Bifidenone: Structure-Activity Relationship and Advanced Preclinical

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Bifidenone: Structure-Activity Relationship and Advanced Preclinical Candidate Zhongping Huang, Russell B. Williams, Steven M. Martin, Julie A. Lawrence, Vanessa L. Norman, Mark O'Neil-Johnson, Jim Harding, John E. Mangette, Shuang Liu, Peter Guzzo, Courtney M. Starks, and Gary R. Eldridge J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01644 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Journal of Medicinal Chemistry

Bifidenone: Structure-Activity Relationship and Advanced Preclinical Candidate Zhongping Huang,*,† Russell B. Williams,*,‡ Steven M. Martin,‡,§ Julie A. Lawrence,‡ Vanessa L. Norman,‡ Mark O’Neil-Johnson,‡ Jim Harding,† John E. Mangette,† Shuang Liu,† Peter R. Guzzo,† Courtney M. Starks,‡ and Gary R. Eldridge‡ †

Albany Molecular Research Inc., 1001 Main Street, Buffalo, New York 14203, United States

‡

Sequoia Sciences, Inc., 1912 Innerbelt Business Center Drive, St. Louis, Missouri 63114,

United States

ACS Paragon Plus Environment

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ABSTRACT

Bifidenone is a novel natural tubulin polymerization inhibitor that exhibits antiproliferative activity against a range of human cancer cell lines, making it an attractive candidate for development. A synthetic route was previously developed to alleviate supply constraints arising from its isolation in microgram quantities from a Gabonese tree. Using that previously published route, we present here 42 analogues that were synthesized to examine the structure-activity relationship of bifidenone derivatives. In addition to in vitro cytotoxicity data, data from murine xenograft and pharmacokinetic studies were used to evaluate the analogues. Compounds 45b and 46b were found to demonstrate promising efficacy in murine xenograft experiments and 46b had significantly more potent in vitro antiproliferative activity against taxane-resistant cell lines than paclitaxel.


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INTRODUCTION Since FDA approval of paclitaxel in 1992, the taxanes have become some of the most prescribed anticancer agents. Although treatment with taxanes improves the duration and quality of life for some cancer patients, many cancers do not respond to treatment, and drug resistance is a major obstacle for all types of cancer.1 The success of targeting tubulin in susceptible cancers has resulted in the development of numerous taxane and vinca binding site antitubulin compounds in the hopes of overcoming resistance. Though various approved antitubulin agents have improved efficacy, they are still prone to resistance mechanisms, especially those involving expression of p-glycoprotein efflux pumps.2–4 Antitubulin agents that act via the colchicine binding site have been the focus of considerable research because they are typically not affected by the resistance mechanisms relevant to taxane and vinca binding site agents (Figure 1).5–7 O

O

OH

NH

O

O

O

H N

S

O O

O

Colchicine

O

N

Combretastatin A-4

Nocodazole

Cl O

S

N

O

N

O Indibulin

O NH

O

O

H N

O

O H OH

O

N

H Curacin A

RPR112378

Figure 1. Representative colchicine binding site tubulin polymerization inhibitors. Recently, several natural products with a dihydrobenzodioxolone core were isolated from a plant of the genus Beilschmiedia (Lauraceae) collected from the Kwassa region of Gabon.8


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Among these compounds, bifidenone (Figure 2) was found to exhibit submicromolar antiproliferative activity against a range of human cancer cell lines and shown to inhibit tubulin polymerization, making it an attractive candidate for development. Other analogues isolated from the same plant provided initial SAR information; for example, the cis-orientation of the allyl and aromatic-containing groups with respect to the cyclohexenone ring was required for activity, while an additional methoxy group at the R6 position (Figure 2) abolished activity. Subsequent work described the total synthesis of bifidenone and determination of its absolute stereochemistry via single crystal X-ray analysis of a derivatized intermediate.9 Achieving the synthesis of bifidenone set the stage for a medicinal chemistry effort to improve its potency and pharmacological properties. To that end, over 300 analogues have been synthesized to date. The work described here details the synthesis and biological screening of 42 of those analogues and the selection of several preclinical candidates with improved potency and in vivo efficacy over bifidenone.

Figure 2. Bifidenone (left) and positions targeted for SAR investigation (right).

Chemistry A large number of colchicine binding site tubulin polymerization inhibitors have been identified, and while there is some overlap in their structural features, the overall diversity makes


4

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it difficult to predict which modifications will be most advantageous.10,11 The initial focus was to retain the dihydrobenzodioxolone core and determine the key stereochemical elements of the scaffold. A second area of interest was optimization of the aromatic ring, as well as exploration of R4 of bifidenone (Figure 2). developed

for

bifidenone.9

Analogue synthesis was achieved using the methodology Synthesis

started

with

commercially

available

1,4-

dioxaspiro[4.5]decan-8-one (Scheme 1), which was reacted with isopropenylmagnesium bromide, or an R4 analogue thereof, to provide 1. Subsequent Heck reaction of 1 with substituted bromobenzene

or

substituted

iodobenzene

2,

catalyzed

by

palladium

acetate/tri(o-

tolyl)phosphine or palladium acetate/dicyclohexylamine,12 afforded 3. This strategy allowed the creation of a large number of aromatic ring analogues and R4 analogues using the previously published synthesis, with only small changes in protection/deprotection as required by the substituents on the aromatic ring.


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Scheme 1. General Synthetic Route for Bifidenone Analoguesa R2

R1 R4 OH

R5 R4

R6

R1 R6

R2

R3

R5

a

OH

R3

+ O

O 1

X

O

2 X = Br or I

3 R2

R2 R1

R5

R1 R4

R6

O

R3

+

R5

R4

O

R3

R5

R1 R4

R6

O +

O a

R2

R2 R1

R6

O

O

O +

O b

R4

R6

O R3

R5

O O

R3

O c

O d

4-6, 16-24, 28, 31-32, 34-39, 44-49 a

Reagents and conditions: (a) Pd(OAc)2, tri(o-MePh)3, NEt3, reflux or Pd(OAc)2,

dicyclohexylamine, dioxane, H2O, 105 °C.

This approach provided a mixture of diastereomers. The letters a-d appended to the compound numbers herein denote the configurations as drawn in Scheme 1. The four diastereomers and their corresponding enantiomers were separated by silica gel chromatography and chiral chromatography. Analogues with NH2, NHAc, CN, and morpholine substituents at R2 were prepared as shown in Scheme 2. Deprotection of intermediate 7 by hydrogenation provided 8, which was treated with PhNTf2 (N-phenyl-bis(trifluoromethanesulfonimide)) to afford the intermediate 9. Compound 9 was dehydrogenated by palladium-catalyzed aerobic dehydrogenation to afford the common intermediate 10.9 Compounds 27, 30, 39-40 and Fmoc protected 33 and 41 were


6

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synthesized from the intermediate 10 by standard coupling procedures, subsequent acylation, and palladium-catalyzed decarboxylation-allylation. Compounds 33 and 41 were prepared by Fmoc deprotection under the conditions of dicyclohexylamine.

Scheme 2. Synthesis of R2 Analogues with NH2, NHAc, CN and Morpholinea OBn

OH

MeO

OTf

MeO Me O

MeO Me O

a

Me O

b

O

O

R3

R3

R3

O

O

7

O 9

8

R2

R2

OTf

MeO

MeO

MeO Me O

Me O

Me O

d

O

O

e

O

R3

R3

R3

O O

O

O 10

R2

R2 MeO Me O

Me O O

R3

O g

R3

O

O

27, 29-30, 40, 42

33, 41

R2 = NHAc, CN, morpholine, NHFmoc

a

O 12

11

MeO

f

c O

R2 = NH2

Reagents and conditions: (a) Pd(OH)2/C, H2, EtOAc; (b) PhNTf2, NEt3, DCM; (c) Pd(OAc)2,

diazafluoren-9-one, DMSO, 80 °C; (d) For R2 = CN: Zn(CN)2, Pd(PPh3)4, DMF, 120 °C; For


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NH2, NHAc and morpholine: NHFmoc (NHAc, morpholine), Pd2(dba)3, 2-di-tertbutylphosphino-3,4,5,6-tetramethyl-2’,4’,6’-triisopropyl-1,1’-biphenyl, K3PO4, dioxane, heat; (e) LHMDS, allyl carbonocyanidate, THF, -78 °C to rt; (f) Pd(PPh3)4, DMF; (g) dicyclohexylamine, DMF.

Compounds 25, 26 and 43 were synthesized from the intermediate 10 by palladium-catalyzed carbonylation13 and subsequent hydrolysis, standard amide coupling procedures, acylation and palladium-catalyzed decarboxylation-allylation as shown in Scheme 3. Scheme 3. Synthesis of R2 Analogues with –CONH2, -CONHMe a

a

Reagents and conditions: (a) 1. Pd(OAc)2, 1,3-bis(diphenylphosphine)propane, CO(g), NEt3,

DMSO, MeOH; 2. LiOH, THF, H2O; (b) hexafluorophosphate benzotriazole tetramethyl uronium, N,N-diisopropylethylamine , DCM, NH4Cl (or NH2Me); (c) LHMDS, allyl carbonocyanidate, THF, -78 °C to rt; (d) Pd(PPh3)4, DMF.


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Biological Results and Discussion All newly prepared bifidenone analogues were initially tested in antiproliferative assays in the M14 (amelanotic melanoma) and NCI-H460 (large-cell lung carcinoma) cell lines. Table 1 summarizes the four diastereomers of bifidenone prepared. The cis-arrangement (4a and 4b) is preferred over the trans-arrangement (4c and 4d). Interestingly, the configuration of R4 did not significantly impact activity as demonstrated by comparing the potencies of compounds 4a and 4b (Table 1). With this information in hand, the cis-isomers were targeted exclusively for evaluation.

