Design, synthesis, and SAR studies of 4-substituted methoxylbenzoyl-aryl- thiazoles analogues as potent and orally bioavailable anticancer agents

ARTICLE

pubs.acs.org/jmc

Design, Synthesis, and SAR Studies of 4-Substituted

Methoxylbenzoyl-aryl-thiazoles Analogues as Potent and

Orally Bioavailable Anticancer Agents

Yan Lu,^†,§Chien-Ming Li,^‡,§Zhao Wang,^†Jianjun Chen,^†Michael L. Mohler,^‡Wei Li,^†

James T. Dalton,^‡and Duane D. Miller*^,†,‡

^†Department of Pharmaceutical Sciences, University of Tennessee, Health Science Center, Memphis, Tennessee 38163, United States

^‡GTx Inc., Preclinical R&D, Memphis, Tennessee 38163, United States

S Supporting Information

b

ABSTRACT: In a continued eﬀort to improve upon the previously published

4-substituted methoxybenzoyl-aryl-thiazole (SMART) template, we explored

chemodiverse “B” rings and “B” to “C” ring linkage. Further, to overcome the

poor aqueous solubility of this series of agents, we introduced polar and ionizable

hydrophilic groups to obtain water-soluble compounds. For instance, based on

in vivo pharmacokinetic (PK) studies, an orally bioavailable phenyl-amino-

thiazole (PAT) template was designed and synthesized in which an amino

linkage was inserted between “A” and “B” rings of compound 1. The PAT

template maintained nanomolar (nM) range potency against cancer cell lines via

inhibiting tubulin polymerization and was not susceptible to P-glycoprotein mediated multidrug resistance in vitro, and markedly

improved solubility and bioavailability compared with the SMART template (45aꢀc (PAT) vs 1 (SMART)).

’ INTRODUCTION

Microtubules are cytoskeletal ﬁlaments consisting of Rβ-

tubulin heterodimers and are involved in a wide range of cellular

functions, including shape maintenance, vesicle transport, cell

motility, and division. Tubulin is the major structural component

of the microtubules and a well veriﬁed target for a variety of

highly successful anticancer drugs. Anticancer drugs like pacli-

taxel and vinblastine that are able to interfere with microtubuleꢀ

tubulin equilibrium in cells are extensively used in cancer

chemotherapy.¹There are two major classes of antimitotic

agents: microtubule-stabilizing agents, which inhibits the micro-

tubule depolymerization by binding to and stabilizing micro-

tubule, are represented by taxanes and epothilones. Another class

is microtubule-destabilizing agents, such as vinca alkaloids and

colchicine, which cause microtubule disassembling and inhibit

tubulin polymerization into microtubules. Vinblastine represents

Figure 1. Structures of Colchicine-Binding Site Tubulin Inhibitors.

one of vinca alkaloids. Colchicine and colchicine-site binders are

all deﬁned as microtubule-destabilizing antimitotic agents.²

(MDR), namely ATP binding cassette (ABC) transporter

protein-mediated drug eﬄux, limits their eﬃcacy.^5ꢀ7P-glyco-

While no colchicine-site binders are currently approved for

cancer chemotherapy, both the taxanes and vinca alkaloids are

proteins (P-gp, encoded by the MDR1 gene) is an important

member of the ABC superfamily.⁸P-gp prevents the intracellular

widely used to treat human cancers. However, colchicine binding

agents like combretastatin A-4 (CA-4) and N-(2-(4-hydroxy

accumulation of many cancer drugs by increasing their eﬄux out

phenylamino)pyridin-3-yl)-4-methoxybenzenesulfonamide (ABT-

of cancer cells as well as contributing to hepatic, renal, or

intestinal clearance pathways. Attempts to coadminister P-gp

751, 47) (Figure1) are now under clinical investigation as potential

new chemotherapeutic agents.^2,3,4

Unfortunately, microtubule-interacting anticancer drugs in

clinical use share two major problems, resistance and high

lipophilicity. A common mechanism of multidrug resistance

Received: March 23, 2011

Published: May 10, 2011

r

2011 American Chemical Society

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|

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Scheme 1^a

^a(a) MeOH, CH₃COCl, 83%; (b) ethyl benzimidate hydrochloride, CH₂Cl₂, Et₃N, 96%; (c) LiOH, MeOH, H₂O, 65%; (d) EDCI, HOBt, NMM,

CH₃OCH₃NH HCl, 61%; (e) 3,4,5-trimethoxyphenylmagnesium bromide, THF, 48ꢀ71%; (f) CBrCl₃, DBU, CH₂Cl₂, 56%.

3

Scheme 2^a

^a(a) EDCI, HOBt, NMM, CH₃OCH₃NH HCl, CH₂Cl₂, 51ꢀ95%; (b) 3,4,5-trimethoxyphenyl-magnesium bromide, THF, 48-78%; (c) LAH, ꢀ78 °C,

3

THF, 85%; (d) DessꢀMartin reagent, CH₂Cl₂, 81%; (e) EDCI, HOBt, NMM, 3,4,5-trimethoxybenzoic acid, CH₂Cl₂, 58%.

modulators or inhibitors to increase cellular availability by

blocking the actions of P-gp have met with limited success.^8,9

The other major problem with taxanes, as with many biologically

active natural products, is its high lipophilicity and lack of

solubility in aqueous systems. This leads to the use of emulsiﬁers

like Cremophor EL and Tween 80 in clinical preparations.

A number of biologic eﬀects related to these drug formula-

tion vehicles have been described, including acute hypersensi-

tivity reactions and peripheral neuropathies.^10,11Compared

to compounds binding the paclitaxel- or vinca alkaloid-binding

site, colchicine-binding agents usually exhibit relatively simple

structures. Thus, it provides a better opportunity for oral

bioavailability via structural optimization, as reported herein for

compounds with improved solubility and pharmacokinetic (PK)

parameters. In addition, most of these new agents appear to

circumvent P-gp-mediated MDR. Therefore, these novel colchi-

cine binding site targeted compounds hold great promise as

therapeutic agents, particularly because they have improved

aqueous solubility and overcome P-gp mediated MDR.

4-Substituted methoxybenzoyl-aryl-thiazole (SMART, 1,

Figure 1) is a potential anticancer agent that was discovered

recently in our laboratory which targets tubulin by binding to its

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Scheme 3^a

^a(a) MeOH/pH = 6.4 phosphate buﬀer, RT; (b) EDCI, HOBt, NMM, HNCH₃OCH₃; (c) CBrCl₃, DBU, CH₂Cl₂; (d) 3,4,5-trimethoxyphenylmagne-

sium bromide, THF; (e) isopropyl triphenylphosphonium iodide, n-BuLi, THF; (f) LAH, THF; (g) for 13a and 13b, NH₂OH HCl, C₂H₅OH, H₂O,

3

NaOH; for 13e and 13f, NH₂OMe HCl, pyridine; (h) TsCl, NaH, basic Al₂O₃; (i) NH₂NH₂xH₂O, CH₂Cl₂, C₂H₅OH; (j) diethyl cyanomethyl-

3

phosphonate, n-BuLi, THF; (k) bis-trimethylsilylcarbodiimide, TiCl₄, CH₂Cl₂; (l) EDCI, HOBt, Et₃N, 3,4,5-trimethoxyaniline, CH₂Cl₂.

colchicine binding site.¹²The structureꢀactivity-relationship

(SAR) studies in the “A” and “C” rings were discussed based

on synthesized analogues. Whereas, alternatives to the thiazole

“B” ring and carbonyl linker were not investigated. In this article,

we synthesized and evaluated the biological properties of “B” ring

and carbonyl linker modiﬁed derivatives to extend the SAR

studies beyond just the SMART template and found some more

potent compounds. “A” ring modiﬁcations and introducing a NH

linkage between “A” and “B” rings produced potent water-

soluble compounds including the phenyl amino thiazole

(PAT) compounds 45aꢀc. We demonstrated that these com-

pounds can eﬀectively inhibit cancer cell growth in vitro by

interfering with tubulin polymerization. In experiments de-

scribed herein, we have examined the antiproliferative eﬀects of

these novel compounds on a drug-sensitive ovarian cancer cell

line (OVCAR-8) and its P-gp-overexpressing drug-resistant

counterpart (NCI/ADR-RES). Tested compounds did not de-

monstrate susceptibility to P-gp mediated drug resistance. We

also compared the oral bioavailability of the PAT compounds in

rats. Compounds 45a and 45c showed a signiﬁcant improvement

in bioavailability compared with SMART compound 1. Thus, the

new PAT compounds and other molecules reported herein

represent new families of compounds that may be very useful

in the treatment of cancer with improved PK properties.

’ RESULTS AND DISCUSSION

Chemistry. Design of novel chemotypes of SMART agents

that target tubulin polymerization were investigated mainly by

preparing three series of modifications based upon compound 1.

The first series of derivatives (5, 6, 9aꢀ9h) were characterized by

the replacement of the thiazole in SMART molecules with a

panel of different B rings as illustrated in Schemes 1ꢀ2. ^L-Sertine

methyl ester hydrochloride was obtained from ^L-serine stirred in

acetyl chloride/MeOH solution. Condensation of the methyl

ester with ethyl benzimidate hydrochloride led to oxazoline

methyl ester 2 in excellent yield (Scheme 1).¹³Hydrolyzation

of the methyl ester gave carboxylic acid 3 that was in turn coupled

to N,O-dimethylhydroxylamine to provide Weinreb amide 4.

Compound 4 was reacted with appropriate Grignard reagents in

anhydrous THF to give the oxazoline 5. Oxidation of 5 with

BrCCl₃/DBU gave the oxazole product 6.¹⁴Other B ring variants

9aꢀ9f (except 9e) were obtained from different acids 7aꢀ7f

with a similar method as described above (Scheme 2). Pure

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Scheme 4^a

^a(a) Bromine, EtOH; (b) benzothioamide, EtOH, reﬂux; (c) EDCI, HOBt, NMM, HNCH₃OCH₃, CH₂Cl₂; (d) CBrCl₃, DBU, CH₂Cl₂; (e) LAH,

THF; (f) 5-(bromomethyl)-1,2,3-trimethoxybenzene, Ph₃P, THF; (g) n-BuLi, THF; (h) (1) HCl, H₂O; (2) NaNO₂, H₂O, 0 °C; (i) ethyl potassium

xanthate; (j) KOH/EtOH; (k) H₂O, HCl; (l) 5-iodo-1,2,3-trimethoxybenzene, CuI, t-BuONa; (m) 2 equiv or 1 equiv m-CPBA, CH₂Cl₂; (n) 3,4,5-

trimethoxyaniline, NEt₃, DMF.

compound 9e with thiophene in B ring position can not be

separated from the mixture of 9e and a Grignard reagent coupling

byproduct 3,4,5,3⁰,4⁰,5⁰-hexamethoxybiphenyl using chromatog-

raphy. So we used an alternative method to prepare 9e: Weinreb

amide 8e was converted into an aldehyde and then reacted with

3,4,5-trimethoxyphenylmagnesium bromide to afford the alcohol

10e, which can be easily separated from 3,4,5,3⁰,4⁰,5⁰-hexa-

methoxybiphenyl after flash column purification. Oxidation with

pyridinium dichromate (PDC) or DMSO did not afford 9e from

secondary alcohol 10e with good yields, but using DessꢀMartin

periodinane reagent as oxidant successfully formed the desired

ketone compound 9e with 81% yield.¹⁵9g and 9h were prepared

from alcohol 10gꢀ10h using a similar method. Compound 9i

was obtained via a coupling reaction from piperidine 11 and

3,4,5-trimethoxybenzoic acid.

