Discovery of a clinical stage multi-kinase inhibitor sodium (E)-2-{2-methoxy-5-[(2′,4′,6′-trimethoxystyrylsulfonyl)methyl] phenylamino}acetate (ON 01910.Na): Synthesis, structure-activity relationship, and biological activity

ARTICLE

pubs.acs.org/jmc

Discovery of a Clinical Stage Multi-Kinase Inhibitor Sodium (E)-2-{2-

Methoxy-5-[(2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl]phenylamino}-

acetate (ON 01910.Na): Synthesis, StructureꢀActivity Relationship,

and Biological Activity

M. V. Ramana Reddy,*^,†Padmavathi Venkatapuram,^†,§Muralidhar R. Mallireddigari,^‡,§Venkat R. Pallela,^‡,§

Stephen C. Cosenza,^†,§Kimberly A. Robell,^†Balaiah Akula,^‡Benjamin S. Hoﬀman,^†and

E. Premkumar Reddy*^,†

^†Department of Oncological Sciences, Icahn Medical Institute, Mount Sinai School of Medicine, 1425 Madison Avenue, New York,

New York 10029-6514, United States

^‡Department of Medicinal Chemistry, Onconova Therapeutics Inc., 375 Pheasant Run, Newtown, Pennsylvania 18940-3423,

United States

S Supporting Information

b

ABSTRACT: Cyclin D proteins are elevated in many cancer

cells, and targeted deletion of cyclin D1 gene in the mammary

tissues protects mice from breast cancer. Accordingly, there is an

increasing awareness of this novel nonenzymatic target for

cancer therapeutics. We have developed novel, nonalkylating

styrylbenzylsulfones that induce cell death in wide variety of

cancer cells without aﬀecting the proliferation and survival of

normal cells. The development of derivatized styrylbenzylsul-

fones followed logically from a tumor cell cytotoxicity screen

performed in our laboratory that did not have an a priori target proﬁle. Modiﬁcations of some of the precursor molecules led to lead

optimization with regard to tumor cell cytotoxicity. In this report we describe the synthesis and structureꢀactivity relationships of

novel, nonalkylating (E)-styrylbenzylsulfones and the development of the novel anticancer agent sodium (E)-2-{2-methoxy-

5-[(2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl]phenylamino}acetate (ON 01910.Na), which is in phase III trials for myelodysplastic

syndromes (MDS) associated with aberrant expression of cyclin D proteins.

’ INTRODUCTION

inhibitors of serine/theronine and tyrosine kinases, providing a

rationale for further expansion of this chemotype for applications

related to cancer therapy.

In a recent paper,¹we described the synthesis of a group of

styrylbenzylsulfones that induce apoptotic death of a wide variety

of human tumor cell lines at nanomolar concentrations while

exhibiting relatively low toxicity to normal human cells. Our

studies showed that the cytotoxic activity of styrylbenzylsulfones

is completely dependent on the nature and position of the sub-

stituents on the two aromatic rings. Structureꢀfunction studies

showed that positions of functional groups on the styryl aromatic

ring play a critical role in determining the biochemical and

biological activity of these molecules. Biological evaluation of

the activity of these compounds showed that these compounds

are highly active against a wide variety of human tumor cell lines

including those that are resistant to the activity of many of the

currently used chemotherapeutic agents. The low toxicity pro-

ﬁle, both in vitro and in vivo, and their potent tumor inhibi-

tory activity as seen in soft agar and nude mouse xenograft

assays pointed to the potential value of these compounds as safe

therapies for cancer, lacking many of the side eﬀects normally

associated with current chemotherapeutic agents. Many of the

compounds described in this study were found to act as allosteric

The present report describes the synthesis and structureꢀactivity

relationships of novel, nonalkylating (E)-styrylbenzylsulfones and

the development of the novel anticancer agent sodium (E)-2-

{2-methoxy-5-[(2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl]phenyla-

mino}acetate (ON 01910.Na, 28).²The development of deri-

vatized styrylbenzylsulfones followed logically from a tumor cell

cytotoxicity screen performed in our laboratory. Precursors of 28

were identiﬁed speciﬁcally based upon their ability to target

cancer cells while leaving nonmalignant cell cultures virtually

unaﬀected. Modiﬁcations of some of the precursor molecules led

to lead optimization with regard to tumor cell cytotoxicity.

Structureꢀactivity studies conﬁrmed that the nature, number,

and position of substituents on the two aromatic rings of the

parent molecule are the determining factor in the tumor cell

cytotoxicity of these compounds.

Received: May 5, 2011

Published: August 03, 2011

r

2011 American Chemical Society

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Journal of Medicinal Chemistry

ARTICLE

Scheme 1. Synthesis of (E)-Styrylbenzylsulfones from

’ CHEMISTRY

Benzylsulfonylacetic Acids^a

Multiple synthetic routes for synthesis of the (E)-styrylben-

zylsulfone scaﬀold were explored. The initial method (method A)

involved the synthesis of substituted (E)-styrylbenzylsulfones by the

reaction of benzyl bromides (2) with mercaptoacetic acid in the

presence of a strong base, sodium hydroxide in methanol, to obtain

benzylthioacetic acids (5) in quantitative yields (Scheme 1). Oxida-

tion of 5 with 30% hydrogen peroxide (H₂O₂) in glacial acetic acid

aﬀorded benzylsulfonylacetic acids 6.⁷Knovenagel condensation of

6 with aromatic aldehydes 7 in either benzylamine/acetic acid⁷or

piperidine/benzoic acid⁸in toluene aﬀorded (E)-styrylbenzylsul-

fones 8 in good yields.

Some of the substituted benzyl bromides 2 that are not com-

mercially available were synthesized as shown in the Scheme 2.

Substituted toluenes 1 were brominated with N-bromosuccini-

mide (NBS) in the presence of a catalytic amount of benzoyl

peroxide in carbon tetrachloride (CCl₄) to obtain 2.⁹Some of

these nitro substituted benzyl bromides 2 were also made start-

ing from substituted nitrobenzaldehydes 3 which were reduced

with sodium borohydride (NaBH₄)¹⁰and on subsequent bro-

mination of the resulting alcohol 4 with phosphorus tribromide

(PBr₃) (Scheme 2).¹¹

In method B, 3-nitro-4-methoxybenzylmercaptan (9) was

treated with 4-methoxyphenacyl bromide (10) to obtain 4-

methoxyphenacyl-3-nitro-4-methoxybenzyl sulﬁde 11, which on

oxidation with 30% H₂O₂gave 4-methoxyphenacyl-3-nitro-4-

methoxybenzylsulfone 13.⁷

^aReagents and conditions: (a) HSCH₂COOH, MeOH, NaOH, room

temp, 3 h; (b) 30% H₂O₂, ACOH, room temp, 24 h; (c) benzylamine,

AcOH, reﬂux, 2ꢀ8 h; (d) piperidine, benzoic acid, toluene, 2ꢀ4 h.

Scheme 2. Synthesis of Benzyl Bromides^a

Sequential reduction of 13 with sodium borohydride¹and

subsequent dehydration with p-toluenesulfonic acid (p-TSA)

aﬀorded the desired 8 in moderate yields (Scheme 3).¹²Method

A is superior to method B for the synthesis of 8, as it involves

steps with relatively higher yields. The 3-nitro-4-methoxyben-

zylmercaptan 9 was in turn synthesized from 2a as outlined in

Scheme 4. 2a on treatment with thiourea in water gave an

intermediate isothiouronium salt (12) which on reduction with

ammonia yielded 9 in low to moderate yields.¹³

To see the eﬀect of trans sulﬁde and sulfoxide on the biological

activity of these (E)-styrylbenzylsulfones, we made (Z) (16) and

(E) (17) 4-methoxy-3-nitrobenzyl- 2⁰,4⁰,6⁰-trimethoxystyryl sul-

ﬁdes by the reaction of 3-nitro-4-methoxybenzylmercaptan (9)

and 2,4,6-trimethoxyphenylacetylene (15) in the presence of

triethylboraneꢀhexane (Et₃B) in benzene (Scheme 5).¹⁴In this

reaction, (Z) to (E) ratio of 40:60 resulted in mainly trans

isomers. 17 on controlled oxidation with 1,1,1,3,3,3-hexaﬂuoro-

2-propanol and 30% H₂O₂at room temperature resulted

in sulfoxide 20,¹⁵which on reduction with sodium hydrosulﬁte

in acetoneꢀwater mixture at 50 °C aﬀorded the corresponding

amine 21.⁹

The sulfoxide 20 was oxidized to sulfone 22 with 30% hydro-

gen peroxide in glacial acetic acid⁷which was later reduced with

sodium hydrosulﬁte to the corresponding sulfone 23.⁹The nitro

sulﬁde 17 was also converted to the corresponding amino sulﬁde

24 which on further oxidation with m-chloroperoxybenzoic acid

(m-CPBA) aﬀorded sulfone 23. The acetylene 15 was synthe-

sized starting from 2,4,6-trimethoxybenzaldehyde 18 and tetra-

bromomethane (CBr₄) in the presence of triphenyphosphine

(Ph₃P) in dichloromethane.¹⁶The resulting 2⁰,2⁰-dibromovinyl-

1,3,5-trimethoxybenzene 19 on treatment with n-BuLi in THF

at ꢀ78 °C gave 15¹⁶in high yields. The synthesis of nitro-(E)-

styrylbenzyl sulfoxide 20 and amino-(E)-styrylbenzyl sulfoxide

21 was also achieved from 4-methoxy-3-nitrobenzylthioacetic

^aReagents and conditions: (a) NBS, benzoyl peroxide, CCl₄, 18 h; (b)

NaBH₄, MeOH, 0ꢀ5 °C, 1 h; (c) PBr₃, toluene, 100 °C, 30 min.

In previous studies in our laboratory, 28 displayed desirable

pharmacokinetic and pharmacodynamic properties and was able

to reduce tumor size and increase survival in mice carrying tumor

cell xenografts.²28 received orphan drug status for the myelo-

dysplastic syndrome, a heterogeneous hematopoietic stem cell

disorder that aﬀects cell proliferation, diﬀerentiation, and func-

tion. MDS is characterized by dyspoiesis, hyperproliferative bone

marrow, and peripheral blood cytopenias involving one or more

lineages.^3ꢀ5Most of the untreated patients with high risk MDS

die from progressive bone marrow failure within 1 year because

of hemorrhage and/or infection. In vitro studies with 28 showed

that incubation of human leukemic cells with this compound

results in the inhibition of the PI3K/AKT pathway, down-

regulation of cyclin D1, induction of NOXA and BIM, and

activation of the JNK pathway.⁶Treatment of MDS patients with

28 results in a dramatic reduction of cytogenetically abnormal

cells with a minimal inhibition of normal hematopoiesis. This

drug is currently in phase III clinical trials.

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Scheme 3. Synthesis of (E)-Styrylbenzylsulfones from Phenacylbenzylsulfones^a

^aReagents and conditions: (a) NaOH, MeOH, room temp, 2 h; (b) 30% H₂O₂, ACOH, room temp, 24 h; (c) NaBH₄, tetrahydrofuran, 0 °C, 1 h;

(d) p-toluenesulfonic acid, toluene, 2ꢀ4 h.

Scheme 4. Synthesis of 4-Methoxy-3-nitrobenzylmercaptan^a

^aReagents and conditions: (a) thiourea, H₂O, reﬂux, 2 h; (b) ammonia, hexane, reﬂux, 30 min.

acid 5a by oxidation to sulfoxide 25¹⁷and then condensation

with 2,4,6-trimethoxybenzaldehyde 18 in the presence of ben-

zylamine and acetic acid (Scheme 7).⁷The resulting 20 was

reduced to 21 as described in Scheme 5.

and dichloromethane followed by methyl ethyl ketone washing

aﬀorded crystalline 28 in high yields with two molecules of water of

hydration as determined by Karl Fischer analysis (Scheme 9).

To enhance the solubility and bioavailability of these (E)-

styrylbenzylsulfones, several 3-amino substituted esters and acids

were made from (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-ami-

nobenzylsulfone (8p) and (E)-3⁰,4⁰,5⁰-trimethoxystyryl-4-meth-

oxy-3-aminobenzylsulfone (8q) (Scheme 8). 8p and 8q were

treated with diﬀerent R-bromo esters in the presence of mild base

sodium acetate in ethanol for 48 h to give amine esters (26)

which on subsequent hydrolysis with sodium hydroxide in

ethanol aﬀorded the corresponding acids 27.

The lead compound of our investigation, 28, was synthe-

sized from 8p by its reaction with methyl 2-bromoacetate

in the presence of a mild base sodium acetate in methanol for

4ꢀ6 h to give amino substituted methyl ester 26a which on

subsequent hydrolysis with sodium hydroxide in aqueous ethanol

’ STRUCTUREꢀACTIVITY RELATIONSHIPS (SAR)

After the synthesis of these compounds, their in vitro cyto-

toxicity was assessed using two diﬀerent human tumor cell lines

derived from human prostate (DU145) and leukemic (K562)

cancers. The results of the study are presented in Table 1. These

studies reveal that the cytotoxicity of the (E)-styrylbenzylsul-

fones is totally dependent on the nature and position of the

substituents present on the two aromatic rings. For the purpose

of structureꢀactivity relationship, we have selected a few com-

pounds from a library of 2000 (E)-styrylbenzylsulfones synthe-

sized from our laboratory. In most of the selected compounds

described here, we have kept a methoxy group at the fourth

position and an amino group at the third position of benzylsul-

fonyl aromatic ring and one or more methoxy groups on styryl

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Scheme 5. Synthesis of (Z)- and (E)-Styrylbenzyl Sulﬁdes from 4-Methoxy-3-nitrobenzylmercaptan and 2,4,6-Trimethoxyphe-

nylacetylene and (E)-Styrylbenzyl Sulfoxide and Sulfone^a

^aReagents and conditions: (a) Et₃Bꢀhexane, benzene, 25 °C, 2 h; (b) 1,1,1,3,3,3-hexaﬂuoro-2-propanol, 30% H₂O₂, 25 °C, 2 h; (c) acetone/water

(2:1), sodium hydrosulﬁte, 50 °C, 30 min; (d) 30% H₂O₂, AcOH, room temp, 24 h; (e) m-CPBA, CH₂Cl₂, 0 °C to room temp, 3 h.

Scheme 6. Synthesis of 2,4,6-Trimethoxyphenylacetylene^a

^aReagents and conditions: (a) triphenylphosphine, tetrabromomethane, CH₂Cl₂, 5 °C, 30 min; (b) n-BuLi, dry THF, ꢀ78 °C, 15 min.

aromatic ring at diﬀerent positions. A moderate cytotoxicity in

tumor cells was seen in a compound when an amino group and

methoxy group are at third and fourth positions of the benzyl ring

and a methoxy group at the fourth position of the styryl ring (8d).

By introduction of a second methoxy group on the styryl ring of

8d, the cytotoxicity of the resulting compounds 8g, 8h, and 8i

was further enhanced by several fold. Whereas the results are

quite surprising for the molecules that are disubstituted with

methoxy groups on the styryl aromatic ring compared to 8d, the

results obtained in cytotoxicity assays using these compounds

(8g, 8h, 8i, 8j, and 8k) clearly show that the methoxy group,

when present at 2 and 6 positions (8i), enhances the activity

of the molecule by greater than 10-fold when compared to

other disubstituted methoxy groups (8g and 8h). It is also clear

from the cytotoxicity proﬁle of the compounds 8j and 8k that

when amino and methoxy groups are placed at other than third

and fourth positions, the compounds exhibit decreased cell

killing activity: 15- to 500-fold. Because the introduction of

two methoxy groups on the styryl aromatic ring enhanced the

biological activity, we have synthesized some trimethoxystyryl

analogues to determine if this further enhances their cytotoxic

properties. Analysis of these compounds (8n, 8o, 8p, 8q, and 8r)

in cell-killing assays showed that (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-

methoxy-3-aminobenzylsulfone (8p) exhibited the best activity

in the entire series. This compound, 8p, is almost 11-fold more

active than 8i in both cell lines. These results also show that when

2, 4, and 6 positions of the styryl ring are occupied by methoxy

groups (8p), the molecule attains optimum biological activity

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Scheme 7. Synthesis of (E)-Styryl-4-methoxy-3-nitrobenzyl Sulfoxide from 4-Methoxy-3-nitrobenzyl Sulfoxide Acetic Acid^a

^aReagents and conditions: (a) NaOH/deionized H₂O, NaHCO₃, acetone, Oxone solution, sodium bisulﬁte, 6 N HCl; (b) benzylamine, AcOH, reﬂux,

2ꢀ8 h; (c) acetone/water (2:1), sodium hydrosulﬁte, 50 °C, 30 min.

Scheme 8. Synthesis of Amine Esters and Acids of 4-Methoxy-3-aminostyrylbenzylsulfones (8p and 8q)

Scheme 9. Synthesis of 28

compared to other trimethoxy substituted styrylsulfones (8n, 8o,

8q, and 8r). To validate the critical requirement of the methoxy

group at the fourth position of the 2⁰,4⁰,6⁰-trimethoxystyryl

moiety, we have replaced the methoxy group at the fourth

postion by a hydroxy (8t) and a carboxypropoxy group (8u).

Both replacements in 8p caused severe loss of cytotoxicity in the

molecules, indicating the indispensability of a methoxy group at

the fourth position of the ring. Further replacing the methoxy

group with halogen atoms or changing the position of the amino

group in the benzyl aromatic ring resulted in molecules (8w, 8x,

8y, and 8aa) that suﬀered substantial loss of cytotoxicity. Also the

compounds formed by replacing all three methoxy groups of the

styryl ring by ﬂuorine atoms (8ab and 8ac) in 8p have reduced

level of cytotoxicity. The SAR analysis clearly shows that all

the compounds with nitro substitutions on the benzylic ring

(8a, 8c, 8e, 8f, 8l, 8m, 8s, 8v, and 8z) are far less active than

the corresponding amino compounds, indicating that the amino

group in that position is critical for the interaction of the

compounds with their target and activity.

Once we established that 3-amino-4-methoxy groups on

benzyl aromatic ring and 2,4,6-trimethoxy groups on styryl ring

produced a compound (8p) with the best cytotoxic activity, we

then analyzed the role of sulfone in the activity of the molecule

(Table 2). To understand the oxidative state of sulfur in the

molecule, we have made sulﬁde (24) and sulfoxide (21) analo-

gues of 8p. Both sulﬁde (24) and sulfoxide (21) analogues are

10-fold less active than sulfone (8p), indicating complete oxida-

tive state of sulfur in the compound is required for optimum

activity.

The compound 8p, which has a high potency, has very low

solubility in aqueous buﬀers and solutions. In order to enhance

its water solubility and bioavailability, the amino group in third

position was converted to amino acids (27aꢀl). From Table 3, it

is clear that all 3-substituted amino acids (27aꢀl) are more active

than the corresponding esters (26aꢀl). As the 3-glycine substituted

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Table 1. In Vitro Cytotoxicity of (E)-Styrylbenzylsulfones 8

Table 2. In Vitro Cytotoxicity of (E)-Styrylbenzyl Sulﬁdes

(24) and Sulfoxides (21)

IC₅₀(μM)

compd

R

R₁

DU145

K562

compd

R

R₁

n

DU145

K562

8a

8b

8c

8d

8e

8f

3-NO₂, 4-CH₃O

3-NH₂, 4-CH₃O

3-NO₂, 4-CH₃O

3-NH₂, 4-CH₃O

3-NO₂, 4-CH₃O

2-NO₂, 4-CH₃O

3-NH₂, 4-CH₃O

2-NH₂, 4-CH₃O

3-NH₂, 6-CH₃O

3-NO₂, 4-CH₃O

2-NO₂, 4-CH₃O

2-NH₂, 4-CH₃O

3-NH₂, 4-CH₃O

3-NH₂, 6-CH₃O

3-NO₂, 4-CH₃O

3-NH₂, 4-CH₃O

2-CH₃O

4-CH₃O

20

17

20

21

24

3-NO₂, 4-OCH₃

3-NH₂, 4-OCH₃

2,4,6-(OCH₃)₃

0

1

0

5.0

7.5

25

7.5

15

25

0.04

0.05

0.02

0.03

2.5

20

2.5

2,6-(CH₃O)₂

2,4-(CH₃O)₂

2,5-(CH₃O)₂

2,6-(CH₃O)₂

2,4,6-(CH₃O)₃

2,4,5-(CH₃O)₃

2,4,6-(CH₃O)₃

3,4,5-(CH₃O)₃

2,4,6-(CH₃O)₃

2,6-(CH₃O)₂, 4-OH

2,6-(CH₃O)₂, 4-O-

(CH₂)₃COOH

2,4,6-(CH₃O)₃

2,4,6-F₃

20

50

8g

8h

8i

0.5

0.75

0.05

0.75

25

0.5

Developmental Therapeutics Program (DTP) against their panel

of 60 human cancer cell lines.¹⁸Their results showed that these

compounds exhibited broad-spectrum activity and inhibited the

growth of all of the tested cell lines, including drug-resistant cell

lines, at nanomolar concentrations. The finding that such a large

number of tumor cell lines are sensitive to these compounds

suggests that the target is essential for the proliferation and

survival of cancer cells. Statistical comparison (using the NCI

algorithm COMPARE) revealed that these drugs are mitotic

blockers of tumor cells. This statistical observation was further

substantiated by flow cytometry analysis shown in Figure 2.

8p and 28 Compounds Are Highly Active against Drug

Resistant Tumor Cell Lines. Development of multidrug resis-

tance (MDR) to classical chemotherapeutic agents is a clinical

problem oncologists face as patients fail first round treatment or

become resistant during or following recurrent tumor growth.