Table 1. SAR of Bifidenone Diastereomers IC50 (µM) Compound

M14

bifidenone (4a)

0.11 ± 0.06 0.26 ± 0.08

4b

0.3 ± 0.2

0.32 ± 0.06

4c

>4

>4

4d

>4

>4

NCI-H460

While the configuration of R4 was not critical for activity, R4 replacement studies demonstrated that the size of the R4 group was important (Table 2). Replacement of the methyl group with hydrogen or with a larger group such as ethyl significantly reduced potency. Based on those results, subsequent SAR studies focused on analogues with a methyl group at the R4 position. Table 2. SAR of different sized substituents at the R4 position.


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OMe MeO R4

O O

O

IC50 (µM) a Compound

R4

M14

NCI-H460

bifidenone (4a)

Me

0.11 ± 0.06

0.26 ± 0.08

5

H

2.0

3.2

6b

Et

2.6

11

a

IC50 values without standard deviation (SD) are from single experiments.

b

Mixture of

diastereomers.

Bifidenone has two methoxy groups on the phenyl ring, and the anti-proliferative results of their substitution are shown in Table 3. Comparison of those compounds revealed that the methoxy at the R1 position was important for potency while the methoxy at R2 position was dispensable. Removal of the R2 methoxy (16a, 16b) resulted in a 2- to 5-fold increase in potency. However, removal of the R1 methoxy (17a) caused a 40- to 80-fold drop in potency. A small survey of R1 substitutions indicated that the methoxy group was well suited for maintaining potency. Smaller substituents such as hydroxy (19a) or electronegative fluoro (21a) substitution resulted in a severe drop in potency. Likewise, the larger ethoxy substitution (20a) also offered no advantage, and saw a 6-fold decrease in potency in the M14 assay. Consequently, the decision was made to maintain the methoxy substitution at R1 and focus efforts to improve


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potency on other areas of the compound. One may also observe that a 4-methoxyphenyl group can be found in a number of known colchicine binding site compounds.5,10,11

Table 3. SAR of the R1 Position

IC50 (µM)a Compound

R1

bifidenone (4a) OMe

R2

M14

NCI-H460

OMe 0.11 ± 0.06

0.26 ± 0.08

16a

OMe

H

0.020 ± 0.008 0.09 ± 0.04

16b

OMe

H

0.081± 0.112

17a

H

OMe 8.8

10.6

18a

H

H

5.1

9.7

19a

OH

H

>20

>20

20a

OEt

H

0.65

0.28

21a

F

H

>20

>20

22a

OAc

H

>20

>20

23a

OCF3 H

>20

3.8

a

0.12 ± 0.07

IC50 values without SD are from single experiments.


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Efforts to optimize the R2 position indicated that it was more tolerant to modification as shown in Table 4. Even though the methoxy group is a strongly electron-donating group, it could be replaced by hydrogen (16a and 16b) and still maintain potency. Certain electron-withdrawing groups such as the primary amide (25a), cyano (27a), and chloro (31a) also retained potency roughly equivalent to the parent compound bifidenone (4a). The anilino moiety was tolerated well (33), but the more sterically demanding morpholino (30) substitution was not well-tolerated. Similarly, the primary amide (25a) was 4- to 9-fold more potent than bifidenone, but the larger secondary amide (26a) was 4- to 12-fold less potent. The most beneficial substitutions at this position were the phenol (34a) and the fluoro isostere (32a) which were 20-fold more active against the NCI-H460 cancer cell line and 4-fold more active against the M14 cancer cell line compared to the parent compound bifidenone. As was the case with the 4-methoxyphenyl substitution, compounds with a 3-fluoro or 3-hydroxy substitution have previously been reported as active.5,10,11 Table 4. SAR of the R2 Position


R2

bifidenone (4a) OMe 24a

Me

M14

NCI-H460

0.11 ± 0.06

0.26 ± 0.08

0.41

0.63


12

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R2

M14

25a

CONH2

0.012± 0.004 0.066± 0.004

26a

CONHMe

1.4

1.1

27a

CN

0.18

0.34

28

OAc

0.016± 0.008 0.11± 0.04

29a

NHAc

0.085

30b

Morpholine >20

>20

31a

Cl

0.24

0.64

32a

F

0.008± 0.004 0.09 ± 0.02

33b

NH2

0.049

34a

OH

0.008± 0.003 0.08± 0.02

a

NCI-H460

0.31

0.18

IC50 values without SD are from single experiments. bMixture of diastereomers.

Pyridine moieties are commonly used to modify the physicochemical properties, specifically LogP of molecules, which in turn can alter their absorption, distribution and metabolism.14 Consequently, pyridyl replacement of the phenyl ring was explored. However, introduction of the pyridine nitrogen as shown in Table 5 proved to be poorly tolerated, yielding either inactive or only moderately active analogues as shown in Table 5.


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Table 5. SAR of Pyridine Analogues

IC50 (µM)a Compound R3

R6

M14

NCI-H460

35a

H

H

0.18

0.33

36a

F

H

8

>20

37a

H

OMe >20

a

>20

IC50 values are from single experiments.

With the success of identifying highly potent compounds, the focus shifted to improving the pharmacokinetic properties of the series. Fluorine substitution is commonly used as a tool to block metabolic activation of compounds and in the development of safer drugs.15,16 For this reason, a variety of combinations of the best tolerated R2 groups with fluorine substitution at other positions on the phenyl ring were explored, and the results are summarized in Table 6. In general, fluorine substitution around the phenyl ring was well tolerated at the R3 position. When fluorine was installed at the R3 position, compounds with R2 groups consisting of hydrogen (44a), fluorine (45a) or hydroxy (46b) demonstrated improved tumor concentration (SI Table S1), while also either maintaining or increasing in potency.

On the other hand, fluorine

substitution at the R5 or R6 positions (47a, 48a, and 49a) was poorly tolerated.


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Table 6. SAR of Aryl Substitution

IC50 (µM)a Compound R2

R3 R6 R5 M14

NCI-H460

OMe

H

H

H

0.11 ± 0.06

0.26 ± 0.08

38a

OMe

F

H

H

0.014 ± 0.001 0.12 ± 0.01

39a

Me

F

H

H

0.23

0.65

40a

CN

F

H

H

0.037

0.15

41a

NH2

F

H

H

0.029

0.16

42a

NHAc

F

H

H

0.052

0.071

43a

CONH2 F

H

H

0.004

0.33

44a

H

F

H

H

0.014 ± 0.005 0.094 ± 0.02

45a

F

F

H

H

0.03 ± 0.02

45b

F

F

H

H

0.010 ± 0.004 0.035± 0.009

46a

OH

F

H

H

0.009 ± 0.008 0.033± 0.001

46b

OH

F

H

H

0.017 ± 0.006 0.015± 0.002

47a

F

H

H

F

1.6

2.7

48a

H

F

H

F

0.32

2.5

Bifidenone (4a)

0.059± 0.006


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IC50 (µM)a Compound R2 49a a

F

R3 R6 R5 M14

NCI-H460

H

3.8

F

H

3.3

IC50 values without SD are from single experiments.

In anticipation of mouse xenograft efficacy studies, an ip formulation was initially optimized for bifidenone. Mouse plasma concentrations (1 h post-injection) of bifidenone greater than its IC50 against NCI-H460 human lung cancer cells were initially achieved with a 40 mg/kg injection in a vehicle consisting of 40% propylene glycol, 20% Tween-20, 20% PEG400, 20% ethanol (v/v). Plasma concentrations were not diminished when this formulation was diluted with up to 20% water. Additional experiments demonstrated that plasma levels were diminished when propylene glycol was decreased to 25%, but increased or were not diminished when propylene glycol was increased to 45% and Tween-20 was decreased to 6 times the IC50 at 30 min, > 3 times the IC50 at 1 h, and greater than the IC50 at 3 h. With larger doses (75-100 mg/kg), plasma concentrations were > 4 times the IC50 at 6 h. Additional limited pharmacokinetic studies were conducted on selected analogues, in parallel with continued vehicle optimization. Supporting Information Table S1 lists concentrations of compound in mouse plasma and xenografted tumor tissue after single ip injections of compound. Of the compounds tested, 46b reliably demonstrated tumor concentrations many fold over its IC50. It was thus selected as the lead compound for further in vitro and in vivo studies.


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The optimized compound 46b demonstrated in vitro potency against an expanded panel of human-derived cancer cell lines (SI Table S2). Notably, it was active against multiple taxaneresistant cell lines: for seven cell lines, 46b was more potent than paclitaxel when tested in the same assay (Table 7). These include the NCI/ADR-RES (adriamycin-resistant ovarian cancer) cell line, which overexpresses the p-glycoprotein multidrug efflux pump. This suggests that 46b could be active against paclitaxel-resistant tumors, including those that express p-glycoprotein. Like bifidenone, the optimized compound 46b also inhibits tubulin polymerization in vitro (Figure 3).

Figure 3. Tubulin polymerization kinetics in the presence of 46b, paclitaxel, or vinblastine. Tubulin polymerization is monitored as an increase in absorbance (340 nm) over time.


