Discovery of Potent Orally Active Protease-Activated Receptor 1 (PAR1) Antagonists Based on Andrographolide

Article abstract of DOI:10.1021/acs.jmedchem.7b00951

Protease-activated receptor-1 (PAR1), a G-protein-coupled receptor, plays a critical role in thrombin-mediated platelet aggregation. It is regarded as a promising antithrombosis target that is unlikely to result in bleeding. Here, we describe the synthesi

Full text of DOI:10.1021/acs.jmedchem.7b00951

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Article
Discovery of Potent Orally Active Protease-Activated
Receptor 1 (PAR1) Antagonists Based on Andrographolide
Jun Liu, Bin Sun, Xiaoyu Zhao, Jie Xing, Yanhui Gao, Wenqiang Chang,
Jianbo Ji, Hongbo Zheng, Changyi Cui, Aiguo Ji, and Hong-Xiang Lou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00951 • Publication Date (Web): 26 Jul 2017
Downloaded from http://pubs.acs.org on July 27, 2017
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Discovery of Potent Orally Active Protease-Activated Receptor 1
(
PAR1) Antagonists Based on Andrographolide
†
,§
⊥
,§
†
†
†
†
Jun Liu, Bin Sun, Xiaoyu Zhao, Jie Xing, Yanhui Gao, Wenqiang Chang,
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†
†
†
,†,‡
,†
Jianbo Ji, Hongbo Zheng, Changyi Cui, Aiguo Ji,* Hongxiang Lou*
†
School of Pharmaceutical Sciences, Shandong University, Jinan, China
⊥
National Glycoengineering Research Center, Shandong University, Jinan, China
‡
Weihai International Biotechnology Research and Development Centre, Shandong
University, Weihai, China
Author Information
Corresponding Author
*
L.H.:
louhongxiang@sdu.edu.cn.
J.A.: Tel: +86ꢀ531ꢀ8838ꢀ2346.
Tel:
+86ꢀ531ꢀ8838ꢀ2012.
Fax:
Fax:
+86ꢀ531ꢀ8838ꢀ2019.
+86ꢀ531ꢀ8838ꢀ2019.
Eꢀmail:
Eꢀmail:
*
jiaiguo@sdu.edu.cn.
Author Contributions
§
L.J. and S.B. contributed equally to this work.
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ABSTRACT: Proteaseꢀactivated receptorꢀ1 (PAR1), a G proteinꢀcoupled receptor,
plays a critical role in thrombinꢀmediated platelet aggregation. It is regarded as a
promising antithrombosis target that is unlikely to result in bleeding. Here, we
describe the synthesis of a series of novel PAR1 antagonists by borrowing the chiral
fragment of andrographolide, an easily accessible natural molecule from
Andrographis paniculata, to produce natural product/synthesis hybrids. An in vitro
PAR1 inhibition assay and an in vivo pharmacokinetic profile led to the identification
of compound 39 as the best PAR1 inhibitor. The further in vitro and ex vivo
antiꢀplatelet aggregation assays of compound 39 indicated that compound 39 was a
potent antiꢀplatelet agent. In addition, this compound is metabolically stable, and
displays a favorable pharmacokinetic profile with an elimination halfꢀlife of 3.1 h,
which could be treated as a promising candidate for further clinical development.
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KEYWORDS: Andrographolide analogs; proteaseꢀactivated receptorꢀ1; antagonists;
orally active.
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INTRODUCTION
Proteaseꢀactivated receptorꢀ1 (PAR1), a Gꢀproteinꢀcoupled thrombin receptor, is
1
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the most potent cell surface inducer of platelet activation.
Mechanistically,
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thrombin activates PAR1 by cleaving its Nꢀterminal ectodomain to reveal a new
Nꢀterminus, which serves as a tethered peptide ligand that binds to the heptahelical
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domain to trigger G protein activation and lead to the aggregation of platelets.
Given the critical role of PAR1, a PAR1 antagonist could be used as potent
5ꢀ7
antiplatelet agent for the prevention of thrombosis and restenosis. Additionally,
because PAR1 antagonists target the cellular effector of thrombin, thrombinꢀmediated
fibrin generation is not affected, and therefore normal hemostasis should be
maintained when platelet activation is induced. Therefore, a PAR1 antagonist is likely
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to result in a lower risk of bleeding than conventional antithrombotic agents.
1
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Several novel PAR1 antagonists such as vorapaxar (SCH530348),
atopaxar
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14ꢀ16
(E5555),
SCH79797,
and others have been reported to have good efficacy in
inhibiting the thrombin receptor (Fig. 1). So far, only vorapaxar, derived from the
natural product himbacine, has been approved by the US Food and Drug
Administration as the firstꢀinꢀclass antiꢀplatelet drug targeting PAR1 to reduce the
risk of recurrent cardiovascular events. It binds to PAR1 with high affinity, has good
platelet inhibitory activity, and is rapidly absorbed via the gastrointestinal tract, with a
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terminal plasma halfꢀlife of 126 ꢀ 269 h.
The excellent antiꢀplatelet activity of
vorapaxar makes it an attractive molecule, and many series of its analogues were also
reported to date. Based on the SAR analysis of vorapaxar as well as its analogues, it
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was found that (3ꢀfluorophenyl)pyridineꢀ2ꢀvinyl moiety is a critical pharmacophore
for the PAR1 inhibitory activity by binding to the pocket of PAR1 involving residues
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Tyr 183, Leu 237, Phe 271, Leu 333 and Tyr 337.
In addition, the tricyclic ring of
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vorapaxar was also proved to be very important for the PAR1 inhibitory activity, and
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chemists invested considerable efforts and expense to synthesize this moiety,
because of its multiꢀchiral center. Recently, it was found that the tricyclic ring of
vorapaxar could be simplified to bicyclic ring while maintaining the potent PAR1
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inhibitory activity,
which provided new ways, and of course inspired us to design
and synthesis of novel vorapaxar analogues targeting PAR1.
Natural products are considered to be one of the main sources of medicines, and
their scaffolds have been well recognized as ‘privileged’ structures due to their
25ꢀ28
chemical diversity, structural complexity and conformation immobilization.
An
attractive concept of naturalꢀproduct hybridization is becoming popular as an
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emerging structural modification tool to design novel and complex molecules.
The advantage of this concept over other approaches is the high diversity and the
31,32
inherent biological activity of the hybrids.
By this means, it may be possible to
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improve the probability of finding new lead structures. In addition, this concept
could be used in the construction and preparation of complex molecules in a more
convenient and efficient way.
Andrographolide, an easily accessible molecule isolated from the leaves of
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Andrographis paniculata, is an important active natural product (Fig. 2).
The two
fused sixꢀmembered rings of andrographolide adopt the chair conformation and,
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importantly, the stereostructure of this bicyclic ring is consistent with that of ring B
and ring C of vorapaxar. This bicyclic ring could be “borrowed” to facilitate the
efficient and rapid construction of vorapaxar analogues. Meanwhile, we found that
there are two potential “hybrid points” at Cꢀ4 and Cꢀ9 of the andrographolide, which
have the same conformation as that of Cꢀ9 of vorapaxar. Either of these two “hybrid
points” could be converted to aldehyde group individually, and then be coupled with
the (3ꢀfluorophenyl)pyridineꢀ2ꢀvinyl phamacophore of vorapaxar, resulting in the
formation of two novel series of analogues with a stereostructure consistent with that
of vorapaxar (Fig. 2). The preliminary molecular modeling of these two series
analogues was also performed, and it was found that these novel compounds could fit
nicely to the vorapaxar binding pocket in PAR1, with the orientation similar to that of
vorapaxar (Fig. S1).
