Uncommon Sugars and Their Conjugates to Natural Products - ACS

Chapter 2

Uncommon Sugars and Their Conjugates to Natural Products 1

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Downloaded by COLUMBIA UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: March 13, 2007 | doi: 10.1021/bk-2007-0960.ch002

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Wenlan Chen , Guisheng Zhang , Lizhi Zhu , Lanyan Fang , Xianhua Cao , James Kedenburg , Jie Shen , Duxin Sun,*, and Peng George Wang1,* 1

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Department of Chemistry and Biochemistry and Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210

Indolocarbazoles (such as rebeccamycin, J-107088 and NSC 655649) and anthracyclines (such as doxorubicin and daunorubicin) are two important classes of anticancer drugs. Both natural and synthetic indolocarbazoles and anthracyclines contain mono-, or di-saccharides, which are critical parts of the molecules for binding to DNA or for inhibiting topoisomerases. Moreover, both natural and synthetic analogs containing disaccharides exhibit higher antitumor efficacy with unique DNA binding and topoisomerase poisoning characteristics. Thus, varying the uncommon sugar structure of indolocarbazole and anthracycline drugs will provide insightful information on the Structure-Activity Relationship (SAR) of topoisomerase I & II poisoning and DNA binding, and will potentially produce analogs for overcoming drug resistance in cancer therapy.

© 2007 American Chemical Society In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Numerous lead compounds in modem drug discovery are directly derived from natural products, many of which are glycosylated metabolites. Uncommon sugars, which are derived from common sugars by replacement of at least one hydroxyl group or hydrogen atom with another functional group, are frequently found in the secondary metabolites of microorganisms and plants, such as cardioglycosides, antibiotics, and anticancer agents (1,2). The sugar moieties of these pharmaceutical^ important metabolites often play critical roles in determining the biological and pharmacological activities. Thus, modification or alteration of sugar structures has been validated as a powerful strategy in modem drug discovery (3-5).

Uncommon Sugar Library Deoxysugars and their oligosaccharides arefrequentlyfound in the structure of bioactive drugs. These uncommon sugars play very important roles in maintaining some drugs' activity. There have been continuing efforts on the synthesis of uncommon monosaccharides, starting from either carbohydrates or noncarbohydrate precursors (6).

Systematic syntheses of uncommon sugars Traditionally uncommon sugars were synthesized through multistep transformations of relatively inexpensive common sugars (7). One major disadvantage of this approach is the long reaction sequence for protection and deprotection manipulations. An alternative approach is using noncarbohydrate precursors. In this case, many chemists have come up with different synthetic approaches (8-11). In 2002, our group developed a strategy for generating sixmember P,y-unsaturated lactones using Ring-Closing-Metathesis (RCM) (12), as shown in Scheme 1. The major advantage of this synthetic strategy is that it provides a systematic synthesis pathway for producing desired uncommon sugar units. In addition, we developed an alternative synthetic pathway by using an inexpensive starting material, fnms-4-phenyl-3-buten-2-one (13). The key intermediates for the uncommon sugars were achieved by this chemo-enzymatic synthetic pathway (Scheme 2). By using this method, four different types of deoxysugars were successfully prepared from one a, P-unsaturated lactone in moderate to good yields (Scheme 3). The major advantage of this method is that it allows the formation of uncommon sugars with both 3,4-cw- and 3,4-transdifunctionalities.

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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M eM s '" Y ^" M M , es N

N

V

C I

R,

R R

O

^ k A

"J or I C I- I

2 2

0

A ^

Ri, R = H o r C H 2

>

n

P(Cy) RCM

3

Xf Xf

3

Y

°


Sharpless AD

%

R(

3

2

HO^S< ^ R, OH

OH

R R =HorCH 1(

Na(CN)BH

c/s-3,4-dihydroxy group were introduced

Ri, R

3

= 2

H or C H

3

Scheme 1. Synthesis of2,6-dideoxysugar by RCM.

1. Ru(ll), Lipase, Et N isopropenyl acetate 3

NaBH

4

Q

Ph P DEAD^

H

3

t

OH 2. DIBAL-H (2.0 eq.)

95%

83%, > 97% ee

20:1

2 steps

L-digitoxose

'"../OÔ

,

9 H0CZ^°H 85% HO R

H0^>^ 6H 9 5 %

tens only


2

s t e p s

L-canarose

T B S 0 ^ " 45% TBSO* 80%

(

J

A x

TBSO'

88% < z i ^ r o H

1

N

3

ScAeroe 3. ^ # , P ^ C ; 6; DIBAL-H; c) NaCIO, Pyridine; d) NaBH PhSeSePh, AcOH; e) NaBH CN;f) TBSCl, imidazole; g) TBAF then NaBH CN; h) DPP A, PPh DEAD; i) TBAF then DIBAL-H. 2

4>

3

3

3t

Lipase

n-\

NBS

n~i\

O'T^

isopropyl

C r Y '

d)

acetate

QH

H

1. Cr0 NH CI 3

VÔvÔ

4

2. NaBH , CeCI 4

3

'

J L J H C T ^

0

VÔÔ +

U HO

o V s

VÔÔH

^

\

Q

V^VOH R 2,6-deoxysugars

Scheme 4. Alternative enzymatic synthetic pathway of 2,6-dideoxysugar.

