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

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 267

A facile synthesis of ﬂ uoro - oleﬁ ns through Pd(0) - catalyzed reductive deﬂ uorination

of allylic gem - diﬂ uorides, in particular γ , γ - diﬂ uoro - α , β - enoates was recently reported (see

Scheme 10.16 ) [26] . Stereoselectivities were moderate to high ( E : Z = 1 : 1 – 1 : 9).

The reductive deﬂ uorination – alkylation method shown in Scheme 10.12 was success-

fully applied to the synthesis of N - Boc - Sta - Ψ [CF = CH] - Ala ethyl ester 51 , which mimics

the central Sta - Ala unit of pepstatin, a naturally occurring inhibitor of aspartyl proteases

including renin, pepsin, HIV - 1 and HIV - 2 proteases (see Figure 10.3 ) [27] .

γ , γ - Diﬂ uoro - α , β - enoate 56 , the key substrate for the reductive deﬂ uorination – alkyla-

tion reaction, was synthesized in optically pure form using N - Boc leucinol 52 as the start-

ing material (see Scheme 10.17 ). The Reformatsky reaction of bromodiﬂ uoroacetate with

aldehyde 53 , having natural statin conﬁ guration, gave a 3 : 1 diastereomeric mixture of β -

hydroxy ester 54 , which was subjected to radical deoxygenation reaction to give diﬂ uoro

ester 55 . DIBAL reduction of 55 , followed by the HWE reaction using phosphonoacetate

provided the key substrate 56 .

As expected, Me

2

CuLi - mediated reductive deﬂ uorination of 56 and subsequent meth-

ylation with methyl iodide proceeded smoothly to give methylated product 57 in 96%

yield. Regarding the stereoselectivity, a moderate Z - selectivity ( Z : E = 6.4 : 1) was observed

for the oleﬁ n moiety, but the C - 2 - methylation proceeded in nonselective manner. Fortu-

nately, two diastereomers were readily separable by medium - pressure liquid chromatog-

raphy (MPLC) as shown in Scheme 10.18 . Structural assignments were made based on

the x - ray crystallographic analysis of (2 S ) - 57 .

As described above, an organocopper reagent derived from alkyl lithium, such as

Me

2

CuLi, readily reacts with γ , γ - diﬂ uoro - α , β - enoate 42 to form a reductive deﬂ uorinated

metal species 43 , exclusively (see Scheme 10.12 ). To facilitate the reactivity of ﬂ uorine

as a leaving group, we also investigated the effects of alkylaluminum on this reaction as

Scheme 10.16

Figure 10.3 Pepstatin and Sta - ψ [CF = CH] - Ala.

268 Fluorine in Medicinal Chemistry and Chemical Biology

Scheme 10.17

Scheme 10.18

an additive and/or alkylating reagent, since it is well documented that aluminum, in addi-

tion to its high oxygenophilicity (O - Al 511 ± 3 kJ/mol), has exceedingly high afﬁ nity

toward ﬂ uorine (F - Al 663 ± 3 kJ/mol) [28] . We found that, in the presence of Cu(I), the

reaction of γ , γ - diﬂ uoro - α , β - enoate 42b having a free hydroxyl group at the δ - position with

trialkylaluminum (5 equivalents) gave allylic alkylation product 44 , whereas γ , γ - diﬂ uoro -

α , β - enoate having amino group 42a or hydrogen derivative 42c did not give the alkylated

products (see Scheme 10.19 ) [22, 23] . With the hydroxyl substrate 44b , the reaction pro-

ceeded in a Z - selective manner but without or with very low diastereoselectivity.

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 269

Scheme 10.19

Excellent diastereoselectivity was realized on using the substrate of allylic alcohol

substrates 59 instead of α , β - unsaturated ester substrate 42 (Y = OH) mentioned above.

Thus, in the presence of CuI · 2LiCl, the reaction of the E - isomer of 5 - hydroxy - 4,4 - diﬂ uoro -

2 - alken - 1 - ol ( E ) - 59 with trialkylaluminum proceeded smoothly to give the allylic substitu-

tion product 60 with complete 2,5 - syn - and Z - selectivity (see Scheme 10.20 ) [29, 30] . With

the Z - isomer substrate Z - 59 , reaction proceeded in a manner that favored the 2,5 - anti -

product, although the reactivity of the Z - isomer was clearly low compared with that of the

E - isomer. It should be noted that the C - 5 free hydroxyl functionality is crucial for the

reaction to occur, since the lack of this OH group (i.e., protection as its ether or amine

derivative in place of the OH group), resulted in no reaction under similar conditions,

recovering the starting material.