through three steps described previously.¹⁷1 was converted to

oxime isomers (13aꢀd) upon reaction with hydroxylamines,

NH₂OH or NH₂OCH₃. Assignments were made on the basis of

chemical and spectral data as described infra. An improved

Beckmann rearrangement¹⁸readily produced the rearranged

amides 13e and 13f from the two geometric stereoisomers 13a

and 13b via their reaction with tosyl chloride and subsequent

basic aluminum oxide column. Hydrazide derivatives 14a and

14b were prepared by mixing 1 with hydrazine hydrate in

ethanol/CH₂Cl₂and reﬂuxing for 24 h. Acrylonitriles 15aꢀ

15b were obtained from Wittig reaction of 1 with diethyl cyano-

methylphosphonate.¹⁹Cyanoimine 16 was prepared using the

procedure described by Cuccia.²⁰The carbonyl group in com-

pound 1 was also reduced to a secondary alcohol (17) or

converted to an alkene (18) as illustrated. We also tried to

remove the carbonyl group between B and C rings in compound

1 and thus obtained compound 19 in Scheme 4. Introducing

cis- and trans- double bonds into the carbonyl position formed

compounds 20 and 21, which were synthesized from a

Wittig reaction with 2-phenylthiazole-4-carbaldehyde. We also

In the second series of novel templates, structure modiﬁca-

tions focused on alternatives to the carbonyl group to avoid

potential metabolic problems caused by ketone reduction

(Scheme 3 and Scheme 4).¹⁶SMART compound 1 was synthe-

sized from 2-phenyl-4,5-dihydro-thiazole-4-carboxylic acid 12a

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Scheme 5^a

^a(a) L-Cysteine, EtOH, 65 °C; (b) EDCI, HOBt, NMM, HNCH₃OCH₃, CH₂Cl₂; (c) TBDMSCl, imidazole, THF; (d) 3,4,5-trimethoxyphenylbro-

mide, BuLi, THF; (e) TBAF, THF; (f) SOCl₂, Et₂O; (g) NH₃, MeOH; (h) POCl₃; (i) PhSO₂Cl, Bu₄NHSO₄, toluene, 50% NaOH; (j) 1N NaOH,

EtOH, reﬂux; (k) Boc₂O, 1N NaOH, 1,4-dioxane; (l) CBrCl₃, DBU, CH₂Cl₂; (m) 2N HCl in 1,4-dioxane; (n) NaH, DMF, MeI; (o) HCHO, NaBH₃-

CN, Et₃N.

prepared sulﬁde compound 23, sulfone 24 and sulfoxide 25 using

3-aminobiphenyl as starting material through an initial Sand-

meyer reaction to yield carbonodithioate 22, followed by CuI

catalyzed coupling reaction and m-CPBA oxidation.²¹Sulfon-

amide linked compound 26 was prepared from reaction of

3-biphenylsulfonyl chloride with 3,4,5-trimethoxyaniline in the

presence of NEt₃in DMF.

A third aim was to improve aqueous solubility and oral

bioavailability of these colchicine site targeted agents. As illu-

strated in Scheme 5, we introduced hydroxyl and aminomethyl at

the para-position of the phenyl A-ring, as well as replacing phenyl

with 5-indolyl and 2-indolyl rings. Weinreb amides 27, 31, 35,

and 37 were prepared by the procedure described before using

aryl nitriles as starting materials.¹²2-Cyano-indole 30 was

prepared with reported method.²²Protections of hydroxyl

(TBDMSCl), indolyl (PhSO₂Cl), and amino (Boc₂O) groups

were used in preparations. Deprotection of TBDMS and oxida-

tion from thiazoline (28) to thiazole (29) were ﬁnished in one-

step using TBAF/THF solution. We reported this thiazolineꢀ

thiazole oxidation can happen spontaneously in the reaction of

thiazoline Weinreb amide and Grignard reagent.¹²We also

observed the same phenomena during preparing indole com-

pounds 32 and 36. Compound 32 was separated as a pure

thiazole compound after reaction with 3,4,5-trimethoxphenyl-

lithium and needed no further oxidation. Compound 36 was

obtained after removing phenylsulfonyl protecting groups in hot

NaOH ethanol solution. para-OH and NH₂on the A ring of 29

and 39 were obtained from similar Grignard reactions from

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Scheme 6^a

^a(a) EtOH, 65 °C; (b) NaOH, C₂H₅OH, reﬂuxing; (c) EDCI, HOBt, NMM, HNCH₃OCH₃, CH₂Cl₂; (d) 3,4,5-trimethoxyphenylbromide, BuLi,

THF; (e) 2 N HCl in 1, 4-dioxane.

Figure 2. ¹H NMR of Beckmann rearrangement product from 13a isomer (A) is the same with previous prepared 4-carbonyl amide 13e (C) while

rearrangement product from isomer 13b showed reversed 4-amino amide NMR signals (B).

Weinreb amides 27 and 37. Compound 38 was further converted

to HCl salt of monomethyl amine 40 via NaH/MeI conditions,

and compound 39 was converted to HCl salt of dimethylamine

42 under HCHO/NaBH₃CN conditions. To improve bioavail-

ability, we introduced an NH linker between A phenyl and B

thiazole rings. We synthesized this new series of compounds as

shown in Scheme 6. Reaction of 3-bromo-2-oxopropanoic acid

ethyl ester and arylthiourea in ethanol under 65 °C produced

2-(arylamino)-thiazole-4-carboxylic acids 43aꢀc with high

yields. Then these acids were converted to Weinreb amides

44aꢀc, followed by reactions with 3,4,5-trimethoxphenyllithium

yielded aniline linked free bases 45aꢀc, which can be converted

into HCl salts 46aꢀc.

Structural Identification of syn-/anti- Isomers. Oximes

13aꢀ13b, hydrazides 14aꢀ14b, and acrylonitriles 15aꢀ15b

were obtained from flash column as separated isomer pairs.

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Table 1. SAR of Alternate B ring Compounds

IC₅₀( SEM (μM)

B ring

B16-F1

A375

DU 145

PC-3

LNCaP

PPC-1

1

2,4-thiazole

2,4-oxazoline

2,4-oxazole

1,3-phenyl

0.055 ( 0.005

6.5 ( 0.8

0.6 ( 0.2

0.5 ( 0.2

>30

0.028 ( 0.005

0.5 ( 0.1

0.071 ( 0.004

1.2 ( 0.1

0.021 ( 0.001

1.2 ( 0.1

0.028 ( 0.004

1.2 ( 0.1

0.043 ( 0.005

1.1 ( 0.1

5

6

0.3 ( 0.1

0.292 ( 0.01

0.171 ( 0.01

6.9 ( 5.2

0.292 ( 0.02

0.124 ( 0.02

8.3 ( 4.7

0.331 ( 0.06

0.052 ( 0.01

7.0 ( 2.9

0.343 ( 0.04

0.080 ( 0.01

3.7 ( 0.2

9a

9b

9c

9d

9e

9f

9g

9h

9i

0.087 ( 0.015

>30

4,6-pyrimidine

2,6-pyridine

2,5-furan

0.039 ( 0.012

0.151 ( 0.024

0.072 ( 0.015

0.245 ( 0.032

12.5 ( 5.2

>30

0.030 ( 0.014

0.027 ( 0.008

0.015 ( 0.006

0.100 ( 0.018

13.6 ( 3.8

>30

0.033 ( 0.003

0.035 ( 0.004

0.026 ( 0.005

0.145 ( 0.01

>10

0.032 ( 0.002

0.021 ( 0.002

0.012 ( 0.001

0.101 ( 0.02

>10

0.027 ( 0.002

0.023 ( 0.001

0.017 ( 0.001

0.101 ( 0.01

>10

0.025 ( 0.001

0.020 ( 0.001

0.015 ( 0.001

0.084 ( 0.01

>10

2,4-thiophene

3,5-pyrazol

2,5-thiazole

3,5-isoxazole

1,4-piperidine

>10

>30

>20

Table 2. SAR of Alternative to the Carbonyl Linker

IC₅₀( SEM (μM)

X linker

CdO

B16-F1

A375

WM-164

DU 145

PC-3

LNCaP

PPC-1

1

0.055 ( 0.005

0.3 ( 0.1

11.4 ( 2.1

3.8 ( 1.6

>10

0.028 ( 0.005

0.2 ( 0.1

7.8 ( 1.2

2.9 ( 1.2

>10

0.064 ( 0.004

ND^a

0.071 ( 0.004

0.103 ( 0.04

>10

0.021 ( 0.001

0.120 ( 0.05

>10

0.028 ( 0.004

0.169 ( 0.06

>10

0.043 ( 0.005

0.144 ( 0.01

>10

13a

13b

13c

13d

13e

13f

14a

14b

15a

15b

16

syn-CdN-OH

anti-CdN-OH

syn-CdN-OMe

anti-CdN-OMe

CONH

ND

3.4 ( 1.8

>10

>30

ND

>10

NHCO

>30

ND

>10

syn-CdN-NH2

anti-CdN-NH₂

syn-CdC-CN

anti-CdC-CN

CdN-CN

CHOH

2.0 ( 0.8

1.8 ( 0.7

5.4 ( 2.1

1.2 ( 0.3

0.060 ( 0.021

>30

0.9 ( 0.3

0.6 ( 0.2

4.6 ( 1.5

1.2 ( 0.4

0.028 ( 0.012

>30

ND

1.210

2.28

1.120

1.040

0.89 ( 0.34

∼10

0.027 ( 0.001

>10

1.800

0.872

ND

1.300

0.966

4.9 ( 1.3

1.0 ( 0.2

0.027 ( 0.013

ND

0.58 ( 0.12

1.99

0.90 ( 0.1

∼10

0.020 ( 0.001

>10

∼10

0.042 ( 0.002

>10

0.023 ( 0.002

>10

17

18

CdCMe2

none

3.8 ( 1.3

>10

1.9 ( 0.8

>10

3.7 ( 1.2

>10

2.65

2.47

1.39 ( 0.39

>10

2.04

19

>10

20

cis-CdC

11.0 ( 2.8

32.8 ( 13

2.4 ( 0.9

>10

46.5 ( 23.3

>100

10.6 ( 5.8

30.8 ( 12

2.0 ( 1.2

>10

21

trans-CdC

S

>10

23

1.6 ( 0.4

>10

2.3 ( 0.2

>10

2.3 ( 0.1

>10

24

SO₂

>10

25

SO

>10

26

SONH₂

>10

^aND = not determined.

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Table 3. Antiproliferative Activity of Modiﬁed Compounds with Improved Aqueous Solubility

IC₅₀( SEM (μM)

apart

B16-F1

A375

DU 145

PC-3

LNCaP

0.549

PPC-1

28

29

32

36

38

39

40

42

4-OTBDMSPh

4-OHPh

0.5 ( 0.2

0.11 ( 0.02

0.043 ( 0.021

0.025 ( 0.013

2.9 ( 0.4

0.038 ( 0.011

>10

0.7 ( 0.3

0.10 ( 0.01

0.019 ( 0.009

0.008 ( 0.001

7.9 ( 0.5

0.041 ( 0.013

>10

0.434 ( 0.030

0.116 ( 0.014

0.032 ( 0.001

0.013 ( 0.001

4.3 ( 3.7

0.183 ( 0.024

0.087 ( 0.005

0.024 ( 0.004

0.007 ( 0.001

3.1 ( 1.7

0.246 ( 0.008

0.076 ( 0.002

0.028 ( 0.002

0.008 ( 0.001

2.7 ( 1.5

0.103 ( 0.009

0.028 ( 0.003

0.010 ( 0.001

2.6 ( 0.9

2-indolyl

5-indolyl

4-BocNHCH₂Ph

4-NH₂CH₂Ph

4-NHMeCH₂Ph

4-NMe₂CH₂Ph

0.025 ( 0.001

∼10

0.080 ( 0.007

>10

0.013 ( 0.001

1.14 ( 0.08

1.025 ( 0.2

0.034 ( 0.001

∼10

>10

away from C ring protons while in anti- isomer (14b) is short, the

2D NOESY spectrum of compound 14b showed strong NOE

correlations between NH₂and C ring protons and demonstrated

14b is anti- conﬁrmation. Compound 14a which has a syn-

conformation did not show any NOE on 2D NOESY. Acrylo-

nitriles 15a and 15b were identiﬁed with the similar 1D NOE

spectrum. There are no NOE correlations observed among

protons from B ring (H_b)/C ring (H_c) and acrylonitrile linker

(H_a), thus compound 15a is an anti- isomer. Compound 15b

showed NOE between H_aand H_cprotons, which corresponding

to a syn- conﬁrmation (Supportin Information).

