Ojima I. (ed.) Fluorine in Medicinal Chemistry and Chemical Biology

Fluorinated Inhibitors of Matrix Metalloproteinases 113

Acknowledgment

We thank the European Commission (IHP Network grant “ FLUOR MMPI ” HPRN - CT -

2002 - 00181), MIUR (Coﬁ n 2004, Project “Polipeptidi Bioattivi e Nanostrutturati”),

Politecnico di Milano, and C.N.R. for economic support. We thank Professor Wolfram

Bode (Max Planck Institute, Martinsried, Germany) for very useful discussions and

suggestions.

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5

Fluoro - Taxoid Anticancer Agents

Antonella Pepe , Larisa Kuznetsova , Liang Sun , and Iwao Ojima

5.1 Introduction

The importance of ﬂ uorine in bioorganic and medicinal chemistry has been demonstrated

by a large number of ﬂ uorinated compounds approved by the FDA for medical use [1, 2] .

According to our most up - to - date survey, 138 ﬂ uorine - containing drugs have received

FDA approval for human diseases (of which 23 have been discontinued from the market,

however), while 33 are currently in use for veterinary applications (see the Appendix ).

These statistics make ﬂ uorine the “ second - favorite heteroatom ” after nitrogen in drug

design [3] . Small atomic radius, high electronegativity, nuclear spin of ½, and low polariz-

ability of the C – F bond are among the special properties that render ﬂ uorine so attractive.

Those atomic properties translate widely into equally appealing attributes of ﬂ uoroorganic

compounds. Higher metabolic stability, often increased binding to target molecules, and

increased lipophilicity and membrane permeability are some of the properties associated

with the replacement of a C – H or C – O bond with a C – F bond in biologically active

compounds. Because of the recognized value of ﬂ uorine, it is now a common practice in

drug discovery to study ﬂ uoro - analogues of lead compounds under development [4] .

Although medicinal chemists have long introduced ﬂ uorine into bioactive molecules on

the basis of experience and intuition, it is only recently that experimental and computa-

tional studies have been conducted to better understand how the introduction of ﬂ uorine

into small drug molecules results in higher binding afﬁ nities and selectivity [5] . An under-

standing of how the replacement of H with F affects the electronic nature and conformation

of small molecules is crucial for predicting the interaction of ﬂ uoroorganic molecules with

proteins and enzymes. As a result, the rational design of ﬂ uoroorganic molecules will lead

to the generation of new and effective biochemical tools. In addition,

19

F NMR has found

Fluorine in Medicinal Chemistry and Chemical Biology Edited by Iwao Ojima

118 Fluorine in Medicinal Chemistry and Chemical Biology

numerous applications to molecular imaging and promoted the development of molecular

probes for imaging. The sensitivity of

19

F NMR spectroscopy, along with large

19

F –

1

H

coupling constants and the virtual absence of

19

F in living tissues, makes incorporation of

ﬂ uorine into bioactive compounds a particularly powerful tool for the investigation of

biological processes [6, 7] .

This chapter gives an account of our research on the use of ﬂ uorine, exploiting the

unique nature of this element, in the medicinal chemistry and chemical biology of taxoid

anticancer agents (i.e., “ Taxol - like ” compounds).

5.2 Paclitaxel, Taxoids, and Second - generation Taxoids

Paclitaxel (Taxol

®

) and its semisynthetic analogue docetaxel (see Figure 5.1 ) are two of

the most important chemotherapeutic drugs, currently used for the treatment of advanced

ovarian cancer, metastatic breast cancer, melanoma, non - small - cell lung cancer, and Kar-

posi ’ s sarcoma [8, 9] . More recently, these drugs have been used for the treatment of neck,

prostate, and cervical cancers [8, 9] .

The mechanism of action of paclitaxel involves its binding to the β - subunit of α , β -

tubulin dimer, accelerating the formation of microtubules. The resulting paclitaxel - bound

microtubules are much more stable and less dynamic than the natural GTP - bound micro-

Paclitaxel: R

1

= Bz, R

2

= Ac

Docetaxel: R

1

= t-Boc, R

2

= H

IDN 5390

OH

OAc

O

HO

NH

HO

OH

O

OAc

O

HO

R

1

HN

HO

OH

R

2

O

Second-generation taxoids

O

OAc

O

R

3

O

HO

NH

HO

OH

R

2

O

C-seco taxoids

OH

OAc

O

HO

NH

HO

OH

O

X

t-Boc

Figure 5.1 Structures of paclitaxel, docetaxel, second - generation taxoids, and

C - seco - taxoids .