The MDR phenotype is caused by the overexpression of ATP-

binding cassette (ABC) transporters divided into seven subfam-

ily members. The overexpression of various members of this

family enables the tumor cells to actively pump out a wide range

of amphipathic drugs such that the intracellular concentrations

are no longer cytotoxic. To further investigate the activity of

these compounds against MDR positive tumor types, we deter-

mined the IC₅₀values of 8p and 28 using two classical MDR

positive cell lines (Table 5). The results shown in Figure 1A show

a 96 h dose response of the uterine sarcoma cell line MES-SA and

the multidrug resistant subline MES-SA/DX5¹⁹treated with 28

compared to a dose response against paclitaxel, a known sub-

strate for P-glycoprotein. This cell line has been shown to express

high levels of P-glycoprotein (ABCB1) and is resistant to a

number of hydrophobic compounds including doxorubicin,

paclitaxel, vincristine, vinblastine, etoposide, mitoxantrone, dac-

tinomycin, and daunorubucin. Our results show that the parental

cell line was very sensitive to paclitaxel (IC₅₀= 4 nM) but the

0.75

0.05

0.75

25

8j

8k

8l

2.5

20

2.5

8m

8n

8o

8p

8q

8r

8s

8t

20

0.025

0.25

0.004

3.0

7.5

5.0

0.2

25

0.025

0.25

0.003

0.25

7.5

5.0

0.3

8u

20

8v

3-NO₂, 4-Br

3-NH₂, 4-Br

3-NH₂, 4-Cl

2-NH₂, 4-Cl

3-NO₂, 4-F

25

8w

8x

0.75

0.01

2.5

25

0.75

0.01

2.5

25

8y

8z

8aa

8ab

8ac

3-NH₂, 4-F

2.5

2.0

5.0

2.5

0.8

15

3-NH₂, 4-CH₃O

2,4,5-F₃

compound 27a has superior activity over other molecules (27bꢀl)

and as it does not have a chiral center at glycine carbon, we have

made a water-soluble sodium analogue of it (28) to carry out our

preclinical and clinical studies. Compound 28 has 28 mg/mL

solubility in water and other aqueous buﬀers, which makes it suitable

for intravenous, intraperitoneal, and oral administration of the

compound.

MDR positive subline was greater than 100-fold resistant (IC₅₀

=

750 nM). When the two cell lines were treated with 28, both the

parental and the MDR positive cell lines were equally sensitive to

the cell killing activity of the compound with an IC₅₀of 0.1 μM

(Table 5). A second MDR cell line, derived from an ovarian

tumor, was also tested. Once again, both the parental and the resistant

cell lines were equally sensitive to compound 28 (Table 5). We also

investigated as to whether atypical multidrug resistant cells are sensi-

tive to 28. For these studies, we employed the parental leukemic cell

line CEM and its MDR subline CEM/C2 (Figure 1B).²⁰CEM/C2

’ BIOLOGICAL RESULTS AND DISCUSSION

In Vitro Antitumor Effects of 8p and 28 Compounds. We

next tested the activity of two of the most active compounds (8p

and 28) listed in Tables 1ꢀ3 against 94 different human tumor

cell lines, and surprisingly, they were found to induce apoptosis

of all of these cell lines with very similar GI₅₀values (selected data

shown in Table 4). These compounds (such as 8p, and 28) were

also tested by the National Cancer Institute, USA, through its

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Table 3. In Vitro Cytotoxicity of 3-Amino Substituted Esters

26 and Acids 27 and Sodium Salt 28

Table 4. Evaluation of 8p and 28 against a Panel of Human

Tumor Cell Lines and Normal Fibroblasts

IC₅₀(μM)

cell line

BT20

tumor type

breast (ERꢀ)

8p

28

0.03

0.08

0.17

0.075

0.05

0.025

0.075

0.06

0.1

T47D

breast (ER+)

breast (ERꢀ)

breast (ER+)

breast (triple neg)

breast

0.003

0.001

0.003

0.002

ND

MCF-7

SK-BR-3

BT474

MDA-MB-231

MDA-MB-157

Hcc70

ND

IC₅₀(μM)

ND

HCC1428

DU145

PC-3

breast

ND

compd

R₁

R₂

DU145

K562

prostate (ARꢀ)

prostate (AR+)

ovarian

0.005

0.006

0.003

ND

26a

26b

26c

26d

26e

26f

26g

26h

26i

26j

26k

26l

2,4,6-(CH₃O)₃

3,4,5-(CH₃O)₃

2,4,6-(CH₃O)₃

3, 4, 5-(CH₃O)₃

2,4,6-(CH₃O)₃

ON 01910.Na

CH₂

0.1

0.15

0.075

0.05

0.075

0.04

0.08

0.09

0.08

0.05

0.1

75

30

OV-CAR-3

MIA-Paca2

BxPC-3

PANC-1

U87

CH₂CH₂

1.5

1.0

pancreatic

CH(CH₃)

CF₂

0.75

0.25

2.5

0.2

pancreatic

0.25

0.80

0.03

0.8

pancreatic

ND

CH(CF₃)

glioblastoma

NSCLC

0.003

0.004

0.003

0.004

0.003

0.002

0.001

ND

C(CH₃)₂

H157

CH(C₆H₅)

CH(4-FC₆H₄)

CH(4-ClC₆H₄)

CH(4-BrC₆H₄)

CH(4-MeOC₆H₄)

CH₂

0.075

0.3

A549

NSCLC

H1975

NSCLC

0.4

0.2

H187

SCLC

0.3

0.2

N417

SCLC

0.25

0.075

5.0

0.15

0.0075

3.0

AGS

gastric

27a

27b

27c

27d

27e

27f

27g

27h

27i

27j

27k

27l

RF1

gastric

CH₂

RF48

gastric

CH₂CH₂

5.0

HELA

cervical

CH(CH₃)

CF₂

0.025

0.15

0.02

0.15

0.02

0.15

0.4

0.015

0.15

0.075

0.0075

0.015

0.08

0.25

0.2

COLO-205

DLD-1

colorectal

0.005

0.003

0.005

ND

0.15

0.075

0.09

0.06

0.025

0.04

0.075

0.1

colorectal

CH(CF₃)

HCT-116

HCT-15

COLO-320

SW480

colorectal

C(CH₃)₂

colorectal

CH(C₆H₅)

CH(4-FC₆H₄)

CH(4-ClC₆H₄)

CH(4-BrC₆H₄)

CH(4-OMeC₆H₄)

colorectal

RPMI-7951

WM983A

WM3211

WM1341D

WM278

WM239A

WM-793

451-LU

DND-1A

K562

melanoma

CML

0.3

ND

0.1

0.015

ND

28

0.1

ND

0.15

0.075

0.025

0.075

0.015

0.04

0.075

0.1

was selected and subcloned for resistance to camptothecin and has

cross-resistance to etoposide, dactinimycin, bleomycin, mitoxantrone,

doxorubicin, and daunorubicin. As shown in Figure 1B and Table 5,

compound 28 was active against both the parental and the camp-

tothecin resistant subclone, with the CEM/C2 clone being 2-fold

more sensitive than the parental. These studies clearly demonstrate

that this chemotype will not be a substrate for ABC transport proteins

and therefore be a suitable treatment option for tumors expressing

both classical and atypical MDR resistant markers.

ND

0.0025

0.004

0.003

ND

MOLT-4

Z138C

T-lymphoblastic: all

mantle cell lymphoma

hepatoma

GRANTA-519

Bel-7402

KB

Effects of 28 on Cell Cycle Progression of Normal and

Tumor Cells. We next examined the effect of these compounds

on normal and tumor cell cycle progression using FACS analysis.

Figure 2A shows the effect of 28 on the cell cycle progression of

normal diploid human fetal lung (HFL-1) and DU145 (prostate

cancer) cells (Figure 2B). The results of this study show that the

addition of the 28 to DU145 cells resulted in a block of their cell

cycle progression in G2/M phase of the cell cycle in a dose

nasopharyngeal

cervical

ND

0.07

0.1

HELA

ND

U937

lymphoma

multiple myeloma

ND

0.01

0.03

0.025

0.015

LP-1

0.003

U266

OPM-2

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ARTICLE

Table 4. Continued

(BT20) was xenografted into athymic nude mice. The animals

were treated with 200 mg/kg of 28 using Q2D ꢁ 20 schedules.

The animals were treated when the tumors were approximately

70 mm³in size. Figure 5A shows that an intraperitoneal (ip)

injection of 28 was able to significantly inhibit the growth of the

tumors. The tumors of vehicle treated mice, on average, in-

creased in volume over the 22 day period by 5-fold (70ꢀ

480 mm³), while the Q2D 28 treated tumors increased in volume

by only 2.5-fold (70ꢀ180 mm³). 28 was well tolerated at these

doses as determined by body weights and physical observations

(Figure 5B). These studies show that 28 is efficacious against

human tumor xenografts while showing no signs of toxicity at the

schedules tested under this study.

IC₅₀(μM)

cell line

tumor type

8p

28

RPMI-8266

HL-60

multiple myeloma

AML M3

0.003

ND

0.01

0.02

KG1a

AML M1

ND

0.03

HEL

AML M6

ND

0.05

Daudi

Burkitt’s lymphoma (B-cell)

PS-41

0.003

0.002

0.005

ND

0.15

Raji

0.075

>100

Namalwa

ﬁbroblast

endothelial

Hu-VEC

ND

’ CONCLUSION

In this communication, we describe the synthesis of a group of

(E)-styrylbenzylsulfones that induce apoptotic death of a wide

variety of human tumor cell lines at subnanomolar concentra-

tions while exhibiting relatively low toxicity to normal human

cells. Our studies show that the cytoxic activity of the (E)-

styrylbenzylsulfones is completely dependent on the nature and

position of the substituents on the two aromatic rings. These

structureꢀfunction studies show that a molecule with a benzyl

moiety having 3-amino and 4-methoxy groups and a styryl ring

with methoxy groups at 2-, 4-, and 6-positions showed optimum

biological activity (8p). Biological evaluation of the activity of

these compounds shows that these compounds are highly active

against a wide variety of human tumor cell lines including those

that are resistant to the activity of many of the currently used

chemotherapeutic agents.

The low toxicity proﬁle, both in vitro and in vivo, and their

potent tumor inhibitory activity as seen in nude mouse xenograft

assays point to the potential value of these compounds as safe

therapies for cancer, lacking many of the side eﬀects normally

associated with current chemotherapeutic agents. Recent studies

with 28 show that this compound altered the growth and cell

cycle status of mantle cell lymphoma cell lines and potently

inhibited the expression of several important proteins, including

cyclin D1, p-mTOR, and PI3-K.⁶Since 28 is highly eﬀective in

various combinations with conventional chemotherapy,⁶the lack

of overt hematotoxicity of this compound is beneﬁcial for testing

novel combinations for advanced cancers, including tumors

resistant to conventional chemotherapy. In addition, its safety

proﬁle seen with normal hematopoietic cells suggests that these

compounds have a potential use in in vitro purging of tumor cells

from patient bone marrow for use in autologous bone marrow

transplantation. Clinical studies in MDS (phase III) and pan-

creatic (phase II/III) patients currently underway will reveal the

best way to utilize this compound in cancer therapy.

Table 5. Evaluation of 8p and 28 against a Panel of Multi-

drug-Resistance Human Tumor Cell Lines

IC₅₀(μM)

cell line

MES-SA

MES-SA/DX5^a

tumor type

sarcoma

8p

28

0.004

0.01

0.1

resistant sarcoma

leukemic

0.1

CEM

0.1

CEM/C2^a

2008

resistant leukemic

ovarian

0.01

0.05

0.15

0.1

0.003

2008/17/4

resistant ovarian

0.003

^aThese cell lines constitute multidrug-resistance cell lines and show up-

regulation of MDR and in the case of CEM/C2, additional mutations in

the Topo-2 gene.^19,20

dependent manner. In addition, treatment of the tumor cells

resulted in an accumulation of cells containing subG1 content of

DNA, which is an indication of cell death. On the other hand,

treatment of normal diploid fibroblasts cells (HFL-1) with 28

resulted in a block of their cell cycle progression in the G₁and

G2/M phases of the cell cycle, without the corresponding

induction of cell death. We further investigated the selective

nature of cell killing by running studies aimed at studying the

activation of the apoptotic pathway. Tumor and normal cell lines

were then treated with 28 and activation of apoptotic pathways,

as judged by PARP [poly(ADP-ribose) polymerase-1] cleavage,²¹is

shown in (Figure 3). Treatment of 28 selectively induced PARP

cleavage in the tumor cell line, while there was no PARP cleavage

in the treated normal cell line. Since it is well-known that PARP

cleavage is the result of activation of caspase 3, we further inves-

tigated the activation of the apoptotic pathway by looking at cellular

viability together with the activity of caspases 3 and 7. A dual viability

and caspase activation assay was performed in A549 cells treated

with 28 which showed the concentrations at which 28 activated the

apoptotic machinery to induce a cytotoxic effect (Figure 4). These

data show that treatment with 0.25 μM induced loss of viability with

the concomitant induction of caspase 3/7 within 24 h of tumor cell

treatment. Taken together, these data show that 28 selectively

induces G2/M cell cycle block with the induction of apoptosis in

tumor cells.

’ EXPERIMENTAL SECTION

Chemistry: General Experimental Procedures. All reactions

requiring anhydrous conditions were run under an atmosphere of

dry nitrogen, and solvents were dried using standard procedures.

Reagents and solvents were obtained in the highest available purity

and used without further purification unless indicated. Reactions were

monitored by thin layer chromatography (TLC) on preloaded silica gel

60 glass-backed plates with F²⁵⁴plates as the indicator (Sigma-Aldrich),

developed using mobile phases of varying compositions of ethyl acetate/

hexane and methanol/chloroform, and the spots were visualized by a UV

indicator. Column chromatography was performed with Merck 70ꢀ320

In Vivo Antitumor Effect of 28. In order to determine in vivo

efficacy, we utilized the nude mouse model system. A highly

aggressive human estrogen negative breast carcinoma cell line

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Figure 1. 28 is active against multidrug resistant cells (MDR). (A, left) The MDR-1 positive MES-SA/DX5 clone and an atypical MDR resistant to

topoisomerase inhibitors CEM/C2 (B, right) and their respective sensitive parental controls were plated into six-well dishes and treated with increasing

concentrations of 28 or a representative chemotherapeutic agent, paclitaxel (PTX) or camptothecin (CPT), for 96 h. The number of viable cells from

duplicate plates was determined by trypan blue exclusion. As expected, the parentals are sensitive to both 28 and the chemotherapeutic agent, but while

the resistant clones are over 500-fold resistant to PTX or CPT, they remain sensitive to the cytotoxic action of 28.

Figure 2. 28 selectively induces mitotic G2/M arrest and apoptosis in cancer cells. (A) Normal diploid ﬁbroblasts HFL-1 cells or (B) human prostate

cells (DU145) were treated with increasing concentrations of 28 and incubated in medium containing 10% fetal bovine serum for 24 h. The cells were

ﬁxed, stained with propidium iodide, and then subjected to FACS analysis and analyzed for their DNA content. 28 induces a mitotic arrest and induction

of a subG1 population (blue) indicative of apoptosis in the cancer cell line while only inducing a small mitotic block in the normal cells.

mesh silica gel. Melting points were determined using an Electrothermal

MEL-Temp 3.0 micro melting point apparatus with a Fluka 51 K/J

electronic thermometer and are uncorrected. Nuclear magnetic reso-

nance spectra for proton (¹H NMR) were recorded on Bruker Avance

III (300 MHz), Varian INOVA (400 MHz), and GE (500 MHz)

spectrometers. ¹³C NMR spectra (75 MHz) were obtained on a Bruker

Avance III 300 MHz spectrometer. The chemical shift values are

expressed in ppm (parts per million) relative to tetramethylsilane as

an internal standard: s, singlet; d, doublet; dd, doublet of a doublet; t,

triplet; m, multiplet; br s, broad singlet. Coupling constants (J) were

measured in hertz (Hz). Compounds purity was determined by ele-

mental analyses (0.4%) or LC/MS analysis and was confirmed to be

>95% for all compounds. All LC/MS data were gathered on an Agilent

1200 LC with Agilent 6410 triple quadrupole mass spectrometer

detectors. The compound solution was infused into the electron spray

ionization source operating in positive and negative modes.

General Procedure for the Preparation of Benzyl Bro-

mides (2). Method A (Scheme 2). To a well stirred solution of

substituted toluene 1 (40 mmol) in carbon tetrachloride (150 mL) were

added benzoyl peroxide (4.0 mmol) andN-bromosuccinimide (48 mmol).

The reaction mixture was heated at reflux for 18 h. After completion of

the reaction (TLC monitoring, hexane/ethyl acetate, 9:1, on silica gel

plate), the contents were cooled to room temperature, water was added,

and the product was isolated by extraction with dichloromethane. The

organic phase was washed with water, brine, dried over anhydrous

sodium sulfate, filtered, and concentrated under vacuum to obtain the

desired crude product 2. The pure compound 2 was obtained following

purification by silica gel flash column chromatography (hexane/ethyl

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acetate, 9:1). The following benzyl bromides 2 were prepared using the

above procedure.

(d, J = 8.4 Hz, 1H, ArꢀH), 7.89 (d, J = 2.0 Hz, 1H, ArꢀH). HRMS: m/z

calcd [M + H] 295.8667; found 295.8662. Anal. (C₇H₅Br₂NO₂) C, H, N.

4-Chloro-3-nitrobenzyl Bromide (2d). Radical benzylic bromi-

nation of 4-chloro-3-nitrotoluene yielded the corresponding 4-chloro-

3-nitrobenzyl bromide. The yield of this reaction was 87%, giving a pale

yellow liquid with a boiling point of 115ꢀ120 °C (0.25 mmHg). ¹H NMR

(CDCl₃, 300 MHz): δ 4.60 (s, 2H, CH₂), 7.42 (dd, J = 8.4, 2.4 Hz, 1H,

ArꢀH), 7.68 (d, J = 8.4 Hz, 1H, ArꢀH), 7.86 (d, J = 2.4 Hz, 1H, ArꢀH).

HRMS: m/z calcd [M + H] 249.9192; found 249.9186. Anal.

(C₇H₅BrClNO₂) C, H, N.

4-Methoxy-3-nitrobenzyl Bromide (2a). Radical benzylic bro-

mination of 4-methyl-2-nitroanisole yielded the corresponding 4-meth-

oxy-3-nitrobenzyl bromide. The yield of this reaction was 68%, giving a

light yellow solid with a melting point of 106ꢀ108 °C. ¹H NMR

(CDCl₃, 300 MHz): δ 3.97 (s, 3H, OCH₃), 4.47 (s, 2H, CH₂), 7.08 (d,

J = 8.7 Hz, 1H, ArꢀH), 7.58 (dd, J = 8.7, 2.1 Hz, 1H, ArꢀH), 7.89 (d, J =

2.4 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 245.9688; found

245.9682. Anal. (C₈H₈BrNO₃) C, H, N.

4-Methoxy-2-nitrobenzyl Bromide (2b). Radical benzylic bro-

mination of 4-methyl-3-nitroanisole yielded the corresponding 4-meth-

oxy-2-nitrobenzyl bromide. The yield of this reaction was 65%, giving a

yellow solid with a melting point of 60ꢀ62 °C. ¹H NMR (CDCl₃, 300

MHz): δ 3.89 (s, 3H, OCH₃), 4.81 (s, 2H, CH₂), 7.14 (dd, J = 8.4, 2.7

Hz, 1H, ArꢀH), 7.48 (d, J = 8.7 Hz, 1H, ArꢀH), 7.56 (d, J = 2.7 Hz, 1H,

ArꢀH). HRMS: m/z calcd [M + H] 245.9688; found 245.9681. Anal.

(C₈H₈BrNO₃) C, H, N.

4-Bromo-3-nitrobenzyl Bromide (2c). Radical benzylic bromi-

nation of 4-bromo-3-nitrotoluene yielded the corresponding 4-bromo-

3-nitrobenzyl bromide. The yield of this reaction was 78%, giving a

yellow solid with a melting point of 62ꢀ63 °C. ¹H NMR (CDCl₃, 500

MHz): δ 4.45 (s, 2H, CH₂), 7.47 (dd, J = 8.4, 2.4 Hz, 1H, ArꢀH), 7.73

4-Chloro-2-nitrobenzyl Bromide (2e). Radical benzylic bromi-

nation of 4-chloro-2-nitrotoluene yielded the corresponding 4-chloro-2-

nitrobenzyl bromide. The yield of this reaction was 69%, giving a pale

yellow solid with a melting point of 40ꢀ42 °C. ¹H NMR (CDCl₃, 500

MHz): δ 4.80 (s, 2H, CH₂), 7.54 (d, J = 8.3 Hz, 1H, ArꢀH), 7.61 (dd, J =

8.3, 2.1 Hz, ArꢀH), 8.07 (d, J = 2.1 Hz, ArꢀH). HRMS: m/z calcd [M +

H] 249.9192; found 249.9189. Anal. (C₇H₅BrClNO₂) C, H, N.

4-Fluoro-3-nitrobenzyl Bromide (2f). Radical benzylic bromi-

nation of 4-fluoro-3-nitrotoluene yielded the corresponding 4-fluoro-3-

nitrobenzyl bromide. The yield of this reaction was 84%, giving a yellow

solid with a melting point of 50ꢀ52 °C. ¹H NMR (CDCl₃, 300 MHz): δ

4.49 (s, 2H, CH₂), 7.32(dd, J = 8.7, 1.8 Hz, 1H, ArꢀH), 7.66ꢀ7.71 (m,

1H, ArꢀH), 8.11 (dd, J = 6.9, 2.4 Hz, 1H, ArꢀH). HRMS: m/z calcd

[M + H] 233.9488; found 233.9481. Anal. (C₇H₅BrFNO₂) C, H, N.

General Procedure for the Preparation of Benzyl Alcohols

(4). Method B (Scheme 2). Sodium borohydride (20 mmol) was

added in small portions to an ice-cold solution of aldehyde 3 (20 mmol)

in dry methanol (100 mL) with stirring. The reaction mixture was left at

0ꢀ5 °C for 1 h. After completion of the reaction (TLC monitoring,

hexane/ethyl acetate, 9:1 on silica gel plate), the solvent was evaporated.

Then the chloroform (100 mL) was added to the residue obtained. The

organic layer was washed with 5% sodium bicarbonate (50 mL) and

water (75 mL), dried over anhydrous sodium sulfate, filtered, and

concentrated under vacuum to obtain the desired crude product 4.

The pure compound 4 was obtained after recrystallization from ben-

zene/hexane. The following benzyl alcohols 4 were prepared using the

above procedure.