Biological Evaluation: In Vitro Cell Growth Inhibtions. All

of the reported compounds were first evaluated for cytotoxicity

in a mouse melanoma cell line B16-F1, human melanoma cell

lines (A375 and WM-164), and prostate cancer cell lines

(DU145, PC-3, LNCaP, and PPC-1). Compounds 1 (Tables 1,

2, and 4) and 47 (E7010, Abbott Laboratories/Eisai Co Ltd.,

Table 4), which has entered phase II clinical studies in treating

patients with different cancers, were included in the assays as

examples of colchicine-site binding agents. IC₅₀values for cell

growth inhibition are shown in Tables 1ꢀ4.

Figure 3. Aqueous solubility of novel anti-tubulin compounds. Com-

pounds 45a and 45c showed improved solubility compared with

compound 1. Compound 39 is soluble at 35 μg/mL concentration.

Their configurations were identified using a chemistry method,

NMR NOE spectrum and quantum chemical shift calculations.

Improved Beckmann rearrangement was used to distinguish

oxime isomers 13a and 13b as the group anti- position to the

departing OTs that migrates to nitrogen (Figure 2). The syn-

isomer 13a rearranged by a 1,2- shift of 3,4,5-trimethoxyphenyl

(TMP) to yield amide 13e, whereas the anti-isomer 13b under-

went a 1, 2-thiazolyl shift to yield 13f. An alternative coupling

method was also used to prepare compound 13e via intermediate

12c (see Scheme 3),¹²which was characterized and compared

with two rearrangement products 13e and 13f. The NMR spectra

showed that alternatively prepared 13e was identical with

Beckmann rearrangement product of syn- isomer 13a. Quantum

chemical calculations based on ¹H NMR also corroborated the

conformations of these two isomers.

SAR of Alternative “B” Ring Molecules. The first series was

targeted to alternatives to the thiazole “B” ring. Accordingly, a

series of heterocyclic “B” rings was examined. As shown in Table 1,

the successful replacements of the thiazole were pyridine 9c, furan

9d, and thiophene 9e. The IC₅₀s (12ꢀ35 nM against prostate

cancer cells) are close to the thiazole compound 1. Introducing

oxazoline (5), oxazole (6), phenyl (9a), and pyrazole (9f) main-

tained activity in the hundreds of nanomolar range. But introduc-

tion of pyrimidine (9b, IC₅₀3.7ꢀ8.3 μM), a reversed 2,5-thiazole

or 3,5-isoxazole (9g and 9h, IC₅₀> 10 μM) caused obvious losses

of potency. Modification of “B” ring to the saturated ring of

piperidine (9i) also totally abolished activity (9c, IC₅₀>20 μM).

SAR of Alternative Linkers between “B” and “C” Rings. In

vitro hepatic metabolic stability studies revealed that the carbonyl

linker between “B” and “C” rings in SMART compounds caused

short half-lives (5ꢀ17 min) primarily due to carbonyl

reduction.¹⁶For the sake of blocking this carbonyl reduction to

the inactive hydroxyl linker compound 17, we modified the

carbonyl linker in the second series of compounds (Table 2).

2D NOESY NMR and 1D NOE NMR were used to identify

syn/anti- isomers of hydrazides 14aꢀ14b, and acrylonitriles

1

15aꢀ15b, respectively. The H NMR, 2D NOESY, and 1D

NOE NMR spectra of 14aꢀ14b, 15aꢀ15b are shown in

Supporting Information . From chemical structures, we can see

the distance between amino in syn- isomer (14a) hydrazine is far

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ARTICLE

Table 4. Antiproliferative Activity of Phenyl Amino Thiazole Compounds

IC₅₀( SEM (μM)

R

B16-F1

A375

DU 145

PC-3

LNCaP

PPC-1

45a

46b

45c

1

H

0.065 ( 0.012

ND^a

0.045 ( 0.008

ND

0.070 ( 0.004

0.035 ( 0.001

0.063 ( 0.001

0.071 ( 0.004

0.839 ( 0.719

0.057 ( 0.003

0.038 ( 0.002

0.043 ( 0.001

0.021 ( 0.001

0.786 ( 0.089

0.051 ( 0.001

0.035 ( 0.001

0.041 ( 0.001

0.028 ( 0.004

0.658 ( 0.117

0.054 ( 0.001

0.036 ( 0.001

0.037 ( 0.001

0.043 ( 0.005

0.701 ( 0.307

4-CH₃

4-F

ND

0.055 ( 0.005

2.127 ( 0.351

0.028 ( 0.005

1.111 ( 0.108

47

^aND = not determined.

Table 5. Pharmacokinetic Parameters for Compounds Tested in Vivo

1

39

46a

46c

route

IV

PO

IV

PO

IV

PO

IV

PO

N^a

4

3

4

3

dose (mg/kg)

CL^b(mL/min/kg)

Vss^c(L/kg)

AUC^d(min μg/mL)

C_max^e(ng/mL)

F ^f(%)

2.5

10

2.5

5

10

5

10

7.7 ( 1.0

4.9 ( 1.9

279 ( 53

3816 ( 509

22 ( 13

17 ( 3

1.4 ( 0.2

296 ( 46

4198 ( 438

13 ( 2

1.4 ( 0.2

381 ( 65

0.33 ( 0.25

139 ( 77

37 ( 20

0.4

65 ( 20

814 ( 255

160 ( 13

3

212 ( 65

3794 ( 1580

3.2 ( 1.6

3349 ( 686

1262 ( 362

3.3

0.2

11

21

^aNumbers of rats. ^bSystemic clearance. ^cVolume of distribution following intravenous dosing. ^dArea under the curve following intravenous dosing,

integrated drug concentration with respect to time and integrated drug concentration with respect to time following oral dosing. ^eMaximum plasma

concentration following intravenous dosing. ^fPercent oral bioavailability.

The carbonyl linker was replaced with double bonds (18, 20, 21),

amides (13e, 13f), oximes (13aꢀ13d), hydrazide (14a, 14b),

acrylonitriles (15a, 15b), cyanoimine (16), sulfonyl amide (26),

sulfur ether (23), and sulfonyl and sulfinyl compounds (24, 25).

A direct link compound 19 without any linker between “B” and

“C” rings was also prepared. Among these linker modifications,

only cyanoimine linkage (16) showed promising activity (20ꢀ60

nM) compared with carbonyl compound 1, but an in vitro

metabolism study showed that the half-life of 16 in human liver

microsome was less than 5 min (data not shown). This result

suggested that although we blocked the ketone reduction, it might

introduce another new metabolic liability in compound 16. We

separated the isomer pairs of compounds containing double

bonds, oximes, and hydrazides. Compound 20 was designed to

mimic the structure of CA-4, which contain a cis-CdC between

two aryl rings, unfortunately 20(Z) and other isomer 21(E) lost

activity after replacing the CdO linker. One interesting phenom-

enon is syn-isomer of 13a (0.1ꢀ0.3 μM) showed 10-fold more

activity than its anti isomer 13b (>10 μM). The half-life of 13a in

human liver microsomes is extended to 35 min, while the half-lives

of compounds 14aꢀb were prolonged to 55 min, however,

activities were much lower (∼1 μM) than compound 1.

Captex200/Tween80 (1/4, IP) to study in vivo SMART beha-

vior and obtained favorable results.²³But these surfactants are

biologically active and are responsible for many side effects.¹⁰In

addition, it was thought²⁴that low aqueous solubility of 1

resulted in low oral bioavailability (3.3%, in Table 5). In our

third series of compounds, we successfully increased aqueous

solubility without impacting the potency by introducing polar

groups like hydroxyl and indolyls. In addition, we also designed

ionizable groups like amino and alkylamino groups into “A” ring

para-position. As shown in Scheme 5 and Table 3, introducing

indolyl groups to the “A” ring, especially 5-indolyl (36, 7ꢀ

25 nM), increased the potency compared with the 4-OH

compound 29 (76ꢀ116 nM). Aminomethyl at the “A” ring para

position also maintained potency (39, 13ꢀ80 nM), but p-NHMe

(40) or p-NMe₂(42) abrogated activity. As shown in Figure 3,

analytical measurement to estimate aqueous solubility showed

that indolyl compound 36 increased solubility in PBS from

1.1 μg/mL (compound 1) to 3.8 μg/mL. Aminomethyl com-

pound 39 was converted to the HCl salt, which increased

solubility over 35-fold (>35 μg/mL). Although compound 39

showed satisfactory aqueous solubility, the pharmacokinetic

studies showed this compound still had very poor bioavailability

(F = 0.2%, Table 5).

Introducing Polar and Ionizable Groups into the SMART

Agents. One major limitation of the SMART agents was low

aqueous solubility. We used surfactant formulation strategies like

Modifications to Improve Oral Bioavailability. Many estab-

lished tubulin targeting anticancer drugs like taxanes and

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Figure 5. Structureꢀactivity relationship of novel anti-tubulin

compounds.

Figure 4. Compounds inhibit tubulin polymerization in vitro.

bioavailability. This data suggested that these new synthesized

amino linked compounds have appropriate potency and PK

proﬁles to be developed as a new class of orally bioavailable

anticancer agents.

Table 6. Antiproliferative Activity of Selected Compounds

Against P-gp Overexpressed MDR Cell Lines

Compounds Inhibit in Vitro Tubulin Polymerization. We

investigated the inhibition of tubulin polymerization of selected

potent compounds 9c, 16, 36, and 45a from all three design stra-

tegies (alternative B-rings, novel linkers, and solubilizing moieties)

and compared them with 1. Bovine brain tubulin (>97% pure) was

incubated with the individual compounds (5 μM) to test their effect

on tubulin polymerization (Figure 4). After 20 min incubation,

tubulin polymerization was inhibited 45% by 1, as compared to

vehicle. Compound 16 inhibited 33% of polymerization at 20 min

with different inhibition patterns. Compounds 9c and 45a provided

similar inhibitions of 56% and 58%, respectively. Compound 36,

whichshowedlowaverageIC₅₀of 10.1 nM, inhibited 69% of tubulin

polymerization. These data suggest that these compounds exhibit

strong antitubulin polymerization activity that corresponds well with

their antiproliferative potency.

Compounds Overcome P-Glycoprotein Mediated Multi-

drug Resistance. The P-glycoprotein (P-gp) system appears to

be a primary physiological mechanism of multidrug resistance

(MDR) which acts as an ATP-dependent drug efflux pump,

actively removing a variety of structurally diverse cytotoxic com-

pounds.^8,26Enhanced efflux of these compounds reduces their

intracellular accumulation and so reduces their cytotoxicity.

Therefore, novel compounds which are not susceptible to drug

resistance could be of high therapeutic and economic value. In

addition to P-gp, clinically used antitubulin agents have other

resistance mechanisms such as changes in microtubule dyna-

mics²⁷and mutations in β-tubulin which are known to limit

sensitivity to the taxanes.²⁸We tested our selected compounds

against an ovarian cancer cell line OVCAR-8 (parent) and P-gp

overexpressing NCI/ADR-RES cell line (Table 6). Notably,

tested compounds demonstrated equipotent antiproliferative

effects against OVCAR-8 and NCI/ADR-RES cell lines, suggest-

ing that they are not P-gp substrates and that they function in a

P-gp-independent manner. This feature is distinct from that of

paclitaxel, vinblastine, and colchicine in NCI/ADR-RES cells

which demonstrate 1333-, 149-, and 65-fold resistance.