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Table 7. In Vitro Antiproliferation Activity of 46b and Paclitaxel in Taxane-resistant Cell Lines IC50 (nM) Cell Line

Paclitaxel 46b

Fold Difference

NCI/ADR-RES

>9000

9

>1000

OVCAR-4 (ovarian cancer) >600

8±2

> 75

UO-31 (kidney cancer)

240

7

34

EKVX (NSCLC)

150

7

21

HCT-15 (colon cancer)

190 ± 30

14 ± 5

14

ACHN (renal cancer)

80 ± 30

12 ± 3

7

SF295 (glioblastoma)

56 ± 6

9.6 ± 0.5 6

Tumor growth inhibition of 46b was measured in several murine xenograft models. Tumors were initiated by subcutaneous injection of either tumor cells from cell culture or tumor fragments from continuous in vivo passage. Compound or vehicle was administrated by ip injection in an ethanol/PEG400/citrate vehicle optimized as described above, using an optimized injection volume of 5-10 mL/kg. Initial experiments were conducted to optimize the dosages and dosing schedules. For most tumor types, the optimized schedule consisted of three consecutive days of dosing at 50-70 mg/kg followed by two days of rest, for 3-4 cycles. An optimized schedule of 5-7 consecutive days of dosing at 30-40 mg/kg was used for the rapidly growing LOX-IMVI (melanoma) tumors.

Under these optimized dosing regimens, 46b effected

significant tumor growth inhibition (>60% TGI) against a variety of tumors derived from human lung cancer and melanoma. The data from several experiments are summarized in Table 8, and the growth curves from one experiment are shown in Figure 4.


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Table 8. Antitumor Activity of 46b Against Human Tumor Xenografts Max. mean Dose

n total

bodyweight loss %

Cell Line

(mg/kg)

Schedule

(deaths)

(day)

%TGI

NCI-H460

40

qd×3, 2 days rest, 3

10 (1)

7 (7)

71

7 (0)

12 (8)

67

6 (0)

3 (7)

54

6 (0)

8 (7)

69

6 (0)

9 (3)

82

10 (0)

4 (17)

76

10 (0)

2 (2)

63

10 (0)

6 (3)

77

10 (0)

9 (3)

84

cycles 60

qd×3, 2 days rest, 3 cycles

NCI-H522

40

(NSCLC)


50


60


Calu-6

(lung

50

cancer)


60


NCI-H69 (small-

60

cell lung cancer)

day 1, 4 days rest; then qd×3, 2 days rest, 2 cycles

70

day 1, 4 days rest; then qd×3, 2 days rest, 2


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cycles LOX-IMVI

a

30

qd×7

11 (0)

9 (6)

63

40

qd×5

11 (0)

50 (5)

63

Changes in tumor weight (delta weights) for each treated (T) and control (C) group are

calculated for each day tumors are measured by subtracting the median tumor weight on the first day of treatment (staging day) from the median tumor weight on the specified observation day. These values are used to calculate a percent TGI as follows: % TGI = [1-(delta T/delta C)] x 100. The optimal % TGI is the maximum value obtained after the first treatment.

Figure 4. Treating mice with 46b slows growth of NCI-H69 tumors. Points represent the median tumor volume from groups of 10 mice each. Mice received ip injections of 46b on days 0, 5, 6, 7, 10, 11, and 12.

CONCLUSION


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Bifidenone is an exciting natural product lead, but as is often the case the initial activity and bioavailability were inadequate for use as a potential anticancer agent. A medicinal chemistry effort was used to determine the moieties that were amenable to modification. That knowledge was used to produce several promising clinical candidates via the modification of bifidenone. Compounds 45b and 46b have improved potency and excellent bioavailability in mouse PK experiments. The subsequent xenograft experiments demonstrated significant inhibition of tumor growth. Additionally, compound 46b shows much better in vitro antiproliferative activity against taxane-resistant cell lines than paclitaxel, suggesting the potential for application in the treatment of cancers that are no longer responsive to the taxanes.

EXPERIMENTAL SECTION Chemistry. 1H NMR spectra were recorded on Bruker 300, 500, or 600 MHz spectrometers. Structures of selected compounds were verified with 2D experiments (COSY, ROESY, HSQC, and HMBC). Chemical shifts are reported in parts per million on the scale from an internal standard of tetramethylsilane.

Mass spectra were recorded on an Agilent 6100 Single

Quadrupole instrument. HRMS spectra were recorded on a Waters LCT time-of-flight mass spectrometer with an electrospray interface. Purity analysis was performed on Luna C18 (4.6 x 250 mm, 5 µm, flow 1.15 mL/min, detection UV 254 nm). Chiral analysis and preparative HPLC isolations were performed on Chiralpak AD (4.6 x 250 mm, 5 µm, flow 1.0 mL/min, detection UV 254 nm) or Chiralpak AD (50 x 250 mm, 10 µm, flow 100 mL/min, detection UV 254 nm), respectively. All commercially available reagents and solvents were used without further purification. All yields reported were not optimized.


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General Procedure. The general procedure used for the synthesis of compounds 5-6, 16-24, 31-32, 35-39, 44-49 and the physical properties of intermediates and bifidenone (4a) were previously reported.9 Compounds 6, 30, and 33 were obtained as a mixture of diastereomers. Compound purity was determined by HPLC. Compounds 26a, 43a, and 48a were obtained at 93%, 92%, and 79% purity, respectively. All other compounds (4, 5, 16-25, 27-29, 31, 32, 3442, 44-47, and 49) were obtained with a purity of 95% or greater. ((6S,7aS)-6-Allyl-7a-(3,4-dimethoxyphenethyl)-7,7a-dihydrobenzo[d][1,3]dioxol-5(6H)one) (5). Off-white solid, mp 40-42 °C. 1H NMR (500 MHz, CD3OD): δ 6.90 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 6.79 (d, J = 8.0 Hz, 1H), 5.96–5.92 (m, 1H), 5.67 (s, 1H), 5.60 (s, 1H), 5.40 (s, 1H), 5.22–5.18 (m, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 2.80–2.75 (m, 3H), 2.69 (d, J = 13.5 Hz, 1H), 2.64–2.60 (m, 1H), 2.29–2.24 (m, 2H), 2.15–2.12 (m, 1H), 1.82–1.78 (m, 1H). ESI MS m/z 345 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H25O5 345.1702, found 345.1711. ((6S,7aR)-6-Allyl-7a-(1-(3,4-dimethoxyphenyl)butan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (6). Colorless oil.

1

H NMR (500 MHz, CD3OD): δ

6.88–6.66 (m, 3H), 5.85–5.65 (m, 1H), 5.66 (s, 1H), 5.60 (s, 1H), 5.48 (s, 1H), 5.12-5.08 (m, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 2.95–2.72 (m, 2H), 2.63–2.52 (m, 3H), 2.16–2.08 (m, 2H), 1.95– 1.85 (m, 1H), 1.62–1.35 (m, 2H), 0.92–0.89 (m, 3H). ESI MS m/z 373 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C22H29O5 373.2015, found 373.2016. ((6S,7aR)-6-Allyl-7a-((S)-1-(4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (16a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.09 (d, J = 8.5 Hz, 2H), 6.86 (dd, J = 7.0, 2.5 Hz, 2H), 5.92–5.85 (m, 1H), 5.66 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.18–5.12 (m, 2H), 3.76 (s, 3H), 3.10 (dd, J = 13.5, 3.5 Hz, 1H), 2.90–2.87 (m, 1H), 2.70 (d, J = 14.0 Hz, 1H), 2.62–2.60 (m, 1H), 2.45 (t, J = 11.0 Hz, 1H), 2.25–2.20 (m, 1H),


22

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2.14 (dd, J = 13.5, 9.5 Hz, 1H), 1.96–1.93 (m, 1H), 0.83 (d, J = 6.0 Hz, 3H). ESI MS m/z 329 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H25O4 329.1753, found 329.1769. ((6S,7aR)-6-Allyl-7a-((R)-1-(4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (16b). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 6.98 (d, J = 8.5 Hz, 2H), 6.83 (dd, J = 6.5, 2.0 Hz, 2H), 5.83–5.78 (m, 1H), 5.68 (s, 1H), 5.63 (s, 1H), 5.54 (s, 1H), 5.08–5.02 (m, 2H), 3.75 (s, 3H), 2.80–2.78 (m, 2H), 2.64–2.58 (m, 1H), 2.55 (d, J = 14.0 Hz, 1H), 2.33 (t, J = 11.5 Hz, 1H), 2.11 (dd, J = 14.0, 10.0 Hz, 1H), 2.05–2.02 (m, 1H), 1.89–1.85 (m, 1H), 0.95 (d, J = 6.5 Hz, 3H). ESI MS m/z 329 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H25O4 329.1753, found 329.1758. ((6S,7aR)-6-Allyl-7a-((S)-1-(3-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (17a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.21 (t, J = 8.0 Hz, 1H), 6.77–6.73 (m, 3H), 5.92–5.87 (m, 1H), 5.67 (s, 1H), 5.61 (s, 1H), 5.50 (s, 1H), 5.18–5.12 (m, 2H), 3.77 (s, 3H), 3.15–3.13 (m, 1H), 2.95–2.88 (m, 1H), 2.70 (d, J = 9.5 Hz, 1H), 2.65–2.62 (m, 1H), 2.48 (dd, J = 14.5, 11.0 Hz, 1H), 2.25–2.22 (m, 1H), 2.15 (dd, J = 14.0, 9.5 Hz, 1H), 2.02–1.98 (m, 1H), 0.97 (d, J =7.0 Hz, 3H). ESI MS m/z 329 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H25O4 329.1753, found 329.1739. ((6S,7aR)-6-Allyl-7a-((S)-1-(phenyl)propan-2-yl)-7,7a-dihydrobenzo[d][1,3]dioxol-5(6H)one) (18a). Colorless oil.