Figure 3. DU145 and HFL-1 (normal human ﬁbroblasts) cells were

treated with increasing concentrations of 28 or DMSO (vehicle) for

48 h. Cells were harvested, and total protein was resolved by 10%

SDSꢀPAGE, Western blotted and hybridized to anti-PARP (Cell

Signaling no. 542) antibody. The blot was then washed and treated

with anti-rabbit secondary labeled with infrared dye (700) and scanned

using the Odyssey scanner (LiCor).

4-Methoxy-3-nitrobenzyl Alcohol (4a). Reduction of 4-meth-

oxy-3-nitrobenzaldehyde yielded the corresponding 4-methoxy-3-nitro-

benzyl alcohol. The yield of this reaction was 78%, giving a yellow solid

Figure 4. Cellular viability and the activity of caspases 3/7 were assayed concomitantly in A549 cells treated with 28 for 24 h (n = 3). Viability was

measured after addition of CellTiter-Blue reagent (Promega Corporation). Caspase 3/7 activity was measured after addition of Caspase-Glo 3/7 reagent

(Promega Corporation). Fluorescence and luminescence were plotted together to reveal the concentrations at which 28 activated the apoptotic

machinery to induce a cytotoxic eﬀect.

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Figure 5. Human breast cancer cells (BT20) were injected subcutaneously into female nude mice. The mice were grouped and then treated with

200 mg/kg 28 by intraperitoneal injections Q2D ꢁ 20 formulated in phosphate buﬀered saline (PBS) or placebo (PBS) alone. Tumor volumes (A, left)

and body weights (B, right) were determined, and the average ((SEM) for each group (N = 9) was determined and plotted. 28 signiﬁcantly inhibited the

in vivo growth of tumors. Placebo tumors doubled in 6 days, while it took over 18 days for the 28 treated tumors to double in size. There was no sign of

toxicity or body weight loss.

with a melting point of 69ꢀ70 °C. ¹H NMR (CDCl₃, 300 MHz): δ 2.65

(br s, 1H, OH), 3.96 (s, 3H, OCH₃), 4.64 (s, 2H, CH₂), 7.12 (d, J = 8.4

Hz, 1H, ArꢀH), 7.61 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 7.84 (d, J = 2.1

Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 184.0532; found 184.0522.

Anal. (C₈H₉NO₄) C, H, N.

alcohol. The yield of this reaction was 77%, giving a white solid with a

melting point of 123ꢀ125 °C. ¹H NMR (CDCl₃, 300 MHz): δ 2.31 (br s,

1H, OH), 3.97 (s, 3H, OCH₃), 4.73 (d, J = 4.1 Hz, 2H, CH₂), 6.93 (d, J =

9.0 Hz, 1H, ArꢀH), 8.19 (dd, J = 9.0, 2.8 Hz, 1H, ArꢀH), 8.26 (d, J = 2.8

Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 184.0532; found 184.0521.

Anal. (C₈H₉NO₄) C, H, N.

4-Methoxy-2-nitrobenzyl Alcohol (4b). Reduction of 4-meth-

oxy-2-nitrobenzaldehyde yielded the corresponding 4-methoxy-2-nitro-

benzyl alcohol. The yield of this reaction was 73%, giving colorless

General Procedure for the Preparation of Benzyl Bromides

(2). Method B (Scheme 2). Phosphorus tribromide (24 mmol) was

added to a stirred solution of alcohol 4 (20 mmol) and toluene (40 mL) at

40 °C. The solution was heated to 100 °C for 30 min, and after completion

of the reaction (TLC monitoring, hexane/ethyl acetate, 9:1 on silica gel

plate), the contents were cooled to ambient temperature. The liquid was

decanted and washed with water (2 ꢁ 50 mL) and brine (50 mL). The

combined aqueous washes were extracted with ether (2 ꢁ 75 mL), and

the combined organic fractions were dried and evaporated to give crude

residue. The residue was dissolved in ether (100 mL) and washed with

water (2 ꢁ 50 mL) and brine (50 mL). The organic phase was dried over

anhydrous sodium sulfate, filtered, and evaporated to get the crude product

2, which on silica gel flash chromatography (hexane/ethyl acetate, 9:1)

resulted in pure benzyl bromide 2. The following benzyl bromides 2 were

prepared using the above procedure.

4-Methoxy-3-nitrobenzyl Bromide (2a). Bromination of the

alcohol 4a with phosphorus tribromide yielded the corresponding 2a.

The yield of this reaction was 52%. The analytical data are in accord with

above method A procedure.

4-Methoxy-2-nitrobenzyl Bromide (2b). Bromination of the

alcohol 4b with phosphorus tribromide yielded the corresponding 2b.

The yield of this reaction was 58%. The analytical data are in accord with

above method A procedure.

4-Bromo-3-nitrobenzyl Bromide (2c). Bromination of the

alcohol 4c with phosphorus tribromide yielded the corresponding 2c.

The yield of this reaction was 62%. The analytical data are in accord with

above method A procedure.

4-Chloro-3-nitrobenzyl Bromide (2d). Bromination of the

alcohol 4d with phosphorus tribromide yielded the corresponding 2d.

The yield of this reaction was 59%. The analytical data are in accord with

above method A procedure.

4-Chloro-2-nitrobenzyl Bromide (2e). Bromination of the

alcohol 4e with phosphorus tribromide yielded the corresponding 2e.

The yield of this reaction was 63%. The analytical data are in accord with

above method A procedure.

1

needles with a melting point of 80ꢀ82 °C. H NMR (CDCl₃, 400

MHz): δ 2.53 (t, J = 6.8 Hz, 1H, OH), 3.88 (s, 3H, OCH₃), 4.86 (d, J =

6.7 Hz, 2H, CH₂), 7.19 (dd, J = 8.6, 2.7 Hz, 1H, ArꢀH), 7.58 (d, J = 8.6

Hz, 1H, ArꢀH), 7.60 (d, J = 2.7 Hz, 1H, ArꢀH). HRMS: m/z calcd

[M + H] 184.0532; found 184.0523. Anal. (C₈H₉NO₄) C, H, N.

4-Bromo-3-nitrobenzyl Alcohol (4c). Reduction of 4-bromo-3-

nitrobenzaldehyde yielded the corresponding 4-bromo-3-nitrobenzyl

alcohol. The yield of this reaction was 89%, giving a yellow solid with a

melting point of 60ꢀ62 °C. ¹H NMR (CDCl₃, 300 MHz): δ 2.61 (br s,

1H, OH), 4.71 (s, 2H, CH₂), 7.38 (d, J = 8.5 Hz, 1H, ArꢀH), 7.67 (d, J =

8.3 Hz, 1H, ArꢀH), 7.81 (s, 1H, ArꢀH). HRMS: m/z calcd [M + H]

231.9531; found 231.9533. Anal. (C₇H₆BrNO₃) C, H, N.

4-Chloro-3-nitrobenzyl Alcohol (4d). Reduction of 4-chloro-3-

nitrobenzaldehyde yielded the corresponding 4-chloro-3-nitrobenzyl

alcohol. The yield of this reaction was 81%, giving a yellow solid with

a melting point of 63ꢀ65 °C. ¹H NMR (CDCl₃, 300 MHz): δ 2.63 (br s,

1H, OH), 4.49 (s, 2H, CH₂), 7.48 (dd, J = 8.0, 2.0 Hz, 1H, ArꢀH), 7.53

(d, J = 8.0 Hz, 1H, ArꢀH), 7.85 (d, J = 2.0 Hz, 1H, ArꢀH). HRMS: m/z

calcd [M + H] 188.0036; found 188.0031. Anal. (C₇H₆ClNO₃) C, H, N.

4-Chloro-2-nitrobenzyl Alcohol (4e). Reduction of 4-chloro-2-

nitrobenzaldehyde yielded the corresponding 4-chloro-2-nitrobenzyl

alcohol. The yield of this reaction was 78%, giving a pale yellow solid

with a melting point of 89ꢀ91 °C. ¹H NMR (CDCl₃, 500 MHz): δ 2.91

(br s, 1H, OH), 4.92 (s, 2H, CH₂), 7.47(d, J = 8.7 Hz, 1H, ArꢀH), 7.72

(dd, J = 8.7, 2.4 Hz, ArꢀH), 8.09 (d, J = 2.1 Hz, ArꢀH). HRMS: m/z

calcd [M + H] 188.0036; found 188.0029. Anal. (C₇H₆ClNO₃) C, H, N.

4-Fluoro-3-nitrobenzyl Alcohol (4f). Reduction of 4-fluoro-

3-nitrobenzaldehyde yielded the corresponding 4-fluoro-3-nitrobenzyl

alcohol. The yield of this reaction was 91%, giving a colorless solid

with a melting point of 42ꢀ44 °C. ¹H NMR (CDCl₃, 300 MHz): δ 2.69

(br s, 1H, OH), 4.76 (s, 2H, CH₂), 7.28 (dd, J = 10.7, 8.6 Hz, 1H,

ArꢀH), 7.61ꢀ7.66 (m, 1H, ArꢀH), 8.06 (dd, J = 10.7, 8.6 Hz, 1H,

ArꢀH). HRMS: m/z calcd [M + H] 172.0732; found 172.0713. Anal.

(C₇H₆FNO₃) C, H, N.

6-Methoxy-3-nitrobenzyl Alcohol (4g). Reduction of 6-methoxy-

3-nitrobenzaldehyde yielded the corresponding 6-methoxy-3-nitrobenzyl

4-Fluoro-3-nitrobenzyl Bromide (2f). Bromination of the

alcohol 4f with phosphorus tribromide yielded the corresponding 2f.

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The yield of this reaction was 54%. The analytical data are in accord with

above method A procedure.

HRMS: m/z calcd [M ꢀ H] 244.0158; found 244.0143. Anal.

(C₉H₈FNO₄S) C, H, N.

6-Methoxy-3-nitrobenzyl Bromide (2g). Bromination of the

6-methoxy-3-nitrobenzyl alcohol 4g with phosphorus tribromide yielded

the corresponding 6-methoxy-3-nitrobenzyl bromide 2g. The yield of this

reaction was 55%, giving a white solid with a melting point of 76ꢀ78 °C. ¹H

NMR (CDCl₃, 300 MHz): δ 3.97 (s, 3H, OCH₃), 4.73 (s, 2H, CH₂), 6.93

(d, J=9.0Hz, 1H, ArꢀH), 8.19 (dd, J=9.0, 2.8Hz, 1H, ArꢀH), 8.26 (d, J=

2.8 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 245.9688; found

245.9674. Anal. (C₈H₈BrNO₃) C, H, N.

6-Methoxy-3-nitrobenzylthioacetic Acid (5g). Condensation

of 6-methoxy-3-nitrobenzyl bromide 2g with mercaptoacetic acid yielded

the corresponding 6-methoxy-3-nitrobenzylthioacetic acid. The yield of

this reaction was 86%, giving a pale yellow solid with a melting point of

76ꢀ78 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.08 (s, 2H, ꢀSCH₂), 3.74

(s, 2H, CH₂S), 3.98 (s, 3H, OCH₃), 7.29 (d, J = 9.0 Hz, 1H, ArꢀH), 8.21

(m, 1H, ArꢀH), 8.30 (d, J = 3.0 Hz, 1H, ArꢀH). HRMS: m/z calcd [M ꢀ

H] 256.0358; found 256.0349. Anal. (C₁₀H₁₁NO₅S) C, H, N.

General Procedure for the Preparation of Benzylthioacetic

Acids (5). (Scheme 1). The following benzylthioacetic acids were pre-

pared according to the procedure reported in the literature.⁷

General Procedure for the Preparation of Benzylsulfony-

lacetic Acids (6). (Scheme 1). The following benzylsulfonylacetic

acids were prepared according to the procedure reported in the

literature.⁷

4-Methoxy-3-nitrobenzylsulfonylacetic Acid (6a). Oxida-

tion of 4-methoxy-3-nitrobenzylthioacetic acid 5a with 30% hydrogen

peroxide yielded the corresponding 4-methoxy-3-nitrobenzylsulfonyla-

cetic acid. The yield of this reaction was 51%, giving a yellow solid with a

melting point of 137ꢀ139 °C. ¹H NMR (DMSO-d₆, 500 MHz): δ 3.79

(s, 2H, SCH₂), 3.86 (s, 3H, OCH₃), 4.48 (s, 2H, CH₂S), 7.08 (d, J = 9.0

Hz, 1H, ArꢀH), 7.59 (dd, J = 8.5, 2.5 Hz, 1H, ArꢀH), 7.86 (d, J = 2.5

Hz, 1H, ArꢀH), 13.42 (br s, 1H, COOH). HRMS: m/z calcd [M ꢀ H]

288.0256; found 288.0251. Anal. (C₁₀H₁₁NO₇S) C, H, N.

4-Methoxy-2-nitrobenzylsulfonylacetic Acid (6b). Oxida-

tion of 4-methoxy-2-nitrobenzylthioacetic acid 5b with 30% hydrogen

peroxide yielded the corresponding 4-methoxy-2-nitrobenzylsulfonyla-

cetic acid. The yield of this reaction was 63%, giving a pale yellow solid

with a melting point of 158ꢀ161 °C. ¹H NMR (DMSO-d₆, 300 MHz):

δ 3.87 (s, 3H, OCH₃), 4.27 (s, 2H, ꢀSCH₂), 5.02 (s, 2H, CH₂S), 7.36

(dd, J = 8.4, 2.7 Hz, 1H, ArꢀH), 7.53 (d, J = 8.7 Hz, 1H, ArꢀH), 7.57 (d,

J = 2.4 Hz, 1H, ArꢀH), 13.53 (br s, 1H, COOH). HRMS: m/z calcd

[M ꢀ H] 288.0256; found 288.0244. Anal. (C₁₀H₁₁NO₇S) C, H, N.

4-Bromo-3-nitrobenzylsulfonylacetic Acid (6c). Oxidation

of 4-bromo-3-nitrobenzylthioacetic acid 5c with 30% hydrogen peroxide

yielded the corresponding 4-bromo-3-nitrobenzylsulfonylacetic acid.

The yield of this reaction was 62%, giving a yellow solid with a melting

point of 172ꢀ174 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 4.30 (s, 2H,

ꢀSCH₂), 4.81(s, 2H, CH₂S), 7.65 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 8.01

(d, J = 8.4 Hz, 1H, ArꢀH), 8.08 (d, J = 1.8 Hz, 1H, ArꢀH), 13.58 (br s,

1H, COOH). HRMS: m/z calcd [M ꢀ H] 335.9256; found 335.9252.

Anal. (C₉H₈BrNO₆S) C, H, N.

4-Chloro-3-nitrobenzylsulfonylacetic Acid (6d). Oxida-

tion of 4-chloro-3-nitrobenzylthioacetic acid 5d with 30% hydrogen

peroxide yielded the corresponding 4-chloro-3-nitrobenzylsulfonylace-

tic acid. The yield of this reaction was 68%, giving a yellow solid with a

melting point of 161ꢀ163 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 4.27

(s, 2H, ꢀSCH₂), 4.77 (s, 2H, CH₂S), 7.61 (dd, J = 8.4, 2.1 Hz, 1H,

ArꢀH), 7.98 (d, J = 8.4 Hz, 1H, ArꢀH), 8.04 (d, J = 1.8 Hz, 1H, ArꢀH),

13.60 (br s, 1H, COOH). HRMS: m/z calcd [M ꢀ H] 291.9761; found

291.9755. Anal. (C₉H₈ClNO₆S) C, H, N.

4-Methoxy-3-nitrobenzylthioacetic Acid (5a). Condensation

of 4-methoxy-3-nitrobenzyl bromide 2a with mercaptoacetic acid yielded

the corresponding 4-methoxy-3-nitrobenzylthioacetic acid. The yield of this

reaction was 96%, giving a pale yellow solid with a melting point of

129ꢀ133 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 3.12 (s, 2H, ꢀSCH₂),

3.82 (s, 2H, CH₂S), 3.90 (s, 3H, OCH₃), 7.32 (d, J = 8.4 Hz, 1H, ArꢀH),

7.59 (dd, J = 8.7, 2.4 Hz, 1H, ArꢀH), 7.82 (d, J = 2.1 Hz, 1H, ArꢀH), 12.60

(br s, 1H, COOH). HRMS: m/z calcd [M ꢀ H] 256.0358; found

256.0346. Anal. (C₁₀H₁₁NO₅S) C, H, N.

4-Methoxy-2-nitrobenzylthioacetic Acid (5b). Condensa-

tion of 4-methoxy-2-nitrobenzyl bromide 2b with mercaptoacetic

acid yielded the corresponding 4-methoxy-2-nitrobenzylthioacetic acid.

The yield of this reaction was 86%, giving a light yellow solid with a

1

melting point of 86ꢀ88 °C. H NMR (CDCl₃, 300 MHz): δ 3.05

(s, 2H, ꢀSCH₂), 3.81 (s, 3H, OCH₃), 4.11 (s, 2H, CH₂S), 7.04 (dd, J =

8.4, 2.7 Hz, 1H, ArꢀH), 7.33 (d, J = 8.7 Hz, 1H, ArꢀH), 7.49 (d, J = 2.7

Hz, 1H, ArꢀH), 12.41 (br s, 1H, COOH). HRMS: m/z calcd [M ꢀ H]

256.0358; found 256.0349. Anal. (C₁₀H₁₁NO₅S) C, H, N.

4-Bromo-3-nitrobenzylthioacetic Acid (5c). Condensation of

4-bromo-3-nitrobenzyl bromide 2c with mercaptoacetic acid yielded the

corresponding 4-bromo-3-nitrobenzylthioacetic acid. The yield of this

reaction was 92%, giving a yellow solid with a melting point of

133ꢀ135 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 3.15 (s, 2H, ꢀSCH₂),

4.05 (s, 2H, CH₂S), 7.51 (d, J = 8.1, Hz, 1H, ArꢀH), 7.91 (dd, J = 8.4, 2.1

Hz, 1H, ArꢀH), 8.21 (d, J = 1.8 Hz, 1H, ArꢀH), 12.60 (br s, 1H,

COOH). HRMS: m/z calcd [M ꢀ H] 303.9357; found 303.9348. Anal.

(C₉H₈BrNO₄S) C, H, N.

4-Chloro-3-nitrobenzylthioacetic Acid (5d). Condensation of

4-chloro-3-nitrobenzyl bromide 2d with mercaptoacetic acid yielded the

corresponding 4-chloro-3-nitrobenzylthioacetic acid. The yield of this

reaction was 90%, giving a yellow solid 110ꢀ114 °C. ¹H NMR (DMSO-

d₆, 300 MHz): δ 3.15 (s, 2H, ꢀSCH₂), 3.86 (s, 2H, CH₂S), 7.42 (d, J =

8.1 Hz, 1H, ArꢀH), 7.69 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 7.92 (d, J = 1.8

Hz, 1H, ArꢀH), 12.70 (br s, 1H, COOH). HRMS: m/z calcd [M ꢀ H]

259.9863; found 259.9856. Anal. (C₉H₈ClNO₄S) C, H, N.

4-Chloro-2-nitrobenzylthioacetic Acid (5e). Condensation

of 4-chloro-2-nitrobenzyl bromide 2e with mercaptoacetic acid yielded

the corresponding 4-chloro-2-nitrobenzylthioacetic acid. The yield of

this reaction was 92%, giving a yellow solid with a melting point of

103ꢀ105 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.13 (s, 2H, ꢀSCH₂),

4.22 (s, 2H, CH₂S), 7.47 (d, J = 8.1 Hz, 1H, ArꢀH), 7.57 (dd, J = 8.1, 2.1

Hz, 1H, ArꢀH), 8.04 (d, J = 2.1 Hz, 1H, ArꢀH), 12.73 (br s, 1H,

COOH). HRMS: m/z calcd [M ꢀ H] 259.9863; found 259.9851. Anal.

(C₉H₈ClNO₄S) C, H, N.

4-Fluoro-3-nitrobenzylthioacetic Acid (5f). Condensation of

4-fluoro-2-nitrobenzyl bromide 2f with mercaptoacetic acid yielded the

corresponding 4-fluoro-3-nitrobenzylthioacetic acid. The yield of this

reaction was 88%, giving a pale yellow solid with a melting point of

74ꢀ76 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.12 (s, 2H, ꢀSCH₂), 3.92

(s, 2H, CH₂S), 7.28 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 7.61ꢀ7.68 (m, 1H,

ArꢀH), 8.07 (dd, J = 6.9, 2.4 Hz, 1H, ArꢀH), 12.61 (br s, 1H, COOH).

4-Chloro-2-nitrobenzylsulfonylacetic Acid (6e). Oxidation

of 4-chloro-2-nitrobenzylthioacetic acid 5e with 30% hydrogen peroxide

yielded the corresponding 4-chloro-2-nitrobenzylsulfonylacetic acid. The

yield of this reaction was 64%, giving a yellow solid with a melting point of

113ꢀ115 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 4.36 (s, 2H, ꢀSCH₂),

5.00 (s, 2H, CH₂S), 7.65 (dd, J = 8.4, 3.0 Hz, 1H, ArꢀH), 7.80 (d, J =

8.4 Hz, 1H, ArꢀH), 8.18 (d, J = 1.8 Hz, 1H, ArꢀH), 13.70 (br s, 1H,

COOH). HRMS: m/z calcd [M ꢀ H] 291.9761; found 291.9753. Anal.

(C₉H₈ClNO₆S) C, H, N.

4-Fluoro-3-nitrobenzylsulfonylacetic Acid (6f). Oxidation of

4-fluoro-3-nitrobenzylthioacetic acid 5f with 30% hydrogen peroxide

yielded the corresponding 4-fluoro-3-nitrobenzylsulfonylacetic acid.

The yield of this reaction was 69%, giving a yellow solid with a melting

point of 120ꢀ122 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 4.24 (s, 2H,

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ꢀSCH₂), 4.75 (s, 2H, CH₂S), 7.64 (dd, J = 8.4, 2.7 Hz, 1H, ArꢀH),

7.79ꢀ7.84 (m, 1H, ArꢀH), 8.20 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 13.40

(br s, 1H, COOH). HRMS: m/z calcd [M ꢀ H] 276.0056; found

276.0046. Anal. (C₉H₈FNO₆S) C, H, N.

6-Methoxy-3-nitrobenzylsulfonylacetic Acid (6g). Oxida-

tion of 6-methoxy-3-nitrobenzylthioacetic acid 5g with 30% hydrogen

peroxide yielded the corresponding 6-methoxy-3-nitrobenzylsulfonyla-

cetic acid. The yield of this reaction was 67%, giving a pale yellow solid

with a melting point of 166ꢀ168 °C. ¹H NMR (DMSO-d₆, 300 MHz):

δ 3.93 (s, 3H, OCH₃), 4.28 (s, 2H, ꢀSCH₂), 4.79 (s, 2H, CH₂S), 7.30

(d, J = 9.0 Hz, 1H, ArꢀH), 8.26ꢀ8.28 (m, 1H, ArꢀH), 8.31 (d, J = 3.0

Hz, 1H, ArꢀH), 13.40 (br s, 1H, COOH). HRMS: m/z calcd [M ꢀ H]

288.0256; found 288.0248. Anal. (C₁₀H₁₁NO₇S) C, H, N.