IC₅₀(nM)

compd

9c

OVCAR-8

NCI/ADR-RES

resistance factor

33 ( 3

34 ( 2

10 ( 3

26 ( 2

46 ( 6

28

13 ( 0.8

14 ( 1

4 ( 2

0.4

0.6

0.8

0.6

0.4

1333

149

65

16

36

39

11 ( 2

27

45a

45b

21

45c

44 ( 3

35 ( 2

4.7 ( 0.1

3.9 ( 0.1

17 ( 1

25 ( 6

13 ( 1

6263 ( 634

582 ( 57

1113 ( 79

1

paclitaxel²³

vinblastine

colchicine

vinblastine require intravenous administration because of low

oral bioavailability. Oral bioavailability is a complex parameter

involving many chemical and physiological processes, such as

solubility, permeability, and metabolic stability. Our initial thought

was that the low bioavailability of 1 might be caused by poor

solubility. However, the water-soluble compound 39 failed to

improve oral bioavailability. We further improved the solubility of

these tubulin inhibitors by inserting an amino linker between the

“A” and “B” rings (phenyl amino thiazole, PAT) as in 45aꢀc

(Scheme 6), whichis similarto the reported orallyactive colchicine

binding anticancer agent 47 (Figure 1, clinical phase II trial).^3,25

IC₅₀values (Table 4) demonstrate that these compounds (45a,

46b, and 45c) had similar potency (35ꢀ65 nM) as 1 with

increased solubility (15 and 19 μg/mL for 45a and 45c, respec-

tively (Figure 3), and they are over 20-fold more active than 47.

Rat pharmacokinetic studies were performed to study whether

these new compounds exhibited improved bioavailability com-

pared to compound 1 (Table 5). The data clearly showed that

46c (HCl salt of 45c) exhibited more than 4.3-fold increased

exposure (AUC, 160 vs 37 min μg/mL) by the oral route as

3

’ CONCLUSION

compared to 1, suggesting that improved aqueous solubility by

the amino linker successfully improved oral bioavailability. In

addition, the maximal concentration (C_max) of 46a and 46c by

oral administration were 814 and 1262 ng/mL, respectively,

while C_maxof 1 was only 212 ng/mL. Overall, the bioavailability

of 46a and 46c were increased from 3.3% of 1 to 11% and 21%,

respectively (Table 5). Compound 46c exhibited moderate

clearance, moderate volume of distribution, and acceptable oral

A new series of tubulin polymerization inhibitors with accep-

table oral bioavailability and equipotent activity in multidrug

resistant tumor cell lines has been discovered. Medicinal chem-

istry eﬀorts improved upon SMART compound 1. Chemical

modiﬁcations were include alternative “B” ring and alternative

linkages between “B” and “C” rings with regard to in vitro

cytotoxicity against cancer cells (Figure 5) based on biological

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Journal of Medicinal Chemistry

ARTICLE

evaluation against cancer cells in vitro. SAR studies revealed that

optimal “B” rings include pyridine (9c), thiophene (9e), and

furan (9d), which maintain excellent in vitro potency. Replacing

carbonyl linker with cyanoimine (16) between “B” and “C” ring

also increased the activity. Structure modiﬁcations to increase

aqueous solubility and bioavailability were performed. Introdu-

cing an amino between “A” and “B” rings gave us PAT com-

pounds 45aꢀc, which showed similar in vitro antiproliferative

potency against tested cancer cells as well as resistant cancer cell

lines, furthermore, the solubility and in vivo bioavailability were

improved greatly over those of 1. Therefore, these new com-

pounds represent a new family of antimitotic agents that may be

very useful in the treatment of cancer.

9a as a white solid (43.8%); mp 87ꢀ89 °C. ¹H NMR (CDCl₃) δ 8.02 (t, 1

H), 7.84ꢀ7.74 (m, 2 H), 7.64ꢀ7.38 (m, 6 H), 7.11 (s, 2 H), 3.95 (s, 3 H),

3.88 (s, 6 H). MS (ESI) m/z371.1 (M þ Na)^þ. Anal. (C₂₂H₂₀O₄) C, H, N.

(6-Phenylpyrimidin-4-yl)(3,4,5-trimethoxyphenyl)methanone (9b).

To a solution of 8b (0.243 g, 1 mmoL) in 5 mL of THF was added a THF

solution of 3,4,5-trimethoxyphenylmagnesiumbromide (0.5 N, 5.6 mL,

1.4 mmol) at 0 °C. The mixture was allowed to stir for 30 min and

quenched with satd NH₄Cl, extracted with ethyl ether, and dried with

MgSO₄. The solvent was removed under reduced pressure to yield a

crude product, which was purified by column chromatography to obtain

1

pure compound 9b (52.3%); mp 132ꢀ133 °C. H NMR (CDCl₃) δ

9.40 (d, 1 H, J = 1.5 Hz), 8.29 (d, 1 H, J = 1.5 Hz), 8.22ꢀ8.18, 7.57ꢀ7.54

(m, 5 H), 7.46 (s, 2 H), 3.96 (s, 3 H), 3.91 (s, 6 H). MS (ESI) m/z 351.1

(M þ H)^þ. Anal. (C₂₀H₁₈N₂O₄) C, H, N.

(6-Phenylpyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (9c).

To a solution of 8c (0.210 g, 0.86 mmoL) in 5 mL of THF was added

a THF solution of 3,4,5-trimethoxyphenylmagnesiumbromide (0.5 N,

3.5 mL, 1.73 mmol) at 0 °C. The mixture was allowed to stir for 30 min

and quenched with water, extracted with ethyl acetate, and dried with

MgSO₄. The solvent was removed under reduced pressure to yield a

crude product, which was purified by column chromatography to obtain

pure compound 9c as white needle crystals (78%); mp 116ꢀ117 °C. ¹H

NMR (CDCl₃) δ 8.10 (d, br, 2 H), 8.02ꢀ8.00 (m, 1 H), 7.97ꢀ7.96 (m,

2 H), 7.66 (s, 2 H), 7.49ꢀ7.43 (m, 3 H), 3.97 (s, 3 H), 3.89 (s, 6 H). MS

(ESI) m/z 372.6 (M þ Na)^þ. Anal. (C₂₁H₁₉NO₄) C, H, N.

’ EXPERIMENTAL SECTION

General. All reagents were purchased from Sigma-Aldrich Chemical

Co., Fisher Scientific (Pittsburgh, PA), AK Scientific (Mountain View,

CA), Oakwood Products (West Columbia, SC), etc. and were used

without further purification. Moisture-sensitive reactions were carried

under an argon atmosphere. 47 was prepared according methods

reported by Yoshino et al.²⁹Routine thin layer chromatography

(TLC) was performed on aluminum backed Uniplates (Analtech,

Newark, DE). Melting points were measured with Fisher-Johns melting

point apparatus (uncorrected). NMR spectra were obtained on a Bruker

AX 300 (Billerica, MA) spectrometer or Varian Inova-500 (Vernon

Hills, Illinois) spectrometer. Chemical shifts are reported as parts per

million (ppm) relative to TMS in CDCl₃. Mass spectral data was

collected on a Bruker ESQUIRE electrospray/ion trap instrument in

positive and negative ion modes. Elemental analyses were performed by

Atlantic Microlab Inc. (Norcross, GA). Unless specified, all the tested

compounds described in the article present >95% purity established

through combustion analysis.

(2R)-(2-Phenyl-4,5-dihydro-oxazol-4-yl)-(3,4,5-trimethoxy-phenyl)-

methanone (5). To a solution of n-BuLi (1.6 M, 0.713 mL) in 8 mL of

THF was added a solution of 3,4,5-trimethoxybromobenzene (1.09

mmol) in 3 mL of THF under ꢀ78 °C. The mixture was allowed to stir

for 2 h, and a solution of Weinreb amide 4 (1.14 mmol) in 3 mL of THF

was charged. The temperature was allowed to increase at RT and stirred

overnight. The reaction mixture was quenched with satd NH₄Cl,

extracted with ethyl ether, and dried with MgSO₄. The solvent was

removed under reduced pressure to yield a crude product, which was

purified by column chromatography to obtain pure compound 5 as a

white solid (47.9%); mp 60ꢀ62 °C. ¹H NMR (CDCl₃) δ 7.97 ꢀ7.94

(m, 2 H), 7.62 (s, 2 H), 7.54ꢀ7.37 (m, 3 H), 5.61 (q, 1 H, J = 7.5 Hz, 9.9

Hz), 5.12 (t, 1 H, J = 7.5 Hz), 4.57 (q, 1 H, J = 7.8 Hz, 9.9 Hz), 3.96 (s, 6

H), 3.95 (s, 3 H). MS (ESI) m/z 364.1(M þ Na)^þ, 340.1 (M ꢀ H)^ꢀ.

Anal. (C₁₉H₁₉NO₄S) C, H, N.

(2R)-(2-Phenyl-oxazol-4-yl)-(3,4,5-trimethoxy-phenyl)-methanone

(6). A mixture of 5 (1.48 mmol), CBrCl₃(2.59 mmol), and DBU (2.97

mmol) in CH₂Cl₂(20 mL) was stirred overnight. The reaction mixture

was absorbed on silica gel and purified by column chromatography to

yield pure compound 6 as desired (61.6%); mp 138ꢀ139 °C. ¹H NMR

(CDCl₃) δ 8.37 (s, 1 H), 8.14ꢀ8.12 (m, 2 H), 7.74 (s, 2 H), 7.52ꢀ7.49

(m, 3 H), 3.97 (s, 9 H). MS (ESI) m/z 362.1 (M þ Na)^þ. Anal.

(C₁₉H₁₇NO₅) C, H, N.

Biphenyl-3-yl(3,4,5-trimethoxyphenyl)methanone (9a). To a solu-

tion of 8a (0.174 g, 0.72 mmoL) in 5 mL of THF was added a THF

solution of 3,4,5-trimethoxyphenylmagnesiumbromide (0.5 N, 1.08

mmol) at 0 °C. The mixture was allowed to stir for 30 min and quenched

with satd NH₄Cl, extracted with ethyl ether, and dried with MgSO₄. The

solvent was removed under reduced pressure to yield a crude product,

which was purified by column chromatography to obtain pure compound

(5-Phenylfuran-2-yl)(3,4,5-trimethoxyphenyl)methanone (9d). To

a solution of 8d (0.231 g, 1 mmoL) in 5 mL of THF was added a THF

solution of 3,4,5-trimethoxyphenylmagnesiumbromide (0.5 N, 4.0 mL,

2 mmol) at 0 °C. The mixture was allowed to stir for 30 min and

quenched with water, extracted with ethyl acetate, and dried with

MgSO₄. The solvent was removed under reduced pressure to yield a

crude product, which was purified by column chromatography to obtain

1

pure compound 9d as white crystals (35.5%); mp 114ꢀ116 °C. H

NMR (CDCl₃) δ 7.85ꢀ7.82 (m, 1 H), 7.48ꢀ7.36 (m, 4 H), 7.35 (s, 2

H), 7.25 (d, 1 H, J = 4.0 Hz), 6.86 (d, 1 H, J = 4.2 Hz), 3.96 (s, 3 H), 3.95

(s, 6 H). MS (ESI) m/z 339.1 (M þ H)^þ. Anal. (C₂₀H₁₈O₅) C, H.

(5-Phenylthiophen-3-yl)(3,4,5-trimethoxyphenyl)methanone (9e).

To a solution of 10e (0.260 g, 0.73 mmoL) in 20 mL of anhydrous

CH₂Cl₂was added DessꢀMartin reagent (0.465 g, 1.36 mmol). The

mixture was allowed to stir for 30 min and quenched with satd Na₂S₂O₃

solution, extracted with ethyl acetate, and dried with MgSO₄. The

solvent was removed under reduced pressure to yield a crude product,

which was purified by column chromatography to give pure compound

9e as light-yellow crystals (81.0%); mp 140ꢀ141 °C. ¹H NMR (CDCl₃)

δ 7.97 (d, 1 H, J = 1.5 Hz), 7.82 (d, 1 H, J = 1.5 Hz), 7.59ꢀ7.57 (m, 2 H),

7.45ꢀ7.34 (m, 3 H), 7.19 (s, 2 H), 3.95 (s, 3 H), 3.93 (s, 6 H). MS (ESI)

m/z 355.1 (M þ H)^þ. Anal. (C₂₀H₁₈O₄S) C, H.