1

H NMR (500 MHz, CD3OD): δ 7.30–7.17 (m, 5H), 5.95–5.78 (m,

1H), 5.67 (s, 1H), 5.61 (s, 1H), 5.50 (s, 1H), 5.18–5.12 (m, 2H), 3.16–3.13 (dd, J = 3.5, 2.0 Hz, 1H), 2.95–2.80 (m, 1H), 2.72 (d, J = 14.5 Hz, 1H), 2.64–2.62 (m, 1H), 2.51 (dd, J = 13.5, 11.5 Hz, 1H), 2.28–2.22 (m, 1H), 2.16 (dd, J = 14.0, 10.0 Hz, 1H), 2.09–2.01 (m, 1H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 299 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C19H23O3 299.1647, found 299.1656.


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((6S,7aR)-6-Allyl-7a-((S)-1-(4-hydroxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (19a). Off-white solid, mp 125-127 °C. 1H NMR (500 MHz, CD3OD): δ 6.98 (d, J = 7.5 Hz, 2H), 6.71 (d, J = 7.5 Hz, 2H), 5.92–5.85 (m, 1H), 5.64 (s, 1H), 5.59 (s, 1H), 5.48 (s, 1H), 5.17–5.11 (m, 2H), 3.05 (dd, J = 14.0, 4.0 Hz, 1H), 2.88–2.86 (m, 1H), 2.69 (d, J = 14.0 Hz, 1H), 2.67–2.60 (m, 1H), 2.40 (dd, J = 14.0, 11.0 Hz, 1H), 2.25–2.20 (m, 1H), 2.12 (t, J = 10.0 Hz, 1H), 1.99–1.93 (m, 1H), 0.82 (d, J = 7.0 Hz, 3H). ESI MS m/z 315 [C19H22O4 + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C19H23O4 315.1596, found 315.1604. ((6S,7aR)-6-Allyl-7a-(1-(4-ethoxyphenyl)propan-2-yl)-7,7a-dihydrobenzo[d][1,3]dioxol5(6H)-one) (20a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.08–6.95 (m, 2H), 6.83–6.80 (m, 2H), 5.92-5.81 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.18–5.13 (m, 2H), 4.01 (q, J = 7.0 Hz, 2H), 3.09–2.75 (m, 2H), 2.70 (d, J = 14.0 Hz, 1H), 2.65–2.58 (m, 1H), 2.53–2.44 (m, 1H), 2.28-2.22 (m, 1H), 2.14 (dd, J = 14.0, 10.0 Hz, 1H), 1.99–1.93 (m, 1H), 1.37 (t, J = 7.0 Hz, 3H), 0.83 (d, J = 6.0 Hz, 3H). ESI MS m/z 343 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H27O4 343.1909, found 343.1904. ((6S,7aR)-6-Allyl-7a-(1-(4-fluorophenyl)propan-2-yl)-7,7a-dihydrobenzo[d][1,3]dioxol5(6H)-one) (21a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.20 (dd, J = 5.5, 2.0 Hz, 2H), 7.00 (t, J = 4.0 Hz, 2H), 5.92–5.82 (m, 1H), 5.66 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.17–5.12 (m, 2H), 3.14 (dd, J = 14.0, 4.0 Hz, 1H), 2.90–2.76 (m, 1H), 2.69 (d, J = 14.0 Hz, 1H), 2.66–2.63 (m, 1H), 2.53 (d, J = 11.0 Hz, 1H), 2.28–2.22 (m, 1H), 2.14 (dd, J = 14.0, 9.5 Hz, 1H), 2.00–1.93 (m, 1H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 317 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C19H21FO3 317.1553, found 317.1562. (4-((S)-2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3ayl)propyl)phenylacetate) (22a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.22 (d, J = 7.5


24

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Hz, 2H), 7.03 (d, J = 7.5 Hz, 2H), 5.92–5.85 (m, 1H), 5.67 (s, 1H), 5.61 (s, 1H), 5.50 (s, 1H), 5.14–5.11 (m, 2H), 3.16 (dd, J = 14.5, 4.0 Hz, 1H), 2.90–2.87 (m, 1H), 2.68 (d, J = 14.0 Hz, 1H), 2.67–2.60 (m, 1H), 2.53 (t, J = 11.0 Hz, 1H), 2.25 (s, 3H), 2.25–2.20 (m, 1H), 2.16 (t, J = 9.5 Hz, 1H), 2.02–1.95 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H). ESI MS m/z 357 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H25O5 357.1702, found 357.1699. ((6S,7aR)-6-Allyl-7a-((S)-1-(4-(trifluoromethoxy)phenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (23a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.29 (d, J = 6.5 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 5.92–5.85 (m, 1H), 5.66 (s, 1H), 5.61 (s, 1H), 5.51 (s, 1H), 5.16–5.12 (m, 2H), 3.15 (d, J = 13.5 Hz, 1H), 2.90–2.87 (m, 1H), 2.68 (d, J = 14.5 Hz, 1H), 2.67–2.60 (m, 1H), 2.59 (dd, J = 14.0, 9.0 Hz, 1H), 2.25–2.20 (m, 1H), 2.16 (t, J = 10.0 Hz, 1H), 2.04–2.01 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H). ESI MS m/z 383 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H22F3O4 383.1470, found 383.1452. ((6S,7aR)-6-Allyl-7a-((S)-1-(4-methoxy-3-methylphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (24a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 6.96–6.92 (m, 2H), 6.82 (d, J = 7.0 Hz, 1H), 5.92-5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.15–5.12 (m, 2H), 3.78 (s, 3H), 3.07 (dd, J = 13.5, 3.5 Hz, 1H), 2.90–2.87 (m, 1H), 2.70 (d, J = 14.0 Hz, 1H), 2.62–2.60 (m, 1H), 2.37 (t, J = 11.0 Hz, 1H), 2.25–2.20 (m, 1H), 2.15 (s, 3H), 2.14–2.11 (m, 1H), 1.96–1.93 (m, 1H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 343 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H27O4 343.1909, found 343.1917. (5-((S)-2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)-2methoxybenzamide) (25a). Off-white semisolid. 1H NMR (500 MHz, CD3OD): δ 7.84 (d, J = 3.0 Hz, 1H), 7.36 (d, J = 6.0 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 5.92-5.85 (m, 1H), 5.66 (s, 1H), 5.61 (s, 1H), 5.50 (s, 1H), 5.22–5.13 (m, 2H), 3.96 (s, 3H), 3.13 (dd, J = 11.0, 3.0 Hz, 1H), 2.90–


25


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2.85 (m, 1H), 2.70 (d, J = 10.5 Hz, 1H), 2.65–2.64 (m, 1H), 2.53 (t, J = 11.5 Hz, 1H), 2.18–2.11 (m, 1H), 2.15 (dd, J = 13.5, 9.5 Hz, 1H), 2.03–1.95 (m, 1H), 0.85 (d, J = 7.0 Hz, 3H). ESI MS m/z 372 [M + H]+. HRMS (ESI) m/z: [M + Na]+ calcd for C21H25NO5Na 394.1630, found 394.1633. (5-((S)-2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)-2methoxy-N-methylbenzamide) (26a). Off-white semisolid.

1


7.78 (s, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 5.95–5.85 (m, 1H), 5.66 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.18–5.11 (m, 2H), 3.94 (s, 3H), 3.10 (dd, J = 14.0, 3.5 Hz, 1H), 2.94 (s, 3H), 2.92–2.89 (m, 1H), 2.70 (d, J = 14.0 Hz, 1H), 2.68–2.65 (m, 1H), 2.52 (dd, J = 14.0, 10.0 Hz, 1H), 2.27–2.22 (m, 1H), 2.15 (dd, J = 14.0, 9.5 Hz, 1H), 2.03–1.95 (m, 1H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 386 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C22H28NO5 386.1943, found 386.1955. (5-((S)-2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)-2methoxybenzonitrile) (27a). Colorless oil.

1

H NMR (500 MHz, CD3OD): δ 7.48–7.43 (m,

2H), 7.14 (d, J = 14.5 Hz, 1H), 5.92-5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.18– 5.11 (m, 2H), 3.92 (s, 3H), 3.11 (dd, J = 14.0, 10.0 Hz, 1H), 2.92–2.85 (m, 1H), 2.66–2.58 (m, 2H), 2.53 (t, J = 14.0 Hz, 1H), 2.25–2.21 (m, 1H), 2.13 (dd, J = 14.0, 9.5 Hz, 1H), 2.03–1.95 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H). ESI MS m/z 354 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H24NO4 354.1705, found 354.1697. (5-(2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)-2methoxyphenyl acetate) (28). Light brown semisolid. 1H NMR (500 MHz, CD3OD): δ 7.04– 7.00 (m, 2H), 6.68 (s, 1H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.59 (s, 1H), 5.49 (s, 1H), 5.13–5.11 (m, 2H), 3.79 (s, 3H), 3.08 (dd, J = 13.5, 3.5 Hz, 1H), 2.92-2.87 (m, 1H), 2.68 (d, J = 14.5 Hz,


26

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1H), 2.65–2.60 (m, 1H), 2.47 (t, J = 11.5 Hz, 1H), 2.24 (s, 3H), 2.23–2.17 (m, 1H), 2.14 (dd, J = 14.0, 9.5 Hz, 1H), 1.95–1.90 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H). ESI MS m/z 387 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C22H27O6 387.1808, found 387.1819. (N-(5-((S)-2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)2-methoxyphenyl)acetamid (29a). Off-white semisolid. 1H NMR (500 MHz, CD3OD): δ 7.88 (s, 1H), 6.95–6.92 (m, 2H), 5.95–5.85 (m, 1H), 5.66 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.19–5.11 (m, 2H), 3.85 (s, 3H), 3.11 (dd, J = 23.0, 5.5 Hz, 1H), 2.92–2.85 (m, 1H), 2.72 (d, J = 24.7 Hz, 1H), 2.66–2.59 (m, 1H), 2.42 (t, J = 11.5 Hz, 1H), 2.28–1.98 (m, 1H), 2.11 (s, 3H), 2.10–1.95 (m, 2H), 0.84 (d, J = 11.5 Hz, 3H). ESI MS m/z 386 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C22H28NO5 386.1967, found 386.1979. ((6S,7aR)-6-Allyl-7a-((S)-1-(2-fluoro-4-methoxy-5-morpholinophenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (30).