Recently, we developed another synthetic pathway by enzymatic resolution of l-(2-furyl)ethanol followed by Achmatowicz rearrangement (Scheme 4) (14). In this pathway, furanaldehyde, an agrobased product, is used as starting material for preparation of the chiral a,p-unsaturated lactones. As an alternative way to construct the desired a,|3-unsaturated lactones, this strategy is more economic and more practical for large-scale manipulations than that one shown in scheme 2. So far, 20 uncommon sugars were obtained in large quantity (Figure 1) by using these two strategies in our group.


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H O ^ T

O

H

~p>T°H

&

^QTOH

N 3

HO (W2)

&

-^QTOH

(W3)

@

HO°

H

N

Q

OH

N


©

N ^°J 3

r v

H (

OH

3

N

W25

HoCglS7"

W

2

6

HOC^97"

OH

3

^

0

W19

H

2

o h

-pgÔH

N

3

W

2

7

-pipyoH H

@

°

O H

W20

OH

-pgTOH

N î97-OH

H

1

3

(wil)

°

W

N

3

3

(wi}

3

W35

3

W28

-j^TOH °

H

W36

(a)

HO

N

(W5>

W6

W7

H O ' ^ . O H

(W13l

OH(wJ4|

HO&ÔH

"%ÔH

• t e ^ O H

N

W29

N

H

[W37|

H0

^

3

0

0

^

H

W23

N

W30

W31 HO

^SwOH

O

H

3 W24

H O ^ O H

\

HfoSÔH

OHW16 H

fW22|

' - ^ O H °

\

'

° - ^ O H N3

(W2ll

^ ~ O H

W16 HO

H

(W8~l HO

HO " f o ^ - O H

3

^ O H

100 40

1 5 3

2 2 4

3 24 26

4 34 19

5 >100 >100

6 13 12

7 32 12

8 >100 34

9 76 53

10 >100 55

Reb 4 6

SOURCE: Reproduced with permission from reference 37. Copyright 2005.)

and Reb) are more active than compounds with 2-deoxyglucose (6 and 7) or with 2,6-dideoxyglucose (3 and 4) against colon cancer cells (SW620) and leukemia cells (K562). Compounds with 2,3,6-trideoxyglucose (8) are the least active. These data indicate that 2-OH, 3-OH and 6-OH groups in the sugar moieties, rather than the modifications in the imide structure, play crucial roles in maintaining their anticancer activity. The better activities of compounds 1, 2 and 6 indicate that the 6-OH may be more important than other hydroxyl groups in the sugar moiety. The 6-OH group on the carbohydrate residue may form a hydrogen bond with the indole NH group, which would maintain the



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Scheme 5. Reagents and conditions: a) Et 0/toluene, 88 °C, 24 h; b) Boc 0, DMAP, THF, rt, 1 h; c) TBAF THF, reflux, 8 h; d) LiBrH 0, AG 50W-X2 resin CH CN, rt, 15 min; e) Ph P, DEAD, THF, -78 °C, 15 h ( a:0 =1:2.5); f) 88% HC0 H, rt, 6 h; g) hv, air, I , benzene, 8 h; h) TBAF, THF, reflux, 15 h i) EtOH/toluene (1:3), 48% KOH, rt, overnight; then 10% citric acid; j)N H H 0. 2

f

t

2

2

3

3

2

2

2

4

2

carbohydrate in a fixed conformation for optimal activity and interaction with the topoisomerase I-DNA complex. As shown in Figure 4, compound 1 with glucose moiety showed similar strong topoisomerase inhibition as that of CPT at the same molar concentration, However, compound 6 with 2-deoxy-D-glucose moiety gave a very weak topo I inhibition while compound 3 with 2,6-dideoxy glucose moiety was inactive. This indicates that the 2-OH and 6-OH of the sugar moiety also modulate the topoisomerase I activity in cancer cells, which is worthy of further investigation with various modifications in the sugar moiety. Therefore, the modifications of rebeccamycin with uncommon sugars may provide a new class of anticancer compounds.

Daunorubicin analogs with uncommon sugars The anthracycline quinine antibiotics daunorubicin (DNR) and doxorubicin (DOX) (Figure 5) are potent antitumor agents against a variety of human solid


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0.01 0.05 0.1 0.5 1


5 (|ig DNA)

Mnnr,,n Nourug

(10

CPT o^M)

1 (100îM)

3 (100^M)

6 (100^M)

1 (250nM)

Figure 4. Standard ICT assay in HeLa cells comparing camptothecin (CPT) with Rebeccamycin-sugar derivatives 1, 3, and 6. Exponentially growing HeL cells were incubated with the compounds indicated to the right of the blot fo 30 min at 37 °Q followed by sarkosyl lysis and a standard ICT assay (CsCl gradient separation ofDNA, recovery ofDNA and spottingfrom0.01 to 5 ig ofDNA on the blot). The blot was probed with anti-topo I antibody. Signals were developed using ECL and a short (1 min) exposure. (Reproduced with permissionfromreference 37. Copyright 2005.)