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In this study, based on the naturalꢀproduct hybrid theory, the chiral fragment of
andrographolide was combined with the (3ꢀfluorophenyl)pyridineꢀ2ꢀvinyl
pharmacophore, producing two series of novel vorapaxar analogues (series 1 and
series 2), and the following in vitro PAR1 inhibitory assay resulted in the lead 13a
(Scheme 1). To optimize the drugꢀlike properties beyond the basic PAR1 inhibitory
activity, 13a was further modified and ultimately resulted in compound 39, which also
exhibited in vitro and ex vivo antiꢀplatelet activity, and could be considered as a
candidate for further clinical development.
RESULTS AND DISCUSSION
Synthesis of Hybrid 13a and Its Derivatives (Series 1). The synthetic procedure
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for hybrid 13a and its derivatives (12a-14b) is shown in Scheme 1. The synthesis
started with andrographolide (5), which was dehydrated with activated alumina in
refluxing anhydrous pyridine to generate dehydroandrographolide 6 with a yield of
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5 %. The double bond of compound 6 was cleaved with O to give aldehyde 7,
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which failed to react with the phosphonate 4a by HornerꢀWadsworthꢀEmmons
reaction, presumably owing to the hydrogen bond between aldehyde group and
betaꢀcarbonyl group, which inactivated the aldehyde group. For this reason, the
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ꢀexoꢀmethylene group of compound 6 was selectively epoxidized through the
mꢀCPBAꢀmediated Prilezhaev reaction to prevent the hydrogen bond from forming.
To our delight, this reaction not only occurred preferentially at the 8ꢀexoꢀmethylene
rather than the 11ꢀene but also formed an 8,19ꢀepoxide ring stereoselectively from the
less sterically hindered βꢀface to exclusively give 8Sꢀepoxide 9. The acetonide 10 was
obtained by reaction with 2,2ꢀdimethoxypropane in the presence of pyridinium
pꢀtoluenesulfonate (PPTS). Double bond cleavage by ozonolysis gave aldehyde 11,
which was subjected to the HornerꢀWadsworthꢀEmmons reaction using the
phosphonates 4a and 4b to successfully yield the 3ꢀfluorophenyl analog 12a and
3ꢀtrifluoromethylphenyl analog 12b, respectively. Phosphonate 4a was synthesized in
three steps from 2ꢀmethylꢀ5ꢀhydroxypyridine 1 as shown in Scheme 1. Triflate
formation followed by Suzuki coupling gave 3a which was converted into 4a via
deprotonation followed by treatment with diethyl chlorophosphate, and 4b was
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obtained using the same method. The protecting group was then removed by
hydrolysis with Amberlyst 15 to produce the diols 13a and 13b. The structure of 13a
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was also unequivocally determined by Xꢀray crystallographic analysis (Fig. S2). The
double bonds were selectively reduced by titanocene dichloride and Mn in THF to
give compounds 14a and 14b, respectively.
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Synthesis of Hybrid 21 and the Derivatives (Series 2). The synthetic strategy
above was mainly dependent on cleavage of the double bond of andrographolide to
give an aldehyde group at Cꢀ9 point, which was then hybridized with the
(3ꢀfluorophenyl)pyridineꢀ2ꢀvinyl moiety of vorapaxar. All the key chiral carbon
atoms in the two fused sixꢀmembered ring were the same as those of vorapaxar. Next,
we intended to hybridize with the moiety of vorapaxar from the Cꢀ4 point of
andrographolide by selectively oxidizing the primary hydroxyl group. Meanwhile, a
fiveꢀmembered lactone ring was also introduced for structural diversity, and the SAR
was analyzed. As shown in Scheme 2, target compounds were synthesized starting
from compound 10, and the double bond was cleaved by ozonolysis to give aldehyde
1
1, which was then oxidized to carboxylic acid 15 under the condition of
NaClO ꢀNaH PO in isopentane. Titanoceneꢀcatalyzed reductive epoxide opening
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stereoselectively gave compound 17. In this step, the intermediate 16 was only formed
from the βꢀface of the Bꢀring because of the steric effect. Furthermore, a dehydration
condensation reaction of compound 17 with DCC/DMAP in 1,4ꢀDioxane resulted in
the formation of the fiveꢀmembered lactone 18. The protecting group was then
removed by hydrolysis with Amberlyst 15 to produce diol 19, which was selectively
oxidized with the TEMPOꢀNCS system to give aldehyde 20. Aldehyde 20 was
coupled with phosphonate 4a to give compound 21. Unexpectedly, the target
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compound 21 had four diastereoisomers (21a - 21d) which were then separated by
HPLC, and their stereostructures were determined by HMBC and NOESY
experiments (shown in Fig. S3). The structure of 21c was also unequivocally
determined by Xꢀray crystallographic analysis (Fig. S2). The process of the
generation of the diastereoisomers most likely occurred through a reversible aldol
reaction, and the proposed underlying mechanism is shown in Fig S4. Briefly, the
betaꢀhydroxyl group was initially deprotonated under the basic condition, followed by
electron transfer and cleavage of the covalent bond between Cꢀ3 and Cꢀ4, and the
resulting dialdehyde then underwent an aldol reaction to yield the isomers. To study
the effect of substitution on the hydroxyl at Cꢀ3, the resultant lactone 21 was
subsequently derivatized with sulfonyl chloride, acetic anhydride, isocyanate, and
butyric anhydride to produce the corresponding sulfonate, acetate, carbamate, and
butyrate, respectively. All of the diastereoisomers were separated by HPLC, their
PAR1 inhibition activities were evaluated, and the SAR was explored.
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Identification of Lead Compound 13a. Twentyꢀone andrographolide analogs were
initially obtained and subjected to a biological assay to determine their potency
against PAR1 (Table 1). The PAR1 inhibitory effect of the test compounds was
measured in 384ꢀwell plates by a functional calcium mobilization assay with the
recombinant stable cell line HEK293ꢀGα15ꢀPAR1 using haTRAP as the agonist.
Vorapaxar was purchased from MedChemexpress (Princeton, USA), and served as
37
the positive control. In this assay, the highest concentration of tested drugs was set
at 100 ꢁM: compounds that did not show activity at this concentration were regarded
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as inactive. The results showed that acetonides 12a and 12b were essentially inactive,
with IC50 value more than 100 ꢁM. To our surprise, when the acetonides were cleaved,
the resulting compounds 13a and 13b, as well as their reductive products 14a and 14b,
exhibited potent PAR1 inhibitory activities, with IC values ranging from 9.1 to 14
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M, which were obvious higher than vorapaxar (IC50 = 0.064 ꢁM). Compounds 21a,
4a, 24b and 24c exhibited moderate PAR1 inhibitory activities with IC50 values
ranging from 12 ꢁM to 21 ꢁM, while the other lactone derivatives (21bꢀ21d, 22a, 22c,
3aꢀ23c, 24d and 25a, 25c) were inactive. The results suggested that the substitution
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on the hydroxyl at Cꢀ3 with carbamate was preferable to the other substitutions.
Additionally, the compounds hybridized with vorapaxar through selective oxidation
of the primary hydroxyl group were less active than those hybridized through double
bond cleavage of andrographolide. This could have been due to the steric effect of the
lactone ring and changes in the conformation of two fused sixꢀmembered ring system.
Given the increased lipophilicity that resulted from the CF ꢀsubstituted moiety of 13b,
3
compound 13a with the mꢀFꢀsubstituted diol was chosen as a candidate for further
research on its drugꢀlike properties.
It is well known that drug metabolism and pharmacokinetic (DMPK) studies have
always played a critical role in helping to optimize the bioavailability and duration of
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action of new drugs and thereby increasing the success rate of drug development.
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The application of DMPK principles to drug discovery is thus not a new concept.
Here, we applied concept to the new molecules as part of the lead optimization
process.
