for 1000 nM PMA in the same experiment. For comparison, the EC50 values for PMA in HEK cells overexpressing RasGRP3 are also included. All values represent the mean ± SEM (n = 3 independent experiments). EC50 values above 10 µM represent extrapolation and are therefore approximate, since the maximal 96 concentration tested was 10 µM.

In the cells over-expressing RasGRP1 or RasGRP3, the EC50 of 96 for Ras activation ranged from 0.64 µM to 1.83 µM. The overexpressed RasGRP1 and RasGRP3 behaved similarly to one another, consistent with the previous observations on their in vitro binding behavior. No


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difference was seen between the HEK293 and the LNCaP cells for sensitivity to activation of RasGRP1, despite the presence of the PTEN mutation in the LNCaP cells, which should have favored membrane association of RasGRP1 in those cells.18 The endogenously expressed RasGRP3 in the Ramos cells behaved similarly to the overexpressed RasGRP1/3 in the other cell types. As was typical, all of the in vivo values reflected weaker potencies than those found in vitro, presumably reflecting issues of uptake, metabolism, and intracellular context. We conclude that the sensitivity of RasGRP1/3 does not appear to depend to a large degree on the specific cell in which it is present.

(A)


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(B)

Figure 6. Selectivity of 96 for activation of endogenous Ras. A) Comparison of the EC50 values from Table 4. B) Relative EC50 values for activation of Ras compared to those for PKCδ and ERK (from Table 4). These experiments also provided an opportunity to compare the selectivity of 96 for RasGRP1/3 relative to PKCδ (Figure 6, Table 4). PKCδ was clearly less sensitive, with EC50 values for PKCδ S299 phosphorylation all above 8 µM. While those EC50 values above 10 µM represent extrapolation, since 10 µM was the maximum concentration of 96 tested in the dose response curves, the approximate selectivities for RasGRP1/3 over PKCδ ranged from 8.3 to 28.6-fold. For comparison, the EC50 values for activation of Ras and PKC in response to PMA in the HEK293 cells overexpressing RasGRP3 yielded a 3.5-fold difference. Previously, we have examined phosphorylation of Erk1/2 downstream of RasGRP as a surrogate for Ras activation in response to DAG-lactones.12,17 Here, we observe that the EC50 values for Erk1/2 phosphorylation in response to 96 were intermediate between those for Ras and PKC activation (Table 4), presumably reflecting that Erk1/2 phosphorylation reflects both activation of RasGRP as well as one or more pathways independent of RasGRP.12,17 In conclusion, we confirm here that 96 shows selectivity in vivo for activation of RasGRP1/3 relative to PKCδ. Despite the possibility that a PTEN mutation might have a substantial effect on


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RasGRP1 responsiveness and correspondingly on response of RasGRP1 versus RasGRP3, only quite modest differences were seen in the various cellular contexts examined.

Conclusion In this study, we investigated the structure activity relationship of a series of α-arylidene DAG-lactones employing representative sn-1 and sn-2 substituents to explore RasGRP3 selectivity over PKC isoforms. Through our systematic effort, we concluded that O-pivaloyl αN-methylindolidene DAG-lactone (96) showed the highest selectivity to RasGRP3 with a ratio of PKCα/RasGRP3 = 72.6 and PKCε/RasGRP3 = 45.1. Compared to PDBu, the high selectivity of 96 was derived from the reduction in the binding affinity for PKC isoforms while retained the affinity for RasGRP3. Starting with characterization to confirm its binding potency for RasGRP1 and RasGRP3 versus PKC isozymes (PKCα and PKCε), we evaluated its in vivo activity with different cell lines. Finally, although we confirmed the selectivity of 96 for activation of RasGRP1/3 compared to PKCδ in vivo, the contribution to this result of the complex responses downstream of the DAG-activated pathways remains to be defined.

Experimental Section General. All chemical reagents were commercially available. Melting points were determined on a melting point Buchi B-540 apparatus. Silica gel column chromatography was performed on silica gel 60, 230–400 mesh, Merck. H-NMR were recorded on a JEOL JNM-LA 300 at 300 MHz, Bruker Analytik, DE/AVANCE Digital 400 at 400 MHz, and Bruker Analytik DE/AVANCE Digital 500 at 500 MHz C-NMR were recorded on Bruker Analytik DE/AVANCE Digital 500 at 125 MHz, and JEOL JNM-ECA-600 at 150 MHz. Chemical shifts


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are reported in ppm units with Me4Si as a reference standard. Mass spectra were recorded on a VG Trio-2 GC−MS instrument and a 6460 Triple Quad LC−MS instrument. All final compounds were assessed for purity by high performance liquid chromatography (HPLC) on Agilent 1120 Compact LC (G4288A) system via the following conditions. Column: Agilent TC-C18 column (4.6 mm × 250 mm, 5 µm). Mobile phase A: 0.1% TFA water. Mobile phase B: MeOH (10:90, v/v). Wavelength: 254 nM. Flow: 1.0 mL/min. According to the HPLC analyses, all final compounds showed a purity of ≥95%.

General Procedure for Alkylation. A stirred solution of lactone 36 (or 37) (1 eq) in anhydrous THF (5 mL/mmol) was cooled to -78 °C under nitrogen and treated dropwise with LiHMDS (3 eq, 2 M solution in THF). The mixture was stirred at -78 °C for 30-60 min and lithium enolate formation was detected by TLC (usual system, Hex:EtOAc = 4:1). A solution of the corresponding aldehyde (1.4 to 4 eq) in anhydrous THF (1 mL/mmol) was added dropwise to the enolate at the same temperature and stirring at -78 °C was continued for 1.5-2 h. The reaction was warmed to room temperature for 15 h and quenched by the slow addition of a saturated aqueous solution of ammonium chloride. The aqueous layer was extracted three times with Et2O, and the combined organic extracts were washed three times with water, twice with brine, dried over MgSO4. After filtration, the filtrate was concentrated in vacuo, the obtained mixture of βhydroxy-lactone diastereomers were typically used directly in the following step without further purification.

General procedure for Mesylation-Olefination. A solution of the alkylation product (1 eq) in CH2Cl2 at 0 °C was treated with methanesulfonyl chloride (2 eq) and triethylamine (4 eq). The


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mixture was stirred at 0 °C for 30 min and then for 2 h at room temperature. 1,8Diazabicyclo[5.4.0]undec-7-ene (DBU, 5 eq) was added at 0 °C, and the resulting solution was stirred overnight at ambient temperature. The reaction mixture was concentrated to a brown syrup and the residue was extracted with EtOAc followed by 1 N HCl. The combined organic extracts were washed three times with water, twice with brine, dried over MgSO4, and filtered. The combined organics were purified by flash column chromatography after eluting with the appropriate solvent.

General procedure for Deprotection. A. TBDPS deprotection. TBAF (2 eq) was added slowly to a stirred solution of the olefination product (1 eq) in THF at 0 °C and stirring continued as the temperature was allowed to rise to room temperature. The reaction was monitored by TLC and the solution was concentrated in vacuo upon completion without further work-up. Purification by silica gel column chromatography (gradient elution) gave the desired deprotected diol-lactone. B. PMP deprotection. A stirred solution of the olefination product (1 eq) in acetonitrile/water (4:1) was treated with CAN (3 eq) while under nitrogen at 0 °C, and the reaction was monitored by TLC. After 30 min, the reaction was quenched by addition of aqueous NaHCO3 solution and the resulting solution was extracted with CH2Cl2, dried over MgSO4, and concentrated in vacuo. The residue was purified by column chromatography to give the deprotected product. C. Benzyl deprotection. BCl3 (3 eq) was added slowly to a stirred solution of the olefination product (1 eq) in CH2Cl2 at -78 °C. The reaction was monitored by TLC and quenched by addition of MeOH at 0 °C. The reaction mixture was concentrated in vacuo and purified by silica gel column chromatography to give the deprotected product.