(d, J = 2.0 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 364.0777; found

364.0765. Anal. (C₁₇H₁₇NO₆S) C, H, N.

(E)-4⁰-Methoxystyryl-4-methoxy-3-aminobenzylsulfone (8d).

The title compound was obtained by the reduction of (E)-4⁰-methoxystyryl-

4-methoxy-3-nitrobenzylsulfone 8c following the procedure as described for

compound 8b. Yield, 49%; yellow solid, mp 152ꢀ154 °C. ¹HNMR(CDCl₃,

300 MHz): δ 3.89 (s, 3H, OCH₃), 3.99 (s, 3H, OCH₃), 4.27 (s, 2H, CH₂),

6.92ꢀ6.99 (m, 2H, ArꢀH), 7.01 (d, J = 15.6 Hz, 1H, dCH), 7.12 (d, J = 8.7

Hz, 1H, ArꢀH), 7.35ꢀ7.45 (m, 2H, ArꢀH), 7.62 (d, J = 15.6 Hz, 1H,

CHd), 7.63 (dd, J = 8.7, 2.4 Hz, 1H, ArꢀH), 7.87 (d, J = 2.4 Hz, 1H,

ArꢀH). HRMS: m/z calcd [M + H] 334.1035; found 334.1068. Anal.

(C₁₇H₁₉NO₄S) C, H, N.

(E)-2⁰,6⁰-Dimethoxystyryl-4-methoxy-3-nitrobenzylsulfone

(8e). The title compound was obtained from 4-methoxy-3-nitrobenzyl-

sulfonylacetic acid 6a and 2,6-dimethoxybenzaldehyde following the

procedure as described in method A. Yield, 53%; yellow solid, mp

188ꢀ190 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.78 (s, 6H, 2 ꢁ

OCH₃), 3.90 (s, 3H, OCH₃), 4.17 (s, 2H, CH₂), 6.48 (d, J = 8.7 Hz,

2H, ArꢀH), 7.03 (d, J = 8.7 Hz, 1H, ArꢀH), 7.13 (d, J = 15.6 Hz,

1H, dCH), 7.26 (t, J = 8.4 Hz, 1H, ArꢀH), 7.56 (dd, J = 8.7, 2.4 Hz,

1H, ArꢀH), 7.82 (d, J = 2.4 Hz, 1H, ArꢀH), 7.84 (d, J = 15.6 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 394.0882; found 394.0889. Anal.

(C₁₈H₁₉NO₇S) C, H, N.

(E)-2⁰,6⁰-Dimethoxystyryl-4-methoxy-2-nitrobenzylsulfone

(8f). The title compound was obtained from 4-methoxy-2-nitrobenzyl-

sulfonylacetic acid 6b and 2,6-dimethoxybenzaldehyde following the

procedure as described in method A. Yield, 52%; yellow solid, mp

176ꢀ178 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.78 (s, 6H, 2 ꢁ OCH₃),

3.80 (s, 3H, OCH₃), 4.74 (s, 2H, CH₂), 6.47 (d, J = 8.7 Hz, 2H, ArꢀH),

7.09 (dd, J= 8.4, 2.7 Hz, 1H, ArꢀH), 7.16(d, J =15.6Hz, 1H,dCH), 7.25

(t, J = 8.4 Hz, 1H, ArꢀH), 7.45 (m, 2H, ArꢀH), 7.72 (d, J = 15.6 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 394.0882; found 394.0869. Anal.

(C₁₈H₁₉NO₇S) C, H, N.

(E)-2⁰,4⁰-Dimethoxystyryl-4-methoxy-3-aminobenzylsul-

fone (8g). Step 1: (E)-2⁰,4⁰-Dimethoxystyryl-4-methoxy-3-

nitrobenzylsulfone. The condensation of 4-methoxy-3-nitroben-

zylsulfonylacetic acid 6a with 2,4-dimethoxybenzaldehyde following

the procedure as described in method A resulted in the desired product (E)-

2⁰,4⁰-dimethoxystyryl-4-methoxy-3-nitrobenzylsulfone. Yield, 50%; yellow

solid, mp 148ꢀ150 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.78 (s, 6H, 2 ꢁ

OCH₃), 3.90 (s, 3H, OCH₃), 4.17 (s, 2H, CH₂), 6.48 (d, J = 8.7 Hz, 2H,

ArꢀH), 7.03 (d, J = 8.7 Hz, 1H, ArꢀH), 7.13 (d, J = 15.6 Hz, 1H, dCH),

7.26 (t, J = 8.4 Hz, 1H, ArꢀH), 7.56 (dd, J = 8.7, 2.4 Hz, 1H, ArꢀH), 7.84

(d, J = 2.4 Hz, 1H, ArꢀH), 7.82 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z

calcd [M + H] 394.0882; found 394.0877. Anal. Calcd for C₁₈H₁₉NO₇S: C,

54.95%; H, 4.87%; N, 3.56%. Found: C, 55.06%; H, 4.82%; N, 3.43%.

Step 2: (E)-2⁰,4⁰-Dimethoxystyryl-4-methoxy-3-aminobenzyl-

sulfone (8g). The title compound was obtained by the reduction of (E)-

2⁰,4⁰-dimethoxystyryl-4-methoxy-3-nitrobenzylsulfone following the proce-

dure as described for compound 8b. Yield, 47%; yellow solid, mp

136ꢀ138 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.86 (s, 6H, 2 ꢁ OCH₃),

3.98(s, 3H, OCH₃), 4.25(s, 2H, CH₂), 6.46(d, J= 2.4 Hz, 1H, ArꢀH), 6.85

(d, J = 15.3 Hz, 1H, dCH), 7.12 (d, J = 8.7 Hz, 1H, ArꢀH), 7.29 (d, J = 8.7

Hz, 1H, ArꢀH), 7.51 (d, J = 15.3 Hz, 1H, CHd), 7.63 (dd, J = 8.7, 2.4 Hz,

1H, ArꢀH), 7.86 (d, J = 2.4 Hz, 1H, ArꢀH). HRMS: m/z [M + H]

364.1140; found 364.1129. Anal. (C₁₈H₂₁NO₅S) C, H, N.

General Procedure for the Preparation of (E)-Styrylben-

zylsulfone (8). Method A (Scheme 1). A mixture of benzylsulfo-

nylacetic acid 6 (10 mmol), araldehyde 7 (10 mmol), glacial acetic acid

(15 mL), and a catalytic amount of benzylamine (200 μL) was refluxed

for about 2ꢀ8 h. After completion of the reaction (TLC monitoring,

chloroform on silica gel plate), with the contents cooled to room

temperature, the precipitated product was filtered and washed with

2-propanol. If solid was not formed, the reaction mixture was diluted

with ether and washed successively with saturated sodium bicarbonate,

dilute hydrochloric acid, and water. The organic layer was dried over

anhydrous sodium sulfate, filtered, and concentrated under vacuum to

obtain the desired crude product 8. The crude product was recrystallized

in 2-propanol to yield an analytically pure sample of 8. The following

(E)-styrylbenzylsulfones 8 were prepared using the above procedure.

(E)-2⁰-Methoxystyryl-4-methoxy-3-nitrobenzylsulfone

(8a). The title compound was obtained from 4-methoxy-3-nitrobenzyl-

sulfonylacetic acid 6a and 2-methoxybenzaldehyde following the pro-

cedure as described in method A. Yield, 56%; yellow solid, mp

1

169ꢀ171 °C. H NMR (CDCl₃, 300 MHz): δ 3.89 (s, 3H, OCH₃),

3.99 (s, 3H, OCH₃), 4.27 (s, 2H, CH₂), 6.92ꢀ6.99 (m, 2H, ArꢀH), 7.01

(d, J = 15.6 Hz, 1H, dCH), 7.12 (d, J = 8.7 Hz, 1H, ArꢀH), 7.35ꢀ7.45

(m, 2H, ArꢀH), 7.62 (d, J = 15.6 Hz, 1H, CHd), 7.63 (dd, J = 8.7, 2.4

Hz, 1H, ArꢀH), 7.87 (d, J = 2.4 Hz, 1H, ArꢀH). HRMS: m/z calcd

[M + H] 364.0777; found 364.0780. Anal. (C₁₇H₁₇NO₆S) C, H, N.

(E)-2⁰-Methoxystyryl-4-methoxy-3-aminobenzylsulfone

(8b). (E)-2⁰-Methoxystyryl-4-methoxy-3-nitrobenzylsulfone 8a (900

mg, 2.5 mmol) was dissolved in acetone/water (40:20 mL) and heated

to 50 °C. After 30 min sodium hydrosulfite (8.79 g, 50.0 mmol) was

added slowly, and temperature was maintained at 50 °C for a further

30 min. After completion of reaction (TLC monitoring, chloroform on

silica gel plate), the contents were cooled to room temperature, water

was added, and the product was isolated by extraction with ethyl acetate.

The organic phase was washed with water (3 ꢁ 100 mL), brine (50 mL),

dried over anhydrous sodium sulfate, filtered, and concentrated under

vacuum to obtain the desired crude product 8b. The pure compound 8b

was obtained following purification by silica gel flash column chroma-

1

tography (chloroform). Yield, 51%; white solid, mp 140ꢀ142 °C. H

NMR (CDCl₃, 300 MHz): δ 3.78 (s, 3H,OCH₃), 3.83 (s, 3H, OCH₃),

4.15 (s, 2H, CH₂), 6.95ꢀ7.02 (m, 2H, ArꢀH), 6.98 (d, J = 15.6 Hz, 1H,

dCH), 7.12 (d, J = 8.7 Hz, 1H, ArꢀH), 7.37ꢀ7.45 (m, 2H, ArꢀH),

7.62 (d, J = 15.6 Hz, 1H, CHd), 7.63 (dd, J = 8.7, 2.4 Hz, 1H, ArꢀH),

7.87 (d, J = 2.4 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 334.1035;

found 334.1066. Anal. (C₁₇H₁₉NO₄S) C, H, N.

(E)-4⁰-Methoxystyryl-4-methoxy-3-nitrobenzylsulfone (8c).

The title compound was obtained from 4⁰-methoxy-3-nitrobenzylsulfony-

lacetic acid 6a and 4-methoxybenzaldehyde following the procedure as

described in method A. Yield, 58%; yellow solid, mp 172ꢀ174 °C. ¹HNMR

(CDCl₃, 500 MHz): δ 3.88 (s, 3H, OCH₃), 3.98 (s, 3H, OCH₃), 4.27 (s,

2H, CH₂), 6.93ꢀ6.98 (m, 2H, ArꢀH), 6.95 (d, J = 15.5 Hz, 1H, dCH),

7.12 (d, J = 9.0 Hz, 1H, ArꢀH), 7.36 (dd, J = 7.5, 1.5 Hz, 1H, ArꢀH),

7.42ꢀ7.49 (m, 2H, ArꢀH), 7.61 (d, J = 15.5 Hz, 1H, CHd), 7.86

(E)-2⁰,5⁰-Dimethoxystyryl-4-methoxy-3-aminobenzylsul-

fone (8h). Step 1: (E)-2⁰,5⁰-Dimethoxystyryl-4-methoxy-3-

nitrobenzylsulfone. The condensation of 4-methoxy-3-nitrobenzyl-

sulfonylacetic acid 6a with 2,5-dimethoxybenzaldehyde following the

procedure as described in method A resulted in the desired product (E)-

2⁰,5⁰-dimethoxystyryl-4-methoxy-3-nitrobenzylsulfone. Yield, 52%; yellow

solid, mp 174ꢀ175 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.80 (s, 6H, 2 ꢁ

OCH₃), 3.94 (s, 3H, OCH₃), 4.19 (s, 2H, CH₂), 6.54 (d, J = 8.7 Hz,

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2H, ArꢀH), 7.13 (d, J = 8.7 Hz, 1H, ArꢀH), 7.17 (d, J = 15.6 Hz,

1H, dCH), 7.30 (t, J = 8.4 Hz, 1H, ArꢀH), 7.59 (dd, J = 8.7, 2.4 Hz,

1H, ArꢀH), 7.78 (d, J = 2.4 Hz, 1H, ArꢀH), 7.82 (d, J = 15.6 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 394.0882; found 394.0881. Anal.

Calcd for C₁₈H₁₉NO₇S: C, 54.95%; H, 4.87%; N, 3.56%. Found: C,

54.86%; H, 4.75%; N, 3.47%.

Step 2: (E)-2⁰,5⁰-Dimethoxystyryl-4-methoxy-3-amino-

benzylsulfone (8h). The title compound was obtained by the

reduction of (E)-2⁰,5⁰-dimethoxystyryl-4-methoxy-3-nitrobenzylsulfone

following the procedure as described for compound 8b. Yield, 49%;

yellow solid, mp 130ꢀ132 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.78 (s,

3H, OCH₃), 3.83 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 4.18 (s, 2H,

CH₂), 6.68ꢀ6.77 (m, 3H, ArꢀH), 6.86 (d, J = 9.0 Hz, 2H, ArꢀH), 6.91

(d, J = 15.6 Hz, 1H, dCH), 6.93ꢀ7.02 (m, 1H, ArꢀH), 7.64 (d, J = 15.9

Hz, 1H, CHd). HRMS: m/z [M + H] 364.1140; found 364.1131. Anal.

(C₁₈H₂₁NO₅S) C, H, N.

(E)-2⁰,6⁰-Dimethoxystyryl-4-methoxy-3-aminobenzylsul-

fone (8i). The title compound was obtained by the reduction of (E)-

2⁰,6⁰-dimethoxystyryl-4-methoxy-3-nitrobenzylsulfone 8e following the

procedure as described for compound 8b. Yield, 48%; pale yellow solid,

mp 106ꢀ108 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.83 (s, 3H, OCH₃),

3.86 (s, 6H, 2 ꢁ OCH₃), 4.16 (s, 2H, CH₂), 6.52ꢀ6.59 (m, 2H, ArꢀH),

6.71ꢀ6.79 (m, 2H, ArꢀH), 7.17 (t, J = 8.1 Hz, 1H, ArꢀH), 7.23 (d, J =

15.9 Hz, 1H, dCH), 7.32 (t, J = 8.4 Hz, 1H, ArꢀH), 7.94 (d, J = 15.9 Hz,

1H, CHd). HRMS: m/z [M + H] 364.1140; found 364.1131. Anal.

(C₁₈H₂₁NO₅S) C, H, N.

(E)-2⁰,6⁰-Dimethoxystyryl-4-methoxy-2-aminobenzylsul-

fone (8j). The title compound was obtained by the reduction of (E)-

2⁰,6⁰-dimethoxystyryl-4-methoxy-2-nitrobenzylsulfone 8f following the

procedure as described for compound 8b. Yield, 49%; yellow solid, mp

160ꢀ164 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.70 (s, 3H, OCH₃), 3.79

(s, 6H, 2 ꢁ OCH₃), 4.19 (s, 2H, CH₂), 6.24ꢀ6.29 (m, 2H, ArꢀH), 6.49

(d, J = 8.4 Hz, 2H, ArꢀH), 6.91 (d, J = 8.1 Hz, 1H, ArꢀH), 7.23 (d, J =

15.9 Hz, 1H, dCH), 7.29 (t, J = 6.3 Hz, 1H, ArꢀH), 7.94 (d, J = 15.6 Hz,

1H, CHd). HRMS: m/z [M + H] 364.1140; found 364.1151. Anal.

(C₁₈H₂₁NO₅S) C, H, N.

the procedure as described in method A. Yield, 56%; yellow solid, mp

184ꢀ186 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.84 (s, 6H, 2 ꢁ OCH₃),

3.86 (s, 3H, OCH₃), 3.98 (s, 3H, OCH₃), 4.23 (s, 2H, CH₂), 6.09 (s, 2H,

ArꢀH), 7.03 (d, J = 15.6 Hz, 1H, dCH), 7.10 (d, J = 8.7 Hz, 1H, ArꢀH),

7.63 (dd, J = 8.7, 2.4 Hz, 1H, ArꢀH), 7.80 (d, J = 15.6 Hz, 1H, CHd), 7.85

(d, J = 2.1 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 424.0988; found

424.0982. Anal. (C₁₉H₂₁NO₈S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-2-nitrobenzyl-

sulfone (8m). The title compound was obtained from 4-methoxy-2-

nitrobenzylsulfonylacetic acid 6b and 2,4,6-trimethoxybenzaldehyde

following the procedure as described in method A. Yield, 54%; yellow

solid, mp 158ꢀ160 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.84 (s, 6H, 2 ꢁ

OCH₃), 3.85 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 4.80 (s, 2H, CH₂),

6.10 (s, 2H, ArꢀH), 7.05 (d, J = 15.6 Hz, 1H, dCH), 7.16 (dd, J = 8.7,

2.7 Hz, 1H, ArꢀH), 7.50ꢀ7.53 (m, 2H, ArꢀH), 7.70 (d, J = 15.6 Hz,

1H, CHd). HRMS: m/z calcd [M + H] 424.0988; found 424.0972.

Anal. (C₁₉H₂₁NO₈S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-2-aminobenzyl-

sulfone (8n). The title compound was obtained by the reduction of

(E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-2-nitrobenzylsulfone 8m fol-

lowing the procedure as described for compound 8b. Yield, 50%; pale

yellow solid, mp 147ꢀ149 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.78 (s,

3H, OCH₃), 3.85 (s, 6H, 2 ꢁ OCH₃), 3.86 (s, 3H, OCH₃), 4.25 (s, 2H,

CH₂), 6.11 (s, 2H, ArꢀH), 6.32ꢀ6.36 (m, 2H, ArꢀH), 6.98 (d, J = 8.1

Hz, 1H, ArꢀH), 7.13 (d, J = 15.6 Hz, 1H, dCH), 7.93 (d, J = 15.6 Hz,

1H, CHd). HRMS: m/z calcd [M + H] 394.1246; found 394.1261.

Anal. (C₁₉H₂₃NO₆S) C, H, N.

(E)-2⁰,4⁰,5⁰-Trimethoxystyryl-4-methoxy-3-aminobenzyl-

sulfone (8o). Step 1: (E)-2⁰,4⁰,5⁰-Trimethoxystyryl-4-meth-

oxy-3-nitrobenzylsulfone. The condensation of 4-methoxy-3-

nitrobenzylsulfonylacetic acid 6a with 2,4,5-trimethoxybenzaldehyde

following the procedure as described in method A resulted in the desired

product (E)-2⁰,4⁰,5⁰-trimethoxystyryl-4-methoxy-3-nitrobenzylsulfone.

Yield, 56%; pale yellow solid, mp 200ꢀ202 °C. ¹H NMR (CDCl₃, 300

MHz): δ 3.85 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃),

3.99 (s, 3H, OCH₃), 4.26 (s, 2H, CH₂), 6.49 (s, 1H, ArꢀH), 6.79 (d, J =

15.6 Hz, 1H, dCH) 6.84 (s, 1H, ArꢀH), 7.14 (d, J = 8.7 Hz, 1H, ArꢀH),

7.56 (d, J = 15.6 Hz, 1H, CHd), 7.63 (dd, J = 8.7, 2.1 Hz, 1H, ArꢀH),

7.86 (d, J = 2.1 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 424.0988;

found 424.1001. Anal. Calcd for C₁₉H₂₁NO₈S: C, 53.89%; H, 5.00%; N,

3.31%. Found: C, 53.76%; H, 4.91%; N, 3.25%.

Step 2: (E)-2⁰,4⁰,5⁰-Trimethoxystyryl-4-methoxy-3-amino-

benzylsulfone (8o). The title compound was obtained by the

reduction of (E)-2⁰,4⁰,5⁰-trimethoxystyryl-4-methoxy-3-nitrobenzylsul-

fone following the procedure as described for compound 8b. Yield, 48%;

pale yellow solid, mp 112ꢀ114 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.84

(s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 3.94 (s, 3H,

OCH₃), 4.17 (s, 2H, CH₂), 6.50 (s, 1H, ArꢀH), 6.69 (d, J = 2.1 Hz, 1H,

ArꢀH), 6.73 (dd, J = 8.4, 2.1 Hz, 2H, ArꢀH), 6.77 (d, J = 2.1 Hz, 1H,

ArꢀH), 6.81 (d, J = 15.0 Hz, 1H, dCH), 7.61 (d, J = 15.6 Hz, 1H,

dCH). HRMS: m/z calcd [M + H] 394.1246; found 394.1268. Anal.