(3-Phenyl-1H-pyrazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (9f).

Compound 9f was prepared using the same method as used of compound

9c from 3-phenyl-1H-pyrazole-5-carboxylic acid 7f via 8f as intermidiate.

¹H NMR (500M, CDCl₃) δ 10.97 (br, 1 H), 7.77 (s, br, 2 H), 7.48ꢀ7.38

(m, 5 H), 7.14 (s, br, 1 H), 3.96 (s, 3 H), 3.94 (s, 6 H). MS (ESI) m/z

361.1 (M þ Na)^þ, 337.0 (M ꢀ H)^ꢀ. Anal. (C₁₉H₁₈N₂O₄) C, H, N.

(2-Phenylthiazol-5-yl)(3,4,5-trimethoxyphenyl)methanone (9g).

To a solution of 10g (0.357 g, 1 mmoL) in 40 mL of anhydrous

CH₂Cl₂was added DessꢀMartin reagent (0.848 g, 2 mmol). The

mixture was allowed to stir for 30 min and quenched with satd

Na₂S₂O₃solution, extracted with ethyl acetate, and dried with MgSO₄.

The solvent was removed under reduced pressure to yield a crude

product, which was purified by column chromatography to give pure

compound 9 g (80.1%); mp 147ꢀ148 °C. ¹H NMR (CDCl₃) δ 8.33

(s, 1 H), 8.04 (m, 2 H), 7.51 (m, 3 H), 7.18 (s, 2 H), 3.96 (s, 3 H),

3.93 (s, 6 H). MS (ESI) m/z 378.1 (M þ H)^þ. Anal. (C₁₉H₁₇NO₄S)

C, H, N.

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(5-Phenylisoxazol-3-yl)(3,4,5-trimethoxyphenyl)methanone (9h).

To a solution of 10h (0.110 g, 0.73 mmoL) in 8 mL of anhydrous

CH₂Cl₂was added DessꢀMartin reagent (0.274 g, 0.645 mmol). The

mixture was allowed to stir for 30 min and quenched with satd Na₂S₂O₃

solution, extracted with ethyl acetate, and dried with MgSO₄. The

solvent was removed under reduced pressure to yield a crude product,

which was purified by column chromatography to give pure compound

9h (70.1%); mp 143ꢀ144 °C. ¹H NMR (CDCl₃) δ 7.87ꢀ7.85 (m, 2

H), 7.72 (s, 2 H), 7.53ꢀ7.49 (m, 3 H), 7.05 (s, 1 H), 7.82 (d, 1 H, J = 1.5

Hz), 3.97 (s, 3 H), 3.96 (s, 6 H). MS (ESI) m/z 362.1 (M þ H)^þ. Anal.

(C₁₉H₁₇NO₅) C, H, N.

(4-Phenylpiperidin-1-yl)(3,4,5-trimethoxyphenyl)methanone (9i).

To a mixture of 4-phenylpiperidine 11 (5 mmol), EDCI (6 mmol),

HOBt (5.5 mmol), and NMM (6 mmol) in CH₂Cl₂(50 mL) was added

3,4,5-trimethoxybenzoic acid (5.3 mmol) and stirring continued at RT

for overnight. The reaction mixture was diluted with CH₂Cl₂(100 mL)

and sequentially washed with water, satd NaHCO₃, and brine and dried

over MgSO₄. The solvent was removed under reduced pressure to yield

a crude product, which was purified by column chromatography to

obtain pure compound 9i. (57.9%); mp 141ꢀ142 °C. ¹H NMR

(CDCl₃) δ 7.35ꢀ7.21(m, 5 H), 6.66 (s, 2 H), 4.84 (br, 1 H), 3.95

(br, 1 H), 3.88 (s, 6 H), 3.86 (s, 3 H), 3.20ꢀ2.87 (br, 2 H), 2.85ꢀ2.74

(tt, 1 H, J = 3.6 Hz, J = 15.6 Hz) 1.92 (br, 2 H), 1.70 (br, 2 H). MS (ESI)

m/z 378.1 (M þ Na)^þ. Anal. (C₂₁H₂₅NO₄) C, H, N.

(15 mg); mp 157ꢀ158 °C. ¹H NMR (300 MHz, CDCl₃) δ 9.22 (s, 1H),

8.19 (s, 1 H), 8.02ꢀ7.99 (m, 2 H), 7.52ꢀ7.50 (m, 3 H), 7.07 (s, 2 H),

3.92 (s, 6 H), 3.85 (s, 3 H). MS (ESI) m/z 371.1 (M þ H)^þ. Anal.

(C₁₉H₁₈N₂O₄S) C, H, N.

3,4,5-Trimethoxy-N-(2-phenylthiazol-4-yl)benzamide (13f). To a

solution of 13b (26 mg, 0.07 mmol) in 5 mL of CH₂Cl₂was added

p-toluenesulfonyl chloride (27 mg, 0.14 mmol) and NaH (5 mg, 60% in

light mineral oil). Then the reaction mixture was stirred for 20 min. After

completion of the reaction, the residue was absorbed on silica gel and

purified by Al₂O₃column chromatography to give compound 13f (15

1

mg); mp 154ꢀ156 °C. H NMR (300 MHz, CDCl₃) δ 8.88 (s, 1H),

7.94ꢀ7.91 (m, 2 H), 7.83 (s, 1 H), 7.48ꢀ7.46 (m, 3 H), 7.18 (s, 2 H),

3.97 (s, 6 H), 3.94 (s, 3 H). MS (ESI) m/z 393.1 (M þ Na)^þ. Anal.

(C₁₉H₁₈N₂O₄S) C, H, N.

(Z)-4-(Hydrazono(3,4,5-trimethoxyphenyl)methyl)-2-phenylthiazole

(14a). To a mixture of 1 (230 mg, 0.65 mmol) in 3 mL of CH₂Cl₂and

3 mL of ethanol was added hydrazine hydrate (2 mL). Then the mixture

was refluxed for overnight. After completion of the reaction, the residue

was absorbed on silica gel and purified by column chromatography to

give compounds 14a (80 mg) and 14b (56 mg); mp 117ꢀ119 °C. ¹H

NMR (300 MHz, CDCl₃) δ 8.01ꢀ7.98 (m, 2 H), 7.49ꢀ7.46 (m, 5 H),

7.33 (s, 1 H), 6.82 (s, 2 H), 3.87 (s, 3 H), 3.85 (s, 6 H). MS (ESI) m/z

370.1 (M þ H)^þ. Anal. (C₁₉H₁₉N₃O₃S) C, H, N.

(E)-4-(Hydrazono(3,4,5-trimethoxyphenyl)methyl)-2-phenylthiazole

1

(14b). Melting point: 65ꢀ66 °C. H NMR (300 MHz, CDCl₃) δ

(Z)-(2-Phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone Oxime

(13a). To a suspension of 1 (210 mg, 0.59 mmol) in 10 mL of ethanol

was added an aqueous solution (2 mL) of hydroxylamine hydrochloride

(127 mg, 1.83 mmol). Then 2 mL of 1N NaOH was added dropwise to

the reaction mixture and the mixture was stirred at 55 °C for 3 h. After

completion of the reaction, the residue was absorbed on silica gel and

purified by column chromatography to give compounds 13a (85 mg)

and 13b (50 mg); mp 150ꢀ152 °C. ¹H NMR (300 MHz, DMSO-d₆) δ

11.95 (s, 1 H), 8.35 (s, 1 H), 7.91ꢀ7.89 (m, 2 H), 7.50ꢀ7.44 (br, 3 H), 6.85

(s, 2 H), 3.73 (s, 6 H), 3.70 (s, 3 H). MS (ESI) m/z 393.1 (M þ Na)^þ;

368.9 (M ꢀ H)^ꢀ. Anal. (C₁₉H₁₈N₂O₄S) C, H, N.

8.04ꢀ8.01 (m, 2 H), 7.44ꢀ7.40 (m, 3 H), 6.95 (s, 1 H), 6.65 (s, 2 H),

5.62 (s, 2 H), 3.93 (s, 3 H), 3.87 (s, 6 H). MS (ESI) m/z 370.1 (M þ

H)^þ. Anal. (C₁₉H₁₉N₃O₃S) C, H, N.

(Z)-3-(2-Phenylthiazol-4-yl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile

(15a). To a solution of 0.4 mL of 2.5 N n-BuLi in hexane and 10 mL of

THF was added dropwise a solution of 177 mg (1 mmol) of diethyl

cyanomethylphosphonate in 5 mL of THF at 0 °C under Ar₂. The ice

bath was removed, and the mixture was stirred at 25 °C for 40 min. A

solution of 200 mg (0.56 mmol) of 1 in 10 mL of THF was added

dropwise at 0 °C, and the mixture was stirred for 1 h at RT. The reaction

mixture was treated with saturated NH₄C1 solution. After a conven-

tional workup, column chromatography (silica gel, petroleum ether/

ethyl acetate) gave compounds 15a (83 mg) and 15b (76 mg);

(E)-(2-Phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone oxime

1

(13b). Melting point: 176ꢀ177 °C. H NMR (300 MHz, DMSO-

d₆) δ 11.49 (s, 1 H), 7.92ꢀ7.89 (m, 2 H), 7.64 (s, 1 H), 7.51ꢀ7.49

(m, 3 H), 7.34 (s, 1 H), 6.75 (s, 2 H), 3.75 (s, 6 H), 3.72 (s, 3 H).

MS (ESI) m/z 393.1 (M þ Na)^þ; 368.9 (M ꢀ H)^ꢀ. Anal. (C₁₉H₁₈-

N₂O₄S) C, H, N.

1

mp:192ꢀ193 °C. H NMR (300 MHz, CDCl₃) δ 8.01ꢀ7.99 (m, 2

H), 7.44ꢀ7.40 (m, 3 H), 7.21 (s, 1 H), 6.74 (s, 2 H), 6.67 (s, 1 H), 3.93

(s, 3 H), 3.89 (s, 6 H). MS (ESI) m/z 401.1 (M þ Na)^þ. Anal.

(C₂₁H₁₈N₂O₃S) C, H, N.

(Z)-(2-Phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

O-Methyl Oxime (13c). To a suspension of 1 (110 mg, 0.59 mmol) in

10 mL of pyridine was added O-methylhydroxylamine hydrochloride

(52 mg, 0.63 mmol) and the mixture was stirred at 60 °C for overnight.

The reaction was quenched with 1 N HCl solution, extracted with ethyl

acetate, and dried with MgSO₄. The solvent was removed under reduced

pressure to yield a crude product, which was purified by column

chromatography to give pure compounds 13c (41 mg) and 13d (33

mg); mp 116ꢀ117 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.13 (s, 1 H),

7.96ꢀ7.94 (m, 2 H), 7.45ꢀ7.44 (m, 3 H), 6.94 (s, 2 H), 4.13 (s, 3 H),

3.91 (s, 6 H), 3.88 (s, 3 H). MS (ESI) m/z 407.2 (M þ Na)^þ. Anal.

(C₂₀H₂₀N₂O₄) C, H, N.

(E)-3-(2-Phenylthiazol-4-yl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile

1

(15b). Melting point: 111ꢀ114 °C. H NMR (300 MHz, CDCl₃) δ

8.07ꢀ8.05 (m, 2 H), 7.49ꢀ7.46 (m, 4 H), 6.66 (s, 2 H), 5.64 (s, 1 H),

3.91 (s, 3 H), 3.86 (s, 6 H). MS (ESI) m/z 401.1 (M þ Na)^þ. Anal.

(C₂₁H₁₈N₂O₃S) C, H, N.