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General Procedure for Acylation. Method A. A solution of bis-hydroxy compound (1 eq) in CH2Cl2 was treated with anhydrous pyridine (2 eq) and reacted with the corresponding acid chloride (1.1 eq) at 0 °C. The reaction was stirred at room temperature and monitored by TLC. Upon completion, the reaction was terminated by adding H2O. The organic layer was extracted with CH2Cl2, dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography to give the desired mono-substituted compound along with bis-substituted compound. Method B. A solution of bis-hydroxy compound (1 eq) in CH2Cl2 was treated with EDC (1.1 eq) and dimethylaminopyridine (0.3 eq) and reacted with the corresponding acid (1 eq). The reaction mixture was stirred at room temperature and monitored by TLC. Upon completion, the reaction was terminated by adding H2O. The organic layer was extracted with CH2Cl2, dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel flash column chromatography to give the desired mono-substituted compound along with bis-substituted compound. Method C. A solution of mono-deprotected compound (1 eq) in CH2Cl2 was treated with Et3N (3 eq) and dimethylaminopyridine (2.5 eq) and reacted with the corresponding acid chloride (1.21.5 eq). The reaction was stirred at room temperature and monitored by TLC. Upon completion, the reaction was concentrated in vacuo and purified by column chromatography to give the corresponding product. The final compounds were obtained after the deprotection of benzyl group.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl pivalate (96). Following general Method A, 96 was obtained as a white solid, 52%


25


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yield; mp = 123-125 °C; 1H NMR (400 MHz, CDCl3) δ 7.94 (bt, 1H, >CH=C), 7.82 (d, 1H, J = 7.80 Hz, indole), 7.30-7.36 (m, 2H, indole), 7.26-7.28 (m, 2H, indole), 4.38 (AB d, 1H, J = 11.96 Hz, CO2CHH), 4.23 (AB d, 1H, J = 11.96 Hz, CO2CHH), 3.86 (s, 3H, indole-NCH3), 3.69-3.77 (m, 2H, HOCH2), 3.04 (dd, 1H, J = 17.1, 2.4 Hz, CHH-lactone), 2.82 (dd, 1H, J = 17.1, 2.4 Hz, CHH-lactone), 2.24 (t, 1H, J = 5.4 Hz, HOCH2), 1.15 (s, 9H, -(CH3)3.

13

C NMR (125 MHz,

CDCl3) δ 178.60, 171.27, 135.19, 131.26, 128.92, 127.86, 127.40, 123.75, 118.65, 117.84, 111.42, 110.87, 82.91, 65.72, 64.84, 39.00, 33.72, 33.08, 29.69, 27.06 (2C). Anal. HPLC 98% (Rt = 8.60 min). HRMS (ESI) calc. for C21H25NO5 [M + H]+ 372.1733, found 372.1815.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl 4-chlorobenzoate (97). Following general Method A, 97 was obtained as a yellow solid, 48% yield; mp = 158-159 °C; 1H NMR (500 MHz, CDCl3) δ 7.97 (bt, 1H, >CH=C), 7.87 (d, 2H, J = 8.60 Hz, ClOC6H4CO2), 7.81 (d, 1H, J = 7.90 Hz, indole), 7.69-7.36 (m, 6H, indole and ClOC6H4CO2), 4.63 (AB d, 1H, J =11.95 Hz, CO2CHH), 4.45 (AB d, 1H, J =11.95 Hz, CO2CHH), 3.85 (s, 3H, NCH3), 3.78-3.82 (m, 2H, HOCH2), 3.10 (dd, 1H, J = 16.95, 2.7 Hz, CHH-lactone), 2.93 (dd, 1H, J = 16.95, 2.7 Hz, CHH-lactone), 2.25 (t, 1H, J = 6.7 Hz, HOCH2-). 13

C NMR (125 MHz, CDCl3) δ 169.59, 169.56, 138.56, 138.53, 132.71, 131.24, 128.97 (2C),

127.82 (2C), 127.44 (2C), 127.41, 126.41, 122.35, 122.33, 122.32, 114.92, 86.22, 67.41, 67.39, 33.23, 32.85. Anal. HPLC 96% (Rt = 8.12 min). HRMS (ESI) calc. for C23H20ClNO5 [M + H]+ 426.1035, found 426.1108.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl 4-methoxybenzoate (98). Following general Method A, 98 was obtained as a yellow


26

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solid, 53% yield; mp = 119-121 °C; 1H NMR (300 MHz, CDCl3) δ 7.93 (bt, 1H, >CH=C), 7.86 (d, 2H, J = 8.97 Hz, CH3OC6H4CO2), 7.77 (d, 1H, J = 7.71 Hz, indole), 7.23-7.29 (m, 4H, indole), 6.78 (d, 2H, J = 8.97 Hz, CH3OC6H4CO2), 4.57 (AB d, 1H, J =11.88 Hz, CO2CHH), 4.36 (AB d, 1H, J =11.88 Hz, CO2CHH), 3.80 (s, 3H, NCH3), 3.75 (s, 3H, CH3OC6H4CO2), 3.68-3.74 (m, 2H, HOCH2), 3.07 (d, 1H, J = 16.86 Hz, CHH-lactone), 2.88 (d, 1H, J = 16.86 Hz, CHH-lactone), 1.97 (m, 1H, HOCH2-).

13

C NMR (125 MHz, CDCl3) δ 169.56, 166.27, 163.50,

137.72, 135.57, 132.67, 131.94, 127.44, 126.37, 122.93, 122.33, 121.11, 119.06, 114.92, 113.92, 109.76, 86.22, 67.39, 67.36, 65.13, 65.11, 55.32, 33.26, 32.85. Anal. HPLC 99% (Rt = 7.83 min). HRMS (ESI) calc. for C24H23NO6 [M + H]+ 422.1510, found 422.1611.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl 4-(dimethylamino)benzoate (99). Following general Method A, 99 was obtained as a pale yellow solid, 83% yield; mp = 145-148 °C; 1H NMR (300 MHz, CDCl3) δ 7.99 (bt, 1H, >CH=C), 7.85 (d, 3H, J = 8.79 Hz, indole and (CH3)2NOC6H4CO2), 7.30-7.38 (m, 4H, indole), 6.56 (d, 2H, J = 8.97 Hz, CH3OC6H4CO2), 4.65 (AB d, 1H, J =11.88 Hz, CO2CHH), 4.38 (AB d, 1H, J =11.88 Hz, CO2CHH), 3.87 (s, 3H, NCH3), 3.80 (bs, 2H, HOCH2-), 3.14 (dd, 1H, J = 17.01, 2.58 Hz, CHH-lactone), 3.01 (s, 6H, (CH3)2NOC6H4CO2), 2.93. (dd, 1H, J = 17.01, 2.58 Hz, CHH-lactone), 2.05 (bs, 1H, HOCH2-).

13

C NMR (150 MHz, CDCl3) δ 171.70, 167.08,

153.64, 131.67 (2C), 130.57, 128.78, 127.97, 123.26, 121.25, 118.94, 117.09, 115.46, 111.84, 110.68 (2C), 109.74, 83.14, 65.48, 64.78, 39.98 (2C), 33.48. Anal. HPLC 99% (Rt = 7.30 min). HRMS (ESI) calc. for C25H26N2O5 [M + H]+ 435.1837, found 435.1909.