Fluoro-Taxoid Anticancer Agents 119

tubules, with a growth rate higher than the disassembly rate. The unnatural growth and

stabilization of microtubules causes the arrest of the cell division cycle, mainly at the G

2

/M

stage, activating a cell - signaling cascade that induces apoptosis [10, 11] .

Although paclitaxel and docetaxel possess potent antitumor activity, chemotherapy

with these drugs encounters many undesirable side - effects as well as drug resistance [8,

12 – 14] . It is therefore important to develop new taxoid anticancer drugs as well as efﬁ ca-

cious drug delivery systems with fewer side - effects, superior pharmacological properties,

and improved activity against various classes of tumors, especially against drug - resistant

cancers.

It has been shown that a primary mechanism of drug resistance is the overexpression

of ABC transporters, for example, P - glycoprotein (Pgp), an integral membrane glycopro-

tein that acts as a drug - efﬂ ux pump to maintain the intracellular concentration of drugs

below therapeutically active level [15] . In the course of our extensive studies on the design,

synthesis, and structure – activity relationships (SARs) of taxoid anticancer agents, we

discovered second - generation taxoids that possess one order of magnitude better activity

against drug - sensitive cell lines and more than two orders of magnitude better activity

against drug - resistant cell lines [16] . Several examples are shown in Table 5.1 (see Figure

5.1 for structures).

It was found that meta - substitution of the benzoyl group at the C - 2 position dramati-

cally increases the cytotoxicity of taxoids against drug - resistant cell lines [17] . This class

of the second - generation taxoids is three orders of magnitude more potent than paclitaxel

and docetaxel against drug - resistant cancer cell lines expressing multidrug resistance

(MDR) phenotype [17] (see Table 5.1 ).

Because of the aforementioned advantages of introducing ﬂ uorine into biologically

active molecules, we synthesized ﬂ uorine - containing paclitaxel and docetaxel analogues

to investigate the effects of ﬂ uorine – incorporation on the cytotoxicity and the blockage of

known metabolic pathways. Our earlier studies have been reported in several publications

[18 – 21] . Thus, in this chapter, we describe the synthesis and biological evaluation of

second - generation ﬂ uoro - taxoids bearing a diﬂ uoromethyl, triﬂ uoromethyl, or diﬂ uorovi-

nyl group at the C - 3 ′ position.

Although the overexpression of Pgp and other ABC transporters is the main cause of

multidrug resistance to paclitaxel and other hydrophobic anticancer drugs, it is not the

only mechanism of drug resistance. Overexpression of speciﬁ c tubulin isotypes has recently

received substantial attention in terms of paclitaxel resistance and has not yet been suc-

cessfully addressed [22 – 25] . Different β - tubulin isotypes form microtubules with anoma-

lous behaviors in vitro with regard to assembly, dynamics, conformation, and ligand

binding [23, 24, 26, 27] . Microtubules with altered β - tubulin isotype compositions respond

differently to paclitaxel [28] . Derry and co - workers reported that α β

III

microtubules are

more dynamic than the most common α β

II

or α β

IV

[28] ; hence, higher concentrations of

paclitaxel are required to interfere with their dynamics. Recently Ferlini has reported that

the C - seco - taxoid IDN 5390 is up to 8 - fold more active than paclitaxel against an inher-

ently drug - resistant OVCAR3 ovarian cancer cell line and paclitaxel - resistant human

ovarian adenocarcinoma cell lines A2780TC1 and A2780TC3 (cell lines overexpressing

class III β - tubulin) [29 – 31] . Following observation of the remarkable effects of meta -

substitution at the C - 2 benzoate moiety, mentioned above, we synthesized a series of IDN

5390 analogues with C - 2 - benzoate modiﬁ cations, including a 3 - ﬂ uorobenzoyl group. The