Off-white semisolid.

1

H NMR (500 MHz,

CD3OD): δ 6.90 (d, J = 8.0 Hz, 1H), 6.85 (s, 1H), 6.76 (s, 1H), 5.95–5.90 (m, 1H), 5.67 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.18–5.10 (m, 2H), 3.83 (s, 3H), 3.82–3.80 (m, 4H), 3.09 (d, J = 14.0 Hz, 1H), 3.02–2.98 (m, 4H), 2.8–2.85 (m, 1H), 2.72 (d, J = 14.0 Hz, 1H), 2.64–2.58 (m, 1H), 2.45 (dd, J = 13.5, 9.0 Hz, 1H), 2.22–2.20 (m, 1H), 2.15 (t, J = 10.0 Hz, 1H), 2.03–1.95 (m, 2H), 0.85 (d, J = 7.0 Hz, 3H). ESI MS m/z 414 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C24H32NO5 414.2280, found 414.2280. ((6S,7aR)-6-Allyl-7a-((S)-1-(3-chloro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (31a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.18 (d, J = 2.5 Hz, 1H), 7.10 (dd, J = 7.5, 2.0 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.15–5.13 (m, 2H), 3.85 (s, 3H), 3.08 (dd, J = 14.0, 4.0 Hz, 1H), 2.92–2.87 (m, 1H), 2.67 (d, J = 14.0 Hz, 1H), 2.64–2.62 (m, 1H), 2.47 (dd, J = 14.0,


27


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11.0 Hz, 1H), 2.25–2.20 (m, 1H), 2.15 (dd, J = 14.0, 10.0 Hz, 1H), 1.97–1.95 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H). ESI MS m/z 363 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H24ClO4 363.1363, found 363.1363. ((6S,7aR)-6-Allyl-7a-((S)-1-(3-fluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (32a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.01 (d, J = 7.5 Hz, 1H), 6.93 (s, 1H), 6.91 (d, J = 4.5 Hz, 1H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.17–5.12 (m, 2H), 3.84 (s, 3H), 3.08 (d, J = 13.5 Hz, 1H), 2.90–2.87 (m, 1H), 2.67 (d, J = 14.0 Hz, 1H), 2.62–2.60 (m, 1H), 2.47 (t, J = 11.5 Hz, 1H), 2.25–2.20 (m, 1H), 2.14 (d, J = 9.5 Hz, 1H), 2.03–1.97 (m, 1H), 0.85 (d, J = 6.5 Hz, 3H). ESI MS m/z 347 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H24FO4 347.1659, found 347.1674. ((6S,7aR)-6-Allyl-7a-(1-(3-amino-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (33a).


1

H NMR (500 MHz,

CD3OD): δ 6.76 (dd, J = 13.0, 8.0 Hz, 1H), 6.60 (d, J = 2.0 Hz, 1H), 6.52 (dd, J = 8.5, 2.0 Hz, 1H), 5.92–5.75 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.48 (s, 1H), 5.19–5.12 (m, 2H), 3.81 (s, 3H), 3.01–2.99 (m, 1H), 2.89–2.79 (m, 1H), 2.70 (d, J = 9.0 Hz, 1H), 2.67–2.63 (m, 1H), 2.34–2.29 (m, 2H), 2.12–2.10 (m, 1H), 1.98–1.95 (m, 1H), 0.83 (d, J = 6.5 Hz, 3H). ESI MS m/z 344 [M + H]+. HRMS (ESI) m/z: [M + Na]+ calcd for C20H25NO4Na 366.1681, found 366.1693. ((6S,7aR)-6-Allyl-7a-((S)-1-(3-hydroxy-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (34a).

Light brown solid.

1

H NMR (500 MHz,

CD3OD): δ 6.84 (d, J = 8.0 Hz, 1H), 6.64–6.60 (m, 2H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.15–5.12 (m, 2H), 3.81 (s, 3H), 3.08–3.02 (m, 1H), 2.90–2.87 (m, 1H), 2.69 (d, J = 14.0 Hz, 1H), 2.58–2.53 (m, 1H), 2.38 (t, J = 11.5 Hz, 1H), 2.20–2.15 (m, 1H), 2.13 (dd,


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J = 14.0, 9.5 Hz, 1H), 1.95–1.92 (m, 1H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 345 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H25O5 345.1702, found 345.1697. ((6S,7aR)-6-Allyl-7a-((S)-1-(6-methoxypyridin-3-yl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one)(35a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.95 (d, J = 2.5 Hz, 1H), 7.54 (d, J = 7.5, 2.5 Hz, 1H), 6.77 (d, J = 7.5 Hz, 1H), 5.92–5.85 (m, 1H), 5.66 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.18–5.13 (m, 2H), 3.87 (s, 3H), 3.07 (dd, J = 14.5, 4.0 Hz, 1H), 2.90–2.87 (m, 1H), 2.69 (d, J = 14.0 Hz, 1H), 2.67–2.60 (m, 1H), 2.53 (t, J = 11.0 Hz, 1H), 2.25–2.20 (m, 1H), 2.16 (dd, J = 14.0, 9.5 Hz, 1H), 1.99–1.93 (m, 1H), 0.89 (d, J = 8.0 Hz, 3H). ESI MS m/z 330 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C19H24NO4 330.1705, found 330.1706. (6S,7aR)-6-Allyl-7a-((S)-1-(4-fluoro-6-methoxypyridin-3-yl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one (36a).


1

H NMR (500 MHz,

CD3OD): δ 7.82 (s, 1H), 6.69 (s, 1H), 5.95–5.92 (m, 1H), 5.66 (s, 1H), 5.61 (s, 1H), 5.49 (s, 1H), 5.22–5.12 (m, 2H), 3.92 (s, 3H), 3.12 (dd, J = 13.5, 4.0 Hz, 1H), 2.86–2.82 (m, 1H), 2.70 (d, J = 14.0 Hz, 1H), 2.61–2.54 (m, 2H), 2.39–2.35 (m, 1H), 2.20–2.11 (m, 2H), 0.77 (d, J = 7.0 Hz, 3H). ESI MS m/z 348 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C19H23FNO4 348.1611, found 348.1605. (6S,7aR)-6-Allyl-7a-((S)-1-(5,6-dimethoxypyridin-3-yl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one (37a).

Colorless semisolid.

1

H NMR (500 MHz,

CD3OD): δ 7.52 (s, 1H), 7.05 (s, 1H), 5.98–5.88 (m, 1H), 5.67 (s, 1H), 5.61 (s, 1H), 5.50 (s, 1H), 5.19–5.13 (m, 2H), 3.91 (s, 3H), 3.84 (s, 3H), 3.08 (d, J = 14.0, 3.5 Hz, 1H), 2.90–2.85 (m, 1H), 2.70 (d, J = 14.0 Hz, 1H), 2.64–2.62 (m, 1H), 2.53 (t, J = 11.0 Hz, 1H), 2.27–2.22 (m, 1H), 2.17


29


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(d, J = 10.0 Hz, 1H), 2.15–2.12 (m, 1H), 2.03–1.99 (m, 1H), 0.89 (d, J = 7.0 Hz, 3H). ESI MS m/z 360 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H26NO5 360.1811, found 360.1825. ((6S,7aR)-6-Allyl-7a-((S)-1-(2-fluoro-4,5-dimethoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (38a). Brown oil.

1


6.77 (d, J = 3.5 Hz, 1H), 6.74 (s, 1H), 5.92-5.85 (m, 1H), 5.67 (s, 1H), 5.61 (s, 1H), 5.49 (s, 1H), 5.21–5.10 (m, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.07 (dd, J = 14.0, 3.0 Hz, 1H), 2.86–2.83 (m, 1H), 2.69 (d, J = 14.5 Hz, 1H), 2.62–2.52 (m, 2H), 2.29–2.22 (m, 1H), 2.15 (t, J = 9.0 Hz, 1H), 2.03– 1.99 (m, 1H), 0.86 (d, J = 6.5 Hz, 3H). ESI MS m/z 377 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H26FO5 377.1764, found 377.1777. ((6S,7aR)-6-Allyl-7a-((S)-1-(2-fluoro-4-methoxy-5-methylphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (39a). Brown oil.

1


6.92 (d, J = 7.0 Hz, 1H), 6.67 (d, J = 12.5 Hz, 1H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.18–5.11 (m, 2H), 3.79 (s, 3H), 3.09 (d, J = 14.0 Hz, 1H), 2.82–2.79 (m, 1H), 2.67 (d, J = 14.0 Hz, 1H), 2.62–2.58 (m, 1H), 2.51 (t, J = 11.5 Hz, 1H), 2.28–2.25 (m, 1H), 2.12–2.09 (m, 1H), 2.11 (s, 3H), 2.03–1.95 (m, 1H), 0.82 (d, J = 7.0 Hz, 3H). ESI MS m/z 361 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H26FO4 361.1815, found 361.1826. (5-((S)-2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)-4fluoro-2-methoxybenzonitrile) (40a). Off-white solid, mp 45-47 °C.