(E)-(2-(Hydroxymethyl)-5-oxo-4-(pyridin-2-ylmethylene)tetrahydrofuran-2-yl)methyl


27


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pivalate (100). Following general Method C, 100 was obtained as a yellow oil, 83% yield; 1H NMR (400 MHz, CDCl3) δ 8.67 (bs, 1H, pyridine), 7.72 (t, 1H, J = 7.36 Hz, pyridine), 7.48 (bs, 1H, >CH=C), 7.32 (d, 1H, J = 7.12 Hz, pyridine), 7.24 (m, 1H, pyridine), 4.29 (AB d, 1H, J = 11.8 Hz, CO2CHH), 4.20 (AB d, 1H, J = 11.8 Hz, CO2CHH), 3.68-3.79 (m, 2H, HOCH2), 3.35 (bs, 2H, CH2-lactone), 1.12 (s, 9H, (CH3)3CO). 13C NMR (125 MHz, CDCl3) δ 176.33, 171.32, 153.76, 150.13, 136.65, 134.20, 129.45, 127.85, 127.08, 123.48, 84.40, 71.12, 65.15, 39.02, 29.69, 27.12 (2C). Anal. HPLC 99% (Rt = 7.30 min). HRMS (ESI) calc. for C17H21NO5 [M + H]+ 320.1427, found 320.1499.


4-

chlorobenzoate (101). Following general Method C, 101 was obtained as a white solid, 83% yield; mp = 168-171 °C; 1H NMR (400 MHz, CDCl3) δ 8.68 (bs, 1H, pyridine), 7.87 (d, 2H, J = 8.32 Hz, ClC6H4CO2), 7.69-7.73 (m, 2H, pyridine), 7.50 (bt, 1H, >CH=C), 7.41 (d, 1H, J = 7.44, pyridine), 7.30 (d, 2H, J = 8.32, ClC6H4CO2), 4.59 (AB d, 1H, J =12.08 Hz, CO2C HH), 4.45 (AB d, 1H, J = 12.08 Hz, CO2CHH), 3.74-3.89 (m, 2H, HOCH2), 3.49 (bs, 2H, CH2-lactone). 13

C NMR (125 MHz, CDCl3) δ 176.33, 171.76, 163.98 (2C), 153.76, 132.34, 131.94, 128.89,

127.94, 123.34, 118.74, 116.97, 64.99, 60.41, 33.32, 30.92, 29.69, 27.12 (2C). Anal. HPLC 99% (Rt = 7.60 min). HRMS (ESI) calc. for C19H16ClNO5 [M + H]+ 374.0707, found 374.0779.

(E)-(2-(Hydroxymethyl)-5-oxo-4-(pyridin-2-ylmethylene)tetrahydrofuran-2-yl)methyl-4methoxybenzoate (102). Following general Method C, 102 was obtained as a white solid, 83% yield; mp = 161-163 °C; 1H NMR (400 MHz, CDCl3) δ 8.68 (bs, 1H, pyridine), 7.90 (d, 2H, J = 8.72 Hz, CH3OC6H4CO2), 7.70 (t, 1H, J = 7.12 Hz, pyridine), 7.50 (bt, 1H, >CH=C), 7.41 (d,


28

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1H, J = 7.12 Hz, pyridine), 7.20(m, 1H, pyridine), 6.85 (d, 2H, J = 8.72 Hz, CH3OC6H4CO2), 4.56 (AB d, 1H, J =11.96 Hz, CO2C HH), 4.47 (AB d, 1H, J = 11.96 Hz, CO2CHH), 3.75-3.85 (m, 5H, CH3OC6H4CO2 and HOCH2), 3.49 (irregular q, 2H, CH2-lactone),2.20 (bt, 1H, HOCH2). 13

C NMR (125 MHz, CDCl3) δ 170.19, 170.17, 166.27, 153.32, 148.75, 148.69, 139.10, 131.97

(2C), 131.94, 126.66, 126.59, 113.91 (2C), 86.19, 67.41, 67.39, 55.32, 32.10. Anal. HPLC 99% (Rt = 7.46 min). HRMS (ESI) calc. for C20H19NO6 [M + H]+ 370.1212, found 370.1291.


4-

(dimethylamino)benzoate (103). Following general Method C, 103 was obtained as a pale yellow solid, 80% yield; mp = 158-159 °C; 1H NMR (400 MHz, CDCl3) δ 8.68 (bs, 1H, pyridine), 7.83 (d, 2H, J = 8.92 Hz, (CH3)2NC6H4CO2), 7.70 (t, 1H, J = 7.68 Hz, pyridine), 7.50 (bt, 1H, >CH=C), 7.40 (d, 1H, J = 7.68 Hz, pyridine), 7.20 (m, 1H, pyridine), 6.58 (d, 2H, J = 8.92 Hz, (CH3)2NC6H4CO2), 4.56 (AB d, 1H, J =12.00 Hz, CO2C HH), 4.38 (AB d, 1H, J = 12.00 Hz, CO2CHH), 3.74-3.81 (m, 2H, HOCH2), 3.52 (AB d, 1H, J = 17.01, 2.68 Hz, CHHlactone), 3.42 (AB d, 1H, J = 17.01, 2.68 Hz, CHH-lactone), 3.03 (s, 6H, (CH3)2NC6H4CO2).13C NMR (125 MHz, CDCl3) δ 170.19, 170.17, 153.32, 153.30, 148.69, 131.49, 131.46 (2C), 131.43, 126.66, 126.59, 126.57,123.29, 110.61 (2C), 86.19, 67.41, 67.39, 40.27 (2C), 32.10. Anal. HPLC 99% (Rt = 8.59 min). HRMS (ESI) calc. for C21H22N2O5 [M + H]+ 383.1529, found 373.1605.

(E)-(4-(4-(Dimethylamino)benzylidene)-2-(hydroxymethyl)-5-oxotetrahydrofuran-2yl)methyl pivalate (104). Following general Method C, 104 was obtained as a yellow solid, 83% yield; mp = 152-154 °C; 1H NMR (400 MHz, CDCl3) δ 7.48 (bt, 1H, J = 2.60 Hz, >CH=C), 7.37 (d, 2H, J = 8.92 Hz, (CH3)2NC6H4C=CHC=C), 7.82 (d, 1H, J = 7.84 Hz, indole), 7.37 (m, 3H, indole), 7.28 (m, 1H, indole), 4.32 (d, 1H, J = 11.88 Hz, CO2CHH), 4.24 (d, 1H, J = 11.88 Hz, CO2CHH), 3.88 (s, 3H, CH3N-indole), 3.27 (q, 2H, J = 12.04 Hz, HOCH2), 3.03 (dd, 1H, J = 17.12, 2.60 Hz, CHH-lactone), 2.80 (dd, 1H, J = 17.12, 2.60 Hz, CHH-lactone), 1.82 (m, 2H, cyclohexane), 1.63 (m, 2H, cyclohexane), 1.36 (m, 2H, cyclohexane), 1.22 (m, 2H, cyclohexane). 13C NMR (151 MHz, CDCl3) δ 175.57, 171.26, 136.92, 130.37, 128.66, 128.00, 123.52, 121.49, 119.03, 116.73, 111.90, 109.87, 80.87, 77.31, 77.10, 76.89, 65.55, 43.04, 33.83, 33.65, 29.78, 28.93, 28.90, 25.68, 25.37, 1.10. Anal. HPLC 99% (Rt = 12.05 min). HRMS (ESI) calc. for C23H27NO5 [M + H]+ 398.1889, found 398.1973.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl 2-propylpentanoate (119). Following general Method B, 119 was obtained as a pale yellow solid, 83% yield; mp = 111-112 °C; 1H NMR (300 MHz, CDCl3) δ 7.97 (bt, 1H, >HC=C), 7.85 (d, 1H, J = 7.85 Hz, indole), 7.35 (m, 2H, indole), 7.30 (m, 2H, indole), 4.40 (AB d, 1H, J = 11.88 Hz, CO2C HH), 4.28 (AB d, 1H, J = 11.88 Hz, CO2C HH), 3.88 (s, 3H, H3CN-indole), 3.75 (m, 2H, HOCH2), 3.08 (AB d, 1H, J = 17.19 Hz, CHH-lactone), 2.86 (AB d, 1H, J = 17.19 Hz, CHH-lactone), 2.39 (m, 1H, [CH3(CH2)2]2CHCO2), 1.54 (m, 4H, [CH3(CH2)2]2CHCO2),