120 Fluorine in Medicinal Chemistry and Chemical Biology

Table 5.1 In vitro cytotoxicity ( IC

50

nM)

a

of representative second - generation taxoids

Taxoid R

2

R

3

R

4

MCF7

b

MCF7 - R

c

R/S

d

LCC6 - WT

b

LCC6 - MDR

e

R/S

d

Paclitaxel Ph Ac H 1.7 300 176 3.1 346 112

Docetaxel Ph H H 1.0 235 235 1.0 120 120

SB - T - 1213 Me

2

C = CH - EtCO H 0.18 4.0 22 – –

SB - T - 1103 Me

2

CHCH

2

- EtCO H 0.35 5.1 21 – –

SB - T - 121303 Me

2

C = CH - EtCO MeO 0.36 0.33 0.92 1.0 0.9 0.90

SB - T - 121304 Me

2

C = CH - EtCO N

3

0.9 1.1 1.2 0.9 1.2 1.3

SB - T - 11031 Me

2

CHCH

2

- EtCO F – – – 0.4 2.4 6.0

SB - T - 11033 Me

2

CHCH

2

- EtCO MeO 0.36 0.43 1.19 0.9 0.8 0.89

a

The concentration of compound that inhibits 50% of the growth of a human tumor cell line after 72

h drug exposure.

b

Human breast carcinoma.

c

Multidrug - resistant human breast cancer cell line (currently renamed NCI/ADR).

d

Drug - resistance factor.

e

Multidrug - resistant human breast carcinoma.

Fluoro-Taxoid Anticancer Agents 121

synthesis and biological evaluation of these novel ﬂ uoro - C - seco - taxoids are described

below.

5.3 Second - generation Taxoids and C - seco - Taxoids with Strategic

Incorporation of Fluorine

5.3.1 Synthesis and Biological Evaluation of C - 3 ′ - Diﬂ uoromethyl - and

C - 3 ′ - Triﬂ uoromethyl Taxoids

A series of the second - generation C - 3 ′ - CF

2

H and C - 3 ′ - CF

3

taxoids were synthesized by the

β - lactam synthon method [32, 33] . The synthesis of enantiopure β - lactam 8 , bearing a CF

2

H

group at the C - 4 position is illustrated in Scheme 5.1 . The [2+2] ketene - imine cycloaddition

of acetoxyketene, generated in situ from acetoxyacetyl chloride and triethylamine, with

N - PMP - 3 - methylbut - 2 - enaldimine (PMP = p - methoxyphenyl) gave racemic β - lactam 1.

Enzymatic optical resolution of 1 by PS Amano lipase at 50 ° C gave enantiopure (3 R ,4 S ) -

3 - AcO - 4 - isobutenyl - β - lactam 2(+) [34] . Protection of the 3 - hydroxyl group as TIPS ether

and ozonolysis of the double bond of 2(+) afforded 4 - formyl - β - lactam 5 . Diﬂ uoromethyl-

ation of 5 with diethylaminosulfur triﬂ uoride (DAST) [35] , followed by deprotection of

PMP by cerium ammonium nitrate (CAN) and introduction of tert - butoxycabonyl group to

the β - lactam nitrogen yielded enantiopure (3 R ,4 R ) - 1 - t - Boc - 3 - TIPSO - 4 - CF

2

H - β - lactam 8 .

In a similar manner, enantiopure 1 - t - Boc - 3 - TIPSO - 4 - CF

3

- β - lactam 14 was synthe-

sized from benzyloxyacetyl chloride/triethylamine and N - PMP - CF

3

- aldimine through

[2+2] ketene - imine cycloaddition, enzymatic optical resolution (PS Amano lipase at

Scheme 5.1 Synthesis of enantiopure C - 4 - diﬂ uoromethyl - β - lactam 8 . (i) Et

3

N, CH

2

Cl

2

,

− 78 ° C ∼ r.t., 70%; (ii) PS - Amano, buffer pH 7.0, 10% CH

3

CN, 50 ° C; (iii) KOH, THF, 0 ° C,

100%; (iv) TIPSCl, Et

3

N, DMAP, CH

2

Cl

2

, 85%; (v) O

3

, MeOH/CH

2

Cl

2

, − 78 ° C; Me

2

S, 73%;

(vi) DAST, CH

2

Cl

2

, 86%; (vii) CAN, H

2

O/CH

3

CN, − 15 ° C, 68%; (viii) t - Boc

2

O, Et

3

N, DMAP,

CH

2

Cl

2

, 80%.