1

H NMR (500 MHz,

CD3OD): δ 7.56 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 12.0 Hz, 1H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.18-5.11 (m, 2H), 3.93 (s, 3H), 3.10 (dd, J = 14.0, 8.0 Hz, 1H), 2.85– 2.79 (m, 1H), 2.68–2.58 (m, 3H), 2.25–2.20 (m, 1H), 2.17 (t, J = 10.0 Hz, 1H), 2.05–2.01 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H). ESI MS m/z 372 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H23FNO4 372.1611, found 372.1626.


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((6S,7aR)-6-Allyl-7a-((S)-1-(5-amino-2-fluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (41a). Brown oil.

1


6.65 (d, J = 11.5 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 5.92–5.85 (m, 1H), 5.64 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.18–5.11 (m, 2H), 3.82 (s, 3H), 3.01 (dd, J = 14.0, 10.0 Hz, 1H), 2.85–2.82 (m, 1H), 2.68 (d, J = 13.5 Hz, 1H), 2.63–2.60 (m, 1H), 2.47 (t, J = 10.5 Hz, 1H), 2.25–2.22 (m, 1H), 2.13 (dd, J = 14.0, 9.5 Hz, 1H), 2.03–1.98 (m, 1H), 0.83 (d, J = 7.0 Hz, 3H). ESI MS m/z 362 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H25FNO4 362.1768, found 362.1778. (N-(5-((S)-2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)4-fluoro-2-methoxyphenyl)acetamide) (42a). Off-white solid, mp 40-42 °C.

1

H NMR (500

MHz, CD3OD): δ 7.79 (d, J = 7.5 Hz, 1H), 6.81 (d, J = 11.5 Hz, 1H), 5.95–5.85 (m, 1H), 5.65 (s, 1H), 5.59 (s, 1H), 5.48 (s, 1H), 5.22–5.09 (m, 2H), 3.84 (s, 3H), 3.02 (d, J = 14.0 Hz, 1H), 2.85– 2.79 (m, 1H), 2.69 (d, J = 14.0 Hz, 1H), 2.58–2.50 (m, 2H), 2.25–2.21 (m, 1H), 2.14 (s, 3H), 2.14–2.11 (m, 1H), 2.03–1.99 (m, 1H), 0.83 (d, J = 7.0 Hz, 3H). ESI MS m/z 404 [M + H]+. HRMS (ESI) m/z: [M + Na]+ calcd for C22H27FNO5 404.1873, found 404.1858. (5-(2-((3aR,5S)-5-Allyl-6-oxo-3a,4,5,6-tetrahydrobenzo[d][1,3]dioxol-3a-yl)propyl)-4fluoro-2-methoxybenzamide) (43a). Off-white solid, mp 43-45 °C.

1

H NMR (500 MHz,

CD3OD): δ 7.92 (d, J = 16.5 Hz, 1H), 6.99 (dd, J = 20.0, 14.5 Hz, 1H), 5.95–5.85 (m, 1H), 5.69 (s, 1H), 5.54 (s, 1H), 5.50 (s, 1H), 5.18–5.01 (m, 2H), 3.96 (s, 3H), 2.89–2.79 (m, 2H), 2.68–2.45 (m, 3H), 2.30–1.95 (m, 3H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 390 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C21H25FNO5 390.1717, found 390.1726. ((6S,7aR)-6-Allyl-7a-((S)-1-(2-fluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (44a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 7.13 (t, J = 7.5 Hz, 1H), 6.70 (t, J = 7.5 Hz, 2H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H),


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5.49 (s, 1H), 5.20–5.10 (m, 2H), 3.77 (s, 3H), 3.06 (dd, J = 14.5, 4.0 Hz, 1H), 2.90–2.87 (m, 1H), 2.69 (d, J = 14.0 Hz, 1H), 2.67–2.60 (m, 1H), 2.57 (t, J = 11.0 Hz, 1H), 2.25–2.20 (m, 1H), 2.15 (dd, J = 14.0, 10.0 Hz, 1H), 2.03–1.95 (m, 1H), 0.82 (d, J = 7.0 Hz, 3H). ESI MS m/z 347 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H24FO4 347.1659, found 347.1648. ((6S,7aR)-6-Allyl-7a-((S)-1-(2,5-difluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (45a). Light brown oil. 1H NMR (500 MHz, CD3OD):

δ 6.99 (dd, J = 11.5, 7.0 Hz, 1H), 6.89 (dd, J = 9.0, 4.0 Hz, 1H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.50 (s, 1H), 5.19–5.10 (m, 2H), 3.84 (s, 3H), 3.06 (dd, J = 14.5, 4.0 Hz, 1H), 2.82– 2.79 (m, 1H), 2.66 (d, J = 14.0 Hz, 1H), 2.63–2.51 (m, 2H), 2.26–2.21 (m, 1H), 2.15 (dd, J = 14.0, 10.0 Hz, 1H), 2.03–1.99 (m, 1H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 365 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H23F2O4 365.1564, found 365.1561. ((6S,7aR)-6-Allyl-7a-((R)-1-(2,5-difluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (45b).

Light brown oil.

1

H NMR (500 MHz,

CD3OD): δ 6.89 (dd, J = 11.5, 7.0 Hz, 1H), 6.83 (dd, J = 11.5, 7.0 Hz, 1H), 5.83–5.74 (m, 1H), 5.64 (s, 1H), 5.58 (s, 1H), 5.50 (s, 1H), 5.06–4.99 (m, 2H), 3.80 (s, 3H), 2.79–2.76 (m, 2H), 2.64–2.58 (m, 1H), 2.54 (d, J = 14.0 Hz, 1H), 2.36 (t, J = 11.5 Hz, 1H), 2.07–1.94 (m, 2H), 0.93 (d, J = 7.0 Hz, 3H). ESI MS m/z 365 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H23F2O4 365.1564, found 365.1559. ((6S,7aR)-6-Allyl-7a-((S)-1-(2-fluoro-5-hydroxy-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (46a). Off-white solid. 1H NMR (500 MHz, CD3OD):

δ 6.72 (t, J = 11.5 Hz, 1H), 6.63 (d, J = 7.5 Hz, 1H), 5.92–5.85 (m, 1H), 5.65 (s, 1H), 5.60 (s, 1H), 5.49 (s, 1H), 5.18–5.11 (m, 2H), 3.81 (s, 3H), 3.02 (d, J = 14.0 Hz, 1H), 2.85–2.82 (m, 1H), 2.68 (d, J = 14.0 Hz, 1H), 2.62–2.58 (m, 1H), 2.48 (t, J = 11.5 Hz, 1H), 2.25–2.20 (m, 1H), 2.13


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(dd, J = 14.0, 10.0 Hz, 1H), 2.03–1.95 (m, 1H), 0.83 (d, J = 7.0 Hz, 3H). ESI MS m/z363 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H24FO5 363.1608, found 363.1594. ((6S,7aR)-6-Allyl-7a-((R)-1-(2-fluoro-5-hydroxy-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (46b).


1

H NMR (500 MHz,

CD3OD): δ 6.69 (d, J = 9.0 Hz, 1H), 6.53 (d, J = 7.5 Hz, 1H), 5.85–5.78 (m, 1H), 5.66 (s, 1H), 5.62 (s, 1H), 5.54 (s, 1H), 5.08–5.01 (m, 2H), 3.80 (s, 3H), 2.82–2.79 (m, 2H), 2.64–2.58 (m, 1H), 2.55 (d, J = 14.0 Hz, 1H), 2.33 (t, J = 11.5 Hz, 1H), 2.10 (dd, J = 13.5, 9.5 Hz, 1H), 2.03– 1.95 (m, 2H), 0.96 (d, J = 7.0 Hz, 3H). ESI MS m/z 363 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H24FO5 363.1608, found 363.1620. ((6S,7aR)-6-Allyl-7a-(1-(3,5-difluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (47a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 6.94 (dd, J = 8.5, 2.0 Hz, 1H), 6.87 (d, J = 7.5 Hz, 1H), 5.92–5.80 (m, 1H), 5.65 (s, 1H), 5.61 (s, 1H), 5.50 (s, 1H), 5.19–5.11 (m, 2H), 3.87 (s, 3H), 3.09 (dd, J = 14.0, 4.0 Hz, 1H), 2.87–2.78 (m, 1H), 2.67–2.59 (m, 3H), 2.25–2.18 (m, 1H), 2.16–2.11 (m, 1H), 2.04–2.01 (m, 1H), 0.84 (d, J = 7.0 Hz, 3H). ESI MS m/z 365 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H23F2O4 365.1564, found 365.1560. ((6S,7aR)-6-Allyl-7a-((R)-1-(2,6-difluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (48a). Colorless oil. 1H NMR (500 MHz, CD3OD): δ 6.56 (s, 1H), 6.52 (s, 1H), 5.85–5.78 (m, 1H), 5.67 (s, 1H), 5.63 (s, 1H), 5.54 (s, 1H), 5.09–5.00 (m, 2H), 3.77 (s, 3H), 2.85–2.50 (m, 5H), 2.10–1.95 (m, 3H), 0.97 (d, J = 11.5 Hz, 3H). ESI MS m/z 365 [M + H]+.

HRMS (ESI) m/z:

[M + H]+ calcd for C20H23F2O4 365.1564, found

365.1568.