N-((2-Phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methylene)cyanamide

(16). First, 100 mg of 1 (0.28 mmol, 1 equiv) was dissolved in 10 mL of

methylene chloride. Then titanium tetrachloride in methylene chloride

(1.0 N, 0.7 mL, 2.5 equiv) was added dropwise at 0 °C and stirred for

30 min. Bis-trimethylsilylcarbodiimide (2.4 equiv) in 2 mL of methylene

chloride was added and the reaction stirred overnight protected from air

and moisture. The reaction was treated with iceꢀwater mixture followed

by extraction with methylene chloride. The organic phase was dried over

magnesium sulfate, filtered through Celite, and concentrated to give the

crude acetophenone cyanoimines, which were purified by flash column

as isomers (35 mg) with a ratio of 3:7. ¹H NMR (300 MHz, CDCl₃) δ

8.72 (br, 0.3 H), 8.63 (s, 0.7 H), 8.09ꢀ8.07 (m, 1.4 H), 7.99 (br, 0.6 H),

7.58ꢀ7.56 (br, 3 H), 7.26 (s, 1.4 H), 7.18 (s, 0.6 H), 3.84, 3.83 (s, s, 6 H),

3.82 (s, 3 H). MS (ESI) m/z 402.1 (M þ Na)^þ. Anal. (C₂₀H₁₇N₃O₃S)

C, H, N.

(E)-(2-Phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

O-Methyl Oxime (13d). Melting point: 91ꢀ92 °C. ¹H NMR (500 MHz,

CDCl₃) δ 8.00ꢀ7.98 (m, 2 H), 7.44ꢀ7.43 (m, 3 H), 7.28 (s, 1 H), 6.70

(s, 2 H), 4.08 (s, 3 H), 3.91 (s, 6 H), 3.85 (s, 3 H). MS (ESI) m/z 407.0

(M þ Na)^þ. Anal. (C₂₀H₂₀N₂O₄) C, H, N.

2-Phenyl-N-(3,4,5-trimethoxyphenyl)thiazole-4-carboxamide (13e).

To a solution of 13a (21 mg, 0.06 mmol) in 5 mL of CH₂Cl₂was added

p-toluenesulfonyl chloride (23 mg, 0.12 mmol) and NaH (5 mg, 60% i

n light mineral oil). Then the reaction mixture was stirred for 20 min.

After completion of the reaction, the residue was absorbed on silica gel

and purified by Al₂O₃column chromatography to give compound 13e

(2-Phenylthiazol-4-yl)(3,4,5-trimethoxyphenyl)methanol (17). At

0 °C, to a solution of 104 mg of 12b (0.55 mmol, 1 equiv) in 6 mL of

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THF was added 3,4,5-trimethoxyphenylmagnesium bromide (0.5 N in

THF, 2.9 mL). The mixtures were stirred for 30 min until aldehyde

disappeared on TLC plates. The reaction mixture was quenched with

satd NH₄Cl, extracted with ethyl ether, and dried with MgSO₄. The

solvent was removed under reduced pressure to yield a crude product,

which was purified by column chromatography to obtain pure com-

pound 17; mp 49ꢀ51 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.95ꢀ7.92

(m, 2 H), 7.44ꢀ7.43 (m, 4 H), 6.97 (s, 1 H), 6.76 (s, 2 H), 5.93 (d, 1 H,

J = 3.6 Hz), 3.86 (s, 9 H). MS (ESI) m/z 402.1 (M þ Na)^þ. Anal.

(C₁₉H₁₉NO₄S) C, H, N.

4-(2-Methyl-1-(3,4,5-trimethoxyphenyl)prop-1-enyl)-2-phenylthia-

zole (18). At ꢀ78 °C, to a solution of 223 mg of isopropyl triphenylpho-

sphonium iodide (0.52 mmol) in 5 mL of THF was added dropwise

0.4 mL of 1.6 N n-BuLi in hexane under Ar₂protection. And the mixture

was stirred at 0 °C for 40 min. A solution of 140 mg (0.39 mmol) of 1 in

5 mL of THF was added dropwise at 0 °C. The mixture was stirred for

1 h at RT. The reaction mixture was treated with saturated NH₄C1

solution. After a conventional workup, column chromatography (silica

gel, petroleum ether/ethyl acetate) gave compound 18 (86 mg, 57.3%).

¹H NMR (300 MHz, CDCl₃) δ 7.98ꢀ7.97 (m, 2 H), 7.45ꢀ7.40

(m, 3 H), 6.77 (s, 1 H), 6.48 (s, 2 H), 3.86 (s, 3 H), 3.82 (s, 6 H),

2.15 (s, 3 H), 1.81 (s, 3 H). MS (ESI) m/z 404.1 (M þ Na)^þ. Anal.

(C₂₂H₂₃NO₃S) C, H, N.

2-Phenyl-4-(3,4,5-trimethoxyphenyl)thiazole (19). Bromine (160

mg, 1 mmol) was added dropwise to a stirred solution of 1-(3,4,5-

trimethoxyphenyl)ethanone (210 mg, 1 mmol) in ethanol (30 mL), and

the solution was stirred at 0 °C for 1 h and then poured into water to

form a precipitate. This was recrystallized from ethanol to give bromoa-

cetophenone (70%) and used directly for the next step. A mixture of

bromoacetophenone (288 mg, 1 mmol) and benzothioamide (137 mg, 1

mmol) in ethanol was refluxed for 1 h. The reaction mixture was

concentrated in vacuo and purified with a flash column to give 19 (167

mg, 51.1%); mp: 95ꢀ96 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.05ꢀ8.03

(m, 2 H), 7.48ꢀ7.44 (m, 3 H), 7.41 (s, 1 H), 7.22 (s, 2 H), 3.97 (s, 6 H),

3.89 (s, 3 H). MS (ESI) m/z 350.1 (M þ Na)^þ. Anal. (C₁₈H₁₇NO₃S)

C, H, N.

slowly and stirred for 15 min. The cold diazonium solution was added

slowly to a solution of 1.3 equiv of potassium ethyl xanthate (1.16 g, 1.3

mmol) in water (1.3 mL) at 45 °C. The reaction mixture was stirred for

an additional 30 min at 45 °C and then cooled to RT. The reaction

mixture was extracted with diethyl ether (3 ꢁ 50 mL). The combined

organic extracts were washed with 1 N NaOH solution (100 mL), water

(3 ꢁ 50 mL), brine (50 mL), dried over MgSO₄, filtered, and evaporated

under reduced pressure. The resulting crude xanthate 22 was used

directly in the next step without further purification. MS (ESI) m/z

275.0 (M þ H)^þ.

Biphenyl-3-yl(3,4,5-trimethoxyphenyl)sulfane (23). To a solution of

22 (1.1 g, crude compound) in ethanol (8 mL) was added potassium

hydroxide (2.1 g, 12 mL) and heated to reflux for overnight. The

solution was cooled to RT, and the ethanol was evaporated under

reduced pressure. The residue was dissolved in water and washed with

diethyl ether (10 mL). The aqueous layer was acidified with 2 N HCl and

extracted with diethyl ether (3 ꢁ 50 mL). The organic extracts were

washed with water (50 mL), brine (50 mL), dried over MgSO₄, filtered,

and evaporated under reduced pressure to afford 0.85 g (77.3%) of crude

biphenyl-3-thiol product (overall, 3 steps). Into a round-bottomed flask,

stirred magnetically, were placed 0.1 g (1.04 mmol) of sodium tert-

butoxide and 83 mg of copper iodide (0.43 mmol). After the reaction

vessel was sealed, 0.13 g (0.71 mmol) of 4-methoxybenzenethiol and

0.19 g (0.65 mmol) of 5-iodo-1,2,3-trimethoxybenzene in 3.0 mL of

toluene were injected through the septum. The reaction mixture was

heated for overnight at 110 °C. Purification was performed by flash

1

chromatography and colorless oil 23 was obtained (40% yield). H

NMR (500 MHz, CDCl₃) δ 7.54ꢀ7.52 (m, 3 H), 7.44ꢀ7.41 (m, 3 H),

7.37ꢀ7.33 (m, 2 H), 7.23 (s, br, 1 H), 6.69 (s, 2 H), 3.86 (s, 3 H), 3.80 (s,

6 H). MS (ESI) m/z 353.2 (M þ H)^þ. Anal. (C₂₁H₂₀O₃S) C, H.

3-(3,4,5-Trimethoxyphenylsulfonyl)biphenyl (24). To a solution of

60 mg (0.17 mmol) of compound 23 and 5 mL of dichloromethane was

added very slowly 2 equiv of m-CPBA over 3 h. Sulfoxide formation was

monitored by thin-layer chromatography. Purification was performed

with a flash chromatographic column, and an amorphous powder of 24

was obtained (73% yield); mp 99ꢀ101 °C. ¹H NMR (500 MHz,

CDCl₃) δ 8.14 (br, 1 H), 7.89 (d, 1 H), 7.78 (d, 1 H), 7.59ꢀ7.56 (m,

3 H), 7.49ꢀ7.39 (m, 3 H), 7.19 (s, 2 H), 3.89 (s, 6 H), 3.87 (s, 3 H). MS

(ESI) m/z 385.0 (M þ Na)^þ. Anal. (C₂₁H₂₀O₅S) C, H.

(Z)-2-Phenyl-4-(3,4,5-trimethoxystyryl)thiazole (20). Triphenylpho-

sphine (3.41 g, 13 mmol) was added to a solution of 5-(bromomethyl)-

1,2,3-trimethoxybenzene (2.61 g, 10 mmol) in dry THF (30 mL). The

mixture was refluxed with stirring for 6 h. The resulting white solid was

filtered and washed with ether/hexane to afford the product 3,4,5-

trimethoxybenzyltriphenylphosphonium bromide in 96.4% yield. ¹H

NMR (500 MHz, CDCl₃) δ 7.77ꢀ7.73, 7.65ꢀ7.61 (m, 15 H), 6.44 (d, 2

H, J = 1.5 Hz), 5.37 (d, 2 H, J = 14 Hz), 3.76 (s, 3 H), 3.51 (d, 6 H). MS

(ESI) m/z 443.1 (M ꢀ Br]^þ. At ꢀ78 °C, n-BuLi (0.42 mL, 2.5 N in

hexane) was added to a solution of 3,4,5-trimethoxybenzyltriphenyl-

phosphonium bromide (500 mg, 0.96 mmol) in 10 mL of THF. After

stirring at RT for 2 h, aldehyde 12b (109 mg, 0.58 mmol) in 3 mL of

THF was charged and stirred for 30 min. The reaction mixture was

treated with saturated NH₄C1 solution. After a conventional workup,

column chromatography (silica gel, petroleum ether/ethyl acetate) gave

compounds 20(Z) (57 mg) and 21(E) (99 mg). ¹H NMR (500 MHz,

CDCl₃) δ 7.90ꢀ7.89 (m, 2 H), 7.42ꢀ7.40 (m, 3 H), 7.07 (s, 1 H), 6.71

(s, 2 H), 6.66 (s, 1 H), 3.87 (s, 6 H), 3.75 (s, 3 H). MS (ESI) m/z 376.1

(M þ Na)^þ. Anal. (C₂₀H₁₉NO₃S) C, H, N.

3-(3,4,5-Trimethoxyphenylsulfinyl)biphenyl (25). At 0 °C, to a solu-

tion of 500 mg (1.42 mmol) of compound 23 and 5 mL of dichlor-

omethane was added very slowly 1 equiv of m-CPBA over 3 h. Sulfoxide

formation was monitored by thin-layer chromatography. Purification was

performed with a flash chromatographic column, and an amorphous

1

powder of 25 was obtained (87% yield); mp 108ꢀ109 °C. H NMR

(500 MHz, CDCl₃) δ 7.92 (br, 1 H), 7.71 (d, 2 H), 7.62ꢀ7.60 (m, 3 H),

7.58ꢀ7.40 (m, 4 H), 6.94 (s, 2 H), 3.79 (s, 3 H), 3.74 (s, 6 H). MS (ESI)

m/z 369.1 (M þ H)^þ. Anal. (C₂₁H₂₀O₄S) C, H.