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First, the metabolite profiling of compound 13a in rats was studied using liquid
chromatography tandem highꢀresolution LTQꢀOrbitrap mass spectrometry
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LCꢀHRMS). As shown in Fig. 3A and S5A, the diol compound 13a showed a poor
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metabolic stability in blood plasma, generating a considerable number of metabolites
observed by LCꢀMS/MS analysis, and these metabolites were generated mainly by
oxidation of hydroxyl group. The total concentration of the metabolites was much
higher than that of the parent. Moreover, the plasma concentration of the major
carboxylic acid metabolite (M0+Oꢀ2H), which was subsequently synthesized as
compound 28 (Scheme 3), exceeded that of the parent by 3ꢀfold. Therefore, the
development of 13a as a thrombin receptor antagonist was suspended and this
prompted us to identify a replacement candidate for 13a with an improved metabolic
stability.
Optimization of 13a and Identification of 29. In this pursuit, we adopted two
approaches for the optimization of 13a. The first approach was to explore the
metabolites. There have been several instances in the history of drug discovery
research where a metabolite has served as an improved replacement for the initial
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drug candidate.
The second approach was to selectively block or modify the
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metabolically labile groups.
As shown in Scheme 3, the synthetic work of
modification started with the diol compound 13a, which was reduced by LiAlH in
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selective oxidation of primary hydroxyl group with the TEMPOꢀNCS system. The
formed aldehyde 27 was further oxidized to a carboxyl group with NaClO ꢀNaH PO
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and isopentane, affording a carboxylic acid 28, which was the main metabolite.
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Furthermore, we treated carboxylic acid 28 with CH I in DMF to obtain the methyl
ester 29. To study the effect of substitution on the hydroxyl at Cꢀ3, some derivatives
of 29 were prepared as shown in Scheme 3 using procedures similar to the preparation
of 23ꢀ25 described previously. Additionally, to block the metabolically labile
hydroxyl group, the diol 13a was also methylated using methyl iodide to give the
ethers 31 and 32. The attachment positions of the methyl substituents were
determined using HMBC (Fig. S6). On the other hand, reductive amination of
aldehyde 27 with ammonia and methylamine gave corresponding amines 33a and 33b,
respectively.
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13
14
15
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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59
60
The PAR1 inhibitory activity of each of these derivatives was then evaluated (Table
1
). The derivatives 27, 29, 30b and 31 showed potent activity. Among them, the
aldehyde 27 exhibited the best PAR1 inhibitory activity with an IC50 value of 3.0 ꢁM.
Compound 29 (IC50 = 7.0 ꢁM), with a methyl ester on Cꢀ9, also displayed potent
PAR1 inhibitory activity. The derivatives 32, 33a and 33b showed slightly diminished
PAR1 inhibitory activity, with IC50 values of 14 ꢁM, 23 ꢁM and 17 ꢁM, respectively.
However, the epoxide ringꢀopened derivative 26 and carboxylic acid 28 displayed
obviously decreased activity, with IC50 values of 55 and 29 ꢁM, respectively.
Compound 30b with a carbamate on Cꢀ3 exhibited potent PAR1 inhibitory activity
with IC50 value of 8.7 ꢁM. Surprisingly, when the carbamate group was replaced by
acetate or butyrate, the PAR1 inhibitory activities of the resulting compounds 30a and
3
0c, respectively, were totally lost, with IC50 values of more than 100 ꢁM. Because of
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aldehyde group in 27 was considered unstable in blood plasma, we chose methyl ester
2
9 as a candidate for further metabolic profiling (Fig. 3B). Unfortunately, the tested
results showed that there was a relatively low plasma concentration for the parent (M1)
0
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0
and high plasma levels of metabolites (M1ꢀCH ) (28). As shown in Fig. S5B, the
2
concentration of the M1ꢀCH
2
metabolites was approximately 31 times higher (AUC0ꢀ∞
=
8412.70 h·ꢁg/L) than that of its parent (AUC0ꢀ∞ = 268.93 h·ꢁg/L). On this basis, an
overwhelming majority of methyl ester 29 could be hydrolyzed and converted to
carboxylic acid 28, which in turn would be eliminated from the systemic circulation.
It was therefore important to pay further attention to the generation of compounds that
would increase metabolic stability and retain the desired activity against PAR1.
Further Optimization and Identification of 39. Based on the results described
above, we attempted to further explore the Cꢀ4 and Cꢀ8 region of the bicyclic motif,
which are known to undergo considerable in vivo metabolism. A series of novel
derivatives was therefore designed and synthesized in which the metabolically labile
primary hydroxyl and epoxy groups were replaced with the metabolically stable
methyl group.
These syntheses, outlined in Scheme 4, started with aldehyde 27. To improve the
structural diversity and study the necessity of the pyridine ring, aldehyde 27 was
oxidized with mꢀCPBA to give oxynitride. Through the reversible intramolecular
aldol reaction as described above (Fig. S4), when the oxynitride was treated with
2 3
basic conditions such as K CO , the configuration at Cꢀ9 was partially inverted to
yield isomers 34 and 35, the stereostructures of which were unambiguously
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determined through Xꢀray crystallographic analysis (Fig. S2). The treatment of
aldehyde 27 with 1.05 equiv of ethanedithiol and boron trifluoride etherate then gave
the thioacetal 36, which was further reduced with Raney nickel to generate compound
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25
26
27
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29
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39
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3
7. When 3 equiv of ethanedithiol was used, dithioacetal 38 was obtained. In this step,
the thioacetals 36 and 38 were only formed in a stereoselective manner from the
βꢀface of the Bꢀring at Cꢀ8 due to a steric effect of the Cꢀ9 group. The stereostructure
of compound 38 was determined by HMBC and NOESY experiments (Fig. S7).
These results also suggested that the epoxy group is extremely active under boron
trifluoride etherate conditions and can be easily hydrolyzed. The hydrolysate was then
dehydrated to generate an aldehyde, which immediately reacted with ethanedithiol
due to its lower steric factor than aldehyde group at Cꢀ4 (Fig. S8). Additionally, when
excessive amounts of ethanedithiol were added, the aldehyde group at Cꢀ4 also
reacted with ethanedithiol to generate thioacetals. Initially, the thioacetals were
reduced by Raney nickel in ethyl alcohol or methanol, but the double bond was also
easily reduced. We therefore investigated changing the solvent. To our surprise, when
“
hydrogenꢀfree” acetone was used as solvent, a single product 39 was obtained.
Subsequently, some derivatives of 39 were prepared as shown in Scheme 4 using
procedures similar to the preparation of 22ꢀ25 as described previously. The sulfonyl
group of 40a was further eliminated to produce olefin 41.
The PAR1 inhibitory activities of these newly synthesized derivatives were then
tested. As shown in Table 1, compounds 37 and 39 showed excellent PAR1 inhibitory
activity, with IC50 values of 1.5 and 1.1 ꢁM, respectively, which were approximate
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2
2
2
2
2
2
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3
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3
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fiveꢀfold better than compound 29. However, compounds 40a and 40c exhibited
significantly decreased PAR1 inhibitory activity compared with compound 27, with
IC50 values of 87 ꢁM and 32 ꢁM, respectively. To our surprise, the rest of derivatives
0
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2
3
4
5
6
7
8
9
0
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2
3
4
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8
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9
0
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7
8
9
0
(
34, 35, 36, 38, 40b, 40d, 40e and 41) were inactive, with IC values of more than
50
1
00 ꢁM.
Next, compound 39 was chosen for further metabolic profile research in rats.
Representative EIC chromatograms of 39 and its metabolites are shown in Fig. 3C. A
total of four primary metabolites were identified in rat plasma. More importantly,
unlike compounds 13a and 29, the metabolically labile sites were blocked, and the
main carboxylic acid metabolite was not observed. As shown in Fig. S5C, the total
concentration of the metabolites was lower than the parent, and the main metabolite
(M2+2Oꢀ2H) with NL = 1.36E5 only accounted for approximately 25 % of its parent
(NL = 4.09E5), which suggested a good metabolic stability of compound 39.