33


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((6S,7aR)-6-Allyl-7a-((R)-1-(2,3-difluoro-4-methoxyphenyl)propan-2-yl)-7,7adihydrobenzo[d][1,3]dioxol-5(6H)-one) (49a). Light brown solid, mp 40-42 °C. 1H NMR (500 MHz, CD3OD): δ 6.86–6.83 (m, 2H), 5.81–5.78 (m, 1H), 5.68 (s, 1H), 5.62 (s, 1H), 5.54 (s, 1H), 5.08–5.01 (m, 2H), 3.86 (s, 3H), 2.85–2.80 (m, 2H), 2.64–2.58 (m, 1H), 2.54 (d, J = 14.0 Hz, 1H), 2.46 (t, J = 11.5 Hz, 1H), 2.12 (dd, J = 14.0, 9.5 Hz, 1H), 2.03–1.95 (m, 2H), 0.96 (d, J = 7.0 Hz, 3H). ESI MS m/z 365 [M + H]+. HRMS (ESI) m/z: [M + H]+ calcd for C20H23F2O4 365.1564, found 365.1568.

The general procedure for synthesis of the intermediate 8 using R3 = F as an example.

To a solution of 7 prepared by the general procedure described in our previous work3 (3.4 g, 8.2 mmol) in 65 mL of ethyl acetate was added Pd(OH)2/C (20 wt. % Pd on carbon, 680 mg) under nitrogen atmosphere. The mixture was stirred under one atmosphere of H2 for 1 h. The reaction mixture was filtered through a pad of celite and the filter cake was washed with ethyl acetate (300 mL). The filtrate was concentrated in vacuo to provide 8 as a colorless oil (2.6 g, 100%). The crude product was used without further purification. 1H NMR (500 MHz, CDCl3): δ 6.70 (d, J = 7.0 Hz, 1H), 6.58 (d, J = 10.5 Hz, 1H), 5.31 (s, 1H), 5.10 (s, 1H), 4.78 (s, 1H), 4.35 (t, J = 3.0 Hz, 1H), 3.87 (s, 3H), 2.87 (dd, J = 17.0, 4.5 Hz, 1H), 3.75 (d, J = 14.0 Hz, 1H), 2.57–2.45 (m, 2H), 2.33–2.22 (m, 2H), 2.07–2.01 (m, 2H), 1.90–1.83 (m, 1H), 0.97 (d, J = 7.0 Hz, 3H).



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To a solution of 8 (2.6 g, 8.21 mmol), triethylamine (2.3 mL, 16.42 mmol) and 4(dimethylamnio)pyridine (20 mg) in CH2Cl2 (80 mL) was added N-phenyl bis-trifluoromethane sulfonimide (4.4 g, 12.31 mmol). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated to dryness. The crude residue was purified by silica gel chromatography (0 to 30% ethyl acetate/hexanes) to provide 9 as a colorless oil (3.2 g, 86%). 1H NMR (500 MHz, CD3OD): δ 7.04 (d, J = 7.5 Hz, 1H), 6.77 (d, J = 11.0 Hz, 1H), 5.12 (s, 1H), 4.79 (s, 1H), 4.36 (t, J = 3.0 Hz, 1H), 3.89 (s, 3H), 2.89 (dd, J = 17.5, 3.0 Hz, 1H), 2.83 (d, J = 13.0 Hz, 1H), 2.56–2.51 (m, 1H), 2.48 (dd, J = 17.0, 3.0 Hz, 1H), 2.34–2.25 (m, 2H), 2.06–2.03 (m, 1H), 1.89 (t, J = 4.0 Hz, 1H), 0.99 (d, J = 7.0 Hz, 3H). The general procedure for synthesis of the intermediate 10 using R3 = F as an example.

A mixture of 9 (3.2 g, 7.04 mmol), Pd(OAc)2 (791 mg, 3.52 mmol) and 4,5-diazofluoren-9-one (626 mg, 3.52 mmol) in 75 mL of DMSO was heated to 80 °C under one atmosphere of oxygen for 4 h. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (300 mL). The organic phase was washed with brine, dried (MgSO4) and concentrated to dryness under reduced pressure. The crude residue was purified by silica gel chromatography (0 to 30% ethyl acetate/hexanes) to provide 10 as a colorless gum-like material (1.6 g, 49%). 1H NMR (500 MHz, CD3OD): δ 7.28 (d, J = 7.0 Hz, 1H), 7.06 (d, J = 11.0 Hz, 1H), 5.69 (s, 1H), 5.63 (s, 1H), 5.45 (s, 1H), 3.90 (s, 3H), 3.06 (dd, J = 13.5, 2.0 Hz, 1H), 2.65 (d, J = 12.0 Hz, 1H), 2.57 (t, J = 10.5 Hz, 1H), 2.53–2.46 (m, 2H), 2.26–2.24 (m, 1H), 2.07–2.03 (m, 1H), 0.95 (d, J = 7.0 Hz, H).

The general procedure for synthesis of the intermediate 11-1 using R2 = CN, R3 = F as an example.


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A mixture of 10 (50 mg, 0.11 mmol), Zn(CN)2 (52 mg, 0.44 mmol), Pd(PPh3)4 (19 mg, 0.02 mmol) in 3 mL of DMF in microwave tube was heated to 105 °C for 4 h. The reaction mixture was diluted with ethyl acetate (100 mL), washed with brine, dried (MgSO4) and concentrated to dryness. The crude residue was purified by silica gel chromatography (0 to 40% ethyl acetate/hexanes) to provide 11-1 as a colorless gum-like material (30 mg, 83%). 1H NMR (500 MHz, CD3OD): δ 7.61 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 12.0 Hz, 1H), 5.69 (s, 1H), 5.63 (s, 1H), 5.45 (s, 1H), 3.93 (s, 3H), 3.08 (d, J = 14.0 Hz, 1H), 2.63 (d, J = 14.0 Hz, 1H), 2.54 (t, J = 10.0 Hz, 1H), 2.51–2.48 (m, 2H), 2.25–2.20 (m, 1H), 2.06–2.01 (m, 1H), 0.93 (d, J = 7.0 Hz, 3H).

The general procedure for synthesis of the intermediate 11-2 using R2 = NHFmoc, R3 = F as an example.

A mixture of 10 (100 mg, 0.22 mmol), 9-fluorenylmethyl carbamate (105 mg, 0.44 mmol), Pd2(dba)3 (20 mg, 0.02 mmol), 2-di-tert-butylphosphino-3,4,5,6-tetramethyl-2’,4’,6’triisopropyl-1,1’-biphenyl (42 mg, 0.09 mmol) and K3PO4 (140 mg, 0.66 mmol) in 4 mL of dioxane was heated to 105 °C under nitrogen atmosphere for 4 h. The reaction mixture was diluted with ethyl acetate (100 mL), washed with brine, dried (MgSO4) and concentrated to dryness. The crude residue was purified by silica gel chromatography (0 to 40% ethyl acetate/hexanes) to provide 11-2 as a colorless semisolid (32 mg, 26%). 1H NMR (500 MHz, CD3OD): δ 7.81–7.78 (m, 2H), 7.66–7.65 (m, 1H), 7.41–7.38 (m, 3H), 7.32–7.29 (m, 2H), 6.80 (d, J = 11.5 Hz, 1H), 5.69 (s, 1H), 5.63 (s, 1H), 5.44 (s, 1H), 4.47 (d, J = 7.0 Hz, 2H), 4.32–4.30


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(m, 1H), 3.83 (s, 3H), 3.02 (dd, J = 14.0, 3.0 Hz, 1H), 2.68–2.66 (m, 1H), 2.48–2.46 (m, 3H), 2.25–2.18 (m, 1H), 2.06–1.98 (m, 1H), 0.97 (d, J = 7.0 Hz, 3H).


A mixture of Pd(OAc)2 (52 mg, 0.24 mmol), 1,3-bis(diphenylphosphine)propane (100 mg, 0.24 mmol) and triethylamine (0.67 mL, 4.8 mmol) in 10 mL of DMSO was stirred at room temperature for 10 min. A solution of 10 (440 mg, 0.96 mmol) in MeOH (10 mL) was added. The reaction mixture was transferred to a pressure reactor. The reactor was charged with 50 psi of CO and sealed. The reaction mixture was heated to 45 °C for 12 h. The reaction mixture was diluted with ethyl acetate (100 mL), washed with brine, dried (MgSO4) and concentrated to dryness. The crude residue was purified by silica gel chromatography (0 to 40% ethyl acetate/hexanes) to provide methyl ester product as a colorless solid (150 mg, 88%). 1H NMR (500 MHz, CD3OD): δ 7.72 (d, J = 10.5 Hz, 1H), 6.93 (d, J = 12.5 Hz, 1H), 5.70 (s, 1H), 5.64 (s, 1H), 5.46 (s, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.10 (dd, J = 13.0, 3.0 Hz, 1H), 2.69–2.66 (m, 1H), 2.57–2.45 (m, 3H), 2.27–2.24 (m, 1H), 2.07–2.01 (m, 1H), 0.94 (d, J = 7.0 Hz, 3H).