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1.26 (m, 4H, [CH3(CH2)2]2CHCO2), 0.80 (t, 6H, J = 7.14 Hz, [CH3(CH2)2]2CHCO2).

13

C NMR

(125 MHz, CDCl3) δ 176.62, 130.42(2C), 128.80, 127.92, 123.37, 121.36, 118.94, 116.84 (2C), 109.74, 82.65, 77.25, 76.99, 76.74, 65.31, 65.04, 45.21, 34.40, 33.48, 33.24, 20.60, 13.90, 13.85. Anal. HPLC 99% (Rt = 12.65 min). HRMS (ESI) calc. for C24H31NO5 [M + H]+ 414.2202, found 414.2268.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl 2-isobutyl-4-methylpentanoate (120). Following general Method A, 120 was obtained as oil, 83% yield; 1H NMR (300 MHz, CDCl3) δ 7.97 (bt, 1H, >HC=C), 7.85 (d, 1H, J = 7.85 Hz, indole), 7.35 (m, 2H, indole), 7.30 (m, 2H, indole), 4.40 (AB d, 1H, J = 11.88 Hz, CO2C HH), 4.28 (AB d, 1H, J = 11.88 Hz, CO2C HH), 3.88 (s, 3H, H3CN-indole), 3.75 (m, 2H, HOCH2), 3.08 (AB d, 1H, J = 17.19 Hz, CHH-lactone), 2.86 (AB d, 1H, J = 17.19 Hz, CHHlactone), 2.23 (d, 2H, J =6.52 Hz, [CH2CH(CH3)2]2CHCH2), 1.96 (p, 1H, J = 6.67 Hz, [CH2CH(CH3)2]2CHCH2),

1.63

(m,

2H,

[CH2CH(CH3)2]2CHCH2),

[CH2CH(CH3)2]2CHCH2), 0.86 (m, 12H, [CH2CH(CH3)2]2CHCH2).

1.13

(m,

4H,

13

C NMR (125 MHz,

CDCl3) δ 173.68, 171.73, 136.89,130.66, 128.84, 128.04, 123.44, 121.44, 119.06, 119.00, 117.00, 111.95, 109.86, 82.85, 65.63, 65.09, 44.15, 44.12, 39.44, 33.55, 33.32, 30.71, 25.24, 25.21, 22.96, 22.70, 22.62. Anal. HPLC 99% (Rt = 12.75 min). HRMS (ESI) calc. for C26H37NO5 [M + H]+ 456.2672, found 456.2759.

((E)-2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl cinnamate (121). Following general Method B, 121 was obtained as oil, 83% yield; 1

H NMR (300 MHz, CDCl3) δ 8.00 (bt, 1H, >HC=C), 7.84 (d, 1H, J = 7.89 Hz, indole), 7.68 (d,


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1H, J = 15.93 Hz, ArCH=CHCO2), 7.45 (m, 2H, Ar), 7.30-7.36 (m, 6H, Ar and indole), 6.42 (d, 1H, ArCH=CHCO2), 7.30 (m, 2H, indole), 4.57 (AB d, 1H, J = 11.88 Hz, CO2C HH), 4.36 (AB d, 1H, J = 11.88 Hz, CO2C HH), 3.88 (s, 3H, H3CN-indole), 3.75 (m, 2H, HOCH2), 3.10 (AB d, 1H, J = 17.19 Hz, CHH-lactone), 2.88 (AB d, 1H, J = 17.19 Hz, CHH-lactone), 2.35 (t, 1H, J = 6.21 Hz, HOCH2).

13

C NMR (125 MHz, CDCl3) δ 171.47, 166.50, 144.69, 130.83, 130.81,

130.61, 129.06, 127.93, 124.68 (2C), 124.53 (2C), 123.35, 121.34, 121.05, 119.80, 118.95, 116.63, 109.76, 82.60, 65.64, 65.03, 33.51, 33.29, 29.69. Anal. HPLC 99% (Rt = 12.56 min). HRMS (ESI) calc. for C25H23NO5 [M + H]+ 418.1576, found 418.1656.

((E)-2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl (E)-3-(pyridin-2-yl)acrylate (122). Following general Method B, 122 was obtained as oil, 63% yield; 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H, J = 4.74 Hz, pyridine), 7.99 (bt, 1H, >HC=C), 7.84 (d, 1H, J = 7.89 Hz, indole), 7.70 (m, 2H, ArCH=CHCO2 and pyridine), 7.36–7.29 (m, 6H, indole and pyridine), 4.50 (AB d, 1H, J = 11.88 Hz, CO2C HH), 4.43 (AB d, 1H, J = 11.88 Hz, CO2C HH), 3.89 (s, 3H, H3CN-indole), 3.81 (m, 2H, HOCH2), 3.09 (AB d, 1H, J = 17.30 Hz, CHH-lactone), 2.95 (AB d, 1H, J = 17.19 Hz, CHH-lactone), 2.31 (m, 1H, HOCH2).

13

C NMR (125 MHz, CDCl3) δ 171.47, 166.50, 152.43, 150.18, 144.69, 136.83,

136.81, 130.61, 129.06, 127.93, 124.68, 124.53, 123.35, 121.34, 121.05, 118.95, 116.63, 109.76, 82.60, 65.64, 65.03, 33.51, 33.29, 29.69. Anal. HPLC 99% (Rt = 11.25 min). HRMS (ESI) calc. for C24H22N2O5 [M + H]+ 419.1529, found 419.1600.