R = C H O H Doxorubicin 2

Figure 5. SAR ofDNR andDOX.



24 tumors and leukemia (38). In addition to DNA intercalation, topoisomerase II (topo II) is considered as the primary cellular target of anthracyclines (39,40). The well-accepted mechanism of action is through interaction with DNA-topo II by stabilization of topo II-DNA-drug ternary complex, which also triggers apoptosis. It is noted that DNA binding and interaction are necessary, but not sufficient, for topo II poisoning (41). In fact, the external (non-intercalating) sugar moieties and the cyclohexane ring A are recognized as crucial moieties for therapeutic efficacy of anthracyclines. The sugar moiety of anthracyclines serves as a minor groove binder of DNA (42,43). Indeed, modifications on the sugar structures have led to the second generation of doxorubicin analogs as monosaccharides such as epirubicin, valrubicin and pirarubicin. The third generation anthracycline analogs with disaccharide, such as MEN 10755 (Figure 6) (44-46), possess a 2-deoxy fucose linked to the aglycon and

MEN 10755

Daunosaminyl daunorubicin

Figure 6. Disaccharide anthracycline analogs.

daunosamine as the second sugar unit. Extensive studies for the SAR of MEN 10755 reveal that the axial orientation of C-O-l bond of the second sugar daunosamine is critical for the topo II poisoning ability. Indeed, glycoside with equatorial configuration is ineffective in topo II poisoning (47,48). The presence of 4-methoxy group in the second sugar unit dramatically decreases its cytotoxicity compared to anthracycline monoglycosides due to the reduced topo II poisoning (47,48). It is conceivable that the axial orientation is optimal for the interactions of MEN 10755 with the DNA-topo II complex only in the absence of methoxy group, and this is confirmed by the crystal structure of the complex between MEN 10755 and hexanucleotide (CGATCG) (49). This structure is similar to previously crystallized anthracycline-DNA complexes, while MEN 10755 showed two different DNA binding sites. In one binding site, the disaccharide resides in the DNA minor groove; in the other binding site, the


25 second sugar protrudes from the DNA helix and is linked to guanine of another DNA through hydrogen bonds. This peculiar behavior suggests that the second sugar may interact with other cellular target such as topoisomerase. MENdisaccharides is capable of stimulating topo-I mediated DNA breakages (50).


Monosaccharide daunorubicin analogs

Previous research has indicated that the linkage between the sugar moiety and the aglycon is very important. Only the a-linked monosaccharide daunorubicin analogs are biologically active. Therefore, the uncommon sugars were introduced stereoselectively. Thioglycosides were used as glycosyl donors because of their high stability (51-53). The promoter system AgPFe/TTBP is efficient for the a-selective glycosylation (54). Taking advantage of our uncommon sugar library, six desired a-linked monosaccharide daunorubicin analogs were synthesized using the method as shown in Scheme 6 (55).

Scheme 6. Reagents and conditions: a) 0.2 MHCl, 90 °C, 1 h; b) PhSH, BF •Et 0/CH Cl 0 °C, 4 h; c) TTBP, AgPFJCH Cl 4AMS,0 °C, 4 h; d) 0.1 MNaOH/THF, 0 °C, 6 h. 3

2

2

2t

2

2i

The cytotoxicities of these six compounds (Figure 7) were examined in colon cancer cell line SW620 cells. The results in Table II indicated that the aglycon, DNR-A, exhibited 70 to 100-fold lower cytotoxicity than daunorubicin derivatives with various uncommon sugars. This suggests that sugar structure in daunorubicin plays a critical role in determining its anticancer activity. Compound 14 with 3'-OMe terminal 2,6-dideoxysugar showed very potent cytotoxicity with IC of 0.1 juM. Importantly, compared to compounds 12 and 13 (with ax/a/-3'-OMe or axial-3'-OH group), 14 (with an equatorial-3'-OMe group) showed 10 to 20-fold higher anticancer activity. This suggested that the ax/a/-3'-substituent in sugar (such as in compounds 12 and 13) may interfere daunorubicin binding to DNA. 50



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Table II. Cytotoxicity (IC ) of compounds 11-16 against colon cancer SW620 cells. DNR 11 14 16 DNR-A Compounds 12 15 13 50

IC in SW620 (uM) 50

0.26

>1

>1

0.1

0.35

>1

>2

0.033

Compound 14 with 3'-OMe terminal 2,6-dideoxysugar showed very potent cytotoxicity with IC of 0.1 îM. Importantly, compared to compounds 12 and 13 (with

Uncommon Sugars and Their Conjugates to Natural Products - ACS

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