A mixture of the methyl ester (310 mg, 0.78 mmol) and LiOH (78 mg, 3.91 mmol) in THF (20 mL) and water (10 mL) was stirred at room temperature for 4 h. The reaction mixture was neutralized by HCl (2 M) and extracted with ethyl acetate (100 mL). The organic phase was washed with brine, dried (MgSO4) and concentrated to dryness. The crude residue was purified by silica gel chromatography (0 to 10% MeOH/CH2Cl2) to provide 13 as an off-white solid (250 mg, 84%). 1H NMR (500 MHz, CD3OD): δ 7.72–7.68 (m, 1H), 6.93 (d, J = 7.0 Hz, 1H), 5.71 (s,


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1H), 5.65 (s, 1H), 5.45 (s, 1H), 3.87 (s, 3H), 3.10 (dd, J = 13.0, 3.0 Hz, 1H), 2.66–2.50 (m, 4H), 2.28–2.22 (m, 1H), 2.08–2.01 (m, 1H), 0.95 (d, J = 12.0 Hz, 3H).

The general procedure for synthesis of the intermediate 14 using R2 = CONHMe, R3 = F as an example. To a solution of 13 (22 mg, 0.06 mmol), hexafluorophosphate benzotriazole tetramethyl uronium (46 mg, 0.12 mmol) and N,N-diisopropylethylamine (0.03 mL, 0.18 mmol) in CH2Cl2 (3 mL) was added methylamine (2.0M solution in THF, 0.06 mL, 0.12 mmol). The mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with ethyl acetate (50 mL), washed with brine, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by silica gel chromatography (0 to 70% ethyl acetate/hexanes) to provide a mixture of diastereomers (15 mg). The mixture was further purified by CHIRALCEL OD (20% iPrOH/heptane) to provide 14 (6 mg, 25%) as a colorless gum-like material. 1H NMR (500 MHz, CD3OD): δ 7.85 (t, J = 9.0 Hz, 1H), 6.95 (d, J = 12.0 Hz, 1H), 5.95–5.85 (m, 1H), 5.66 (s, 1H), 5.61 (s, 1H), 5.50 (s, 1H), 5.18–5.11 (m, 2H), 3.95 (s, 3H), 3.09 (d, J = 14.0 Hz, 1H), 2.92 (s, 3H), 2.82–2.79 (m, 1H), 2.70 (d, J = 14.0 Hz, 1H), 2.64–2.58 (m, 2H), 2.25–2.21 (m, 1H), 2.16 (dd, J = 14.0, 9.5 Hz, 1H), 2.03–1.95 (m, 1H), 0.83 (d, J = 8.0 Hz, 3H). ESI MS m/z 404 [M + H]+. In vitro evaluation. Antiproliferation assays were conducted as described previously.8 Each experiment included at least 3 replicate wells per concentration. NCI-H460 cells were obtained from ATCC.

M14, NCI/ADR-RES, OVCAR-4, HCT-15, ACHN, and SF-295 cells were

obtained from the National Cancer Institute. IC50 values in UO-31 and EKVX were determined


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at Southern Research Institute (Birmingham, AL, USA). Tubulin polymerization was assayed using porcine tubulin as described previously.8 Pharmacokinetics. Pharmacokinetic studies were conducted in accordance with Institutional Animal Care and Use guidelines in male Nu/Nu mice (Crl:NU-Foxn1nu, Charles River, Wilmington, MA, USA). For some experiments, mice with xenografted tumors (NCI-H460 or LOX-IMVI) were used.

Compounds were dissolved in the vehicle indicated; for aqueous

vehicles, compound was first dissolved in the organic portion, and then the aqueous portion was added gradually. The resulting solution was administered by ip injection with a volume of 3 mL/kg. After 1h or 3h, mice were euthanized. Blood was collected by cardiac puncture and centrifuged in a plasma separator tube with NaEDTA/NaF as the anticoagulant; the resulting plasma was stored at −80 °C. Tumors were excised and frozen for analysis. For analysis of plasma, an internal standard (loratadine) was added to a 200 µL aliquot of each plasma sample, and the aliquot was extracted with ethyl acetate. The ethyl acetate extract was evaporated, redissolved in a known quantity of methanol, and analyzed by LC-MS. The ratio of test compound to loratadine was calculated and compared to a standard curve prepared by adding known amounts of test compound and internal standard to 200 µL aliquots of blank mouse plasma, and extracting and analyzing as above. For analysis of tumors, duplicate samples (approximately 200 mg each) of each tumor were weighed and then homogenized in 300 µL of 3% SDS. After addition of a loratadine internal standard, samples were extracted twice with 600 µL ethyl acetate. The combined extracts were dried down, and analysis then proceeded as described for plasma. Concentrations are listed in µM, assuming 1g tumor tissue = 1 mL. In Vivo Efficacy. In vivo efficacy studies were conducted in accordance with Institutional Animal Care and Use guidelines in female athymic nude mice (Crl:NU(NCr)-Foxn1nu, Charles


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River, Wilmington, MA, USA) with human xenograft tumors.

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Tumors were initiated by

subcutaneous injection of either tumor cells from cell culture (NCI-H460, Calu-6, NCI-H69, LOX-IMVI) or tumor fragments from continuous in vivo passage (NCI-H522). When tumors reached volumes between approximately 75-200 mm3, the mice were divided into vehicle and treatment groups. Compound was formulated in a vehicle of 22% ethanol, 38% PEG400, 40% 20 mM Na citrate pH 5.0, and administered by ip injection in a volume of 5-10 mL/kg. Tumors were measured with digital calipers, and tumor volumes were calculated using the formula for an ellipsoid sphere: Volume (mm3) = (length × width2)/2. Calculations were made as described previously.9 NCI-H460 and LOX-IMVI experiments were conducted at Sequoia Sciences, NCIH522 and NCI-H69 experiments were conducted at Southern Research Institute (Birmingham, AL, USA), and Calu-6 experiments were conducted at Charles River Laboratories Discovery Services, Morrisville, NC, USA).

ASSOCIATED CONTENT Supporting Information. The following information is available free of charge: Table S1. Pharmacokinetics of Selected Bifidenone Analogues, Table S2. IC50 values for 46b, NMR assignments for selected compounds, and 1H NMR spectra for all compounds and COSY, HSQC, HMBC, and ROESY spectra for selected compounds as a PDF file. Molecular Formula Strings as a CSV file. AUTHOR INFORMATION Corresponding Authors *Email: [email protected]. Phone: +1-716-888-1161;


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*Email: [email protected]. Phone: +1-314-373-5181 Current Address §

Fimbion Therapeutics, Inc. 20 S. Sarah Street, St. Louis, MO 63108.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We thank Dr. M. Stackhouse for leading the xenograft experiments at Southern Research Institute, and W. Durham for leading the xenograft experiments at Charles River Discovery Services. Dr. M. Wolf provided valuable discussion at Albany Molecular Research Inc. ABBREVIATIONS ACHN, renal cancer cell line; Calu-6, lung adenocarcinoma cell line; EKVX, non-small-cell lung carcinoma cell line; HCT-15, colon adenocarcinoma cell line; LOX-IMVI, malignant amelanotic melanoma cell line; M14, amelanotic melanoma cell line; NCI/ADR-RES, adriamycin-resistant ovarian tumor cell line; NCI-H69, small-cell lung carcinoma cell line; NCIH460, large-cell lung carcinoma cell line; NCI-H522, non-small-cell lung adenocarcinoma cell line; OVCAR-4, ovarian cancer cell line; PhNTf2, N-phenyl-bis(trifluoromethanesulfonimide); SD, standard deviation; SF295, glioblastoma cell line; TGI, tumor growth inhibition; UO-31, kidney carcinoma cell line.

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(1) Orr, G. A.; Verdier-Pinard, P.; McDaid, H.; Horwitz, S. B. Mechanisms of Taxol Resistance Related to Microtubules. Oncogene 2003, 22 (47), 7280–7295. (2) Dumontet, C.; Sikic, B. I. Mechanisms of Action of and Resistance to Antitubulin Agents: Microtubule Dynamics, Drug Transport, and Cell Death. J. Clin. Oncol. 1999, 17 (3), 1061–1061. (3) Dumontet, C.; Jordan, M. A. Microtubule-Binding Agents: A Dynamic Field of Cancer Therapeutics. Nat. Rev. Drug Discov. 2010, 9 (10), 790–803. (4) Jordan, M. A.; Wilson, L. Microtubules as a Target for Anticancer Drugs. Nat. Rev. Cancer 2004, 4 (4), 253. (5) Lu, Y.; Chen, J.; Xiao, M.; Li, W.; Miller, D. D. An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site. Pharm. Res. 2012, 29 (11), 2943–2971. (6) Bughani, U.; Li, S.; Joshi, H. C. Recent Patents Reveal Microtubules as Persistent Promising Target for Novel Drug Development for Cancers. Recent Patents Anti-Infect. Drug Disc. 2009, 4 (3), 164–182. (7) Combeau, C.; Provost, J.; Lancelin, F.; Tournoux, Y.; Prod’homme, F.; Herman, F.; Lavelle, F.; Leboul, J.; Vuilhorgne, M. RPR112378 and RPR115781: Two Representatives of a New Family of Microtubule Assembly Inhibitors. Mol. Pharmacol. 2000, 57 (3), 553– 563. (8) Williams, R. B.; Martin, S. M.; Lawrence, J. A.; Norman, V. L.; O’Neil-Johnson, M.; Eldridge, G. R.; Starks, C. M. Isolation and Identification of the Novel Tubulin Polymerization Inhibitor Bifidenone. J. Nat. Prod. 2017, 80 (3), 616–624. (9) Huang, Z.; Williams, R. B.; O’Neil-Johnson, M.; Eldridge, G. R.; Mangette, J. E.; Starks, C. M. A Total Synthesis of Bifidenone. J. Org. Chem. 2017, 82 (8), 4235–4241.


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Table of Contents graphic


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Bifidenone: Structure-Activity Relationship and Advanced Preclinical

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