N-(3,4,5-Trimethoxyphenyl)biphenyl-3-sulfinamide (26). A mixture

of 65 mg of biphenyl-3-sulfonyl chloride (0.25 mmol), 44 mg of 3,4,5-

trimethoxyaniline (0.24 mmol), and 0.3 mmol of triethylamine in 5 mL

of DMF was stirred overnight. The reaction mixture was treated with

water and extracted with ethyl acetate. After a conventional workup,

column chromatography (silica gel, petroleum ether/ethyl acetate) gave

88 mg of 26 (91.7%); mp 48ꢀ50 °C. ¹H NMR (500 MHz, CDCl₃) δ

7.96 (t, 1 H, J = 1.8 Hz), 7.81ꢀ7.74 (m, 2 H), 7.57ꢀ7.40 (m, 6 H), 6.33

(s, 2 H), 3.86 (s, 3 H), 3.80 (s, 6 H). MS (ESI) m/z 422.1 (M þ Na)^þ.

Anal. (C₂₁H₂₁NO₅S) C, H, N.

(E)-2-Phenyl-4-(3,4,5-trimethoxystyryl)thiazole (21). ¹H NMR (500

MHz, CDCl₃) δ 8.03ꢀ8.01 (m, 2 H), 7.52 (d, 1 H, J = 16 Hz),

7.47ꢀ7.44 (m, 3 H), 7.16 (s, 1 H), 7.05 (d, 1 H, J = 16 Hz), 6.79 (s, 2 H),

3.92 (s, 6 H), 3.88 (s, 3 H). MS (ESI) m/z 354.1 (M þ H)^þ. Anal.

(C₂₀H₁₉NO₃S) C, H, N.

S-Biphenyl-3-yl-O-Ethyl-Carbonodithioate (22). To a solution of 1

equiv of biphenyl-3-amine (1 g, 5.92 mmol) in water (7.3 mL) at 0 °C

was added concentrated hydrochloric acid (1 mL). A cold solution of 1.1

equiv of sodium nitrite (450 mg, 6.5 mmol) in water (3 mL) was added

(2-(4-Hydroxyphenyl)thiazol-4-yl)(3,4,5-trimethoxyphenyl)metha-

none (29). At 0 °C, to a solution of 28 (0.2 mmol) in 5 mL of CH₂Cl₂

was added a solution of tetrabutylammonium fluoride in THF (1 N,

0.6 mmol) and stirred at RT for around 14 h until reaction was finished

by TLC monitor; 67.0% yield. ¹H NMR (500 MHz, DMSO-d₆) δ 10.1

(s, 1 H), 8.51 (s, 1 H), 7.85 (d, 2 H, J = 8.50 Hz), 7.62 (s, 2 H), 6.91 (d,

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2 H, J = 8.5 Hz), 3.86 (s, 6 H), 3.79 (s, 3 H). MS (ESI) m/z 394.1 (M þ

Na)^þ, 369.9 (M ꢀ H)^ꢀ. Anal. (C₁₉H₁₇FNO₅S) C, H, N.

0.55 mmol), the reaction mixture was absorbed on silica gel, and free

base was purified after flash column (41 mg, 70.9%). At 0 °C, to a

solution of free base (41 mg) in 5 mL of CH₂Cl₂was added a solution of

HCl in 1, 4-dioxane (4 N, 2 mL) and stirred at RT for overnight. The

precipitate (42) was filtered and washed with diethyl ether. Yield 71.3%;

(2-(1H-Indol-2-yl)thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

(32) Was Synthesized from 31 Using the Same Method As Used for 5.

Yield 45.8%. ¹H NMR (500 MHz, DMSO-d₆) δ 9.26 (s, 1 H), 8.11 (s, 1

H), 7.66 (d, 1 H, J = 8.0 Hz), 7.46 (s, 2 H), 7.42 (d, 1 H, J = 8.0 Hz), 7.29

(t, 1 H, J = 7.5 Hz), 7.16 (t, 1 H, J = 7.5 Hz), 7.10 (s, 1 H), 3.97 (s, 3 H),

3.93 (s, 6 H). MS (ESI) m/z 417.1 (M þ Na)^þ, 392.9 (M ꢀ H)^ꢀ. Anal.

(C₂₁H₁₈N₂O₄S) C, H, N.

1

mp 94ꢀ96 °C. H NMR (500 MHz, CDCl₃) δ 13.0 (s, 1 H), 8.34

(s, 1 H), 8.13 (d, 2 H, J = 7.0 Hz), 7.82 (d, 2 H, J = 7.5 Hz), 7.75

(s, 2 H), 4.24 (s, 2 H), 3.99 (s, 3 H), 3.97 (s, 6 H), 2.83 (s, 6 H). MS

(ESI) m/z 413.1 (M þ H)^þ. Anal. (C₂₂H₂₄N₂O₄S HCl) C, H, N.

3

(2-(1H-Indol-5-yl)thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

(36). To a solution of n-BuLi (1.6 M, 1.7 mL) in 8 mL of THF was added

a solution of 3,4,5-trimethoxybromobenzene (2.47 mmol) in 3 mL of

THF under ꢀ78 °C. The mixture was allowed to stir for 2 h, and a

solution of Weinreb amide 35 (1.24 mmol) in 3 mL of THF was charged.

The temperature was allowed to increase at RT and stirred overnight.

The reaction mixture was quenched with satd NH₄Cl, extracted

with ethyl ether, and dried with MgSO₄. The solvent was removed

under reduced pressure to yield a crude product, which was refluxed in

1 N NaOH in 5 mL of ethanol solution to obtain the deprotected

compound 36 and purified by column chromatography to obtain

General Procedure for the Synthesis of (2-(Arylamino)-

thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanones (45aꢀc).

At ꢀ78 °C, to a solution of 5-bromo-1,2,3-trimethoxybenzene (1.235 g,

5.0 mmol) in 30 mL of THF was charged n-BuLi in hexane (2.5 N,

2.4 mL, 6 mmol) under Ar₂protection and stirred for 10 min. Weinreb

amide 44aꢀc (1 mmol) in 10 mL of THF was added to the lithium

reagent and allowed to stir at RT for 2 h. The reaction mixture was

quenched with satd NH₄Cl, extracted with ethyl ether, and dried with

MgSO₄. The solvent was removed under reduced pressure to yield a

crude product, which was purified by column chromatography to obtain

pure compound 45aꢀc.

1

pure compound as a light-yellow solid (36.3%); mp 162ꢀ164 °C. H

(2-(Phenylamino)thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

(45a). Yield 33.3%; mp 149ꢀ151 °C. ¹H NMR (500 MHz, DMSO-d₆)

δ 10.4 (s, 1 H), 7.85 (s, 1 H), 7.68 (d, 2 H, J = 8.0 Hz), 7.31 (t, 2 H, J =

8.0 Hz), 6.98 (t, 1 H, J = 8.0 Hz), 3.83 (s, 6 H), 3.78 (s, 3 H). MS (ESI)

m/z 393.1 (M þ H)^þ, 368.9 (M ꢀ H)^ꢀ. Anal. (C₁₉H₁₈N₂O₄S) C, H, N.

(2-(p-Tolylamino)thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

NMR (300M, CDCl₃) δ 8.36 (br, s, 1 H), 8.31 (s, 1 H), 8.21 (s, 1 H),

7.92, 7.89 (dd, 1 H, J = 1.8, 2.7 Hz), 7.46 (d, 1 H), 7.62 (s, 2 H, J = 8.7

Hz), 7.29 (t, 1 H, J = 2.7 Hz), 6.64 (br, 1 H), 3.97 (s, 6 H), 3.97 (s, 3 H).

MS (ESI) m/z 417.1 (M þ Na)^þ, 392.9 (M ꢀ H)^ꢀ. Anal. (C₂₁H₁₈-

N₂O₄S) C, H, N.

1

tert-Butyl 4-(4-(3,4,5-Trimethoxybenzoyl)thiazol-2-yl)benzylcarbamate

(38). A mixture of 37 (2.5 mmol), CBrCl₃(3.2 mmol), and DBU (5.0

mmol) in CH₂Cl₂(20 mL) was stirred overnight. The reaction mixture

was absorbed on silica gel and purified by column chromatography to

yield an intermediate thiazole Weinreb amide. To a solution of (3,4,5-

trimethoxyphenyl)magnesium bromide (0.5 M, 5.5 mL) in THF was

added a solution of the intermediate thiazole Weinreb amide (1.83

mmol) in 10 mL of THF under 0 °C and stirred for 30 min. The reaction

mixture was quenched with satd NH₄Cl, extracted with ethyl ether, and

dried with MgSO₄. The solvent was removed under reduced pressure to

yield a crude product, which was purified by column chromatography to

obtain pure compound as a light-yellow solid (32.3%); mp 123ꢀ124 °C.

¹H NMR (300M, CDCl₃) δ 8.27 (s, 1 H), 7.98 (d, 2 H, J = 8.1 Hz), 7.78

(s, 2 H), 7.39 (d, 2 H, J = 8.1 Hz), 4.93 (br, 1 H), 4.37 (br, d, 2 H), 3.96

(s, 3 H), 3.95 (s, 6 H), 1.47 (s, 9 H). MS (ESI) m/z 507.1 (M þ Na)^þ.

Anal. (C₂₅H₂₈N₂O₆S) C, H, N.

(45b). Yield 40.6%; mp 139ꢀ140 °C. H NMR (500 MHz, CDCl₃)

δ 7.48 (s, 1 H), 7.47 (s, 2 H), 7.30 (br, 1 H), 7.27 (d, 2 H, J = 8.5 Hz),

7.17 (d, 2 H, J = 8.5 Hz), 3.93 (s, 3 H). 3.90 (s, 6 H), 2.34 (s, 3 H).

MS (ESI) m/z 385.1 (M þ H)^þ, 382.9 (M ꢀ H)^ꢀ. Anal. (C₂₀H₂₀-

N₂O₄S) C, H, N.

(2-(p-Fluorophenylamino)thiazol-4-yl)(3,4,5-trimethoxyphenyl)-

methanone (45c). Yield 39.6%; mp 129ꢀ130 °C. ¹H NMR (500 MHz,

CDCl₃) δ 7.52 (br, 1 H), 7.49 (s, 1 H), 7.45 (s, 2 H), 7.40ꢀ7.37 (q, 2 H,

J = 4.5 Hz), 7.08ꢀ7.04 (t, 2 H, J = 8.0 Hz), 3.93 (s, 3 H), 3.89 (s, 6H).

MS (ESI) m/z 389.3 (M þ H)^þ, 386.9 (M ꢀ H)^ꢀ. Anal. (C₁₉H₁₇-

FN₂O₄S) C, H, N.

General Procedure for the Synthesis of Hydrochloride

Salts (46aꢀc). At 0 °C, to a solution of compound 45aꢀc (0.1 mmol)

in 5 mL of CH₂Cl₂was added a solution of HCl in 1,4-dioxane (4 N,

2 mL) and stirred at RT for overnight. The precipitates 46aꢀc were

collected and washed with diethyl ether.

(2-(Phenylamino)thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

Hydrochloride Salt (46a). Yield 91.6%; mp 94ꢀ96 °C. ¹H NMR (500

MHz, DMSO-d₆) δ 12.9 (br, 1 H), 7.49ꢀ7.46 (m, 2 H), 7.42ꢀ7.40 (m,

2 H), 7.37ꢀ7.34 (m, br, 2 H), 7.11 (s, 2 H), 3.94 (s, 3 H), 3.92 (s, 6 H),

(2-(4-(Aminomethyl)phenyl)thiazol-4-yl)(3,4,5-trimethoxyphenyl)-

methanone Hydrochloride (39). At 0 °C, to a solution of 38 (200 mg)

in 10 mL of CH₂Cl₂was added a solution of HCl in 1,4-dioxane (4 N,

2 mL) and stirred at RT for 4 h. The precipitate (39) was filtered and

washed with diethyl ether. Yield 81.3%; mp 200ꢀ203 °C. ¹H NMR (500

MHz, DMSO-d₆) δ 8.68 (s, 1 H), 8.38 (br, 3 H), 8.10 (d, 2 H, J = 8.4

Hz), 7.66 (d, 2 H, J = 8.4 Hz), 7.62 (s, 2 H), 4.11 (s, 2 H), 3.87 (s, 6 H),

3.57 (br, H₂O). MS (ESI) m/z 389.1 (M þ H)^þ. Anal. (C₁₉H₁₈N₂O₄S

3

HCl H₂O) C, H, N.