((E)-2-(Hydroxymethyl)-4-((1-methyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl (E)-3-(1-methyl-1H-indol-3-yl)acrylate (123). Following general Method B, 123


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was obtained as a pale yellow solid, 63% yield; mp = 175-177 °C; 1H NMR (500 MHz, CDCl3) δ 7.98 (t, 1H, J = 2.28 Hz, >HC=C), 7.84 (d, 1H, J = 16.02 Hz, indole-CH=CHCO2), 7.83 (m, 2H, indole), 7.24-7.35 (m, 6H, indole), 7.20 (m, 2H, indole), 6.35 (d, 1H, J = 16.08 Hz, indoleCH=CHCO2), 4.56 (AB d, 1H, J = 11.88 Hz, CO2C HH), 4.32 (AB d, 1H, J = 11.88 Hz, CO2C HH), 3.85 (s, 3H, H3CN-indole (sn-2)), 3.81 (m, 2H, HOCH2), 3.73 (s, 3H, H3CN-indole (sn-1)) 3.10 (AB dd, 1H, J = 16.98, 2.76 Hz, CHH-lactone), 2.94 (AB dd, 1H, J = 17.19, 2.76 Hz, CHHlactone), 2.31 (m, 1H, HOCH2). 13C NMR (125 MHz, CDCl3) δ 171.67, 168.36, 139.81, 138.11, 136.80, 133.83, 30.61, 128.74, 127.96, 125.92, 123.29, 123.11, 121.54, 121.30, 120.55, 118.92, 117.04, 111.92, 111.82, 110.73, 109.98, 109.76, 83.06, 65.29, 64.76, 33.50, 33.38, 29.68. Anal. HPLC 99% (Rt = 11.55 min). HRMS (ESI) calc. for C28H26N2O5 [M + H]+ 471.1842, found 471.1920.

(E)-(4-((1-Butyl-1H-indol-3-yl)methylene)-2-(hydroxymethyl)-5-oxotetrahydrofuran-2yl)methyl pivalate (124). Following general Method A, 124 was obtained as a pale yellow solid, 62% yield; mp = 106-108 °C; 1H NMR (300 MHz, CDCl3) δ 7.95 (bt, 1H, >CH=C), 7.82 (d, 1H, J = 6.96 Hz, indole), 7.26-7.38 (m, 4H, indole), 4.38 (AB d, 1H, J = 11.88 Hz, CO2C HH), 4.23 (AB d, 1H, J = 11.88 Hz, CO2C HH), 4.16 (t, 2H, J = 7.14 Hz, CH3CH2CH2CH2N), 3.67-3.76 (m, 2H, HOCH2), 3.04 (dd, 1H, J = 17.96, 2.40 Hz, CHH-lactone), 2.80 (dd, 1H, J = 17.96, 2.40 Hz, CHH-lactone), 1.85 (m, 2H, CH3CH2CH2CH2N), 1.36 (m, 2H, CH3CH2CH2CH2N), 1.14 (s, 9H, (CH3)3CCO2), 0.94 (t, 3H, J = 7.32 Hz, CH3CH2CH2CH2N).

13

C NMR (125 MHz, CDCl3) δ

177.35, 169.56, 136.45, 135.45, 132.78, 128.44, 127.35, 123.32, 120.92, 120.86, 114.38, 109.09, 86.02, 67.93 (2C), 65.11 (2C), 46.30, 38.83, 32.88, 31.49, 19.79 (2C), 13.00. Anal. HPLC 99% (Rt = 12.45 min). HRMS (ESI) calc. for C24H31NO5 [M + H]+ 414.2202, found 414.2289.


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(E)-(2-(Hydroxymethyl)-4-((1-isobutyl-1H-indol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl pivalate (125). Following general Method A, 125 was obtained as a pale yellow solid, 55% yield; mp = 110-112 °C; 1H NMR (300 MHz, CDCl3) δ 7.89 (t, 1H, J = 2.55 Hz, >CH=C), 7.77 (d, 1H, J = 7.14 Hz, indole), 7.20-7.31 (m, 4H, indole), 4.30 (AB d, 1H, J = 11.91 Hz, CO2C HH), 4.19 (AB d, 1H, J = 11.91 Hz, CO2C HH), 3.90 (dd, 2H, J = 7.32, 2.01 Hz, (CH3)2CHCH2N), 3.70 (q, 2H, J = 12.09 Hz, HOCH2), 3.00 (dd, 1H, J = 17.02, 2.73 Hz, CHHlactone), 2.75 (dd, 1H, J = 17.02, 2.73 Hz, CHH-lactone), 2.13-2.20 (m, 1H, (CH3)2CHCH2N), 1.07 (s, 9H, (CH3)3CCO2), 0.89 (d, 3H, J = 4.77 Hz, (CH3)2CHCH2N), 0.87 (d, 3H, J = 4.74 Hz, (CH3)2CHCH2N).

13

C NMR (125 MHz, CDCl3) δ 177.38, 169.59, 136.41, 135.49, 133.17,

128.39, 127.35, 123.31, 121.28, 120.78, 114.28, 109.05, 86.02, 67.98(2C), 65.16(2C), 55.79, 38.83, 32.88(2C), 29.73, 19.92(2C). Anal. HPLC 99% (Rt = 12.65 min). HRMS (ESI) calc. for C24H31NO5 [M + H]+ 414.2202, found 414.2282.

(E)-(4-((1-(Cyclopropylmethyl)-1H-indol-3-yl)methylene)-2-(hydroxymethyl)-5oxotetrahydrofuran-2-yl)methyl pivalate (126). Following general Method A, 126 was obtained as a pale yellow solid, 58% yield; mp = 113-115 °C; 1H NMR (500 MHz, CDCl3) δ 7.95 (bt, 1H, >CH=C), 7.82 (d, 1H, J = 7.75 Hz, indole), 7.43 (d, 2H, J = 7.90 Hz, indole), 7.37 (s, 1H, indole), 7.26 (t, 1H, J = 7.90 Hz, indole), 4.38 (AB d, 1H, J = 11.90 Hz, CO2C HH), 4.21 (AB d, 1H, J = 11.90 Hz, CO2C HH), 3.76 (m, 2H, HOCH2), 3.04 (dd, 2H, J = 17.35, 2.60 Hz, CHH-lactone), 2.80 (dd, 1H, J = 17.35, 2.60 Hz, CHH-lactone), 2.28 (m, 2H, (CH2)2CHCH2N), 1.91-1.95 (m, 5H, (CH2)2CHCH2N), (CH3)3CCO2).

13

2.13-2.20 (m, 1H, (CH3)2CHCH2N), 1.07 (s, 9H,

C NMR (125 MHz, CDCl3) δ 177.38, 169.59, 136.61, 135.49, 133.10, 128.39,


41


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127.35, 123.38, 121.36, 120.84, 114.26, 108.98, 86.02, 67.98, 65.16 (2C), 51.79, 38.83, 32.88 (2C), 11.16,6.90, 5.80 (2C). Anal. HPLC 99% (Rt = 12.59 min). HRMS (ESI) calc. for C24H29NO5 [M + H]+ 412.2094, found 412.2104.

(E)-(2-(Hydroxymethyl)-4-((5-methoxy-1-methyl-1H-indol-3-yl)methylene)-5oxotetrahydrofuran-2-yl)methyl pivalate (127). Following general Method A, 127 was obtained as a pale yellow solid, 63% yield; mp = 121-122 °C; 1H NMR (400 MHz, CDCl3) δ 1H NMR (500 MHz, CDCl3) δ 7.92 (bt, 1H, >CH=C), 7.82 (d, 1H, J = 7.80 Hz, indole), 7.24 – 7.25 (m, 2H, indole), 6.97 (m, 1H, indole), 4.39 (AB d, 1H, J = 11.95 Hz, CO2CHH), 4.24 (AB d, 1H, J = 11.95 Hz, CO2CHH), 3.89 (s, 3H, CH3O-indole), 3.86 (s, 3H, H3CN-indole), 3.72 (m, 2H, HOCH2), 3.03 (dd, 1H, J = 17.02, 2.15 Hz, CHH-lactone), 2.82 (dd, 1H, J = 17.02, 2.15 Hz, CHH-lactone), 2.22 (t, 1H, J = 5.4 Hz, -OH), 1.15 (s, 9H, -(CH3)3). 13C NMR (125 MHz, CDCl3) δ 178.60, 171.27, 135.19, 131.26, 128.92, 127.86, 127.40, 123.75, 118.65, 117.84, 111.42, 110.87, 82.91, 65.72, 64.84 (2C), 39.00, 33.72, 33.08, 29.69, 27.06 (2C). Anal. HPLC 99% (Rt = 11.45 min). HRMS (ESI) calc. for C22H27NO6 [M + H]+ 402.1811, found 402.1831.