Effects of 39 on human platelet aggregation in vitro. Considering the potent
PAR1 inhibitory activity and excellent metabolism profile of compound 39, we
further evaluated the inhibitory effects of 39 on human platelet aggregation induced
by thrombin, TRAP, ADP, or collagen, using vorapaxar as the positive control. As
shown in Fig. 4, 39 inhibited human platelet aggregation induced by thrombin (0.5
U/mL) or TRAP (15 ꢁM) in a concentrationꢀdependent manner, with IC values of
50
0
.65 ꢁM and 1.6 ꢁM, respectively, and the IC50 values of vorapaxar were 0.081 ꢁM
and 0.031 ꢁM, respectively (Table S1). Meanwhile, the platelet aggregation response
following stimulation of human PRP with ADP (10 ꢁM) and collagen (5 ꢁg/mL), was
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not affected by 39, up to a concentration of 10 ꢁM. These data demonstrated that
compound 39 could selectively inhibit PAR1ꢀmediated platelet aggregation.
Effects of 39 on ex vivo platelet aggregation in the guinea pig. Guinea pigs have
been identified as the only small animal species expressing PAR1 on their
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29
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34
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36
37
38
39
40
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46
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49
50
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4
5ꢀ47
platelets.
We therefore chose guinea pigs as an appropriate small animal model to
evaluate PAR1ꢀdependent responses in platelets. The research protocol complied
strictly with the institutional guidelines of Animal Care and Use Committee at
Shandong University. The guinea pigs were given compound 39 at a dosage of 10 or
3
0 mg/kg, vorapaxar 10 mg/kg, or vehicle orally, respectively. After 2 hours, the
animals were anesthetized and blood was drawn from the vena cava. The inhibitory
effects of 39 on ex vivo platelet aggregation induced by thrombin, TRAP, ADP, or
collagen were then evaluated. It was found that compound 39 could significantly
inhibit ex vivo platelet aggregation either induced by thrombin (1 U/mL) or TRAP (15
ꢁ
M) at all doses tested (Table 2). At a dose of 10 mg/kg, however, platelet
aggregation induced by thrombin and TRAP was not completely inhibited, and the
inhibition was 66.9 % and 87.6 %, respectively. At a dose of 30 mg/kg, compound 39
showed an almost complete inhibition of platelet aggregation stimulated by thrombin
and TRAP, and the inhibition was 99.8 % and 98.7 %, respectively. Even at 30 mg/kg,
39 did not inhibit platelet aggregation in response to ADP or collagen.
Pharmacokinetic Studies. Due to the reasonable activity against PAR1 and
excellent metabolic stability of compound 39, it was also subjected to a full rat
pharmacokinetic assay. Male Wistar rats were intragastrically administered 5 mg/kg
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or intravenously administered 1 mg/kg of the compound. The research protocol
complied strictly with the institutional guidelines of Animal Care and Use Committee
at Shandong University. The blood concentrations were measured over a 24 h period.
The animals tolerated the treatment on the basis that no abnormalities in the animals’
behavior were observed. As shown in table 3, compound 39 exhibited an excellent
oral bioavailability of 52.5 % with an AUC(0ꢀ∞) = 1450 h·ꢁg/L. The oral Cmax was 229
0
1
2
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9
0
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9
0
1
2
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4
5
6
7
8
9
0
ꢁ
g/L with a Tmax of 2.5 h. The oral halfꢀlife was 3.1 h and mean residence time was
5
.6 h. The compound showed a moderate volume of distribution (Vz = 20 L/kg) and a
clearance of 4.5 L/h/kg. In summary, the compound 39 presented favorable
pharmacokinetic properties.
SAR Analysis. Preliminary SAR conclusions were proposed on the basis of the
results described above (Fig. 5). The Cꢀ8ꢀepoxy group was less stable in vivo and the
epoxide ringꢀopened compound showed weaker activity (compound 26). Compounds
3
7 and 39, in which a methyl moiety replaced the epoxy group, showed higher
activity and good metabolic stability. The hydroxyl group at Cꢀ3 was important for
the PAR1 inhibitory property. Except for the formation of a methyl ether, any changes
of this group, e.g., its esterification or elimination, led to a decrease or completely loss
of activity. Furthermore, bulky substitutions at Cꢀ3 and Cꢀ4, for example, a lactone or
an ether ring, led to a decrease in activity. Compounds with a methyl group at Cꢀ4
exhibited the best potency and metabolic stability. Any other changes at this position
either impacted the activity or decreased the stability in vivo. The stereochemistry at
Cꢀ9 and the pyridine ring was critical, and minor changes could greatly impact the
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activity. The reduction of the 11,12ꢀdouble bond (compound 14a and 14b) had no
3
significant effect on the activity. In the analogs that contained mꢀCF or mꢀF on the
aryl group, the activity was not affected.
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Molecular Modeling Analysis. Subsequently, the excellent bioactivity of the
andrographolide derivatives encouraged us to investigate the possible mechanism of
action at the molecular level, and more specifically, the binding mode of active
compound 39 to PAR1. Compound 39 was then docked into the vorapaxarꢀbinding
site on PAR1 using the GOLD (Genetic Optimization for Ligand Docking) program.
The binding mode of compound 39 was studied further by energy minimization. In
the resulting hypothetical structure (Fig. 6), compound 39 fit nicely in the active
binding site, and aligned well with the native ligand vorapaxar. In addition, the
bicyclic region of 39 binds to the extracellular loop region formed by residues His 336,
Tyr 353, Tyr 350, Leu 258 and His 255, while the biaryl region binds to a
hydrophobic pocket toward the transꢀmembrane region involving residues Leu 333,
Tyr 183, Leu 237 and Phe 271. Moreover, the pyridine ring of compound 39 may
form a strong hydrogen bond with residue Tyr 337, which is similar to the binding
mode of vorapaxar.
CONCLUSION
Plantꢀderived natural products with unique skeletons and bioactivities provide
considerable drug leads and candidates, thereby constituting a hotspot in the field of
drug discovery. In this manuscript, we prepared two series of novel PAR1 inhibitors
by “naturalꢀproduct hybridization”, and “borrowed” the two fused sixꢀmembered ring
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moiety from the natural product andrographolid, which led the discovery of the lead
compound 13a. The further structural optimization was guided by the metabolic
stability evaluation and finally resulted in the most potent thrombin receptor
antagonist 39, which also displayed potent antiꢀplatelet activity both in vitro and ex
vivo. Although, there was an obvious antiꢀplatelet potency gap between compound 39
and vorapaxar, the unique drugꢀlikeness properties of 39 were underscored by its
metabolic stability, excellent bioavailability and favorable pharmacokinetic profile.
Importantly, with the help of the natural product skeleton, the synthetic route of
compound 39 was relatively short and resulted in a high yield. Therefore, compound
0
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9
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2
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9
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0
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2
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8
9
0
1
2
3
4
5
6
7
8
9
0
3
9 could be obtained at low cost, which is an advantage in developing compound 39
as a PAR1 antagonist. Additionally, we also presented a model of these compounds
docked to the PAR1 based on the Xꢀray crystal structure of vorapaxar bound to PAR1
and the model explains some of the SAR in this series. These studies suggest that
compound 39 has a potential to be developed as a new generation of PAR1
antagonists.
EXPERIMENTAL SECTION
General Material and Methods. All commercially available reagents were used
without further purification. Andrographolide was purchased from Shanghai Yulan,
China. Anhydrous solvents were dried through routine protocols. All reactions were
carried out under a nitrogen atmosphere in dry glassware with magnetic stirring.
Column chromatography was carried out on 200ꢀ300 mesh silica gel (Qingdao
Haiyang Chemical, China). Analytical TLC was carried out employing 0.25 mm
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silicagel plates (GF254) and visualization under UV light. The NMR spectra were
1
13
1
recorded on a Bruker 400 ( H, 400 MHz; C, 101 MHz) or Bruker 600 ( H, 600 MHz;
1
3
C, 150 MHz) spectrometer. Chemical shifts were expressed in ppm and J values
10
11
12
13
14
15
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23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
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50
51
52
53
54
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60
were given in Hz. Highꢀresolution mass spectra were measured using a Thermo Fisher
Finnigan LTQ Orbitrap Elite mass spectrometer. Ionization was achieved using the
positive mode. The purity of the final compounds was verified using an HPLC system
(Agilent Technologies 1200) equipped with a G1311A isopump, a G1322A degasser,
and a G1315D DAD detector using an Eclipse XDBꢀC18 (150 mm × 4.6 mm, 5 µm).