( Source: Reprinted with permission from Ojima, I.; Kuznetsova, L. V., and Sun, L. (2007)

Organoﬂ uorine Chemistry at the Biomedical Interface: A Case Study on Fluoro - Taxoid

Anticancer Agents. ACS Symposium Series 949, Current Fluoroorganic Chemistry , (2007) pp.

288 – 304. Copyright (2007) American Chemical Society.)

N

AcO

OPMP

O

Cl

AcO

N

AcO

OPMP

N

HO

OPMP

3(-)

+

2(+)

47%/50% in theory

2(+)

N

PMP

A

cO

O

N

TIPSO

O

F

8

t-Boc

N

PMP

TIPSO

O

4

N

PMP

i

ii

+

iii, iv

v

N

PMP

CHOTIPSO

O

5

1(±)

vi

N

TIPSO

O

F

PMP

6

NH

TIPSO

O

F

7

vii

viii

122 Fluorine in Medicinal Chemistry and Chemical Biology

N

CF

3

PMP

BnO

Cl

O

N

O

CF

3

BnO

PMP

9(±)

N

O

CF

3

AcO

PMP

N

O

CF

3

HO

PMP

10(+)

11(-)

N

O

CF

3

AcO

PMP

10(+)

N

O

CF

3

TIPSO

PMP

12

N

O

CF

3

AcO

PMP

N

O

CF

3

TIPSO

t-Boc

14

i

v, vi

iv

vii

ii, iii

10(±)

N

O

CF

3

TIPSO

H

13

viii

Scheme 5.2 Synthesis of enantiopure C - 4 - triﬂ uoromethyl - β - lactam 14 . (i) Et

3

N, CH

2

Cl

2

,

40 ° C, 83%; (ii) H

2

, Pd, MeOH, 45 ° C, 98%; (iii) Ac

2

O, DMAP, Py, CH

2

Cl

2

, 74%; (iv) PS -

Amano, buffer pH 7, 10% CH

3

CN, 0 – 5 ° C; (v) KOH, THF, − 5 ° C, 100%; (vi) TIPSCl, Et

3

N,

CH

2

Cl

2

, 95%; (vii) CAN, CH

3

CN/H

2

O, − 10 ° C, 84%; (viii) t - Boc

2

O, Et

3

N, DMAP, CH

2

Cl

2

,

87%.

0 – 5 ° C), deprotection – protection, and t - Boc carbamoylation as illustrated in Scheme

5.2 [34] .

The Ojima – Holton coupling of 4 - CF

2

H - and 4 - CF

3

- β - lactams 8 and 14 , thus obtained,

with 2,10 - modiﬁ ed baccatins 15 was carried out at − 40 ° C in THF using LiHMDS as a

base followed by deprotection of silicon protecting groups with HF/pyridine to give the

corresponding second - generation 3 ′ - CF

2

H - taxoids ( 18 ) and 3 ′ - CF

3

- taxoids (19) in moder-

ate to high overall yields (see Scheme 5.3 ) [20, 34, 36] . 2,10 - Modiﬁ ed baccatins 15 were

prepared by methods we have reported previously [17] .

The second - generation ﬂ uoro - taxoids ( 18 and 19 ) were evaluated for their cytotoxic-

ity in vitro against human breast cancer cell lines (MCF7 - S and LCC6 - WT), their corre-

sponding drug - resistant cell lines (MCF7 - R and LCC6 - MDR), human non - small - cell lung

(NSCL) cancer cell line (H460), and human colon cancer cell line (HT - 29). The IC

50

values

were determined through 72 h exposure of the cancer cells to the ﬂ uoro - taxoids, following

the procedure developed by Skehan et al. [37] . Results are summarized in Table 5.2

(3 ′ - CF

2

H - taxoids 18 ) and Table 5.3 (3 ′ - CF

3

- taxoids 19 ).