(E)-(4-((5-Chloro-1-methyl-1H-indol-3-yl)methylene)-2-(hydroxymethyl)-5oxotetrahydrofuran-2-yl)methyl pivalate (128E). Following general Method A, 128E was obtained as a pale yellow solid, 62% yield; mp = 124-126 °C; 1H NMR (500 MHz, CDCl3) δ 7.79 (t, 1H, J = 2.40 Hz, >CH=C), 7.75 (s, 1H, indole), 7.27 (s, 1H, indole), 7.24 (s, 2H, indole), 4.36 (AB d, 1H, J = 11.95 Hz, CO2CHH), 4.22 (AB d, 1H, J = 11.95 Hz, CO2CHH), 3.84 (s, 3H, H3CN-indole), 3.72 (m, 2H, HOCH2), 3.04 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 2.79 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 2.35 (bt, 1H, -OH), 1.15 (s, 9H, -(CH3)3). 13C NMR (125


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MHz, CDCl3) δ 178.57, 171.34, 135.16, 131.25, 128.90, 127.80, 127.35, 123.70, 118.60 (2C), 117.88, 111.40, 110.84, 82.99, 65.74, 64.83, 38.99, 33.70, 33.03, 29.68, 27.05. Anal. HPLC 99% (Rt = 11.65 min). HRMS (ESI) calc. for C21H24ClNO5 [M + H]+ 406.1343, found 406.1413.

(Z)-(4-((5-Chloro-1-methyl-1H-indol-3-yl)methylene)-2-(hydroxymethyl)-5oxotetrahydrofuran-2-yl)methyl pivalate (128Z). Following general Method A, 128Z was obtained as a pale yellow solid, 58% yield; mp = 133-135 °C; 1H NMR (400 MHz, CDCl3) δ 8.76 (s, 1H, indole), 7.69 (s, 1H, indole), 7.21-7.23 (m, 2H, indole), 7.11 (bt, 1H, >CH=C), 4.33 (AB d, 1H, J = 11.95 Hz, CO2CHH), 4.22 (AB d, 1H, J = 11.95 Hz, CO2CHH), 3.78 (s, 3H, H3CN-indole), 3.72 (m, 2H, HOCH2), 3.04 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 2.79 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 2.35 (bt, 1H, -OH), 1.10 (s, 9H, -(CH3)3). 13C NMR (125 MHz, CDCl3) δ 178.57, 171.34, 135.16, 131.25, 128.90, 127.80, 127.35, 123.70, 118.60 (2C), 117.88, 111.40, 110.84, 82.99, 65.74, 64.83, 38.99, 33.70, 33.03, 29.68, 27.05. Anal. HPLC 99% (Rt = 5.66 min). HRMS (ESI) calc. for C21H24ClNO5 [M + H]+ 406.1343, found 406.1414.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-5-nitro-1H-indol-3-yl)methylene)-5oxotetrahydrofuran-2-yl)methyl pivalate (129E). Following general Method A, 129E was obtained as a pale yellow solid, 61% yield; mp = 124-126 °C; 1H NMR (500 MHz, CDCl3) δ 8.79 (s, 1H, indole), 8.59 (d, 1H, J = 1.90 Hz, indole), 8.15 (dd, 1H, J = 9.00, 1.90 Hz, indole), 7.42 (bt, 1H, >CH=C), 7.38 (d, 1H, J = 9.00 Hz, indole), 4.22 (AB d, 1H, J = 11.98 Hz, CO2CHH), 4.18 (AB d, 1H, J = 11.98 Hz, CO2CHH), 3.90 (s, 3H, H3CN-indole), 3.78 (m, 2H, HOCH2), 3.09 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 2.89 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 1.91 (bt, 1H, -OH), 1.23 (s, 9H, -(CH3)3). 13C NMR (125 MHz, CDCl3) δ 177.38,


43


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169.59, 142.62, 142.61, 132.42, 127.36, 127.34, 127.32, 119.44, 118.33, 115.30, 109.92, 86.05, 67.96, 67.98 38.83, 33.33, 32.85, 27.07 (3C). Anal. HPLC 99% (Rt = 5.52 min). HRMS (ESI) calc. for C21H24N2O7 [M + H]+ 417.1584, found 417.1604.

(Z)-(2-(Hydroxymethyl)-4-((1-methyl-5-nitro-1H-indol-3-yl)methylene)-5oxotetrahydrofuran-2-yl)methyl pivalate (129Z). Following general Method A, 129Z was obtained as a white solid, 60% yield; mp = 126-128 °C; 1H NMR (500 MHz, CDCl3) δ 8.92 (s, 1H, indole), 8.65 (d, 1H, J = 1.90 Hz, indole), 8.17 (dd, 1H, J = 9.00, 1.90 Hz, indole), 7.31 (d, 1H, J = 9.00 Hz, indole), 7.02 (bt, 1H, >CH=C), 4.20 (AB d, 1H, J = 11.99 Hz, CO2CHH), 4.17 (AB d, 1H, J = 11.99 Hz, CO2CHH), 3.90 (s, 3H, H3CN-indole), 3.78 (m, 2H, HOCH2), 3.10 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 2.91 (dd, 1H, J = 17.07, 2.60 Hz, CHH-lactone), 1.23 (s, 9H, -(CH3)3).

13

C NMR (125 MHz, CDCl3) δ 177.38, 169.78, 142.64, 142.63, 132.42, 132.38,

127.36, 127.35, 119.47, 119.45, 119.43, 114.29, 86.05, 67.96, 65.13, 38.83, 33.33, 32.96, 27.07 (3C). Anal. HPLC 99% (Rt = 5.55 min). HRMS (ESI) calc. for C21H24N2O7 [M + H]+ 417.1584, found 417.1608.

(E)-(4-(Benzofuran-3-ylmethylene)-2-(hydroxymethyl)-5-oxotetrahydrofuran-2-yl)methyl pivalate (130E). Following general Method A, 130E was obtained as a white solid, 62% yield; mp = 132-135 °C; 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H, benzofuran), 7.73 (d, 1H, J = 3.08 Hz, benzofuran), 7.72 (bt, 1H, >C=CH), 7.53 (d, 1H, J = 7.80 Hz, benzofuran), 7.32-7.40 (m, 2H, benzofuran), 4.34 (AB d, 1H, J = 11.92 Hz, CO2CHH), 4.22 (AB d, 1H, J= 11.92 Hz, CO2CHH), 3.80 (dd, 1H, J = 12.12, 6.39 Hz, HOCHH-), 3.71 (dd, 1H, J = 12.12, 6.39 Hz, HOCHH-), 3.08 (dd, 1H, J = 17.68, 2.88 Hz, CHH-lactone), 2.85 (dd, 1H, J = 17.68, 2.88 Hz,


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CHH-lactone), 2.15 (bt, 1H, HOCH2-), 1.13 (s, 9H, (CH3)3CO2). 13C NMR (125 MHz, CDCl3) δ 178.27, 170.40, 155.02, 145.23, 126.12, 125.70, 125.21, 124.69, 123.78, 119.52, 117.41, 111.80, 83.51, 65.60, 64.70, 38.96, 33.00, 27.02 (3C). Anal. HPLC 99% (Rt = 8.86 min). HRMS (ESI) calc. for C20H22O6 [M + H]+ 359.1422, found 359.1495.