All compounds evaluated for biological effects were > 95 % pure.
6
-Methylpyridin-3-yl trifluoromethanesulfonate (2). To a stirred solution of
6ꢀmethylpyridineꢀ3ꢀol (2.18 g, 20 mM) in anhydrous CH Cl (20 mL) under a
2
2
nitrogen atmosphere at 0 °C, pyridine (2.5 mL, 30 mM) and trifluoromethanesulfone
anhydride (4.1 mL, 24 mM) were slowly added. After stirring for 2 h at 0 °C, TLC
analysis indicated the starting material had been consumed. Methanol (1 mL) and a
saturated aqueous NaHCO
3
solution were then added to the mixture. The resulting
Cl and the extracts were washed with brine, dried
mixture was extracted with CH
2
2
over MgSO
4
, filtered and concentrated under reduced pressure and then purified by
1
flash chromatography to yield compound 2 (3.86 g, 80 %) as colorless oil. H NMR
(400 MHz, CDCl ) δ 8.47 (d, J = 2.4 Hz, 1H), 7.57 – 7.49 (m, 1H), 7.26 (d, J = 8.6 Hz,
3
13
1
H), 2.61 (t, J = 2.6 Hz, 3H). C NMR (101 MHz, CDCl
3
) δ 159.2, 145.2, 142.1,
1
9
3
129.3, 124.5, 123.7 (q, J = 123.7 Hz), 24.1. F NMR (376 MHz, CDCl ) δ ꢀ72.65.
+
ESIꢀMS m/z 242 [M + H] .
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5
-(3-Fluorophenyl)-2-methylpyridine (3a). To a stirred solution of 2 (4.82 g, 20
mM) in anhydrous toluene (56 mL),
ꢀtrifluoromethylphenylboronic acid (4.5 g, 32 mM), EtOH (14 mL), H
and Pd(PPH ) (232 mg, 0.2 mM) were added. The mixture was heated in a closed
K
2
CO
3
(13.9 g, 100 mM),
3
2
O (28 mL)
0
1
2
3
4
5
6
7
8
9
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2
3
4
5
6
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8
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8
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2
3
4
5
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7
8
9
0
3
4
pressure tube under N
2
at 120 °C for 16 h. After cooling to room temperature, the
mixture was diluted with EtOAc, washed with 5 % NaOH and brine, dried over
MgSO
4
and concentrated in vacuo. The residue was purified by flash chromatography
1
to yield compound 3a as a yellow oil (3.80 g, 80 %). H NMR (400 MHz, CDCl
3
) δ
8
.69 (d, J = 2.2 Hz, 1H), 7.73 (dd, J = 8.0, 2.4 Hz, 1H), 7.40 (td, J = 7.9, 5.9 Hz, 1H),
.33 (dt, J = 7.6, 1.2 Hz, 1H), 7.27 – 7.18 (m, 2H), 7.05 (tdd, J = 8.5, 2.5, 0.9 Hz, 1H),
7
1
3
2.59 (s, 3H). C NMR (101 MHz, CDCl
3
) δ 164.6, 162.1, 158.0, 147.5, 140.3 (d, J =
7
.8 Hz), 134.8, 132.7 (d, J = 2.2 Hz), 130.7 (d, J = 8.4 Hz), 123.4, 122.7 (d, J = 2.9
+
Hz), 114.8, 114.6, 114.1, 113.9, 24.22. ESIꢀMS m/z 188 [M + H] .
2
-Methyl-5-(3-(trifluoromethyl)phenyl)pyridine (3b). The general procedure for
1
the synthesis of 3b was similar to 3a and resulted in a 78 % yield. H NMR (400 MHz,
CDCl
7.8 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 5.8 Hz, 1H), 2.64 (s, 3H). C NMR
101 MHz, CDCl ) δ 158.4, 147.6, 139.0, 135.1, 132.7, 131.9 (d, J = 32.5 Hz), 130.5,
129.8, 125.6 (d, J = 273.6 Hz), 124.73 (q, J = 3.8 Hz), 123.98 (q, J = 3.8 Hz), 123.6,
3
) δ 8.75 (d, J = 2.1 Hz, 1H), 7.81 (m, 2H), 7.76 (d, J = 7.6 Hz, 1H), 7.66 (d, J =
13
(
3
19
+
2
4.33. F NMR (376 MHz, CDCl
3
) δ ꢀ62.69. ESIꢀMS m/z 238 [M + H] .
Diethyl ((5-(3-fluorophenyl)pyridin-2-yl)methyl)phosphonate (4a). A solution of
compound 3a (1.3 g, 5.5 mM) and diisopropylamine (924 ꢁL, 6.6 mM) in anhydrous
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THF (20 mL) under a nitrogen atmosphere at ꢀ78 °C was treated, dropwise, with
nꢀBuLi (4.84 mL, 12.1 mM, 2.0 M in hexane) and the resulting mixture was left to stir
at ꢀ78 °C for 20 min. Then, diethyl chlorophosphate (875 ꢁL, 6.05 mM) was added.
After an additional 20 min, the mixture was allowed to warm to room temperature and
10
11
12
13
14
15
16
17
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
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quenched with saturated NH
4
Cl. The mixture was then extracted with EtOAc, washed
and concentrated under reduced pressure to give a
with brine, dried over MgSO
4
crude product, which was purified by flash chromatography to yield compound 4a as
1
a yellow oil (1.5 g, 74 %). H NMR (400 MHz, CDCl
3
) δ 8.75 (d, J = 2.3 Hz, 1H),
7
.84 (dd, J = 8.1, 2.2 Hz, 1H), 7.79 (s, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 7.8
Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.48 (dd, J = 8.1, 2.0 Hz, 1H), 4.10 (dq, J = 14.2,
1
3
7.1 Hz, 4H), 3.46 (d, J = 22.0 Hz, 2H), 1.28 (t, J = 7.1 Hz, 6H). C NMR (101 MHz,
CDCl ) δ 152.6 (d, J = 8.5 Hz), 147.9 (d, J = 2.5 Hz), 138.5 (d, J = 1.3 Hz), 135.2 (d,
J = 2.8 Hz), 133.6 (d, J = 3.4 Hz), 131.7 (d, J = 32.3 Hz), 130.4, 129.7, 125.4 (d, J =
72.7 Hz), 124.9 (q, J = 3.7 Hz), 124.4 (d, J = 4.9 Hz), 123.9 (q, J = 4.1 Hz), 62.4 (d,
3
2
J = 6.6 Hz), 36.4 (d, J = 135.3 Hz), 35.76 (s), 16.5 (d, J = 6.0 Hz). ESIꢀMS m/z 324
+
[
M + H] .
Diethyl ((5-(3-(trifluoromethyl)phenyl)pyridin-2-yl)methyl)phosphonate (4b).
The general procedure for the synthesis of 4b was similar to 4a and resulted in a 72 %
1
yield as a yellow oil. H NMR (400 MHz, CDCl ) δ 8.77 (d, J = 2.1 Hz, 1H), 7.86 (dd,
3
J = 8.1, 2.1 Hz, 1H), 7.80 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.62 (dt, J = 15.4, 7.7 Hz,
1H), 7.50 (dd, J = 8.1, 1.9 Hz, 1H), 4.23 – 4.02 (m, 2H), 3.48 (d, J = 22.0 Hz, 1H),
13
1
.30 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, CDCl
3
) δ 152.7 (d, J = 8.5 Hz), 147.9
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2
2
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(
d, J = 2.4 Hz), 138.6, 135.3 (d, J = 2.8 Hz), 133.8 (d, J = 3.3 Hz), 131.9 (d, J = 32.6
Hz), 130.5, 129.8, 125.5 (d, J = 273.5 Hz), 125.0 (q, J = 3.7 Hz), 124.5 (d, J = 4.9 Hz),
31
1
24.0 (q, J = 3.7 Hz), 62.5, 62.4, 37.2 (d, J = 135.8 Hz), 16.6, 16.5. P NMR (162
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19
MHz, CDCl ) δ 25.10 – 24.21 (m). F NMR (376 MHz, CDCl ) δ ꢀ62.70 (s). ESIꢀMS
3
3
+
m/z 374 [M + H] .