As Tables 5.2 and 5.3 show, all ﬂ uoro - taxoids 18 and 19 possess substantially higher

potencies than those of paclitaxel and docetaxel, with cytotoxicity in single - digit nanomo-

lar IC

50

values against drug - sensitive MCF7 - S, LCC6 - WT, H460, and HT - 29 cancer cell

lines (except for a few cases). The cytotoxicity of the ﬂ uoro - taxoids 18 and 19 against

multidrug - resistant MCF7 - R and LCC6 - MDR cell lines is more impressive: all ﬂ uoro -

taxoids exhibit single - digit nanomolar IC

50

values (except for a couple of cases) and are

two orders of magnitude more potent than paclitaxel in average. These two series of

ﬂ uoro - taxoids exhibit, in general, comparable cytotoxicity against all cancer cell lines

examined.

However, the potency of 3 ′ - CF

2

H - taxoids 18 against MCF7 - S and LCC6 - WT appears

to be higher and more uniform, with different substitution patterns, than that of 3 ′ - CF

3

-

taxoids 19 , except for two cases (SB - T - 12822 - 1, 0.19 nM, MCF7 - S; SB - T - 12824 - 1,

0.17 nM, MCF7 - S). In contrast, compounds 19 exhibit more uniform potency against

Fluoro-Taxoid Anticancer Agents 123

O

OAc

HO

O

OAc

O

Rf

O

OTIPS

NH

N

IPSO Rf

O Boc-t

LiHMDS, THF, -40

o

C

60-95%

O

OAc

O

Rf

O

HO

NH

HF/pyridine,

CH

3

CN/pyridine

X

15

16: Rf = CF

2

H

17: Rf = CF

3

18: Rf = CF

2

H

19: Rf = CF

3

HO

OH

OTES

RO

O

R = MeCO, EtCO, Me

2

NCO, MeOCO,TES; X = MeO, F, Cl, N

3

; Rf = CF

2

H, CF

3

0

o

C - RT

52-92%

t-Boc

8, 14

Scheme 5.3 Synthesis of C - 3 ′ - CF

2

- taxoids ( 18 ) and C - 3 ′ - CF

3

- taxoids ( 19 ).

( Source: Reprinted with permission from Ojima, I.; Kuznetsova, L. V., and Sun, L. (2007)

Organoﬂ uorine Chemistry at the Biomedical Interface: A Case Study on Fluoro - Taxoid

Anticancer Agents. ACS Symposium Series 949, Current Fluoroorganic Chemistry , (2007) pp.

288 – 304. Copyright (2007) American Chemical Society.)

multidrug - resistant MCF7 - R and LCC6 - MDR cell lines than do 18 . For ﬂ uoro - taxoids 18 ,

cytotoxicity against multidrug - resistant cell lines, MCF7 - R and LCC6 - MDR, depends on

the nature of meta substituents of the C - 2 - benzoate moiety; the potency increases in the

order F < MeO < Cl < N

3

. In contrast, no clear trend is observed for ﬂ uoro - taxoids 19

against these multidrug - resistant cell lines. Among these ﬂ uoro - taxoids examined, SB - T -

12842 - 4 (R = n - propanoyl; X = N

3

) appears to be the most potent compound with a

resistance factor (R/S ratio) of only 2.9 – 3.0 against two sets of human breast cancer cell

lines.

5.4 Synthesis and Biological Evaluation of

C - 2 - (3 - Fluorobenzoyl) - C - seco - Taxoids

As mentioned above, paclitaxel and taxoids stabilize microtubules through their binding

to the β - tubulin subunits, thereby blocking the cell mitosis, which leads to apoptosis [10,

11] . There are eight β - tubulin isotypes, of which class I β - tubulin is the major isotype in

all mammalian tissues [38] . It has been shown that the overexpression of class III β - tubulin

isotype causes paclitaxel drug resistance, which constitutes signiﬁ cant drug - resistance

other than MDR [25] . Recently, a C - seco - taxoid, IDN5390 (see Figure 5.1 ), was reported

to exhibit several times better potency than paclitaxel against drug - resistant ovarian cancer

Ojima I. (ed.) Fluorine in Medicinal Chemistry and Chemical Biology

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