(Z)-(4-(Benzofuran-3-yl-methylene)-2-(hydroxymethyl)-5-oxotetrahydrofuran-2-yl)methyl pivalate (130Z). Following general Method A, 130Z was obtained as a white solid, 60% yield; mp = 128-130 °C; 1H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H, benzofuran), 7.61 (d, 1H, J = 6.84 Hz, benzofuran), 7.32 (p, 2H, J = 6.32 Hz, benzofuran), 7.06 (s, 1H, >C=CH), 4.33 (AB d, 1H, J = 11.84 Hz, CO2CHH), 4.20 (AB d, 1H, J= 11.84 Hz, CO2CHH), 3.77 (dd, 1H, J = 12.04, 6.00 Hz, HOCHH-), 3.67 (dd, 1H, J = 12.04, 6.00 Hz, HOCHH-), 3.21 (dd, 1H, J = 17.08, 1.84 Hz, CHH-lactone), 2.99 (dd, 1H, J = 17.08, 1.84 Hz, CHH-lactone), 2.15 (bt, 1H, HOCH2-), 1.14 (s, 9H, (CH3)3CO2).13C NMR (125 MHz, CDCl3) δ 178.33, 168.47, 165.10, 154.50, 150.03, 126.56, 124.84, 123.27, 123.15, 118.32, 111.84, 82.81, 65.51. 64.66, 38.95, 34.69, 27.05. Anal. HPLC 99% (Rt = 8.28 min). HRMS (ESI) calc. for C20H22O6 [M + H]+ 359.1416, found 359.1479.

(E)-(4-(Benzo[b]thiophen-3-ylmethylene)-2-(hydroxymethyl)-5-oxotetrahydrofuran-2yl)methyl pivalate (131E). Following general Method A, 131E was obtained as a yellow solid, 59% yield; mp = 138-140 °C; 1H NMR (500 MHz, CDCl3) δ 7.98 (d, 1H, J = 8.00 Hz, benzothiophene), 7.92 (bt, 1H, benzothiophene), 7.87 (d, 1H, J = 8.40 Hz, benzophenone), 7.65 (s, 1H, >CH=C), 7.42-7.50 (m, 1H, benzophenone), 4.34 (AB d, 1H, J = 11.95 Hz, CO2CHH), 4.22 (AB d, 1H, J = 11.95 Hz, CO2CHH), 3.79 (dd, 1H, J = 12.15, 6.75 Hz, HOCHH-), 3.73 (dd, 1H, J = 12.15, 6.75 Hz, HOCHH-), 3.21 (dd, 1H, J = 17.70, 2.90 Hz, CHH-lactone), 2.99 (dd,


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1H, J = 17.70, 2.90 Hz, CHH-lactone), 2.12 (t, 1H, J = 6.65 Hz, HOCH2-), 1.12 (s, 9H, (CH3)3CO2).

13

C NMR (125 MHz, CDCl3) δ 177.38, 169.41, 140.23, 131.20, 131.18, 128.60,

125.90, 123.03, 122.96, 122.37, 122.35, 86.05, 67.98, 67.96, 38.83, 32.47, 27.07 (3C). Anal. HPLC 99% (Rt = 7.98 min). HRMS (ESI) calc. for C20H22O5S [M + H]+ 375.1188, found 375.1254.

(Z)-(4-(Benzo[b]thiophen-3-ylmethylene)-2-(hydroxymethyl)-5-oxotetrahydrofuran-2yl)methyl pivalate (131Z). Following general Method A, 131Z was obtained as a yellow solid, 52% yield; mp = 134-136 °C; 1H NMR (500 MHz, CDCl3) δ 9.08 (s, 1H, benzothiophene), 7.61 (d, 1H, J = 6.84 Hz, benzothiophene), 7.32-7.49 (m, 3H, benzothiophene), 7.12 (s, 1H, >CH=C), 4.32 (AB d, 1H, J = 11.98 Hz, CO2CHH), 4.21 (AB d, 1H, J = 11.98 Hz, CO2CHH), 3.79 (m, 2H, HOCHH-), 3.24 (dd, 1H, J = 17.59, 1.98 Hz, CHH-lactone), 2.99 (dd, 1H, J = 17.59, 1.98 Hz, CHH-lactone), 2.12 (t, 1H, J = 6.65 Hz, HOCH2-), 1.12 (s, 9H, (CH3)3CO2). 13C NMR (125 MHz, CDCl3) δ 175.23, 171.44, 152.08, 147.55, 135.12, 131.18, 128.60, 124.84, 123.03, 121.96, 121.37, 121.35, 86.05, 67.98, 67.96, 38.83, 32.47, 27.07 (3C). Anal. HPLC 99% (Rt = 7.53 min); HRMS (ESI) calc. for C20H22O5S [M + H]+ 375.1188, found 375.1201.

(E)-(2-(Hydroxymethyl)-4-((1-methyl-1H-indazol-3-yl)methylene)-5-oxotetrahydrofuran-2yl)methyl pivalate (132). Following general Method A, 132 was obtained as a white solid, 63% yield; mp = 121-123 °C; 1H NMR (600 MHz, CDCl3) δ 7.82-7.84 (m, 2H, >CH=C and indazole), 7.41-7.45 (m, 2H, indazole), 7.27 (m, 1H, indazole), 4.41 (AB d, 1H, J = 11.94 Hz, CO2CHH), 4.22 (AB d, 1H, J = 11.94 Hz, CO2CHH), 4.12 (s, 3H, CH3N-indazole), 3.71-3.79 (m, 2H, HOCHH-), 3.34 (dd, 1H, J = 19.26, 2.76 Hz, CHH-lactone), 2.26 (dd, 1H, J = 19.26,


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2.76 Hz, CHH-lactone), 2.14 (bt, 1H, HOCH2-), 1.12 (s, 9H, (CH3)3CO2). 13C NMR (150 MHz, CDCl3) δ 178.37, 171.23, 140.50, 139.44, 126.83, 124.66, 124.50, 124.24, 121.99, 119.62, 109.31, 83.86, 65.70, 65.15, 38.95, 36.23, 33.97, 29.68, 27.05, 26.99. Anal. HPLC 99% (Rt = 5.67 min). HRMS (ESI) calc. for C20H24N2O5 [M + H]+ 373.1685, found 373.1759.

Biological Study. Materials, cell lines and antibodies. [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) (13.5 Ci/mmol) was custom labelled by Perkin Elmer Life Sciences (Akron, OH). PDBu and phorbol 12myristate 13-acetate (PMA) were purchased from LC Laboratories (Woburn, MA). Phosphatidyl-L-serine was purchased from Avanti Polar Lipids (Alabaster, AL). Dimethyl sulfoxide (DMSO) was purchased from Sigma Aldrich (St. Louis, MO). Human embryonic kidney (HEK) 293 cells, Lymph Node Carcinoma of the Prostate (LNCaP) cells, RAMOS B cell lymphocytes, fetal bovine serum (FBS), RPMI 1640 medium, L-glutamine, and EMEM medium were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The oligonucleotide primers used for polymerase chain reaction (PCR) and Lipofectamine Plus reagent were obtained from Invitrogen (Grand Island, NY). The mouse monoclonal anti-GFP antibody, X-tremeGENE HP DNA transfection reagent, PhosSTOP phosphatase inhibitor, and cOmplete Mini protease inhibitor were purchased from Roche Applied Science (Indianapolis, IN). The rabbit monoclonal anti-PKCδ and anti-PKCδ (phospho S299) antibodies were purchased from Abcam (Cambridge, MA). The rabbit monoclonal anti-phospho ERK antibody was purchased from Cell Signaling Technology (Danvers, MA). The purified human PKC isoforms (PKCα, -ε) were purchased from Invitrogen. The Pan-Ras Activation Assay Kit was purchased from Cell Biolabs, Inc. (San Diego, CA).