This highly stereoselective reaction would provide an efﬁ cient method for the prepa-

ration of ﬂ uoroalkene dipeptide isosteres 62 and related molecules such as depsipeptide

isosteres 61 (see Scheme 10.21 ) [29,30] .

10.2.1.7 Cross - coupling Reaction of Fluorinated Alkenyl Bromides

In 2002, Burton and co - workers demonstrated that the Z - and E - isomers of bromoﬂ uoro-

methylene compound 63 , easily obtained by the Wittig reaction of carbonyl compound

with Ph

3

P - CFBr

3

- Zn, were obtained through efﬁ cient kinetic separation using the palla-

dium - catalyzed carboalkoxylation reaction (see Scheme 10.22 ) [31] . Since the E - isomer

Scheme 10.20

270 Fluorine in Medicinal Chemistry and Chemical Biology

Scheme 10.21

Scheme 10.22

of bromide 63 reacted much faster than the Z isomer, ( Z ) - α - ﬂ uoroenoate ( Z ) - 64 was

preferentially obtained at low temperature. After a complete consumption of the E - isomer

by Pd - catalyzed reduction with HCO

2

H, the Z - isomer of bromide ( Z ) - 63 remaining was

subjected to carboalkoxylation at 70 ° C to give ( E ) - 64 .

Pannecoucke and co - workers developed a highly stereospeciﬁ c synthesis of ( Z ) - and

( E ) - α - ﬂ uoroenones 66 through a kinetically controlled Negishi coupling reaction catalyzed

by palladium (see Scheme 10.23 ) [32] .

Further elaboration of both ( Z ) - and ( E ) - 66 led to the formation of ﬂ uoro - oleﬁ n

ψ [CF = CH] dipeptide isosteres in enantiomerically pure form. For example, chiral imine

( Z ) - 68 prepared by Ti(IV) - promoted condensation of α - ﬂ uoroenone 66a with Ellman ’ s

sulfonamide 67 was reduced in a highly diastereoselective manner to give the reductive

amination product 69 , which was converted to the dipeptide isostere 70 via several steps

(see Scheme 10.24 ) [33] .

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 271

Scheme 10.23

Scheme 10.24

10.2.2 Applications to Biologically Active Peptides and Related Studies

10.2.2.1 Substance P and Thermolysin

As an early example of the application of ﬂ uoro - oleﬁ n Ψ [CF = CH] dipeptide isosteres,

Allmendinger et al. reported the preparation of neuropeptide substance P (SP) analogues

containing the Phe - Ψ [(Z) - CF = CH] - Gly dipeptide unit [16] . In a receptor binding assay

(see Table 10.1 ), SP analogue ( S ) - 30a having the natural S - conﬁ guration in the Phe moiety

was almost as active as SP itself (entry 1 vs. entry 6), while its diastereomer ( R ) - 30a , the

unnatural R - conﬁ gured analogue, was 10 times less active than ( S ) - 30a (entry 2). A similar

difference was also observed in the binding afﬁ nities of the pyro - Glu - containing hexapep-

tide analogues ( S ) - 30b and ( R ) - 30b (entries 3 and 4). Furthermore, compared to the non-

ﬂ uorinated oleﬁ nic analogue ( S ) - 30c , the ﬂ uorinated analogue ( S ) - 30b bound 10 times

more strongly to the receptor (entry 3 vs. entry 5).

Bartlett and Otake experimentally veriﬁ ed the applicability of tripeptide analogues

with the Cbz - Gly - Ψ [( Z ) - CF = CH] - Leu - Xaa structure as ground - state analogue inhibitors

of the zinc endopeptidase thermolysin (see Figure 10.4 ) [19] . These ﬂ uoro - oleﬁ n tripeptide

272 Fluorine in Medicinal Chemistry and Chemical Biology

Table 10.1 Receptor binding of Substance P and ﬂ uoroalkene dipeptide analogues

entry X R

1

R

2

R

3

lC

50

1

( S ) - 30a

F Arg - Pro - Lys - Pro - Gln - Gln - Phe PhCH

2

H 2 nM

2

( R ) - 30a

F Arg - Pro - Lys - Pro - Gln - Gln - Phe H PhCH

2

20 nM

3

( S ) - 30b

F Pyro - Glu - PhCH

2

H

0.8 µ M

4

( R ) - 30b

F Pyro - Glu - H PhCH

2

10 µ M

5

( S ) - 30c

H Pyro - Glu - PhCH

2

H

> 10 µ M

6 Substance P 1.3 nM

Figure 10.4 Tripeptide analogues of the Cbz - Gly - Ψ [CF = CH] - Leu - Xaa structure.

analogues 39 bind to thermolysin about one order of magnitude more tightly than the

substrates, in which the binding mode of the ﬂ uoro - oleﬁ n inhibitor and substrate are not

identical on the basis of K

i

vs. K

m

correlation,. Although these analogues are not particu-

larly potent as enzyme inhibitors, they are useful as substrate models in structural

studies.