(C₁₉H₂₃NO₆S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-aminobenzyl-

sulfone (8p). The title compound was obtained by the reduction of

(E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-nitrobenzylsulfone 8l follow-

ing the procedure as described for compound 8b. Yield, 48%; light

yellow solid, mp 146ꢀ148 °C. ¹H NMR (CDCl₃, 400 MHz): δ 3.77 (s,

3H, OCH₃), 3.84 (s, 6H, 2 ꢁ OCH₃), 3.85 (s, 3H, OCH₃), 4.24 (s, 2H,

CH₂), 4.33 (br s, 2H, NH₂), 6.10 (s, 2H, ArꢀH), 6.31ꢀ6.35 (m, 2H,

ArꢀH), 6.97 (d, J = 8.3 Hz, 1H, ArꢀH), 7.12 (d, J = 15.6 Hz, 1H,

dCH), 7.93 (d, J = 15.6 Hz, 1H, CHd). ¹³C NMR (CDCl₃, 75 MHz): δ

163.7, 161.4, 147.6, 136.4, 135.1, 122.8, 121.1, 121.0, 117.2, 110.2, 103.8,

90.3, 61.7, 55.7, 55.5, 55.4. HRMS: m/z calcd [M + H] 394.1246; found

394.1246. Anal. (C₁₉H₂₃NO₆S) C, H, N.

(E)-2⁰,6⁰-Dimethoxystyryl-6-methoxy-3-aminobenzylsul-

fone (8k). Step 1: (E)-2⁰,6⁰-Dimethoxystyryl-6-methoxy-3-

nitrobenzylsulfone. The condensation of 6-methoxy-3-nitrobenzyl-

sulfonylacetic acid 6g with 2,6-dimethoxybenzaldehyde following the

procedure as described in method A resulted in the desired product (E)-

2⁰,6⁰-dimethoxystyryl-6-methoxy-3-nitrobenzylsulfone. Yield, 55%; pale

yellow solid, mp 189ꢀ191 °C. ¹H NMR (CDCl₃, 500 MHz): δ 3.85 (s,

6H, 2 ꢁ OCH₃), 3.88 (s, 3H, OCH₃), 4.44 (s, 2H, CH₂), 6.55 (d, J = 8.5

Hz, 2H, ArꢀH), 6.93 (d, J = 9.0 Hz, 1H, ArꢀH), 7.27 (d, J = 16.0 Hz,

1H, dCH), 7.33 (t, J = 8.5 Hz, 1H, ArꢀH), 7.79 (d, J = 16.0 Hz, 1H,

CHd), 8.21 (dd, J = 9.0, 3.0 Hz, 1H, ArꢀH), 8.31 (d, J = 3.0 Hz, 1H,

ArꢀH). HRMS: m/z calcd [M + H] 394.0882; found 394.0891. Anal.

Calcd for C₁₈H₁₉NO₇S: C, 54.95%; H, 4.87%; N, 3.56%. Found: C,

55.10%; H, 4.92%; N, 3.63%.

Step 2: (E)-2⁰,6⁰-Dimethoxystyryl-6-methoxy-3-amino-

benzylsulfone (8k). The title compound was obtained by the

reduction of (E)-2⁰,6⁰-dimethoxystyryl-6-methoxy-3-nitrobenzylsulfone

following the procedure as described for compound 8b. Yield, 47%; light

yellow solid, mp 172ꢀ174 °C. ¹H NMR (CDCl₃, 500 MHz): δ 3.63 (s,

3H, OCH₃), 3.84 (s, 6H, 2 ꢁ OCH₃), 4.33 (s, 2H, CH₂), 6.53 (d, J = 8.5

Hz, 2H, ArꢀH), 6.93 (d, J = 9.0 Hz, 1H, ArꢀH), 7.27 (d, J = 16.0 Hz,

1H, dCH), 7.33 (t, J = 8.5 Hz, 1H, ArꢀH), 7.85 (d, J = 16.0 Hz, 1H,

CHd), 8.21 (dd, J = 9.0, 3.0 Hz, 1H, ArꢀH), 8.31 (d, J = 3.0 Hz, 1H,

ArꢀH). HRMS: m/z [M + H] 364.1140; found 364.1134. Anal.

(C₁₈H₂₁NO₅S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-nitrobenzylsul-

fone (8l). The title compound was obtained from 4-methoxy-3-nitro-

benzylsulfonylacetic acid 6a and 2,4,6-trimethoxybenzaldehyde following

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(E)-3⁰,4⁰,5⁰-Trimethoxystyryl-4-methoxy-3-aminobenzyl-

sulfone (8q). Step 1: (E)-3⁰,4⁰,5⁰-Trimethoxystyryl-4-methoxy-

3-nitrobenzylsulfone. The condensation of 4-methoxy-3-nitrobenzyl-

sulfonylacetic acid 6a with 3,4,5-trimethoxybenzaldehyde following the

procedure as described in method A resulted in the desired product (E)-

3⁰,4⁰,5⁰-trimethoxystyryl-4-methoxy-3-nitrobenzylsulfone. Yield, 52%; pale

yellow solid, mp 170ꢀ172 °C. ¹HNMR(CDCl₃, 300 MHz): δ3.88 (s, 6H,

2 ꢁ OCH₃), 3.90 (s, 3H, OCH₃), 3.99 (s, 3H, OCH₃), 4.29 (s, 2H, CH₂),

6.63 (d, J = 15.3 Hz, 1H, dCH), 6.68 (s, 2H, ArꢀH), 7.13 (d, J = 8.7 Hz,

1H, ArꢀH), 7.39 (d, J = 15.3 Hz, 1H, CHd), 7.62 (dd, J = 8.7, 2.4 Hz, 1H,

ArꢀH), 7.86 (d, J = 2.4 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H]

424.0988; found 424.0998. Anal. Calcd for C₁₉H₂₁NO₈S: C, 53.89%; H,

5.00%; N, 3.31%. Found: C, 53.71%; H, 4.93%; N, 3.37%.

(d, J = 9.0 Hz, 1H, ArꢀH), 7.48 (d, J = 15.6 Hz, 1H, CHd), 7.62 (dd, J =

8.7, 2.4 Hz, 1H, ArꢀH), 7.86 (d, J = 2.1 Hz, 1H, ArꢀH). HRMS: m/z

calcd [M + H] 410.08; found 410.09. Anal. (C₁₈H₁₉NO₈S) C, H, N.

(E)-2⁰,6⁰-Dimethoxy-4⁰-hydroxystyryl-4-methoxy-3-ami-

nobenzylsulfone (8t). The title compound was obtained by the

reduction of (E)-2⁰,6⁰-dimethoxy-4⁰-hydroxystyryl-4-methoxy-3-ni-

trobenzylsulfone 8s following the procedure as described for com-

pound 8b. Yield, 45%; light yellow solid, mp 132ꢀ134 °C. ¹H NMR

(CDCl₃, 400 MHz): δ 3.67 (s, 6H, 2 ꢁ OCH₃), 3.76 (s, 3H, OCH₃),

4.07 (s, 2H, CH₂), 5.75 (bs, 1H, OH), 5.95 (s, 2H, ArꢀH), 6.65ꢀ6.69

(m, 3H, ArꢀH), 6.92 (d, J = 15.6 Hz, 1H, dCH), 7.73 (d, J = 15.6 Hz,

1H, CHd). HRMS: m/z calcd [M + H] 380.1090; found 380.0184.

Anal. (C₁₈H₂₁NO₆S) C, H, N.

(E)-2⁰,6⁰-Dimethoxy-4⁰-phenoxybutanoicacidstyryl-4-

methoxy-3-aminobenzylsulfone (8u). Step 1: (E)-2⁰,6⁰-

Dimethoxy-4⁰-phenoxybutanoic acid styryl-4-methoxy-3-

nitrobenzylsulfone. The condensation of 4-methoxy-3-nitrobenzyl-

sulfonylacetic acid 6a with 4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid

following the procedure as described in method A resulted in the desired

product (E)-2⁰,6⁰-dimethoxy-4⁰-phenoxybutanoic acid styryl-4-methoxy-3--

nitrobenzylsulfone. Yield, 56%; pale yellow solid, mp 198ꢀ200 °C. ¹H

NMR (CDCl₃, 400 MHz): δ 2.11ꢀ2.18 (m, 2H, CH₂), 2.61 (t, J = 7.1 Hz,

2H, CH₂), 3.83(s, 6H, 2ꢁ OCH₃), 3.97(s, 3H, OCH₃), 4.08(t, J=6.1Hz,

2H, CH₂), 4.23 (s, 2H, CH₂), 6.09 (s, 2H, ArꢀH), 7.02 (d, J = 15.6 Hz, 1H,

dCH),7.11(d,J=8.7Hz,1H,ArꢀH), 7.63 (dd, J= 8.7, 2.3 Hz, 1H, ArꢀH),

7.78 (d, J=15.6Hz,1H,CHd),7.84(d,J= 2.2 Hz, 1H, ArꢀH). HRMS: m/z

calcd [M + H] 496.1199; found 496.1984. Anal. Calcd for C₂₂H₂₅NO₁₀S: C,

53.33%; H, 5.09%; N, 2.83%. Found: C, 53.42%; H, 5.01%; N, 2.87%.

Step 2: (E)-2⁰,6⁰-Dimethoxy-4⁰-phenoxybutanoic acid styr-

yl-4-methoxy-3-aminobenzylsulfone (8u). The title compound

was obtained by the reduction of (E)-2⁰,6⁰-dimethoxy-4⁰-phenoxybuta-

noic acidstyryl-4-methoxy-3-nitrobenzylsulfone following the pro-

cedure as described for compound 8b. Yield, 48%; light yellow solid,

mp 176ꢀ178 °C. ¹H NMR (CDCl₃, 400 MHz): δ 2.04ꢀ2.10 (m, 2H,

CH₂), 2.53 (t, J = 7.1 Hz, 2H, CH₂), 3.75 (s, 6H, 2 ꢁ OCH₃), 3.77 (s,

3H, OCH₃), 4.00 (t, J = 6.0 Hz, 2H, CH₂), 4.06 (s, 2H, CH₂), 6.02

(s, 2H, ArꢀH), 6.66ꢀ6.70 (m, 3H, ArꢀH), 6.99 (d, J = 15.6 Hz,

1H, dCH), 7.77 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M + H]

466.1457; found 466.1443. Anal. (C₂₂H₂₇NO₈S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-bromo-3-nitrobenzylsulfone

(8v). The title compound was obtained from 4-bromo-3-nitrobenzylsulfo-

nylacetic acid 6c and 2,4,6-trimethoxybenzaldehyde following the proce-

dure as described in method A. Yield, 56%; yellow solid, mp 186ꢀ188 °C.

¹H NMR (CDCl₃, 300 MHz): δ 3.84 (s, 6H, 2 ꢁ OCH₃), 3.86 (s, 3H,

OCH₃), 4.73 (s, 2H, CH₂), 6.10 (s, 2H, ArꢀH), 7.03 (d, J = 15.9 Hz, 1H,

dCH), 7.10 (d, J = 8.7 Hz, 1H, ArꢀH), 7.63 (dd, J = 8.7, 2.4 Hz, 1H,

ArꢀH), 7.80 (d, J = 15.6 Hz, 1H, CHd), 7.85 (d, J = 2.1 Hz, 1H, ArꢀH).

HRMS: m/z calcd [M + H] 471.9987; found 471.9972. Anal.

(C₁₈H₁₈BrNO₇S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-bromo-3-aminobenzylsul-

fone (8w). The title compound was obtained by the reduction of (E)-

2⁰,4⁰,6⁰-trimethoxystyryl-4-bromo-3-nitrobenzylsulfone 8v following the

procedure as described for compound 8b. Yield, 49%; white solid, mp

162ꢀ164 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.80 (s, 6H, 2 ꢁ OCH₃),

3.84 (s, 3H, OCH₃), 4.63 (s, 2H, CH₂), 6.01 (s, 2H, ArꢀH), 6.70ꢀ6.77

(m, 2H, ArꢀH), 7.06 (d, J = 15.9 Hz, 1H, dCH), 7.27 (s, 1H, ArꢀH),

7.85 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M + H] 442.0174;

found 442.0189. Anal. (C₁₈H₂₀BrNO₅S) C, H, N.

Step 2: (E)-3⁰,4⁰,5⁰-Trimethoxystyryl-4-methoxy-3-amino-

benzylsulfone (8q). The title compound was obtained by the

reduction of (E)-3⁰,4⁰,5⁰-trimethoxystyryl-4-methoxy-3-nitrobenzylsul-

fone following the procedure as described for compound 8b. Yield, 48%;

1

light orange solid, mp 108ꢀ110 °C. H NMR (CDCl₃, 400 MHz): δ

3.77 (s, 3H, OCH₃), 3.79 (s, 6H, 2 ꢁ OCH₃), 3.82 (s, 3H, OCH₃), 4.11

(s, 2H, CH₂), 6.55 (d, J = 15.4 Hz, 1H, dCH), 6.58 (s, 2H, ArꢀH), 6.61

(dd, J = 8.2, 2.1 Hz, 1H, ArꢀH), 6.67 (d, J = 8.2 Hz, 1H, ArꢀH), 6.70 (d,

J = 2.1 Hz, 1H, ArꢀH), 7.29 (d, J = 15.4 Hz, 1H, CHd). HRMS: m/z

calcd [M + H] 394.1246; found 394.1246. Anal. (C₁₉H₂₃NO₆S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-6-methoxy-3-aminobenzyl-

sulfone (8r). Step 1: (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-6-meth-

oxy-3-nitrobenzylsulfone. The condensation of 6-methoxy-3-

nitrobenzylsulfonylacetic acid 6g with 2,4,6-trimethoxybenzaldehyde

following the procedure as described in method A resulted in the

desired product (E)-2⁰,4⁰,6⁰-trimethoxystyryl-6-methoxy-3-nitroben-

zylsulfone. Yield, 55%; yellow solid, mp 144ꢀ146 °C. ¹H NMR

(CDCl₃, 500 MHz): δ 3.83 (s, 6H, 2 ꢁ OCH₃), 3.85 (s, 3H, OCH₃),

3.87 (s, 3H, OCH₃), 4.42 (s, 2H, CH₂), 6.08 (s, 2H, ArꢀH), 6.92 (d,

J = 9.0 Hz, 1H, ArꢀH), 7.09 (d, J = 15.5 Hz, 1H, dCH), 7.69 (d, J =

16.0 Hz, 1H, CHd), 8.11 (dd, J = 9.0, 2.5 Hz, 1H, ArꢀH), 8.30 (d, J =

2.5 Hz, 1H, ArꢀH). HRMS: m/z calcd [M + H] 424.0988; found

424.0998. Anal. Calcd for C₁₉H₂₁NO₈S: C, 53.89%; H, 5.00%; N,

3.31%. Found: C, 53.76%; H, 4.94%; N, 3.37%.

Step 2: (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-6-methoxy-3-amino-

benzylsulfone (8r). The title compound was obtained by the reduc-

tion of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-6-methoxy-3-nitrobenzylsulfone

following the procedure as described for compound 8b. Yield, 49%;

orange solid, mp 124ꢀ128 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.57 (s,

3H, OCH₃), 3.76 (s, 6H, 2 ꢁ OCH₃), 3.77 (s, 3H, OCH₃), 4.25 (s, 2H,

CH₂), 6.01 (s, 2H, ArꢀH), 6.59 (dd, J = 8.2, 2.1 Hz, 2H, ArꢀH), 6.77 (d,

J = 8.2 Hz, 1H, ArꢀH), 7.02 (d, J = 15.9 Hz, 1H, dCH), 7.69 (d, J = 15.6

Hz, 1H, CHd). HRMS: m/z calcd [M + H] 394.1246; found 394.1266.

Anal. (C₁₉H₂₃NO₆S) C, H, N.

(E)-2⁰,6⁰-Dimethoxy-4⁰-hydroxystyryl-4-methoxy-3-nitro-

benzylsulfone (8s). A mixture of 4-methoxy-3-nitrobenzylsulfonyla-

cetic acid 6a (2.05 g, 5 mmol), 2,6-dimethoxy-4-hydroxybenzaldehyde

(1.02 g, 5.5 mmol), benzoic acid (92 mg, 0.75 mmol), and piperidine

(55 mg, 0.65 mmol) in toluene (50 mL) was refluxed for 2ꢀ4 h with

continuous removal of water using a DeanꢀStark water separator. Reaction

completion was determined by TLC (9:1 chloroform/methanol on silica

gel plate). The solvent was evaporated. To the residue water was added

and extracted with ethyl acetate. The organic phase was washed with

saturated sodium bicarbonate solution, dilute hydrochloric acid, and

water and dried over anhydrous sodium sulfate. The organic phase was

filtered, and evaporation of the solvent under vacuo yielded a crude

product 8s. The pure compound 8s was obtained following purification

by silica gel flash column chromatography (chloroform). Yield, 48%;

light yellow solid, mp 222ꢀ224 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ

3.77 (s, 6H, 2 ꢁ OCH₃), 3.91 (s, 3H, OCH₃), 4.51 (s, 2H, CH₂), 5.79

(s, 1H, OH), 6.11 (s, 2H, ArꢀH), 6.99 (d, J = 15.6 Hz, 1H, dCH), 7.37

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-chloro-3-aminobenzylsul-

fone (8x). Step 1: (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-chloro-3-

nitrobenzylsulfone. The condensation of 4-chloro-3-nitrobenzyl-

sulfonylacetic acid 6d with 2,4,6-trimethoxybenzaldehyde following the

procedure as described in method A resulted in the desired product (E)-

2⁰,4⁰,6⁰-trimethoxystyryl-4-chloro-3-nitrobenzylsulfone. Yield, 51%; pale

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yellow solid, mp 173ꢀ174 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.76 (s,

6H, 2 ꢁ OCH₃), 3.78 (s, 3H, OCH₃), 4.06 (s, 2H, CH₂), 6.02 (s, 2H,

ArꢀH), 6.59 (dd, J = 8.1, 2.1 Hz, 1H, ArꢀH), 6.77 (d, J = 2.1 Hz, 1H,

ArꢀH), 7.00 (d, J = 15.6 Hz, 1H, dCH), 7.13 (d, J = 8.1 Hz, ArꢀH),

7.75 (d, J = 15.9 Hz, 1H, CHd). HRMS: m/z calcd [M + H] 428.0489;

found 428.0476. Anal. Calcd for C₁₈H₁₈ClNO₇S: C, 50.53%; H, 4.24%;

N, 3.27%. Found: C, 50.72%; H, 4.29%; N, 3.24%.

Step 2: (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-chloro-3-amino-

benzylsulfone (8x). The title compound was obtained by the

reduction of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-chloro-3-nitrobenzylsul-

fone following the procedure as described for compound 8b. Yield,

50%; pale yellow solid, mp 138ꢀ140 °C. ¹H NMR (CDCl₃, 300 MHz):

δ 3.76 (s, 6H, 2 ꢁ OCH₃), 3.78 (s, 3H, OCH₃), 4.00 (br s, 2H, NH₂),

4.06 (s, 2H, CH₂), 6.02 (s, 2H, ArꢀH), 6.59 (dd, J = 8.1, 2.1 Hz, 1H,

ArꢀH), 6.77 (d, J = 2.1 Hz, 1H, ArꢀH), 6.95 (d, J = 15.6 Hz, 1H,

dCH), 7.13 (d, J = 8.1 Hz, ArꢀH), 7.75 (d, J = 15.9 Hz, 1H, CHd).

HRMS: m/z calcd [M + H] 398.0842; found 398.0848. Anal.

(C₁₈H₂₀ClNO₅S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-chloro-2-aminobenzylsul-

fone (8y). Step 1: (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-chloro-2-

nitrobenzylsulfone. The condensation of 4-chloro-2-nitrobenzyl-

sulfonylacetic acid 6e with 2,4,6-trimethoxybenzaldehyde following the

procedure as described in method A resulted in the desired product (E)-

2⁰,4⁰,6⁰-trimethoxystyryl-4-chloro-2-nitrobenzylsulfone. Yield, 54%; pale

yellow solid, mp 206ꢀ208 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.79 (s,

6H, 2 ꢁ OCH₃), 3.84 (s, 3H, OCH₃), 4.27 (s, 2H, CH₂), 6.10 (s, 2H,

ArꢀH), 6.59 (dd, J = 8.1, 2.1 Hz, 1H, ArꢀH), 6.84 (d, J = 2.1 Hz, 1H,

ArꢀH), 7.10 (d, J = 15.6 Hz, 1H, dCH), 7.21 (d, J = 8.1 Hz, ArꢀH),

7.82 (d, J = 15.9 Hz, 1H, CHd). HRMS: m/z calcd [M + H] 428.0489;

found 428.0469. Anal. Calcd for C₁₈H₁₈ClNO₇S: C, 50.53%; H, 4.24%;

N, 3.27%. Found: C, 50.45%; H, 4.20%; N, 3.22%.

Step 2: (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-chloro-2-amino-

benzylsulfone (8y). The title compound was obtained by the reduc-

tion of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-chloro-2-nitrobenzylsulfone fol-

lowing the procedure as described for compound 8b. Yield, 48%; yellow

solid, mp 184ꢀ186 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.86 (s, 6H, 2 ꢁ

OCH₃), 3.88 (s, 3H, OCH₃), 4.27 (s, 2H, CH₂), 4.50 (br s, 2H,

NH₂),6.12 (s, 2H, ArꢀH), 6.74 (dd, J = 8.1, 2.1 Hz, 1H, ArꢀH), 6.79 (d,

J = 2.1 Hz, 1H, ArꢀH), 6.99 (d, J = 8.1 Hz, ArꢀH), 7.11 (d, J = 15.9 Hz,

1H, dCH), 7.92 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M + H]

398.0842; found 398.0836. Anal. (C₁₈H₂₀ClNO₅S) C, H, N.

acid 6a with 2,4,6-trifluorobenzaldehyde following the procedure as de-

scribed in method A resulted in the desired product (E)-2⁰,4⁰,6⁰-trifluor-

ostyryl-4-methoxy-3-nitrobenzylsulfone. Yield, 52%; yellow solid, mp 141ꢀ

143 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.89 (s, 3H, OCH₃), 4.28 (s, 2H,

CH₂), 6.20 (s, 2H, ArꢀH), 6.80ꢀ6.90 (m, 2H, ArꢀH), 6.97ꢀ7.05 (m, 1H,

ArꢀH), 7.10 (d, J = 15.9 Hz, 1H, dCH), 7.62 (d, J = 15.6 Hz, 1H, CHd).

HRMS: m/z calcd [M + H] 388.0388; found 388.0379. Anal. Calcd for

C₁₆H₁₂F₃NO₅S: C, 49.61%; H, 3.12%; N, 3.62%. Found: C, 49.75%; H,

3.17%; N, 3.68%.

Step 2: (E)-2⁰,4⁰,6⁰-Trifluorostyryl-4-methoxy-3-amino-

benzylsulfone (8ab). The title compound was obtained by the

reduction of (E)-2⁰,4⁰,6⁰-trifluorostyryl-4-methoxy-3-nitrobenzylsulfone

following the procedure as described for compound 8b. Yield, 48%; light

yellow solid, mp 110ꢀ112 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.86 (s,

3H, OCH₃), 4.18 (s, 2H, CH₂), 6.67ꢀ6.83 (m, 5H, ArꢀH), 7.01 (d, J =

16.2 Hz, 1H, dCH), 7.50 (d, J = 15.9 Hz, 1H, CHd). HRMS: m/z calcd

[M + H] 358.0646; found 358.0637. Anal. (C₁₆H₁₄F₃NO₃S) C, H, N.