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[3H]PDBu Binding Assay. Binding affinities (Ki) of the DAG lactones were measured by competition of [3H]PDBu binding to the recombinant full-length PKCα, PKCε, human RasGRP1 and RasGRP3 using the polyethylene glycol precipitation assay developed in our laboratory.15 Triton X-100, included in the assays, did not exceed 0.003%. Assays were carried out for 5 min at 37 °C. All values represent the mean ± SEM of at least two independent experiments, as specified, where all points in each dose response curve were measured in triplicate. Expression in BL21 cells and purification of the MBP-tagged full length RasGRP3 - The full length RasGRP3 in the pMAL-c5x plasmid was transformed into BL21 (DE3) One Shot chemically competent E. coli (Invitrogen). Transformants were grown in LB broth medium (K-D Medical) at 37 °C until the optical density of the bacterial suspension reached 0.5-0.6. Expression of the MBP fusion protein was induced with 0.3 mM isopropyl O-Dthiogalactopyranoside (Thermo Fisher Scientific) for 6 h at room temperature. The expressed MBP tagged protein was purified using the pMALTM Protein Fusion and Purification System according to the manufacturer’s instructions. Purification efficiency was evaluated by SDSPAGE analysis. Purified proteins were stored in 20% glycerol at -80oC.

Construction of GFP-fused full-length RasGRP1/3. The full-length RasGRP1 (NCBI Accession # NM_005739.3) and RasGRP3 (NCBI Accession # NM_001139488.1) cDNA was amplified

by

PCR

using

specific

AATaagcttGGCACCCTGGGCAAGGCGAGA-3’

primers and

(forward reverse

primer: primer:

5’3’-

AATggatccCTAAGAACAGTCACCCTGCTCCA-5’; HindIII and BamHI sites, respectively indicated by lower case letters, were incorporated to facilitate cloning of RasGRP1. Forward


48

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primer: 5’- CATctcgagGGATCAAGTGGCCTTGGGAAAG-3’ and reverse primer: 3’AATccgcggTCAGCCATCCTCACCATCCTGT-5’; XhoI and SacII sites, respectively indicated by lower case letters, were incorporated to facilitate cloning of RasGRP3) and subcloned into the pEGFP-C3 plasmid (BD Biosciences Clontech, Palo Alto, CA) generating pEGFP-C3RasGRP1/3 with an N-terminal GFP tag. The DNA fragments of the PCR were purified with the QIAquick PCR purification kit (Qiagene, Inc., Valencia, CA) and afterwards digested with HindIII and BamHI; XhoI and SacII, respectively (New England Biolabs, Beverly, MA). After an additional step of purification, the fragments were finally ligated into the GFP-containing pEGFP-C3 plasmid using the restriction sites. The integrity of the inserts was verified by DNA sequencing, which was performed by the DNA minicore (Center for Cancer Research, NCI, National Institute of Health).

Expression of the GFP-tagged RasGRP1/3 in Live Mammalian Cells. HEK293 and LNCaP cells were plated on tissue culture dishes (100 mm) and cultured at 37 °C in EMEM supplemented with 10% FBS and RPMI-1640 medium supplemented with 10% FBS and 2 mM L-glutamine, respectively. Ramos cells were grown under usual conditions at 37 °C in a 5% CO2 atmosphere in T75 flasks. After 48 h in culture, HEK293 and LNCaP cells were transfected with GFP-tagged RasGRP1/3 constructs, using Lipofectamine Plus reagent (Invitrogen) and XtremeGENE HP DNA transfection reagent (Roche) according to the manufacturer's recommendations, respectively. The expression of the fluorescent protein was detected via fluorescent microscopy after 24-48 h of transfection.


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Pan-Ras activation assay. For Pan-Ras activation assays, HEK293 cells expressing the RasGRP1/3 constructs were lysed in 1X assay buffer (Pan-Ras Activation Assay Kit, Cell Biolabs, Inc.). The cells were incubated with DMSO (negative control), 1000 nM PMA (positive control), and half-log increasing concentrations of 96 at 37 °C for 30 min in a 5% CO2 atmosphere. Lysates were centrifuged at 14000 x g for 10 min at 4 °C. The resulting supernatants were incubated for 60 min at 4 °C with 40 µL Raf1 RBD Agarose beads. After incubation, the beads were collected and washed three times with 1X assay buffer. Proteins were then eluted from the beads with Laemmli sample buffer, separated by electrophoresis, and analyzed by immunoblotting using anti-Pan-Ras antibody (Pan-Ras activation assay kit, Cell Biolabs, Inc.). The signal was developed by enhanced chemiluminescence (Amersham) and imaged on Amersham HyperfilmTM ECL (GE Healthcare, Pittsburgh, PA).

Western blot analysis. The treated cells with DMSO (negative control), 1000 nM PMA (positive control), and half-log increasing concentrations of 96 were lysed in ice cold 1X RIPA Lysis and Extraction Buffer (Thermo Scientific) containing protease and phosphatase inhibitors (Roche). 50 µg lysates (protein concentration measured by TECAN, Pierce protein assay) were added to SDS and beta-mercapthoethanol containing sample buffer (Quality Biological Inc., Gaithersburg, MD), separated on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes (Trans-Blot® Turbo™ Transfer System, BIO-RAD, Hercules, CA). The membranes were blocked with 5% nonfat dry milk, incubated overnight in the primary antiPKCδ, anti-PKCδS299, and anti-phospho-ERK antibodies. RasGRP1/3 levels were detected using anti-GFP antibodies to ensure uniform protein expression in each cell sample. The membranes were washed (three times 10 min in PBS with 0.05% Tweem-20), incubated for 1


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hour in the secondary antibody, and washed (three times 10 min in PBS with 0.05% Tweem-20). The signal was developed by enhanced chemiluminescence (Amersham) and imaged on Amersham HyperfilmTM ECL (GE Healthcare, Pittsburgh, PA). The scanned films were edited using Adobe Photoshop CC (Adobe Systems Inc.).

Quantification of 96 dose response. EC50 analyses were made using ImageJ and GraphPrism softwares and normalized to 1,000 nM PMA. All values are expressed as the mean ± S.E. for three independent experiments.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication website at DOI: molecular modeling methods, docking of compound 96 into the RasGRP3 C1 domain, the synthetic procedure and spectra of intermediate compounds, and molecular formula strings.

AUTHOR INFORMATION Corresponding Author * Phone, 82-2-880-7846; E-mail, [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


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This work was supported by the National Research Foundation of Korea (NRF) grant for the Global Core Research Center (GCRC) funded by the Korea government (MSIP) (No. 20110030001) and in part by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, National Cancer Institute (Project Z1A BC 005270). The project was also funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract HHSN261200800001E.

ABBREVIATIONS DAG-lactone, Diacylglycerol-lactone; PKC, Protein Kinase C; RasGRP, Ras Guanine Releasing Protein

(1)

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