10.2.2.2 Dipeptidyl Peptidase Inhibitors

Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5, CD26) is a serine protease cleaving off

dipeptides from the amino terminus of peptides or proteins having proline or alanine at

the penultimate position. Since prolylamides are known to play a critical role in peptide

structure and function, and because of their high resistance toward nonspeciﬁ c enzymatic

hydrolysis, a few enzymes capable of cleaving this structural motif have attracted consid-

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 273

erable attention. It has been shown that DPP IV inhibition can be used as a new tool for

controlling type II diabetes [34] . Typically, these inhibitors possess a dipeptide skeleton

with a free amine terminus and a pyrrolidine ring attached to an electrophilic site for Ser -

OH scavenging such as a nitrile, a boronic acid, or a hydroxamate (see Figure 10.5 ).

Since the discovery of Xaa - (2 - cyano)pyrrolidines ( 71a , X = CN, Figure 10.5 ) as

potent and reversible inhibitors of DPP IV, the optimization of these “ lead ” structures in

terms of activity and selectivity have been extensively investigated [35] . It was suggested

that for strong enzyme inhibition, reversible interaction of the electrophilic nitrile group

in the inhibitors and the Ser - OH group in the active site of the enzyme would play an

important role. However, these nitrile - containing inhibitors 71a suffer from the inactiva-

tion process to form cyclic amidines 74 and/or hydrolyzed product, diketopiperadine

derivative 75 , due to a facile intramolecular cyclization of N - terminal amine to the nitrile

group through conformational change from trans - amide to cis - amide as shown in Scheme

10.25 .

To solve the inactivation issues mentioned above, the concept of conformationally

restricted ( Z ) - ﬂ uoro - oleﬁ n dipeptide isosteres that mimic the active trans conformation of

the DPP IV inhibitors was applied by Welch and co - workers to the preparation of inhibi-

tors having Ala - Ψ [CF = CH] - Pro structure ( 76 and 78 ) and their inhibitory activities were

evaluated (see Figure 10.6 ) [36, 37] . DPP IV inhibitory activities and the stability of the

inhibitors 76 and 77 , in comparison with those of the model dipeptide Ala - Pro derivative

78 are summarized in Table 10.2 . These ﬂ uoro - oleﬁ n analogues, 76 and 77 , showed better

DPP IV inhibitory activity than that of 78 . In particular, u - 76 having the same relative

stereochemistry as the natural dipeptide (L - Xaa - L - X ′ aa) conﬁ gurations exhibited potent

Figure 10.5 DDP IV inhibitory dipeptides and ﬂ uoro - oleﬁ n analogues.

Scheme 10.25

274 Fluorine in Medicinal Chemistry and Chemical Biology

Figure 10.6 Ala - Ψ [CF = CH] - Pro analogues as DDP IV inhibitors.

Table 10.2 Inhibition of DPP IV and stability of inhibitors

inhibitor Inhibition, %

([l], µ M) K

i

, µ M K

d

× 10

4

, min

− 1

a)

t

1/2

, h

u - 76

42

100

(10)

(250)

0.19 1.1 103

l - 76

4

17

(10)

(500)

14.4 ND ND

u - 77

16

50

(1)

(10)

7.69 –

b)

l - 77

14

47

(1)

(10)

6.03 –

b)

78 29 (1,100) 30.0 13.0 8.8

a)

K

d

: Decomposition rate constant

b)

No detectable degradation at pH 7.6 under buffered conditions.

activity ( K

i

= 0.19 µ M). Moreover, the stability of these ﬂ uoro - oleﬁ n - containing peptide

mimetics was remarkable. For example, no detectable degradation of the cyano derivatives

77 was observed at pH 7.6 under buffered conditions. Although the effects of the replace-

ment of the parent amide bond with the ﬂ uoro - oleﬁ n moiety in these molecules on inhibi-

tory potency and stability might not be fully deduced from these limited examples, good

afﬁ nity of these ﬂ uorine - modiﬁ ed peptide mimetics to the enzyme was experimentally

veriﬁ ed.