-((E)-2-((1R,4aS,5R,6R,8aR)-6-Hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-
3
methylenedecahydronaphthalen-1-yl)vinyl)furan-2(5H)-one (8). To a 100ꢀmL
flameꢀdried roundꢀbottomed flask containing compound 5 (10 g, 28.6 mM) in
anhydrous pyridine (40 mL), Al O (3 g) was added. The mixture was heated to
2
3
1
15 °C and stirred for 5 h. After this time, TLC analysis indicated the consumption of
the starting material. After filtration, the solvent was concentrated under reduced
pressure to give a crude product, which was then purified by flash chromatography to
1
yield compound 8 (7.6 g, 80 %) as a white solid, mp 205ꢀ207 °C. H NMR (400 MHz,
DMSO) δ 7.65 (s, 1H), 6.74 (dd, J = 15.8, 10.1 Hz, 1H), 6.12 (d, J = 15.8 Hz, 1H),
5.05 (d, J = 4.9 Hz, 1H), 4.89 (s, 2H), 4.73 (s, 1H), 4.42 (s, 1H), 4.14 (dd, J = 7.4, 2.7
Hz, 1H), 3.84 (dd, J = 10.9, 2.6 Hz, 1H), 3.31 – 3.14 (m, 2H), 2.36 (d, J = 10.5 Hz,
2H), 2.01 – 1.95 (m, 1H), 1.72 (d, J = 13.0 Hz, 1H), 1.65 – 1.50 (m, 2H), 1.46 – 1.25
13
(m, 2H), 1.23 – 1.11 (m, 2H), 1.09 (s, 3H), 0.76 (s, 3H). C NMR (101 MHz, DMSO)
δ 172.4, 148.9, 146.7, 134.3, 127.1, 121.2, 108.0, 78.6, 70.2, 62.7, 60.6, 53.7, 42.4,
+
3
8.3, 38.0, 36.2, 27.6, 23.2, 23.0, 15.4. ESIꢀMS m/z 333 [M + H] .
3-((E)-2-((1S,2S,4aS,5R,6R,8aR)-6-Hydroxy-5-(hydroxymethyl)-5,8a-dimethylo
ctahydro-1H-spiro[naphthalene-2,2'-oxiran]-1-yl)vinyl)furan-2(5H)-one (9). A
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2 3
magnetically stirred solution of compound 8 (6.0 g, 18 mM) and K CO (3.0 g, 21.6
2 2
mM) in anhydrous CH Cl (100 mL) was maintained at 0 °C under nitrogen. This
mixture was treated, portionꢀwise, with mꢀCPBA (75 % pure, 5.0 g, 21.6 mM), and
the resulting mixture was allowed to warm to room temperature for 3 h. After this
time, TLC analysis indicated the consumption of the starting material. Then the
10
11
12
13
14
15
16
17
18
19
20
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23
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57
58
59
60
2 2
mixture was diluted with CH Cl , washed with 5 % NaOH and brine, dried over
MgSO , filtered and concentrated under reduced pressure to produce a crude product.
4
The crude residue was purified by column chromatography to give compound 9 (5.3 g,
1
8
5 %) as a white solid, mp 186ꢀ188 °C. H NMR (400 MHz, Acetone) δ 7.48 (s, 1H),
6
.49 (dd, J = 15.5, 9.9 Hz, 1H), 6.15 (d, J = 15.6 Hz, 1H), 4.84 (s, 2H), 4.53 (s, 1H),
4.15 (d, J = 10.8 Hz, 1H), 3.69 (s, 1H), 3.43 – 3.31 (m, 2H), 2.72 (d, J = 3.5 Hz, 1H),
.51 (d, J = 4.5 Hz, 1H), 2.20 (d, J = 9.8 Hz, 1H), 1.93 – 1.52 (m, 6H), 1.48 – 1.38 (m,
2
13
2H), 1.23 (s, 3H), 1.20 – 1.17 (m, 1H), 0.99 (s, 3H). C NMR (101 MHz, Acetone) δ
1
73.0, 146.3, 131.7, 129.1, 124.9, 80.8, 70.8, 64.5, 60.1, 58.6, 55.2, 50.8, 43.7, 39.9,
+
39.1, 36.7, 28.5, 23.6, 22.3, 16.5. ESIꢀMS m/z 349 [M + H] .
3
-((E)-2-((4aR,6aR,7S,8S,10aS,10bR)-3,3,6a,10b-Tetramethyloctahydro-1H,6H
-spiro[naphtho[2,1-d][1,3]dioxine-8,2'-oxiran]-7-yl)vinyl)furan-2(5H)-one (10). A
magnetically stirred solution of compound 9 (6.26 g, 18 mM) in anhydrous CH Cl
(100 mL) was maintained at 0 °C under N protection. Pyridinium pꢀtoluenesulfonate
2
2
2
(452 mg, 1.8 mM) and dimethoxypropane (15.5 mL, 126 mM) were added to the
solution, and the resulting mixture was allowed to warm to room temperature for 3 h.
After this time, TLC analysis indicated the consumption of starting material, and the
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reaction was quenched with a 1 % NaHCO
3
solution. The resulting mixture was
4
and filtered, after which the solvent
washed with water and brine, dried over MgSO
was removed under reduced pressure to give the crude compound, which was purified
0
1
2
3
4
5
6
7
8
9
0
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2
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9
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2
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8
9
0
by flash chromatography to obtain compound 10 (5.93 g, 85 %) as a white solid, mp
1
8
1
3
1
1
2
0ꢀ82 °C. H NMR (400 MHz, Acetone) δ 7.49 (s, 1H), 6.54 (dd, J = 15.6, 9.9 Hz,
H), 6.17 (d, J = 15.7 Hz, 1H), 4.85 (d, J = 1.4 Hz, 2H), 4.06 (d, J = 11.6 Hz, 1H),
.45 (dd, J = 9.6, 4.3 Hz, 1H), 3.23 (d, J = 11.6 Hz, 1H), 2.73 (dd, J = 4.7, 1.6 Hz,
H), 2.53 (d, J = 4.7 Hz, 1H), 2.26 (d, J = 9.9 Hz, 1H), 2.09 – 1.96 (m, 2H), 1.92 –
.78 (m, 2H), 1.75 – 1.66 (m, 1H), 1.57 (dd, J = 12.9, 4.0 Hz, 1H), 1.52 – 1.40 (m,
13
H), 1.38 (s, 3H), 1.28 (s, 3H), 1.26 – 1.24 (m, 1H), 1.23 (s, 3H), 1.21 (s, 3H). C
NMR (101 MHz, Acetone) δ 172.9, 146.2, 131.5, 128.9, 125.0, 99.3, 77.7, 70.6, 64.1,
5
9.7, 58.5, 52.5, 50.8, 39.3, 38.3, 36.4, 28.0, 26.7, 26.0, 25.8, 21.4, 17.3. ESIꢀMS m/z
+
389 [M + H] .