(E)-2⁰,4⁰,5⁰-Trifluorostyryl-4-methoxy-3-aminobenzylsul-

fone (8ac). Step 1: (E)-2⁰,4⁰,5⁰-Trifluorostyryl-4-methoxy-3-

nitrobenzylsulfone. The condensation of 4-methoxy-3-nitroben-

zylsulfonylacetic acid 6a with 2,4,5-trifluorobenzaldehyde following

the procedure as described in method A resulted in the desired product

(E)-2⁰,4⁰,5⁰-trifluorostyryl-4-methoxy-3-nitrobenzylsulfone. Yield, 51%;

pale yellow solid, mp 161ꢀ163 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.91

(s, 3H, OCH₃), 4.19 (s, 2H, CH₂), 6.67 (dd, J = 8.1, 2.1 Hz, 1H, ArꢀH),

6.74ꢀ6.77 (m, 2H, ArꢀH), 6.80 (d, J = 15.6 Hz, 1H, dCH), 6.97ꢀ7.05

(m, 1H, ArꢀH), 7.17ꢀ7.24 (m, 1H, ArꢀH), 7.43 (d, J = 15.6 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 388.0388; found 388.0394. Anal.

Calcd for C₁₆H₁₂F₃NO₅S: C, 49.61%; H, 3.12%; N, 3.62%. Found: C,

49.50%; H, 3.16%; N, 3.69%.

Step 2: (E)-2⁰,4⁰,5⁰-Trifluorostyryl-4-methoxy-3-amino-

benzylsulfone (8ac). The title compound was obtained by the

reduction of (E)-2⁰,4⁰,5⁰-trifluorostyryl-4-methoxy-3-nitrobenzylsulfone

following the procedure as described for compound 8b. Yield, 46%;

white solid, mp 132ꢀ134 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.91 (s,

3H, OCH₃), 4.19 (s, 2H, CH₂), 6.67 (dd, J = 8.1, 2.1 Hz, 1H, ArꢀH),

6.74ꢀ6.77 (m, 2H, ArꢀH), 6.80 (d, J = 15.6 Hz, 1H, dCH), 6.97ꢀ7.05

(m, 1H, ArꢀH), 7.17ꢀ7.24 (m, 1H, ArꢀH), 7.43 (d, J = 15.6 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 358.0646; found 358.0627. Anal.

(C₁₆H₁₄F₃NO₃S) C, H, N.

Preparation of 3-Nitro-4-methoxybenzylmercaptan (9)

(Scheme 4). Step 1: 3-Nitro-4-methoxybenzylisothiouro-

nium Salt (12). A solution of 3-nitro-4-methoxybenzyl bromide 2a

(10.0 g, 41 mmol) and thiourea (10.0 g, 131 mmol) in 50 mL water was

heated under reflux for 2 h. The reaction mixture was cooled and stirred

at room temperature for 2 h, and the solid was filtered. The resulting

dried product was used in next step without further purification. The

yield of this reaction was 90%, giving a white solid with a melting point of

174ꢀ176 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.91 (s, 3H, OCH₃), 4.51

(s, 2H, CH₂), 7.39 (d, J = 8.7 Hz, 1H, ArꢀH), 7.72 (dd, J = 8.7, 2.4 Hz,

1H, ArꢀH), 7.98 (d, J = 2.1 Hz, 1H, ArꢀH), 9.12 (br s, 4H, 2 ꢁ NH₂).

HRMS: m/z calcd [M + H] 323.9939; found 323.9929. Anal. (C₉H₁₄

BrN₃O₃S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-fluoro-3-nitrobenzylsulfone

(8z). The title compound was obtained from 4-fluoro-3-nitrobenzylsul-

fonylacetic acid 6f and 2,4,6-trimethoxybenzaldehyde following the

procedure as described in method A. Yield, 53%; light yellow solid, mp

161ꢀ163 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.85 (s, 6H, 2 ꢁ OCH₃),

3.87 (s, 3H, OCH₃), 4.28 (s, 2H, CH₂), 6.10 (s, 2H, ArꢀH), 7.02 (d, J =

15.6 Hz, 1H, dCH), 7.31 (dd, J = 8.7, 1.8 Hz, 1H, ArꢀH), 7.69ꢀ7.74 (m,

1H, ArꢀH), 7.75 (d, J = 15.3 Hz, 1H, CHd), 8.07 (dd, J = 6.9, 2.1Hz, 1H,

ArꢀH). HRMS: m/z calcd [M + H] 412.0791; found 412.0774. Anal.

(C₁₈H₁₈FNO₇S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-fluoro-3-aminobenzylsul-

fone (8aa). The title compound was obtained by the reduction of (E)-

2⁰,4⁰,6⁰-trimethoxystyryl-4-fluoro-3-nitrobenzylsulfone 8z following the

procedure as described for compound 8b. Yield, 46%; pale yellow solid,

Step 2: 3-Nitro-4-methoxybenzylmerccaptan (9). The iso-

thiouronium salt 12 was decomposed by boiling several times with

concentrated ammonia and hexane (100 mL, 15:85). Concentration of

the combined hexane extracts provided crude 9. The pure compound 9

was obtained on silica gel flash column chromatography (hexane/ethyl

acetate, 4:1). The yield of this reaction was 55%, giving a yellow solid

with a melting point of 48ꢀ49 °C. ¹H NMR (CDCl₃, 300 MHz): δ 1.74

(t, J = 7.5 Hz, SH), 3.67 (d, J = 7.5 Hz, 2H, CH₂), 3.89 (s, 3H, OCH₃),

6.98 (d, J = 8.4 Hz, 1H, ArꢀH), 7.45 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH),

7.76 (d, J = 2.4 Hz, 1H, ArꢀH). HRMS: m/z calcd [M ꢀ H] 198.0303;

found 198.0320. Anal. (C₈H₉NO₃S) C, H, N.

1

mp 129ꢀ131 °C. H NMR (CDCl₃, 300 MHz): δ 3.76 (s, 6H, 2 ꢁ

OCH₃), 3.78 (s, 3H, OCH₃), 4.09 (s, 2H, CH₂), 6.02 (s, 2H, ArꢀH),

6.57ꢀ6.62 (m, 1H, ArꢀH), 6.80 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 6.85

(dd, J = 8.4, 2.7 Hz, 1H, ArꢀH), 6.96 (d, J = 15.9 Hz, 1H, dCH), 7.27 (s,

1H, ArꢀH), 7.76 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M +

H] 382.1087; found 382.1105. Anal. (C₁₈H₂₀FNO₅S) C, H, N.

(E)-2⁰,4⁰,6⁰-Trifluorostyryl-4-methoxy-3-aminobenzylsulfone

(8ab). Step 1: (E)-2⁰,4⁰,6⁰-Trifluorostyryl-4-methoxy-3-nitroben-

zylsulfone. The condensation of 4-methoxy-3-nitrobenzylsulfonylacetic

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General Procedure for the Preparation of (E)-Styrylben-

zylsulfone (8). Method B (Scheme 3). Preparation of 4⁰-Meth-

oxyphenacyl-3-nitro-4-methoxybenzyl Sulfide (11). To a cooled

solution of sodium hydroxide (100 mmol) in absolute methanol (50 mL),

taken in a 250 mL round-bottomed flask, 3-nitro-4-methoxybenzylmercap-

tan 9(100 mmol) was added slowly, and the reaction mixture was stirred for

5 min. An appropriate 4-methoxyphenacyl bromide 10 (100 mmol) was

added in portions to the contents of the flask, and the mixture was stirred for

2 h. After completion of the reaction (TLC, monitoring, hexane/ethyl

acetate, 8:2 on silica gel plate), the contents of the flask were poured into

crushed ice and the compound formed was washed with ice-cold water and

dried to get 4⁰-methoxyphenacyl-3-nitro-4-methoxybenzyl sulfide 11. The

yield of this reaction was 95%, giving a yellow solid with a melting point

of 87ꢀ89 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.55 (s, 2H, CH₂), 3.67 (s,

2H, CH₂), 3.81(s, 3H, OCH₃), 3.88(s, 3H, OCH₃), 6.87(d, J=9.0Hz, 2H,

ArꢀH), 6.96 (d, J = 8.4 Hz, 2H, ArꢀH), 7.49 (dd, J = 8.4, 2.1 Hz, 1H,

ArꢀH), 7.79 (d, J = 2.4 Hz, 1H, ArꢀH), 7.85 (d, J = 8.7 Hz, 1H, ArꢀH).

HRMS: m/z calcd [M + H] 348.0827; found 348. 0811. Anal.

(C₁₇H₁₇NO₅S) C, H, N.

the solvent was evaporated under reduced pressure to afford crude

product, which on recrystallization in 2-propanol afforded the desired

product 8c. Yield: 65%; white solid, 184ꢀ186 °C. Analytical data are the

same as for 8c obtained by method A.

General Procedure for the Preparation of 2,4,6-Tri-

methoxyphenylacetylene (15, Scheme 6). Step 1: 2-(2⁰,2⁰-

Dibromovinyl)-1,3,5-trimethoxybenzene (19). To a solution of

2,4,6- trimethoxybenzaldehyde 18 (5.0 g, 25.5 mmol) and triphenylpho-

sphine (13.37 g, 51.0 mmol) in anhydrous dichloromethane (60 mL)

was added a solution of tetrabromomethane (9.8 g, 30.0 mmol) in

dichloromethane (10 mL), keeping the temperature below 5 °C. The

reaction mixture was stirred for an additional 30 min. After completion

of the reaction monitored by TLC (hexane/ethyl acetate, 9:1 on silica

gel plate), the reaction mixture was filtered and concentrated in vacuo.

The residue was purified by flash column chromatography (hexane/

ethyl acetate, 9:1), resulting in pure 19. The yield of this reaction was

72%, giving a white solid with a melting point of 128ꢀ130 °C. ¹H NMR

(CDCl₃, 300 MHz): δ 3.82 (s, 6H, 2 ꢁ OCH₃), 3.83 (s, 3H, OCH₃),

6.12 (s, 2H, ArꢀH), 7.19 (s, 1H, CHd). HRMS: m/z calcd [M +H]

352.9133; found 352.9112. Anal. (C₁₁H₁₂Br₂O₃) C, H.

Step 2: 2,4,6-Trimetoxyphenylacetylene (15). A solution of

2-(2⁰,2⁰-dibromovinyl)-1,3,5-trimethoxybenzene 19 (7.42 g, 21.3

mmol) in dry tetrahydrofuran (135 mL) was cooled to ꢀ78 °C, and

n-butyllithium (18.0 mL, 45.0 mmol) was slowly added. The mixture was

stirred for a further 15 min at ꢀ78 °C. After completion of the reaction

monitored by TLC (hexane/ethyl acetate, 9:1 on silica gel plate), water

was added (60 mL) and extracted with ethyl acetate (2 ꢁ 100 mL). The

organic phase was washed with water, dried over anhydrous sodium

sulfate, filtered, and concentrated in vacuo and the residue was purified

by flash column chromatography (hexane/ethyl acetate, 9:1), resulting

in pure 15. The yield of this reaction was 67%, giving a white solid with a

melting point of 119ꢀ122 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.43 (s,

1H, CH), 3.77 (s, 3H, OCH₃), 3.82 (s, 6H, 2 ꢁ OCH₃), 6.04 (s, 2H,

ArꢀH). HRMS: m/z calcd [M + H] 193.0786; found 193.0764. Anal.

(C₁₁H₁₂O₃) C, H.

Synthesis of (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-ni-

trobenzyl Sulfide (17) and (Z)-2⁰,4⁰,6⁰-Trimethoxystyry-4-

methoxy-3-nitrolbenzyl Sulfide (16) (Scheme 5). A hexane

solution of triethylborane (10.5 mL, 10.1 mmol) was added to a solution

of 2,4,6-trimethoxyphenylacetylene 15 (5.0 g, 10.1 mmol) and 4-meth-

oxy-3-nitrobenzylthiol 9a (2.49 g, 12.12 mmol) in benzene (100 mL) at

25 °C under nitrogen atmosphere. The resulting mixture was stirred for

2 h at 25 °C. After completion of the reaction monitored by TLC

(hexane/ethyl acetate, 9:1 on silica gel plate), the reaction mixture was

quenched with 1 M ammonium chloride solution and the aqueous

portion was extracted with ethyl acetate. The organic phase was washed

with water, dried over anhydrous sodium sulfate, filtered, and concen-

trated in vacuo and the residue was purified by column chromatography

(hexane/ethyl acetate, 9:1, and with gradual increase in polarity),

resulting in a stereoisomeric mixture (E/Z = 60/40).

(E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-nitrobenzyl

Sulfide (17). The yield of this reaction was 60%, giving a white solid

with a melting point of 121ꢀ123 °C. ¹H NMR (CDCl₃, 300 MHz): δ

3.80 (s, 6H, 2 ꢁ OCH₃), 3.81 (s, 3H, OCH₃), 3.93 (s, 2H, CH₂S), 3.96

(s, 3H, OCH₃), 6.10 (s, 2H, ArꢀH), 6.83 (d, J = 15.6 Hz, 1H, CHd),

7.00 (d, J = 15.6 Hz, 1H, dCH), 7.05 (d, J = 8.7 Hz, 1H, ArꢀH), 7.59

(dd, J = 8.7, 2.4 Hz, 1H, ArꢀH), 7.80 (d, J = 2.4 Hz, 1H, ArꢀH). HRMS:

m/z calcd [M + H] 392.1090; found 392.1084. Anal. (C₁₉H₂₁NO₆S)

C, H, N.

Preparation of 4⁰-Methoxyphenacyl-3-nitro-4-methoxy-

benzylsulfone (13). The crude 4⁰-methoxyphenacyl-3-nitro-4-meth-

oxybenzyl sulfide 11 (50 mmol) in glacial acetic acid (100 mL) was taken

in a 250 mL round-bottomed flask, and 30% hydrogen peroxide (60 mL)

was added in portions at frequent intervals. Then the reaction mixture

was kept at room temperature for 24 h. The solid, if any formed, was

separated by filtration, and the filtrate was poured onto crushed ice. The

compound separated was filtered, washed with water, dried, and added

to the first crop, if any. The total product on recrystallization from

methanol afforded pure 4⁰-methoxyphenacyl-3-nitro-4-methoxybenzyl-

sulfone (13). Yield, 76%; white solid, mp 172ꢀ174 °C. ¹H NMR

(CDCl₃, 300 MHz): δ 3.83 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 4.31 (s,

2H, CH₂), 4.45 (s, 2H, CH₂), 6.87 (d, J = 9.0 Hz, 2H, ArꢀH), 7.06 (d,

J = 8.7 Hz, 2H, ArꢀH), 7.67 (dd, J = 8.7, 2.1 Hz, 1H, ArꢀH), 7.90 (d, J =

9.0 Hz, 1H, ArꢀH), 7.98 (d, J = 2.1 Hz, 1H, ArꢀH). HRMS: m/z calcd

[M + H] 380.0726; found 380.0709. Anal. (C₁₇H₁₇NO₇S) C, H, N.

Preparation of 2-(3-Nitro-4-methoxybenzylsulfonyl)-

1-(4⁰-methoxyphenyl)ethanol (14). To anhydrous tetrahydrofur-

an (THF) solution (20 mL) of 4-methoxyphenacylbenzylsulfone 13

(10 mmol) maintained at 0 °C was added NaBH₄(10 mmol) slowly

under N₂atmosphere. The reaction mixture was maintained at 0 °C for 1

h. After completion of the reaction monitored by TLC (hexane/ethyl

acetate, 8:2 on silica gel plate), the contents were poured onto crushed

ice. The solid that separated out was filtered, washed with water, and

dried under vacuum to yield 14. Yield, 56%; white solid, mp

1

152ꢀ154 °C. H NMR (DMSO-d₆, 300 MHz): δ 3.43 (dd, J = 9.9,

4.6 Hz, 2H, CHꢀCH₂), 3.73 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 4.58

(dd, J = 15.7, 13.6 Hz, 2H, SO₂CH₂), 5.05 (m, 1H, CHOH), 6.02 (d, J =

4.2 Hz, 1H, CH), 6.90 (d, J = 8.6 Hz, 2H, ArꢀH), 7.32 (d, J = 8.6 Hz, 2H,

ArꢀH), 7.42 (d, J = 9.0 Hz, 1H, ArꢀH), 7.69 (dd, J = 8,7, 1.8 Hz, 1H,

ArꢀH), 7.93 (d, J = 1.8 Hz, 1H, ArꢀH) . HRMS: m/z calcd [M + H]

382.0882; found 382.0916. Anal. (C₁₇H₁₉NO₇S) C, H, N.

Preparation of (E)-4⁰-Methoxystyryl-3-nitro-4-methoxy-

benzylsulfone (8c). p-Toluenesulfonic acid (1 mmol) was added in

one portion to a mixture of 2-(3-nitro-4-methoxybenzylsulfonyl)-1-(4⁰-

methoxyphenyl)ethanol 14 (5 mmol) in anhydrous toluene (25 mL) at

room temperature and under N₂atmosphere. The temperature was

raised to 120 °C, and the mixture was refluxed for 2ꢀ4 h using a

DeanꢀStark water separator. After completion of the reaction mon-

itored by TLC (chloroform, on silica gel plate), the reaction mixture was

concentrated under reduced pressure and then quenched by the

addition of water (25 mL). The aqueous layer was neutralized with a

saturated aqueous solution of sodium hydrogen carbonate and extracted

with dichloromethane (3 ꢁ 25 mL). The combined organic extracts

were washed with brine (2 ꢁ 25 mL), dried over Na₂SO₄, filtered and

(Z)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-nitrobenzyl Sulfide

(16). The yield of this reaction was 40%, giving a white solid with a melting

point of 102ꢀ104 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.75 (s, 12H, 4 ꢁ

OCH₃), 3.86 (s, 2H, CH₂S), 6.06 (s, 2H, ArꢀH), 6.14 (d, J = 10.2 Hz, 1H,

CHd), 6.49(d, J= 10.5 Hz, 1H, dCH), 6.94(d, J= 8.4 Hz, 1H, ArꢀH), 7.43

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(dd, J= 8.7, 2.1 Hz, 1H, ArꢀH), 7.75 (d, J= 1.8 Hz, 1H, ArꢀH). HRMS: m/z

calcd [M + H] 392.1090; found 392.1084. Anal. (C₁₉H₂₁NO₆S) C, H, N.

Synthesis of (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-

aminobenzyl Sulfide (24). The title compound was obtained by

the reduction of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-nitrobenzyl

sulfide 17 following the procedure as described for compound 8b. Yield,

43%; pale yellow solid, mp 106ꢀ108 °C. ¹H NMR (CDCl₃, 300 MHz):

δ 3.73 (s, 6H, 2 ꢁ OCH₃), 3.76 (s, 6H, 2 ꢁ OCH₃), 3.80 (s, 2H, CH₂S),

6.03 (s, 2H, ArꢀH), 6.63ꢀ6.71 (m, 2H, ArꢀH), 6.74 (d, J = 15.6 Hz,

1H, CHd), 7.01 (d, J = 15.9 Hz, 1H, dCH), 7.30 (s, 1H, ArꢀH).

HRMS: m/z calcd [M + H] 362.1348; found 362.1339. Anal.

(C₁₉H₂₃NO₄S) C, H, N.

Synthesis of (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-ni-

trobenzyl Sulfoxide (20). An aqueous 30% hydrogen peroxide

(0.56 mL, 5.2 mmol) was added to a stirred solution of (E)-2⁰,4⁰,6⁰-

trimethoxystyryl-4-methoxy-3-nitrobenzyl sulfide 17 (1.0 g, 2.6 mmol)

in 1,1,1,3,3,3-hexafluoro-2-propanol (10 mL) at 25 °C. The reaction was

monitored by TLC. After the complete disappearance of the sulfide

(2 h), the excess hydrogen peroxide was quenched with saturated

sodium sulfite (Na₂SO₃) solution (5.0 mL) and the fluorous organic

phase containing the sulfoxide was separated. After removal of the

solvent under vacuo, sulfoxide 20 was obtained as a semisolid. Flash

column chromatography (chloroform) gave pure 20. The yield of this

reaction was 91%, giving a white solid with a melting point of

148ꢀ150 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.75 (s, 6H, 2 ꢁ OCH₃),

3.77 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 3.96ꢀ4.00 (d, J = Hz, 2H,

CH₂S), 6.01 (s, 2H, ArꢀH), 6.96 (d, J = 15.9 Hz, 1H, CHd), 7.02 (d, J =

8.7 Hz, 1H, ArꢀH), 7.36 (d, J = 15.6 Hz, 1H, dCH), 7.46 (dd, J = 8.7,

2.1 Hz, 1H, ArꢀH), 7.71 (d, J = 2.1 Hz, 1H, ArꢀH). HRMS: m/z calcd

[M + H] 408.1039; found 408.1022. Anal. (C₁₉H₂₁NO₇S) C, H, N.

Synthesis of (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-

aminobenzyl Sulfoxide (21). The title compound was obtained

by the reduction of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-nitroben-

zyl sulfoxide 20 following the procedure as described for compound 8b.

Yield, 46%; pale yellow solid, mp 131ꢀ133 °C. ¹H NMR (CDCl₃, 300

MHz): δ 3.77 (br s, 12H, 4 ꢁ OCH₃), 3.81 (br s, 2H, NH₂), 3.92 (d, J =

12.6 Hz, 2H, CH₂S), 6.04 (s, 2H, ArꢀH), 6.60ꢀ6.69 (m, 3H, ArꢀH),

7.08 (d, J = 15.6 Hz, 1H, CHd), 7.43 (d, J = 15.6 Hz, 1H, dCH).

HRMS: m/z calcd [M + H] 378.1297; found 378.1280. Anal.

(C₁₉H₂₃NO₅S) C, H, N.

by column chromatography (chloroform) to obtain the title compound

24. The yield of this reaction was 64%; light yellow solid, mp 146ꢀ

148 °C. The analytical data are in accord with 8p.