3

(2-(p-Tolylamino)thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

Hydrochloride Salt (46b). Yield 39.6%; mp 115ꢀ118 °C. ¹H NMR (500

MHz, CDCl₃) δ 7.30ꢀ7.25 (m, br, 6 H), 7.12 (s, 2 H), 3.94 (s, 3 H),

3.92 (s, 6 H), 2.38 (s, 3 H). MS (ESI) m/z 389.1 (M þ H)^þ. Anal.

3.80 (s, 3 H). MS (ESI) m/z 385.1 (M þ H)^þ. Anal. (C₂₀H₂₀N₂O₄S

3

HCl) C, H, N, Cl.

(2-(4-((Methylamino)methyl)phenyl)thiazol-4-yl)(3,4,5-trimeth-

oxyphenyl)methanone Hydrochloride (40). At 0 °C, to a solution of 41

(60 mg) in 5 mL of CH₂Cl₂was added a solution of HCl in 1,4-dioxane

(4 N, 2 mL) and stirred at RT for overnight. The precipitate (40) was

filtered and washed with diethyl ether. Yield 81.3%; mp 197ꢀ200 °C. ¹H

NMR (500 MHz, CDCl₃) δ 10.0 (s, 1 H), 8.29 (s, 1 H), 8.05 (d, 2 H, J =

6.0 Hz), 7.74 (s, 2 H), 7.72 (d, 2 H, J = 6.0 Hz), 4.15 (s, 2 H), 3.99 (s, 3

H), 3.96 (s, 6 H), 2.61 (s, 3 H). MS (ESI) m/z 399.1 (M þ H)^þ.; Anal.

(C₂₁H₂₂N₂O₄S HCl H₂O) C, H, N.

(C₂₀H₂₀N₂O₄S 2HCl) C, H, N.

3

(2-(p-Fluorophenylamino)thiazol-4-yl)(3,4,5-trimethoxyphenyl)-

methanone Hydrochloride Salt (46c). Yield 89.3%; mp 102ꢀ104 °C.

¹H NMR (500 MHz, DMSO-d₆) δ 10.55 (s, 1 H), 7.85 (s, 1 H),

7.72ꢀ7.69 (q, 2 H, J = 4.5 Hz), 7.50 (s, 2 H), 7.18ꢀ7.15 (t, 2 H, J = 8.5

Hz), 4.30 (br, H₂O), 3.82 (s, 6H), 3.78 (s, 3 H). MS (ESI) m/z 389.3

(M þ H)^þ. Anal. (C₁₉H₁₇FN₂O₄S 1.5HCl 0.5H₂O) C, H, N.

3

Cell Culture and Cytotoxicity Assay of Prostate Cancer

and Melanoma. All cell lines were obtained from American Type

Culture Collection (ATCC, Manassas, VA, USA), while cell culture

supplies were purchased from Cellgro Mediatech (Herndon, VA, USA).

We examined the antiproliferative activity of our antitubulin compounds

3

(2-(4-((Dimethylamino)methyl)phenyl)thiazol-4-yl)(3,4,5-trimeth-

oxyphenyl)methanone Hydrochloride (42). To a solution of 39 (53 mg,

0.14 mmol) in 5 mL of CH₂Cl₂was added formaldehyde solution (37%

in H₂O, 340 mg, 4.2 mmol) and sodium cyanoborohydride (34 mg,

4691

dx.doi.org/10.1021/jm2003427 |J. Med. Chem. 2011, 54, 4678–4693

Journal of Medicinal Chemistry

ARTICLE

in four human prostate cancer cell lines (LNCaP, DU 145, PC-3, and

PPC-1) and three melanoma cell lines (A375, B16ꢀF1 and WM-164).

Human ovarian cell line OVCAR-8 and its resistant cell line that

overexpresses P-gp (NCI/ADR-RES) were used as MDR models. Both

ovarian cell lines were obtained from National Cancer Institutes (NCI).

All cell lines were tested and authenticated by either ATCC or NCI. All

prostate cancer and ovarian cancer cell lines were cultured in RPMI 1640

and supplemented with 10% fetal bovine serum (FBS). Melanoma cells

were cultured in DMEM, supplemented with 5% FBS, 1% antibiotic/

antimycotic mixture (Sigma-Aldrich, Inc., St. Louis, MO, USA), and

bovine insulin (5 μg/mL; Sigma-Aldrich). The cytotoxic potential of

the antitubulin compounds was evaluated using the sulforhodamine

B (SRB) assay after 96 h of treatment.

Aqueous Solubility. The solubility of drugs was determined by a

Multiscreen solubility filter plate (Millipore Corporate, Billerica, MA)

coupled with LC-MS/MS. Briefly, 198 μL of phosphate buffered saline

(PBS) buffer (pH 7.4) was loaded into 96-well plate, and 2 μL of 10 mM

test compounds (in DMSO) was dispensed and mixed with gentle

shaking (200ꢀ300 rpm) for 1.5 h at RT (N = 3). The plate was

centrifuged at 800g for 5 min, and the filtrate was used to determine its

concentration and solubility of test compound by LC-MS/MS as

described below. Concentrations of tested compounds were determined

with individual calibration curve with satisfied linearity (r > 0.995).

Pharmacokinetic Study. Female SpragueꢀDawley rats (n = 3

or 4; 254 ( 4 g) were purchased from Harlan Inc. (Indianapolis, IN). Rat

thoracic jugular vein catheters were purchased from Braintree Scientific

Inc. (Braintree, MA). On arrival at the animal facility, the animals were

acclimated for 3 days in a temperature-controlled room (20ꢀ22 °C)

with a 12 h light/dark cycle before any treatment. Compound 1 was

administered intravenously (IV) into the jugular vein catheters at a dose

of 2.5 mg/kg (in DMSO/PEG300, 2/8), whereas 46a and 46c were

dosed at 5 mg/kg (in DMSO/PEG300, 1/9). An equal volume of

heparinized saline was injected to replace the removed blood, and blood

samples (250 μL) were collected via the jugular vein catheters at 10, 20,

30 min, and 1, 2, 4, 8, 12, 24 h. Compounds 1, 46a, and 46c were given

(PO) by oral gavage at 10 mg/kg (in Tween80/DMSO/H₂O, 2/1/7).

All blood samples (250 μL) after oral administration were collected via

the jugular vein catheters at 30, 60, 90, 120, 150, 180, 210, 240 min, and

8, 12, 24 h. Heparinized syringes and vials were prepared prior to blood

collection. Plasma samples were prepared by centrifuging the blood

samples at 8000g for 5 min. All plasma samples were stored immediately

at ꢀ80 °C until analyzed.

0.5 min before a quick ramp to 15% mobile phase A. Mobile phase A was

continued for another 12 min toward the end of analysis.

A triple-quadruple mass spectrometer, API Qtrap 4000 (Applied

Biosystems/MDS SCIEX, Concord, Ontario, Canada), operating with a

TurboIonSpray source was used. The spraying needle voltage was set at

5 kV for positive mode. Curtain gas was set at 10; gas 1 and gas 2 were set

50. Collision-assisted dissociation (CAD) gas at medium and the source

heater probe temperature at 500 °C. Data acquisition and quantitative

processing were accomplished using Analyst software, Ver. 1.4.1

(Applied Biosystems).

In Vitro Tubulin Polymerization Assay. Bovine brain tubulin

(0.4 mg, >97% pure) (Cytoskeleton, Denver, CO) was mixed with 5 μM

of the test compounds and incubated in 100 μL of general tubulin buffer

(80 mM PIPES, 2.0 mM MgCl₂, 0.5 mM EGTA, and 1 mM GTP) at pH

6.9. The absorbance of wavelength at 340 nm was monitored every 1 min

for 20 min by the SYNERGY 4 Microplate Reader (Bio-Tek Instru-

ments, Winooski, VT). The spectrophotometer was set at 37 °C for

tubulin polymerization.

’ ASSOCIATED CONTENT

S

Supporting Information. 2D NOESY NMR and 1D

b

NOE NMR to identify syn/anti-isomers of hydrazides 14aꢀ14b

and acrylonitriles 15aꢀ15b, synthesis, mass, and NMR of inter-

mediates 1ꢀ4, 8aꢀe, 10eꢀg, 12a, 12b, 27, 30, 31, 34, 35, 37,

38, 41, 43aꢀc, 44aꢀc, and quantum chemical shifts calcula

tions. This material is available free of charge via the Internet at

http://pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: (901)448-6026. Fax: (901)448-3446. E-mail: dmiller@

uthsc.edu.

Author Contributions

^§These authors contributed equally to this work.

’ ACKNOWLEDGMENT

This research was supported by funds from Department of

Pharmaceutical Sciences, College of Pharmacy, and University of

Tennessee Health Science Center; the Van Vleet Endowed

Professorship; USPHS grants 1R15CA125623 and 1RO1CA-

148706. We thank Terrence A. Costello, Katie N. Kail, and

Stacey L. Barnett for providing technical support for animal

studies at GTx, Inc.

Analytes were extracted from 100 μL of plasma with 200 μL of

acetonitrile containing 200 nM of the internal standard ((3,5-

dimethoxyphenyl)(2-phenyl-1H-imidazol-4-yl)methanone). The sam-

ples were thoroughly mixed, centrifuged, and the organic extract was

transferred to autosampler for LC-MS/MS analysis. Multiple reaction

monitoring (MRM) mode, scanning m/z 356 f 188 (compound 1),

m/z 371 f 203 (compound 46a), m/z 389 f 221 (compound 46c),

and m/z 309 f 171 (the internal standard), was used to obtain the most

sensitive signals. The pharmacokinetic parameters were determined

using noncompartmental analysis (WinNonlin, Pharsight Corporation,

Mountain View, CA)

’ ABBREVIATIONS USED

ABC, ATP binding cassette; Boc₂O, tert-butyl dicarbonate;

CA-4, combretastatin A-4; DMSO, dimethyl sulfoxide; MDR,

multidrug resistance; MRM, Multiple reaction monitoring;

NMM, N-methylmorpholine; NMR, nuclear magnetic reso-

nance; PBS, phosphate buﬀered saline; PDC, pyridinium di-

chromate; P-gp, P-glycoprotein; phenylamino-thiazole, PAT;

pharmacokinetic, PK; RT, Room temperature; SAR, Structureꢀ

activity relationship; SMART, 4-substituted methoxybenzoyl-

aryl-thiazole; TBAF, tetrabutylammonium ﬂuoride; TBDMS,

tert-butyldimethylsilyl; THF, tetrahydrofuran; TMP, 3,4,5-tri-

methoxyphenyl

Analytical Method. Sample solution (10 μL) was injected into an

Agilent series HPLC system (Agilent 1100 Series Agilent 1100 Chem-

station, Agilent Technology Co, Ltd.). All analytes were separated on a

narrow-bore C18 column (Alltech Alltima HP, 2.1 mm ꢁ 100 mm,

3 μm, Fisher, Fair Lawn, NJ). Two gradient modes were used. Gradient

mode was used to achieve the separation of analytes using mixtures of

mobile phase A [ACN/H₂O (5%/95%, v/v) containing 0.1% formic

acid] and mobile phase B [ACN/H₂O (95%/5%, v/v) containing 0.1%

formic acid] ata flow rate of 300μL/min. Mobile phase A was usedat15%

from 0 to 1 min, followed by a linearly programmed gradient to 100% of

mobile phase B within 6 min, 100% of mobile phase B was maintained for

4692

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Journal of Medicinal Chemistry

ARTICLE

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Article Doi

Design, synthesis, and SAR studies of 4-substituted methoxylbenzoyl-aryl- thiazoles analogues as potent and orally bioavailable anticancer agents

DOI: 10.1021/jm2003427

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