5
-(3-Fluorophenyl)-2-((E)-2-((4aR,6aR,7S,8S,10aS,10bR)-3,3,6a,10b-tetrameth
yloctahydro-1H,6H-spiro[naphtho[2,1-d][1,3]dioxine-8,2'-oxiran]-7-yl)vinyl)pyri
dine (12a). A solution of compound 10 (2.0 g, 5.15 mM) in CH Cl :MeOH (30
mL:30 mL) was maintained at ꢀ78 °C under N protection. To this mixture, pyridine
3 mL) was added, and ozone was passed into the reaction mixture until a light blue
2
2
2
(
color was obtained. TLC analysis indicated the consumption of starting material. N2
was then bubbled through the solution to remove the excess ozone, and dimethyl
sulfide (10 mL) was added. After 30 min, the ꢀ78 °C bath was allowed to warm to
room temperature, and mixture was stirred for 10 min longer. The resulting mixture
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2 2 2 4
was diluted with CH Cl , washed with H O and brine, dried over MgSO , filtered and
concentrated under reduced pressure to give the crude product. The crude residue was
purified by flash chromatography to yield compound 11 (1.4 g, 88 %) which was
unstable and was quickly used for the next step. To a stirred solution of compound 4a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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59
60
(
3.0 g, 9.1 mM) in dry THF (50 mL), under nitrogen atmosphere at 0 °C, nꢀBuLi (3.7
mL, 9.1 mM, 2.0 M in hexane) was added dropwise, and then the mixture was
allowed to warm to room temperature for 15 min. Compound 11 (1.4 g, 4.55 mM) in
dry THF (20 mL) was added dropwise to the mixture, and the solution was stirred for
an additional 20 min. After this time, TLC analysis indicated that the starting material
was consumed and then the resulting mixture was quenched with saturated NH
extracted 3 times with CH Cl . The combined extracts were washed with brine, dried
over MgSO , and filtered, and the solvent was removed under reduced pressure to
4
Cl and
2
2
4
give the crude product. The residue was subjected to chromatography on silica gel to
1
yield compound 12a (1.7 g, 78 %) as a white solid, mp 191ꢀ193 °C. H NMR (400
MHz, CDCl
.0, 5.9 Hz, 1H), 7.39 – 7.37 (m, 1H), 7.35 – 7.27 (m, 2H), 7.14 – 7.09 (m, 1H), 6.58
– 6.56 (m, 2H), 4.14 (d, J = 11.7 Hz, 1H), 3.54 (dd, J = 10.1, 4.4 Hz, 1H), 3.33 (d, J =
1.6 Hz, 1H), 2.96 (dd, J = 4.4, 1.5 Hz, 1H), 2.65 (d, J = 4.5 Hz, 1H), 2.46 – 2.44 (m,
1H), 2.15 – 1.96 (m, 2H), 1.94 – 1.86 (m, 1H), 1.83 – 1.75 (m, 1H), 1.73 – 1.59 (m,
H), 1.57 (s, 1H), 1.55 – 1.52 (m, 1H), 1.49 (s, 3H), 1.43 (s, 3H), 1.34 (s, 3H), 1.30 –
3
) δ 8.79 (d, J = 2.1 Hz, 1H), 7.82 (dd, J = 8.1, 2.3 Hz, 1H), 7.47 (td, J =
8
1
2
1
3
1.26 (m, 4H). C NMR (101 MHz, CDCl
3
) δ 164.6, 162.2, 154.4, 148.0, 140.2, 135.0
(d, J = 23.4 Hz), 133.7, 130.8 (d, J = 8.4 Hz), 128.7, 122.6 (d, J = 2.8 Hz), 122.1,
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1
3
14.9 (d, J = 21.1 Hz), 113.9 (d, J = 22.3 Hz), 98.9, 77.7, 63.7, 58.5, 58.4, 52.5, 51.2,
8.9, 37.7, 36.0, 35.7, 28.1, 26.6, 25.9, 25.3, 20.9, 17.0. ESIꢀHRMS (m/z): calcd for
+
C
30
H
37FNO
3
[M + H] , 478.2713; found, 478.2742.
0
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2
-((E)-2-((4aR,6aR,7S,10aS,10bR)-3,3,6a,10b-Tetramethyloctahydro-1H,6H-spi
ro[naphtho[2,1-d][1,3]dioxine-8,2'-oxiran]-7-yl)vinyl)-5-(3-(trifluoromethyl)phen
yl)pyridine (12b). The general procedure for the synthesis of 12b was similar to that
1
used for 12a, and produced a yield of 80 % as a white solid, mp 147ꢀ149 °C. H NMR
(400 MHz, CDCl
3
) δ 8.69 (s, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 7.5 Hz, 1H),
.56 (d, J = 7.5 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 6.47 (s,
H), 4.02 (d, J = 11.5 Hz, 1H), 3.43 (dd, J = 9.8, 3.8 Hz, 1H), 3.22 (d, J = 11.7 Hz,
7
2
1H), 2.84 (d, J = 3.4 Hz, 1H), 2.53 (d, J = 4.0 Hz, 1H), 2.34 (d, J = 5.6 Hz, 1H), 2.05
1.84 (m, 2H), 1.78 (d, J = 13.9 Hz, 1H), 1.72 – 1.63 (m, 1H), 1.62 – 1.54 (m, 1H),
–
13
1.50 – 1.40 (m, 2H), 1.37 (s, 3H), 1.30 (d, J = 8.1 Hz, 3H), 1.27 – 1.17 (m, 8H). C
NMR (101 MHz, CDCl ) δ 154.6, 147.9, 138.7, 135.0, 133.5, 131.9, 131.5, 130.3,
3
129.8, 125.5, 124.8, 123.8, 123.8, 122.8, 122.2, 98.9, 77.7, 63.7, 58.5, 58.5, 52.5, 51.3,
3
8.9, 37.7, 36.0, 35.7, 28.1, 26.6, 25.9, 25.3, 20.9, 17.0. ESIꢀHRMS (m/z): calcd for
+
C
31
H
39
F
3
NO
3
[M + H] , 530.2837; found, 530.2773.
(
1S,2S,4aS,5R,6R,8aR)-1-((E)-2-(5-(3-Fluorophenyl)pyridin-2-yl)vinyl)-5-(hydr
oxymethyl)-5,8a-dimethyloctahydro-1H-spiro[naphthalene-2,2'-oxiran]-6-ol
13a). To a stirred solution of compound 12a (500 mg, 1.05 mM) in anhydrous
(
methanol (5 mL) Amberlyst 15 was added, and the mixture was stirred for two days at
room temperature. After filtration and evaporation in vacuo, the crude compound 13a
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was obtained, which was then purified by flash chromatography to give compound
1
1
8
7
4
7
2
1
2
1
3a (366 mg, 80 %) as a white solid, mp 207ꢀ209 °C. H NMR (400 MHz, CDCl
3
) δ
.74 (d, J = 2.1 Hz, 1H), 7.80 (dd, J = 8.1, 2.3 Hz, 1H), 7.45 (td, J = 8.0, 5.9 Hz, 1H),
.38 – 7.33 (m, 1H), 7.31 – 7.23 (m, 2H), 7.14 – 7.06 (m, 1H), 6.57 – 6.42 (m, 2H),
.27 (d, J = 10.6 Hz, 1H), 3.60 – 3.48 (m, 1H), 3.41 (t, J = 9.9 Hz, 1H), 3.21 (d, J =
.6 Hz, 1H), 3.15 (d, J = 3.9 Hz, 1H), 2.96 – 2.86 (m, 1H), 2.62 (d, J = 4.4 Hz, 1H),
.38 (d, J = 9.0 Hz, 1H), 2.04 – 1.92 (m, 2H), 1.90 – 1.78 (m, 1H), 1.77 – 1.69 (m,
H), 1.65 (dt, J = 13.6, 3.3 Hz, 1H), 1.59 – 1.47 (m, 2H), 1.32 (s, 3H), 1.26 – 1.17 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
H), 1.05 (s, 3H). C NMR (101 MHz, CDCl ) δ 164.6, 162.2, 154.3, 147.8, 140.0,
3
35.0, 133.8, 130.79 (d, J = 8.4 Hz), 128.9, 122.64 (d, J = 2.8 Hz), 122.1, 115.0 (d, J
= 21.1 Hz), 113.9 (d, J = 21.1 Hz), 80.7, 77.5, 77.2, 76.8, 64.2, 58.6, 58.5, 54.5, 51.1,
4
3.1, 39.3, 38.2, 35.8, 27.8, 22.9, 21.5, 16.3. ESIꢀHRMS (m/z): calcd for C27H33FNO
3
+
[M + H] , 438.2400; found, 438.2439.