Synthesis of (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-

aminobenzyl Sulfoxide (21, Scheme 7). Step 1: 4-Methoxy-

3-nitrobenzylsulfinylacetic Acid (25). A vigorously stirred solu-

tion of sodium hydroxide (0.58 g, 14.5 mmol) and deionized water

(30 mL) was treated with 4-methoxy-3-nitrobenzylthioacetic acid 5a

(3,30 g, 12.1 mmol). The resulting suspension was stirred at ambient

temperature for 10 min. To this was added sodium bicarbonate (8.00 g,

95 mmol) and acetone (10 mL), and the mixture was cooled to 0 °C.

The Oxone solution (4.85 g in 20 mL of 4 ꢁ 10^ꢀ4M EDTA) was added

over 10 min, keeping the mixture below 5 °C. The suspension was stirred

for 5 min and immediately quenched at 2 °C with sodium bisulfate (3 g

in 6 mL of deionized water) and stirred for 15 min. Ethyl acetate

(75 mL) was added, and the solution was acidified with 6 N (aq) HCl

(18 mL). The aqueous phase was isolated, treated with sodium chloride

(15.0 g), and re-extracted with ethyl acetate (75 mL). The organic layers

were combined and washed with deionized water (50 mL), washed with

brine (30 mL), dried over anhydrous sodium sulfate, filtered, and

concentrated under vacuo. The crystals formed were dried under

vacuum to afford pure 25. The yield of this reaction was 89%, giving a

white solid with a melting point of 122ꢀ124 °C. ¹H NMR (CDCl₃, 300

MHz): δ 3.42 (s, 2H, ꢀSCH₂), 3.90 (s, 3H, OCH₃), 4.02 (s, 2H, CH₂S),

7.42 (d, J = 8.4 Hz, 1H, ArꢀH), 7.69 (dd, J = 8.7, 2.4 Hz, 1H, ArꢀH),

7.92 (d, J = 2.1 Hz, 1H, ArꢀH), 12.90 (br s, 1H, COOH). HRMS: m/z

calcd [M ꢀ H] 272.0307; found 272.0300. Anal. Calcd for

C₁₀H₁₁NO₆S: C, 43.95%; H, 4.06%; N, 5.13%. Found: C, 44.06%; H,

4.09%; N, 5.17%.

Step 2: (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-nitro-

benzyl Sulfoxide (20). The title compound was obtained from

4-methoxy-3-nitrobenzylsulfinylacetic acid 25 and 2,4,6-trimethoxyben-

zaldehyde following the procedure as described in method A and

Scheme 1. The yield of this reaction was 48%. The analytical data are

in accord with 20 as reported in Scheme 5.

Step 3: (E)-2⁰, 4⁰, 6⁰-Trimethoxystyryl-4-methoxy-3-ami-

nobenzyl Sulfoxide (21). The title compound was obtained by the

reduction of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-nitrobenzyl sulf-

oxide 20 following the procedure as described for compound 8b. The

yield of this reaction was 42%. The analytical data are in accord with

21 as reported in Scheme 5.

General Procedure for the Preparation of Amino Esters

(26, Scheme 8). Sodium acetate (32.8 g, 400 mmol) was dissolved in

ethanol (200 mL). Methyl 2-bromoacetate (61.1 g, 400 mmol) was

added to the above solution and refluxed for 10 min. To the cooled

reaction mixture compound (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-

aminobenzylsulfone 8p or (E)-3⁰,4⁰,5⁰-trimethoxystyryl-4-methoxy-3-

aminobenzylsulfone 8q (39.35 g, 100 mmol) was added and then

refluxed for 48 h. After completion of the reaction monitored by TLC

(chloroform/methanol, 9:1 on silica gel plate), the reaction mixture was

concentrated under vacuum and poured into iceꢀwater. The solid

formed was filtered, washed with water, and dried under vacuum. The

crude product on purification from ethanol resulted in analytical pure

product 26. The following amino esters were prepared using the above

procedure.

(E)-Methyl 2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsul-

fonyl)methyl)phenylamino)acetate (26a). Sodium acetate (3.28 g,

39 mmol) was dissolved in methanol (20 mL). Methyl 2-bromoacetate (6.11

g, 40 mmol) was added to the above solution and refluxed for 10 min. To the

cooled reaction mixture compound (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-meth-

oxy-3-aminobenzylsulfone 8p (3.95 g, 10 mmol) was added, and then the

mixture was refluxed for 4ꢀ6 h. The reaction mixture was concentrated

under vacuum and poured into iceꢀwater. The formed precipitate was

filtered, washed with water, and dried under vacuum. The crude product on

Synthesis of (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-ni-

trobenzylsulfone (22 and 8l). The title compound was obtained by

the oxidation of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-nitrobenzyl

sulfoxide 20 according to the procedure reported in the literature.⁷The

yield of this reaction was 62%; yellow solid, mp 184ꢀ186 °C. The

analytical data are in accord with 8l.

Synthesis of (E)-2⁰,4⁰,6⁰-Trimethoxystyryl-4-methoxy-3-

aminobenzylsulfone (23 and 8p). Method A. The title com-

pound was obtained by the reduction of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-

4-methoxy-3-nitrobenzylsulfone 22 following the procedure as de-

scribed for compound 8b. The yield of this reaction was 52%; light

yellow solid, mp 146ꢀ148 °C. The analytical data are in accord with 8p.

Method B. The title compound was obtained by the oxidation of (E)-

2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-aminobenzyl sulfide 24. To a

solution of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-aminobenzyl sul-

fide 24 (903 mg, 2.5 mmol) in anhydrous dichloromethane (20 mL) at

0 °C was added m-chloroperoxybenzoic acid (1.29 g, 7.5 mmol) slowly

under nitrogen atmosphere, and the resulting mixture was stirred at 0 °C

to room temperature for 3 h. After completion of the reaction (TLC

monitoring, chloroform on silica gel plate), 10% sodium hydrogen

carbonate solution (25 mL) was added slowly. The mixture was stirred

for 10 min, and the aqueous layer was separated. The organic layer

washed with water (2 ꢁ 25 mL), dried over sodium sulfate, filtered, and

concentrated under reduced pressure. The resulting residue was purified

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recrystallization from ethanol resulted in pure product 26a. Yield, 70%; white

¹H NMR (CDCl₃, 300 MHz): δ 3.49 (s, 3H, OCH₃), 3.51 (s, 6H, 2 ꢁ

OCH₃), 3.55 (s, 3H, OCH₃), 3.57 (s, 3H, OCH₃), 3.95ꢀ4.16 (m, 1H,

CH), 4.22 (s, 2H, CH₂), 4.85 (t, J = 9.0 Hz, 1H, NH), 5.92 (s, 2H,

ArꢀH), 6.23 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 6.40 (d, J = 8.1 Hz, 1H,

ArꢀH), 6.53 (d, J = 2.1 Hz, 1H, ArꢀH), 6.80 (d, J = 15.6 Hz,

1H, dCH), 7.55 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd

[M + H] 534.1345; found 534.1339. Anal. (C₂₃H₂₆F₃NO₈S) C, H, N.

(E)-Methyl 2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)-2-methylpropanoate (26g). The title com-

pound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-

methoxy-3-aminobenzylsulfone 8p with methyl 2-bromo2-methylpropionate

following the procedure as described for compound 26. Yield, 60%; white solid,

mp 170ꢀ172 °C. ¹H NMR (CDCl₃, 500 MHz): δ 1.52 (s, 6H, 2 ꢁ CH₃),

3.68 (s, 3H, OCH₃), 3.81 (s, 6H, 2 ꢁ OCH₃), 3.82 (s, 3H, OCH₃), 3.83 (s,

3H, OCH₃), 4.11 (s, 2H, CH₂), 6.06 (s, 2H, ArꢀH), 6.41 (s, 1H, ArꢀH),

6.69ꢀ6.71 (m, 2H, ArꢀH), 7.02 (d, J = 15.5 Hz, 1H, dCH), 7.80 (d, J= 15.5

Hz, 1H, CHd). HRMS: m/z calcd [M + H] 494.1770; found 494.1761. Anal.

(C₂₄H₃₁NO₈S) C, H, N.

(E)-Methyl 2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)-2-phenylacetate (26h). The title compound

was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-

aminobenzylsulfone 8p with methyl R-bromophenylacetate following the

procedure as described for compound 26. Yield, 63%; white solid, mp

94ꢀ96 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.71 (s, 3H, OCH₃), 3.83 (s,

6H, 2 ꢁ OCH₃),3.86(s,3H,OCH₃),3.88(s,3H,OCH₃), 4.06 (s, 2H, CH₂),

4.99 (d, J = 6.0 Hz, 1H, CH), 5.43(d, J = 6.3 Hz, 1H, NH), 6.10 (s, 2H,

ArꢀH), 6.36 (d, J=1.8Hz,1H, ArꢀH), 6.67 (dd, J= 8.1, 1.8 Hz, 1H, ArꢀH),

6.73 (d, J = 8.1 Hz, 1H, ArꢀH), 6.99 (d, J = 15.6 Hz, 1H, dCH), 7.29ꢀ7.35

(m, 3H, ArꢀH), 7.42 (dd, J = 8.1, 1.8 Hz, 2H, ArꢀH), 7.77 (d, J = 15.6 Hz,

1H, CHd). HRMS: m/z calcd [M + H] 542.1770; found 542.1778. Anal.

(C₂₈H₃₁NO₈S) C, H, N.

(E)-Methyl 2-(4⁰⁰-Fluorophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-

trimethoxystyrylsulfonyl)methyl)phenylamino)acetate (26i). The

title compound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-

4-methoxy-3-aminobenzylsulfone 8p with methyl 2-bromo-2- (4-fluorophe-

nyl)acetate following the procedure as described for compound 26. Yield, 60%;

white solid, mp 152ꢀ154 °C. ¹H NMR (CDCl₃, 500 MHz): δ 3.72 (s, 3H,

OCH₃), 3.82 (s, 6H, 2 ꢁ OCH₃), 3.86 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃),

4.05 (s, 2H, CH₂), 4.98 (d, J = 2.5 Hz, 1H, CH), 6.10 (s, 2H, ArꢀH), 6.33 (d,

J= 2.0 Hz, 1H, ArꢀH), 6.69 (dd, J= 8.5, 2.0 Hz, 1H, ArꢀH), 6.73 (d, J=8.5Hz,

1H, ArꢀH), 6.96ꢀ6.99 (m, 2H, ArꢀH), 7.00 (d, J = 15.5 Hz, 1H, dCH),

7.37ꢀ7.40 (m, 2H, ArꢀH), 7.76 (d, J = 16.0 Hz, 1H, CHd). HRMS: m/z

calcd [M + H] 560.1676; found 560.1656. Anal. (C₂₈H₃₀FNO₈S) C, H, N.

(E)-Methyl 2-(4⁰⁰-Chlorophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-

trimethoxystyrylsulfonyl)methyl)phenylamino)acetate (26j). The

title compound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-

4-methoxy-3-aminobenzylsulfone 8p with methyl 2-bromo-2- (4-chlorophe-

nyl)acetate following the procedure as described for compound 26. Yield, 64%;

white solid, mp 154ꢀ156 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.72 (s, 3H,

OCH₃), 3.83 (s, 6H, 2 ꢁ OCH₃), 3.86 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃),

4.05 (s, 2H, CH₂), 4.96 (s, 1H, CH), 6.11 (s, 2H, ArꢀH), 6.31 (s, 1H, ArꢀH),

6.67ꢀ6.75 (m, 2H, ArꢀH), 6.99 (d, J = 15.6 Hz, 1H, dCH), 7.25ꢀ7.28 (m,

2H, ArꢀH), 7.54 (d, J= 8.4 Hz, 2H, ArꢀH), 7.76 (d, J= 15.6 Hz, 1H, CHd).

HRMS: m/z calcd [M + H] 576.1381; found 576.1367. Anal.

(C₂₈H₃₀ClNO₈S) C, H, N.

(E)-Methyl 2-(4⁰⁰-Bromophenyl)-2-(2-methoxy-5-((2⁰,4⁰,

6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate

(26k). The title compound was obtained by the alkylation of (E)-

2⁰,4⁰,6⁰-trimethoxystyryl-4-methoxy-3-aminobenzylsulfone 8p with

methyl 2-bromo-2- (4-bromophenyl)acetate following the procedure as

described for compound 26. Yield, 62%; white solid, mp 150ꢀ152 °C.

¹H NMR (CDCl₃, 400 MHz): δ 3.48 (s, 3H, OCH₃), 3.59 (s, 6H, 2 ꢁ

OCH₃), 3.62 (s, 3H, OCH₃), 3.65 (s, 3H, OCH₃), 3.81 (s, 2H, CH₂),

4.71(s, 1H, CH), 5.87 (s, 2H, ArꢀH), 6.07 (d, J = 1.8 Hz, 1H, ArꢀH),

1

solid, mp 150ꢀ152 °C. H NMR (CDCl₃, 300 MHz): δ 3.69 (s, 3H,

OCH₃), 3.75(s, 6H, 2 ꢁ OCH₃), 3.78 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃),

3.81 (d, J = 5.4 Hz, 2H, CH₂), 4.09 (s, 2H, CH₂), 4.74 (t, J = 5.4 Hz, 1H,

NH), 6.01(s, 2H, ArꢀH), 6.41 (d, J = 1.8 Hz, 1H, ArꢀH), 6.61ꢀ6.68 (m,

2H, ArꢀH), 6.97 (d, J = 15.6 Hz, 1H, dCH), 7.73 (d, J = 15.6 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 466.1457; found 466.1446. Anal.

(C₂₂H₂₇NO₈S) C, H, N.

(E)-Methyl 2-(2-Methoxy-5-((3⁰,4⁰,5⁰-trimethoxystyrylsul-

fonyl)methyl)phenylamino)acetate (26b). The title compound

was obtained by the alkylation of (E)-3⁰,4⁰,5⁰-trimethoxystyryl-4-meth-

oxy-3-aminobenzylsulfone 8q with methyl 2-bromoacetate following the

procedure as described for compound 26. Yield, 63%; light brown solid,

mp 82ꢀ84 °C. ¹H NMR (CDCl₃, 400 MHz): δ 3.67 (s, 3H, OCH₃),

3.79 (s, 3H, OCH₃), 3.81 (s, 6H, 2 ꢁ OCH₃), 3.82 (s, 3H, OCH₃), 3.86

(d, J = 5.4 Hz, 2H, CH₂), 4.14 (s, 2H, CH₂), 4.53 (t, J = 5.4 Hz, 1H, NH),

6.52 (d, J = 15.5 Hz, 1H, dCH), 6.57 (s, 2H, ArꢀH), 6.62 (dd, J = 8.2,

2.1 Hz, 1H, ArꢀH), 6.67 (d, J = 8.2 Hz, 1H, ArꢀH), 6.70 (d, J = 2.1 Hz,

1H, ArꢀH), 7.24 (d, J = 15.4 Hz, 1H, CHd). HRMS: m/z calcd [M +

H] 466.1457; found 466.1465. Anal. (C₂₂H₂₇NO₈S) C, H, N.

(E)-Ethyl 3-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfo-

nyl)methyl)phenylamino)propanoate (26c). The title com-

pound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-

4-methoxy-3-aminobenzylsulfone 8p with ethyl 3-bromopropionate

following the procedure as described for compound 26. Yield, 59%;

light green solid, mp 72ꢀ74 °C. ¹H NMR (CDCl₃, 300 MHz): δ 1.26 (t,

J = 7.2 Hz, 3H, CH₃), 2.58 (t, J = 6.3 Hz, 2H, CH₂), 3.41 (t, J = 6.6 Hz,

2H, CH₂), 3.82 (s, 9H, 3 ꢁ OCH₃), 3.84 (s, 3H, OCH₃), 4.04ꢀ4.10 (m,

2H, OCH₂), 4.17 (s, 2H, CH₂), 5.06 (t, J = 5.4 Hz, 1H, NH), 6.09 (s, 2H,

ArꢀH), 6.61ꢀ6.64 (m, 1H, ArꢀH), 6.66ꢀ6.71 (m, 2H, ArꢀH), 7.06

(d, J = 15.6 Hz, 1H, dCH), 7.84 (d, J = 15.9 Hz, 1H, CHd). HRMS: m/

z calcd [M + H] 480.1614; found 480.1647. Anal. (C₂₃H₂₉NO₈S)

C, H, N.

(E)-Methyl 2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsul-

fonyl)methyl)phenylamino)propanoate (26d). The title com-

pound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxystyryl-4-

methoxy-3-aminobenzylsulfone 8p with methyl 2-bromopropionate

following the procedure as described for compound 26. Yield, 60%;

white solid, mp 178ꢀ180 °C. ¹H NMR (CDCl₃, 400 MHz): δ 1.24 (d,

J = 6.9 Hz, 3H, CH₃), 3.50 (s, 3H, OCH₃), 3.61 (s, 6H, 2 ꢁ OCH₃), 3.63

(s, 3H, OCH₃), 3.64 (s, 3H, OCH₃), 3.90ꢀ3,92 (m, 1H, CH), 3.94 (s,

2H, CH₂), 4.52 (br s, 1H, NH), 5.88 (s, 2H, ArꢀH), 6.32 (d, J = 1.7 Hz,

1H, ArꢀH), 6.47 (dd, J = 8.1, 1.7 Hz, 1H, ArꢀH), 6.52 (d, J = 8.1 Hz,

1H, ArꢀH), 6.81 (d, J = 15.6 Hz, 1H, dCH), 7.59 (d, J = 15.6 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 480.1579; found 480.1568. Anal.

(C₂₃H₂₉NO₈S) C, H, N.

(E)-Methyl2⁰⁰,2⁰⁰-Difluoro-2(2-methoxy-5-((2⁰,4⁰,6⁰-trimethox-

ystyrylsulfonyl)methyl)phenylamino)acetate (26e). The title

compound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxys-

tyryl-4-methoxy-3-aminobenzylsulfone 8p with methyl 2-chloro-2,2-

difluoroacetate following the procedure as described for compound

1

26. Yield, 57%; white solid, mp 186ꢀ188 °C. H NMR (CDCl₃, 300

MHz): δ 3.74 (s, 3H, OCH₃), 3.83 (s, 6H, 2 ꢁ OCH₃), 3.85 (s, 3H,

OCH₃), 3.87 (s, 3H, OCH₃), 4.22 (s, 2H, CH₂), 6.09 (s, 2H, ArꢀH),

6.86 (d, J = 8.4 Hz, 1H, ArꢀH), 7.09 (d, J = 15.6 Hz, 1H, dCH), 7.11

(dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 7.19 (s, 1H, ArꢀH), 7.84 (d, J = 15.6

Hz, 1H, CHd), 8.08 (brs, 1H, NH). HRMS: m/z calcd [M + H]

502.1269; found 502.1284. Anal. (C₂₂H₂₅F₂NO₈S) C, H, N.

(E)-Methyl 3⁰⁰,3⁰⁰,3⁰⁰-Trifluoro-2-(2-methoxy-5-((2⁰,4⁰,6⁰-

trimethoxystyrylsulfonyl)methyl)phenylamino)propanoate

(26f). The title compound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-

trimethoxystyryl-4-methoxy-3-aminobenzylsulfone 8p with methyl

2-bromo-3,3,3-trifluoropropanoate following the procedure as de-

scribed for compound 26. Yield, 56%; white solid, mp 192ꢀ194 °C.

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6.44 (dd, J = 8.1, 1.8 Hz, 1H, ArꢀH), 6.50 (d, J = 8.1 Hz, 1H, ArꢀH),

6.75 (d, J = 15.6 Hz, 1H, dCH), 7.05ꢀ7.08 (m, 2H, ArꢀH), 7.16ꢀ7.19

(m, 2H, ArꢀH), 7.53 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M

+ H] 620.0876; found 620.0859. Anal. (C₂₈H₃₀BrNO₈S) C, H, N.

(E)-Methyl 2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)-2-(4⁰⁰-methoxyphenyl)acetate (26l). The

title compound was obtained by the alkylation of (E)-2⁰,4⁰,6⁰-trimethoxystyr-

yl-4-methoxy-3-aminobenzylsulfone 8p with methyl 2-bromo-2-(4-methoxy-

phenyl)acetate following the procedure as described for compound 26. Yield,

61%; white solid, mp 182ꢀ184 °C. ¹H NMR (CDCl₃, 500 MHz): δ 3.70 (s,

3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.83 (s, 6H, 2 ꢁ OCH₃), 3.85 (s, 3H,

OCH₃),3.87(s,3H,OCH₃), 4.06 (s, 2H, CH₂), 4.91(s, 1H, CH), 6.10 (s, 2H,

ArꢀH), 6.37 (d, J=1.5Hz,1H,ArꢀH), 6.68 (dd, J= 8.1, 1.5 Hz, 1H, ArꢀH),

6.73 (d, J = 8.1 Hz, 1H, ArꢀH), 6.84 (d, J = 9.0 Hz, 2H, ArꢀH), 7.00 (d, J =

15.5 Hz, 1H, dCH), 7.33(d, J= 8.0 Hz, 2H, ArꢀH), 7.77 (d, J= 16.0 Hz, 1H,

CHd). HRMS: m/z calcd [M + H] 572.1876; found 572.1896. Anal.

(C₂₉H₃₃NO₉S) C, H, N.

General Procedure for the Preparation of Amino Acids

(27, Scheme 8). To a solution of amine ester 26 (46.5 g, 100 mmol) in

ethanol (200 mL), 20% aqueous sodium hydroxide solution (200 mL)

was added. The reaction mixture was refluxed for 2.5 h. After completion

of the reaction (TLC, monitoring, chloroform/methanol, 9:1 on silica

gel plate), the solvent was removed under vacuum and the remainder

was acidified by acetic acid to pH 4. The solid that formed was filtered

and dried to get the crude amino acid 27 which on crystallization from

acetone (2 ꢁ 25 mL) resulted in analytically pure amino acid 27 as white

crystals.