Augustyns and co - workers reported a structure – activity relationship (SAR) study of

ﬂ uoro - oleﬁ n analogues of N - substituted glycylpyrrolidines 79a , glycylpiperidines 80a , and

glycyl - (2 - cyano)pyrrolidines 81 as well as the corresponding oleﬁ n analogues 79b and

80b for their activities as DPP IV or DPP II inhibitors (Table 10.3 ) [38] . Except for 79c - 1 ,

most of these compounds exhibited a strong preferential binding to DPP II. Recent crystal-

lographic analyses of peptidic inhibitor – DPP IV complexes revealed that the P

2

– P

1

amide

Fluorinated Moieties for Replacement of Amide and Peptide Bonds 275

oxygen atom of inhibitors is involved in hydrogen bonding with the Asp710 and Arg125

residues of the enzyme. Assuming the importance of such a hydrogen bond, the observed

low afﬁ nity of the ﬂ uoro - oleﬁ n isosteres, 79a and 80a , for DPP IV could be attributed to

the fact that they are much weaker hydrogen - bond acceptors than an amide functionality.

In contrast, the hydrogen - bond formation seems less critical for DPP II inhibition. More-

over, 81 , wherein a nitrile group was introduced to ﬂ uoro - oleﬁ n isostere 79a , did not result

in increased DPP IV or DPP II inhibitory activity, although these analogues showed sub-

stantially increased stability in solution over the parent amide compound.

10.2.2.3 Peptide - Nucleic Acid

Peptide - nucleic acid (PNA) is a DNA analogue based on a polyamide backbone. It is

known that PNAs strongly bind to complementary DNA and RNA sequences, obeying the

Watson – Crick hydrogen - bonding rules, by taking advantage of the lack of electrostatic

repulsion between anionic ribose - phosphate moieties in the backbone [39] . The potential

utility of PNAs as gene - therapy agents has been attracting much attention from medicinal

chemists, although their poor cellular uptake, low solubility in water, and self - aggregation

are remaining problems for clinical applications [40, 41] . To realize more speciﬁ c and

higher afﬁ nity to DNA and RNA, modiﬁ cation of PNA backbone has been extensively

studied. Leumann and co - workers reported the synthesis and evaluation of ﬂ uoroalkene

and nonﬂ uorinated alkene isosteres of PNAs as conformationally locked mimics (see

Figure 10.7 ) [42 – 45] .

Table 10.3 Inhibition of DPP IV and DPP II

R n = 1 n = 2

compd.

lC

50

( µ M) lC

50

( µ M)

Compd.

lC

50

( µ M) lC

50

( µ M)

DPP lV DPP ll DPP lV DPP ll

1

79a - 1

> 1000 90 ± 1

80a - 1

> 500 500 ± 50

79b - 1

> 1000 62 ± 12

80b - 1

> 1000 136 ± 14

79c - 1

148 ± 26 276 ± 72

80c - 1

> 1000 177 ± 54

2

79a - 2

> 1000 38 ± 3

80a - 2

> 1000 34 ± 4

79b - 2

> 1000 26 ± 3

80b - 2

> 1000 55 ± 4

79c - 2

1000 ± 10 500 ± 50

80c - 2

> 1000 397 ± 25

3

79a - 3

> 250 1.3 ± 0.2

80a - 3

> 250 1.0 ± 0.1

79a - 3 ND ND 80c - 3

> 1000 3.1 ± 0.1

276 Fluorine in Medicinal Chemistry and Chemical Biology

The thymidyl - PNA monomer 87 having a ﬂ uoroalkene structure was obtained by

multistep reactions using the Wittig reaction for the construction of the ﬂ uoroalkene func-

tionality as shown in Scheme 10.26 . The Wittig reaction of bis - MMTr - protected symmetric

ketone 82 , derived from diethyl 3 - hydroxyglutarate with (EtO)

2

P(O)CHFCO

2

Et, gave

ﬂ uoroalkene 83 in 67% yield. The reaction of 83 with BCl

3

and the subsequent silylation

gave ﬂ uorinated lactone 84 . The introduction of thymidyl group and amino functionality

to 84 was achieved by the chemoselective Mitsunobu reaction of a diol, prepared by 1,2 -

reduction of 84 , with N

3

- benzoyl thymine and Ph

3

P/CBr

4

- mediated S

N

2 - type substitution

of the homoallylic alcohol moiety with LiN

3

to give azide 85 . Conversion of the azide

group of 85 to an MMTr - amino acid functionality in ﬁ ve steps provided monomeric ﬂ uo-

roalkene isostere unit 87 . With the use of essentially the same method, a ﬂ uoroalkene

isostere of an adenyl - PNA was also synthesized. By means of MMTr/acyl solid - phase

peptide synthesis [46] , these ﬂ uoroalkenic monomers were incorporated into the corre-

Figure 10.7 Rotameric forms of PNA and the structures of alkenic isosteres.

Scheme 10.26

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

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