(1S,2S,4aS,5R,6R,8aR)-5-(Hydroxymethyl)-5,8a-dimethyl-1-((E)-2-(5-(3-(triflu
oromethyl)phenyl)pyridin-2-yl)vinyl)octahydro-1H-spiro[naphthalene-2,2'-oxira
n]-6-ol (13b). The general procedure for the synthesis of 13b was similar to that used
1
for 13a, with a yield of 83 % as a white solid, mp 119ꢀ121 °C. H NMR (400 MHz,
CDCl
J = 7.6 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.30 – 7.26 (m, 1H),
.50 (dt, J = 15.3, 12.2 Hz, 2H), 4.27 (d, J = 10.7 Hz, 1H), 3.59 – 3.47 (m, 1H), 3.45 –
3.27 (m, 3H), 2.92 (d, J = 3.6 Hz, 1H), 2.62 (d, J = 4.3 Hz, 1H), 2.39 (d, J = 9.1 Hz,
3
) δ 8.76 (d, J = 1.9 Hz, 1H), 7.83 (dd, J = 8.2, 2.3 Hz, 1H), 7.80 (s, 1H), 7.75 (d,
6
1
H), 2.00 – 1.93 (m, 2H), 1.88 – 1.79 (m, 1H), 1.78 – 1.69 (m, 1H), 1.69 – 1.60 (m,
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H), 1.58 – 1.47 (m, 2H), 1.32 (s, 3H), 1.27 – 1.23 (m, 1H), 1.23 – 1.19 (m, 1H), 1.05
1
3
(
s, 3H). C NMR (101 MHz, CDCl
31.8 (q, J = 32.1 Hz), 130.3, 129.8, 129.2, 124.9 (q, J = 3.6 Hz), 124.2 (d, J = 273.6
Hz), 123.8 (q, J = 3.6 Hz), 122.2, 80.8, 64.3, 58.7, 58.5, 54.6, 51.1, 43.1, 39.3, 38.3,
3
) δ 154.7, 147.9, 138.7, 135.1, 134.8, 133.7,
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19
3
3
5.9, 27.8, 23.0, 21.5, 16.4. F NMR (376 MHz, CDCl ) δ ꢀ62.7. ESIꢀHRMS (m/z):
+
calcd for C28
H
35
F
3
NO
3
[M + H] , 490.2524; found, 490.2559.
(
1S,2S,4aS,5R,6R,8aR)-1-(2-(5-(3-Fluorophenyl)pyridin-2-yl)ethyl)-5-(hydroxy
methyl)-5,8a-dimethyloctahydro-1H-spiro[naphthalene-2,2'-oxiran]-6-ol (14a).
Thoroughly deoxygenated THF (15 mL) was added to a mixture of Cp TiCl (398 mg,
2
2
1
.60 mM) and Mn (282 mg, 5.12 mM) under a N
was stirred at room temperature until it turned lime green (after approximately 15
min). A solution of compound 13a (277 mg, 0.64 mM) and H O (115 ꢁL, 6.40 mM)
2
atmosphere, and the suspension
2
in THF (1 mL) was added, and the mixture was stirred for 24 h, after which the
reaction was quenched with a saturated solution of KHSO and extracted with EtOAc.
4
4
The organic layer was washed with brine, dried with anhydrous MgSO , and
concentrated. The residue was subjected to chromatography on silica gel to yield
1
compound 14a (208 mg, 75 %) as a colorless oil. H NMR (400 MHz, CDCl
3
) δ 8.68
(
d, J = 2.0 Hz, 1H), 7.82 (dd, J = 8.0, 2.3 Hz, 1H), 7.43 (td, J = 8.0, 6.0 Hz, 1H), 7.33
(d, J = 7.8 Hz, 1H), 7.30 – 7.21 (m, 2H), 7.08 (td, J = 8.3, 2.0 Hz, 1H), 4.09 (d, J =
1.2 Hz, 1H), 3.80 – 3.70 (m, 2H), 3.56 (d, J = 11.8 Hz, 1H), 3.48 (dd, J = 11.2, 4.8
Hz, 1H), 3.18 – 3.07 (m, 2H), 2.98 (s, 2H), 2.58 (dd, J = 14.2, 11.7 Hz, 1H), 2.31 –
1
2
.22 (m, 1H), 1.86 – 1.80 (m, 2H), 1.68 – 1.60 (m, 2H), 1.57 – 1.37 (m, 3H), 1.19 (s,
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
5
1
6
H), 0.92 (d, J = 6.4 Hz, 3H), 0.91 – 0.86 (m, 1H), 0.78 – 0.69 (m, 1H), 0.49 (d, J =
1
3
3
.5 Hz, 1H). C NMR (101 MHz, CDCl ) δ 164.6, 162.2, 160.5, 147.1, 139.7, 139.6,
35.8, 133.6, 130.9, 130.8, 123.4, 122.7, 122.7, 115.2, 115.0, 114.1, 113.9, 80.7, 68.8,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.2, 53.5, 43.2, 41.2, 38.6, 37.7, 32.2, 32.1, 29.3, 29.2, 25.4, 22.3, 21.6, 16.9.
+
ESIꢀHRMS (m/z): calcd for C27
H35FNO
3
[M + H] , 440.2556; found, 440.2592.
(
1S,2S,4aS,5R,6R,8aR)-5-(Hydroxymethyl)-5,8a-dimethyl-1-(2-(5-(3-(trifluoro
methyl)phenyl)pyridin-2-yl)ethyl)octahydro-1H-spiro[naphthalene-2,2'-oxiran]-6
-
ol (14b). The general procedure for the synthesis of 14b was similar to that used for
1
1
4a with a yield of 72 % as a colorless oil. H NMR (400 MHz, CDCl ) δ 8.71 (d, J =
3
2
.3 Hz, 1H), 7.85 (dd, J = 8.0, 2.3 Hz, 1H), 7.79 (s, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.66
(d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 4.09 (d, J = 11.2
Hz, 1H), 3.84 – 3.66 (m, 2H), 3.57 (d, J = 11.8 Hz, 1H), 3.55 – 3.48 (m, 1H), 3.21 –
3.08 (m, 2H), 2.83 (s, 2H), 2.60 (dd, J = 14.3, 11.6 Hz, 1H), 2.33 – 2.23 (m, 1H), 1.93
–
1.78 (m, 2H), 1.72 – 1.61 (m, 2H), 1.56 – 1.40 (m, 2H), 1.23 (d, J = 7.0 Hz, 1H),
13
1.19 (s, 3H), 0.93 (s, 3H), 0.83 – 0.72 (m, 2H), 0.49 (d, J = 5.5 Hz, 1H). C NMR
101 MHz, CDCl ) δ 160.9, 147.2, 138.3, 135.9, 133.5, 131.9, 131.6, 130.3, 129.8,
125.4, 124.9, 124.9, 123.9, 123.9, 123.4, 122.7, 80.7, 68.8, 64.2, 53.6, 43.2, 41.2, 38.6,
(
3
3
7.7, 32.2, 32.1, 29.4, 29.2, 25.4, 22.3, 21.6, 16.9. ESIꢀHRMS (m/z): calcd for
+
C H F NO [M + H] , 490.2524; found, 490.2563.
28
35
3
3
(
4aR,6aR,7S,8S,10aS,10bR)-3,3,6a,10b-Tetramethyloctahydro-1H,6H-spiro[na
phtho[2,1-d][1,3]dioxine-8,2'-oxirane]-7-carboxylic acid (15). To a solution of
compound 11 (3.08 g, 10.00 mM), isopentane (17.5 mL), tꢀBuOH (190 mL), and THF
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