(E)-2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)acetic Acid (27a). The title compound

was obtained by the hydrolysis of (E)-methyl 2-(2-methoxy-5-

((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate 26a

following the procedure as described for compound 27. Yield, 50%;

pale yellow solid, mp 172ꢀ174 °C. ¹H NMR (DMSO-d₆, 300 MHz):

δ 3.78 (s, 2H, NHꢀCH₂), 3.86 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃),

3.92 (s, 6H, 2 ꢁ OCH₃), 4.33 (s, 2H, CH₂), 6.35 (s, 2H, ArꢀH), 6.48

(d, J = 1.8 Hz, 1H, ArꢀH), 6.65 (dd, J = 8.1, 1.8 Hz, 1H, ArꢀH), 6.86

(d, J = 8.1 Hz, 1H, ArꢀH), 7.17 (d, J = 15.9 Hz, 1H, dCH), 7.62 (d,

J = 15.6 Hz, 1H, CHd). ¹³C NMR (DMSO-d₆, 75 MHz): δ 172.1,

163.5, 160.8, 146.3, 136.9, 132.7, 123.6, 121.3, 119.2, 111.9, 109.3,

102.5, 90.6, 60.3, 55.9, 55.5, 55.4, 44.4, 30.6. HRMS: m/z calcd

[M ꢀ H] 450.1301; found 450.1311. Anal. (C₂₁H₂₅NO₈S) C, H, N.

(E)-2-(2-Methoxy-5-((3⁰,4⁰,5⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)acetic Acid (27b). The title compound

was obtained by the hydrolysis of (E)-methyl 2-(2-methoxy-5-(-

(3⁰,4⁰,5⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate 26b

following the procedure as described for compound 27. Yield, 55%;

light yellow solid, mp 112ꢀ114 °C. ¹H NMR (CDCl₃, 400 MHz): δ

3.78 (s, 3H, OCH₃), 3.79 (s, 6H, 2 ꢁ OCH₃), 3.81 (s, 3H, OCH₃),

3.87 (s, 2H, CH₂), 4.15 (s, 2H, CH₂), 6.44 (d, J = 1.8 Hz, 1H, ArꢀH),

6.55 (d, J = 15.4 Hz, 1H, dCH), 6.59 (s, 2H, ArꢀH), 6.62ꢀ6.66 (m,

1H, ArꢀH), 6.69 (d, J = 8.2 Hz, 1H, ArꢀH), 7.26 (d, J = 15.5 Hz, 1H,

CHd). HRMS: m/z calcd [M ꢀ H] 450.1301; found 450.1314. Anal.

(C₂₁H₂₅NO₈S) C, H, N.

HRMS: m/z calcd [M ꢀ H] 464.1457; found 464.1471. Anal.

(C₂₂H₂₇NO₈S) C, H, N.

(E)-2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)propanoic Acid (27d). The title com-

pound was obtained by the hydrolysis of (E)-methyl 2-(2-methoxy-

5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)propanoate

26d following the procedure as described for compound 27. Yield, 57%;

light yellow solid, mp 116ꢀ118 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ

1.07 (d, J = 6.6 Hz, 3H,CH₃), 3.57 (s, 3H, OCH₃), 3.60 (s, 3H, OCH₃),

3.64 (s, 6H, 2 ꢁ OCH₃), 3.79ꢀ3.83 (m, 1H, CH), 4.06 (s, 2H, CH₂),

6.08 (s, 2H, ArꢀH), 6.26 (s, 1H, ArꢀH), 6.35(d, J = 7.5 Hz, 1H, ArꢀH),

6.58 (d, J = 8.1 Hz, 1H, ArꢀH), 6.90 (d, J = 15.6 Hz, 1H, dCH), 7.35 (d,

J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M ꢀ H] 464.1457; found

464.1446. Anal. (C₂₂H₂₇NO₈S) C, H, N.

(E)-2⁰⁰,2⁰⁰-Difluoro-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimethoxys-

tyrylsulfonyl)methyl)phenylamino)acetic Acid (27e). The title

compound was obtained by the hydrolysis of (E)-methyl 2⁰⁰,2⁰⁰-difluoro-

2(2-methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)-

acetate 26e following the procedure as described for compound 27. Yield,

59%; white solid, mp 196ꢀ198 °C. ¹H NMR (CDCl₃, 300 MHz): δ 3.85

(s, 6H, 2 ꢁ OCH₃), 3.87 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 4.12 (s, 2H,

CH₂), 6.12 (s, 2H, ArꢀH), 6.58 (d, J = 8.1 Hz, 1H, ArꢀH), 6.89 (d, J = 8.4

Hz, 1H, ArꢀH), 7.06 (dd, J = 8.4, 2.1 Hz, 1H, ArꢀH), 7.29 (d, J = 15.6 Hz,

1H, dCH), 7.84 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M ꢀ H]

486.1112; found 486.1101. Anal. (C₂₁H₂₃F₂NO₈S) C, H, N.

(E)-3⁰⁰,3⁰⁰,3⁰⁰-Trifluoro-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimeth-

oxystyrylsulfonyl)methyl)phenylamino)propanoic Acid (27f).

The title compound was obtained by the hydrolysis of (E)-methyl3⁰⁰,3⁰⁰,3⁰⁰-

trifluoro-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)-

phenylamino)propanoate 26f following the procedure as described for

1

compound 27. Yield, 53%; white solid, mp 180ꢀ182 °C. H NMR

(DMSO-d₆, 300 MHz): δ 3.78 (s, 3H, OCH₃), 3.83 (s, 6H, 2 ꢁ

OCH₃), 3.87 (s, 3H, OCH₃), 4.05 (d, J = 6.3 Hz, CH), 4.12 (s, 2H,

CH₂), 6.28 (s, 2H, ArꢀH), 6.61 (d, J = 8.4 Hz, 1H, ArꢀH), 6.76 (dd, J =

8.4, 2.1 Hz, 1H, ArꢀH), 7.01ꢀ7.09 (m, 1H, ArꢀH), 7.10 (d, J = 15.6

Hz, 1H, dCH), 7.57 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd

[M ꢀ H] 518.1175; found 518.1186. Anal. (C₂₂H₂₄F₃NO₈S) C, H, N.

(E)-2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)-2-methylpropanoic Acid (27g). The title

compound was obtained by the hydrolysis of (E)-methyl 2-(2-methoxy-

5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)-2-methyl-

propanoate 26g following the procedure as described for compound

27. Yield, 61%; white solid, mp 142ꢀ144 °C. ¹H NMR (CDCl₃, 500

MHz): δ 1.48 (s, 6H, 2 ꢁ CH₃), 3.83 (s, 6H, 2 ꢁ OCH₃), 3.84 (s, 3H,

OCH₃), 3.85 (s, 3H, OCH₃), 4.14 (s, 2H, CH₂), 6.07 (s, 2H, ArꢀH),

6.52 (s, 1H, ArꢀH), 6.79 (d, J = 8.0 Hz, 1H, ArꢀH), 6.85 (d, J = 8.1 Hz,

1H, ArꢀH), 7.05 (d, J = 16.0 Hz, 1H, dCH), 7.78 (d, J = 15.5 Hz, 1H,

CHd). HRMS: m/z calcd [M ꢀ H] 478.1614; found 478.1605. Anal.

(C₂₃H₂₉NO₈S) C, H, N.

(E)-2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)-2-phenylacetic Acid (27h). The title

compound was obtained by the hydrolysis of (E)-methyl 2-(2-meth-

oxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)-2-phe-

nylacetate 26h following the procedure as described for compound 27.

Yield, 60%; white solid, mp 124ꢀ126 °C. ¹H NMR (CDCl₃, 400 MHz):

δ 3.75 (s, 6H, 2 ꢁ OCH₃), 3.77 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃),

4.00(s, 2H, CH₂), 4.86 (s, 1H, CH), 6.03 (s, 2H, ArꢀH), 6.32 (d, J = 1.4

Hz, 1H, ArꢀH), 6.66ꢀ6.69 (m, 2H, ArꢀH), 6.94 (d, J = 15.6 Hz,

1H, dCH), 7.22ꢀ7.26 (m, 3H, ArꢀH), 7.32ꢀ7.34 (m, 2H, ArꢀH),

7.69 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z calcd [M ꢀ H] 526.1614;

found 526.1604. Anal. (C₂₇H₂₉NO₈S) C, H, N.

(E)-3-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)propanoic Acid (27c). The title com-

pound was obtained by the hydrolysis of (E)-ethyl 3-(2-methoxy-

5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)propanoate

26c following the procedure as described for compound 27. Yield, 51%;

white solid, mp 130ꢀ132 °C. ¹H NMR (CDCl₃, 300 MHz): δ 2.56 (t,

J = 6.3 Hz, 2H, CH₂), 3.34 (t, J = 6.3 Hz, 2H, CH₂), 3.75 (s, 6H, 2 ꢁ

OCH₃), 3.76 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 4.11 (s, 2H, CH₂),

6.01 (s, 2H, ArꢀH), 6.57 (s, 1H, ArꢀH), 6.64ꢀ6.71 (m, 2H, ArꢀH),

6.99 (d, J = 15.9 Hz, 1H, dCH), 7.76 (d, J = 15.6 Hz, 1H, CHd).

(E)-2-(4⁰⁰-Fluorophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimeth-

oxystyrylsulfonyl)methyl)phenylamino)acetic Acid (27i). The

title compound was obtained by the hydrolysis of (E)-methyl

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2-(4⁰⁰-fluorophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsul-

fonyl)methyl)phenylamino)acetate 26i following the procedure as

described for compound 27. Yield, 60%; light yellow solid, mp

82ꢀ84 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 3.79 (s, 3H, OCH₃), 3.83

(s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.14 (s, 2H,

CH₂), 4.42(s, 1H, CH), 6.29 (s, 2H, ArꢀH), 6.33 (d, J = 2.0 Hz, 1H,

ArꢀH), 6.69 (dd, J = 8.5, 2.0 Hz, 1H, ArꢀH), 6.73 (d, J = 8.5 Hz, 1H,

ArꢀH), 6.96ꢀ6.99 (m, 2H, ArꢀH), 7.00 (d, J = 15.5 Hz, 1H, dCH),

7.37ꢀ7.40 (m, 2H, ArꢀH), 7.76 (d, J = 16.0 Hz, 1H, CHd). HRMS: m/

z calcd [M ꢀ H] 544.1520; found 544.1434. Anal. (C₂₇H₂₈FNO₈S)

C, H, N.

and distillation of the methyl ethyl ketone was at 50 °C. To the

residue, water (10 mL) was added. The resulting mixture was heated to

70 °C and maintained for 30 min. The reaction mixture was cooled to

room temperature and stirred for 2 h at room temperature. The solid

formed was filtered, washed with methyl ethyl ketone (2 ꢁ 20 mL), and

dried to get the title compound 28. Yield, 80%; white solid, mp

1

174ꢀ178 °C. H NMR (D₂O, 300 MHz): δ 3.46 (s, 2H, NHꢀCH₂),

3.63 (s, 6H, 2 ꢁ OCH₃), 3.72 (s, 3H, OCH₃), 3.75(s, 3H, OCH₃), 4.23

(s, 2H, CH₂), 5.89 (s, 2H, ArꢀH), 6.44 (d, J = 1.5 Hz, 1H, ArꢀH), 6.59

(dd, J = 8.4, 1.5 Hz, 1H, ArꢀH), 6.72 (d, J = 8.4 Hz, 1H, ArꢀH), 6.91 (d,

J = 15.6 Hz, 1H, dCH), 7.39 (d, J = 15.6 Hz, 1H, CHd). HRMS: m/z

(E)-2-(4⁰⁰-Chlorophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimeth-

oxystyrylsulfonyl)methyl)phenylamino)acetic Acid (27j). The

title compound was obtained by the hydrolysis of (E)-methyl 2-(4⁰⁰-

chlorophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)-

phenylamino)acetate 26j following the procedure as described for com-

pound 27. Yield, 60%; white solid, mp 172ꢀ174 °C. ¹H NMR (CDCl₃, 400

MHz): δ 3.55 (s, 6H, 2 ꢁ OCH₃), 3.57 (s, 3H, OCH₃), 3.60 (s, 3H,

OCH₃), 3.80 (s, 2H, CH₂), 4.69(s, 1H, CH), 5.83 (s, 2H, ArꢀH), 6.06 (d,

J =1.8 Hz, 1H, ArꢀH), 6.44 (dd, J = 8.1, 1.8 Hz, 1H, ArꢀH), 6.47 (d, J = 8.1

Hz, 1H, ArꢀH), 6.73 (d, J = 15.6 Hz, 1H, dCH), 7.00 (d, J = 7.8 Hz, 2H,

ArꢀH), 7.08 (d, J = 8.5 Hz, 2H, ArꢀH), 7.49 (d, J = 15.6 Hz, 1H, CHd).

HRMS: m/z calcd [M ꢀ H] 560.1224; found 560.1212. Anal.

(C₂₇H₂₈ClNO₈S) C, H, N.

(E)-2-(4⁰⁰-Bromophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimeth-

oxystyrylsulfonyl)methyl)phenylamino)acetic Acid (27k). The

title compound was obtained by the hydrolysis of (E)-methyl 2-(4⁰⁰-

bromophenyl)-2-(2-methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)-

phenylamino)acetate 26k following the procedure as described for com-

pound 27. Yield, 60%; white solid, mp 178ꢀ179 °C. ¹H NMR (DMSO-d₆,

400 MHz): δ 3.93 (s, 6H, 2 ꢁ OCH₃), 3.94 (s, 3H, OCH₃), 3.96 (s, 3H,

OCH₃), 4.25 (s, 2H, CH₂), 4.96 (s, 1H, CH), 6.39 (s, 2H, ArꢀH), 6.41 (d,

J = 1.8 Hz, 1H, ArꢀH), 6.69 (dd, J = 8.1, 1.8 Hz, 1H, ArꢀH), 6.91 (d, J = 8.1

Hz, 1H, ArꢀH), 7.14 (d, J = 15.6 Hz, 1H, dCH), 7.39 (d, J = 8.4 Hz, 2H,

ArꢀH), 7.57 (d, J = 8.4 Hz, 2H, ArꢀH),7.61 (d, J = 15.6 Hz, 1H, CHd).

HRMS: m/z calcd [M ꢀ H] 604.0719; found 604.0731. Anal.

(C₂₇H₂₈BrNO₈S) C, H, N.

(E)-2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)-

methyl)phenylamino)-2-(4⁰⁰-methoxyphenyl)acetic Acid

(27l). The title compound was obtained by the hydrolysis of (E)-

(4⁰⁰-methoxyphenyl)-{2-methoxy-5-[2-(2⁰,4⁰,6⁰-trimethoxyphenyl)ethene-

sulfonylmethyl]phenylamino}acetic acid methyl ester 26l following the

procedure as described for compound 27. Yield, 60%; white solid, mp

174ꢀ175 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 3.72 (s, 3H, OCH₃),

3.84 (s, 3H, OCH₃), 3.86 (s, 6H, 2 ꢁ OCH₃), 3.87 (s, 3H, OCH₃), 4.16 (s,

2H, CH₂), 4.80(s, 1H, CH), 6.31 (s, 2H, ArꢀH), 6.37 (d, J = 1.5 Hz, 1H,

ArꢀH), 6.62 (dd, J = 8.1, 1.5 Hz, 1H, ArꢀH), 6.71 (d, J = 8.1 Hz, 1H,

ArꢀH), 6.82 (d, J = 9.0 Hz, 2H, ArꢀH), 7.08 (d, J = 15.6 Hz, 1H, dCH),

7.27 (d, J= 8.7 Hz, 2H, ArꢀH), 7.54 (d, J=15.6Hz, 1H, CHd). HRMS:m/

z calcd [M ꢀ H] 556.1720; found 556.1731. Anal. (C₂₈H₃₁NO₉S) C, H, N.

Sodium (E)- 2-(2-Methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsul-

fonyl)methyl)phenylamino)acetate (ESTYBON ) (28). The title

compound was obtained by the direct hydrolysis of (E)-methyl 2-(2-

methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyrylsulfonyl)methyl)phenylamino)-

acetate 26a. To a solution of sodium hydroxide (3.95 g, 99 mmol) in

water (11.5 mL) at 20 °C was added ethanol (40 mL), (E)-methyl 2-

(2-methoxy-5-((2⁰,4⁰,6⁰-trimethoxystyryl sulfonyl)methyl)phenylamino)-

acetate 26a and dichloromethane (200 mL). The resulting mixture was

stirred at room temperature for 3ꢀ4 h. After completion of the reaction

monitored by TLC (chloroform/methanol, 9:1 on silica gel plate),

charcoal was added and the mixture was stirred for 30 min. The reaction

mixture was filtered through Celite and washed with ethanol (2 ꢁ 20 mL).

The combined filterate was distilled at 50 °C until most of the solvent

was removed. Methyl ethyl ketone (70 mL) was added to the residue,

calcd [M + H] 510.1365; found 510.1378. Anal. (C₂₁H₂₄NNaO₈S 2H₂0)

C, H, N.

3

Biology. Tissue Culture and Reagents. Paclitaxel was pur-

chased from Sigma. Cell lines were purchased from ATCC. Cell lines

were routinely grown in DMEM or RPM1 (CellGro) supplemented

with 10% fetal bovine serum (Cell Generation Co.) and 1 unit/mL

penicillinꢀstreptomycin (Gibco).

Cytotoxicity Assay. We have tested a number of tumor cell lines

using a dose response end point assay system. The cells were grown in

either DMEM or RPMI supplemented with 10% fetal bovine serum and

1 unit/mL penicillinꢀstreptomycin solution. The tumor cells were

plated into six-well dishes at a cell density of 1.0 ꢁ 10⁵cells/mL/well,

and compounds were added 24 h later at various concentrations. Cell

counts were determined from duplicate wells after 96 h of treatment.

The total number of viable cells was determined by trypan blue

exclusion.

Flow Cytometry. Human prostate tumor cells, DU145 cells, and

normal diploid human lung fibroblasts, HFL-1 cells, were grown in

DMEM (Cellgro) supplemented with 10% fetal bovine serum and 1

unit/mL penicillinꢀstreptomycin. The cells were plated onto 100 mm²

dishes at a cell density of 1.0 ꢁ 10⁶cells/dish, and 24 h later, they were

treated with 2.5 μM compound. The cells were harvested 24 h after

treatment. The cells were removed from the plate by trypsin digestion

and combined with the nonattached cells found in the medium. The cell

pellets were washed in phosphate buffered saline (PBS) and fixed in ice

cold 70% ethanol for at least 24 h. The fixed cells were then washed with

room temperature PBS and stained with propidium iodide (50 mg/mL)

and RNase A (0.5 mg) for 30 min at 37 °C. The stained cells were then

analyzed on a Becton-Dickinson (BD) (FACScan) flow cytometer and

the data analyzed by cell cycle analysis software (Modfit, BD).

PARP Western. DU145 and HFL-1 cells were plated at a density of

3.0 ꢁ 10⁶cells per 150 mm²plate and treated 24 h later with either

DMSO or 28. The cells were collected at 48 h of treatment, and cell

pellets were frozen. The frozen cell pellets were lysed in 1% NP40/PBS

lysis buffer containing protease inhibitors. Equal amounts of total

cellular protein were then resolved on a 10% SDSꢀpolyacrylamide

gel. The gels were transferred onto nitrocellulose paper (S/S), hybri-

dized with anti-PARP antibodies (BD), and developed using ECL

(Perkin-Elmer, MA) solution.

Cellular Viability and Caspase 3/7 Activity. Exponentially

growing A549 cells were seeded in a white walled 96-well plate at a

density of 3600 cells/well in 100 μL of DMEM containing 10% FBS and

1% Pen/Strep. Cells were then allowed to adhere overnight at 37 °C in

an incubator. The next day, cells were treated with varying concentra-

tions of 28 or DMSO and then returned to the incubator. Twenty-four

hours later, plates were removed from the incubator and 20 μL of

CellTiter-Blue Reagent (Promega catalog no. G8080) was added

individually to each well following the manufacturer’s instructions.

Plates were slowly shaken for 0.5 min and then returned to the incubator.

After 3 h, fluorescence was read using a Glomax 96-well plate reader.

Next, 120 μL of Caspase Glo 3/7 reagent (Promega catalog no. G8090)

was added to each well per manufacturer’s instructions. Plates were

slowly shaken for 0.5 min and allowed to develop at room temperature

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Journal of Medicinal Chemistry

ARTICLE

for 2 h. At the end of this period, luminescence was read using a Glomax

96-well plate reader.

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in the hind leg using a 1 mL tuberculin syringe equipped with a 271/2

gauge needle. Approximately 14 days later, mice were paired (N = 9)

and injected with 200 mg/kg 28 or phosphate buffered saline as the

vehicle control. The intravenous injections were performed in the mouse

tail vein using a 1 mL tuberculin syringe equipped with a 30 gauge

needle. The animals were injected following a Q2D ꢁ 20 schedule.

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week using traceable digital vernier calipers (Fisher). Tumor volume was

calculated using the following equation: V = (L(S²)p/6, where L is the

longer and S is the shorter of the two dimensions. Body weight was

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Supporting Information. Elemental analysis data. This ma-

b

terial is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION

Corresponding Author

*For M.V.R.R.: phone, (212) 659-6875; fax, (215) 893-6989;

e-mail: r.reddy@mssm.edu. For E.P.R.: phone, (212) 659-5571;

e-mail, ep.reddy@mssm.edu.

Author Contributions

^§These authors contributed equally.

Notes

Conﬂict of Interest Statement: Dr. E. P. Reddy is a stockholder,

board member, grant recipient, and paid consultant of Onconova

Therapeutics Inc. Dr. M. V. R. Reddy is a stockholder and paid

consultant of Onconova Inc. Dr. S. Cosenza is a paid consultant

of Onconova Therapeutics Inc.

’ ACKNOWLEDGMENT

This work was supported by grants from NIH (Grant CA

109820) and Onconova Therapeutics Inc.

’ ABBREVIATIONS USED

MDS, myelodysplstic syndromes; AML, acute myeloid leukemia;

PI3K, phosphoinositide 3-kinase; BIM, Bcl-2-interacting modu-

lator of cell death; FDA, Food and Drug Administration; DNA,

deoxyribonucleic acid; CDK, cyclin dependent kinase; JNK,

Janus kinase; p-mTOR, mammalian target of rapamycin

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dx.doi.org/10.1021/jm200570p |J. Med. Chem. 2011, 54, 6254–6276

Article Doi

Discovery of a clinical stage multi-kinase inhibitor sodium (E)-2-{2-methoxy-5-[(2′,4′,6′-trimethoxystyrylsulfonyl)methyl] phenylamino}acetate (ON 01910.Na): Synthesis, structure-activity relationship, and biological activity

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