Protein Tyrosine Phosphatases 1B Inhibitors from Traditional Chinese

Chapter 11

Protein Tyrosine Phosphatases1BInhibitors from Traditional Chinese Medicine

Downloaded by YORK UNIV on July 6, 2012 | http://pubs.acs.org Publication Date: January 12, 2006 | doi: 10.1021/bk-2006-0925.ch011

Tianying An, Di Hong, Lihong Hu, and Jia Li National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China

In our research for natural products with antidiabetes activity, we screened our extract bank for inhibitors of PTP1B enzyme and found that some fractions from ethanol extract of Traditional Chinese Medicine showed strong inhibitory bioactivity against PTP1B enzyme. Using the PTP1B enzyme bioassay as a guide, chromatography of the fractions afforded several potential PTP1B inhibitors. Modification of oleanolic acid yielded novel more potent and higher selective PTP1B inhibitors.

Introduction Diabetes is an endemic in industrialized society. A lucrative market is waiting for companies that can find an effective remedy. Diabetes is a common disorder in which blood sugar is ineffectively processed. Type 2 diabetes is the most prolific form, representing - 9 0 % of cases. The primary cause is either insufficient secretion or action of insulin, whose role is to facilitate the passage of glucose into cells. When insulin is scarce or ineffective, extracellular glucose accumulates, starving cells of their energy source and causing a diversity of symptoms that can include blindness and heart disease. Type 2 diabetes can often be controlled by careful lifestyle management, but insulin injections or medication often become necessary. Unfortunately, diabetes drugs often suffer

© 2006 American Chemical Society

In Herbs: Challenges in Chemistry and Biology; Wang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

143


144 from low efficacy as monotherapy and can cause side-effects; therefore, there is a large medical need for an effective treatment. Protein tyrosine phosphatases IB (PTP1B) is a well-validated target in type 2 diabetes drug research. This enzyme modulates the signalling cascade that is activated upon insulin binding to its cell-surface receptor. Specifically, PTP1B dephosphorylates tyrosine residues on the receptor, stopping insulin action. Studies with PTP1B knockout mice have demonstrated that the lack of PTP1B activity resulted in an increased activity to insulin and obesity-resistance (1,2). PTP1B appears to be exemplary target for obesity and diabetes. Today, the obesity epidemic is an important factor in the rise of diabetes. Unfortunately, most of today's diabetes drugs, such as peroxisome proliferator-activated receptor (PPAR)-^ agonist and Metformin™ tend to have the side effect of increasing body weight. Therefore, a drug aiming to target both obesity and hyperglycemia would have enormous advantage. Several companies have been pursuing the development of PTP1B inhibitors as drugs (Figure 1) (3). Wyeth began testing Ertiprotafib/PTPl 12 in Phase II trials in March 2001, but discontinued the trials in June 2002 because of unsatisfactory efficacy, and the occurrence of dose-limiting side effects among several trial participants. Whether these problems stem from this particular chemical serials, speciesrelated differences in PTP1B function, or limited selectivity of the inhibitor for different PTPs is not yet clear. The PTP family of enzymes is large and all are highly specific for the charged phosphotyrosine residue. The chronic dosing required for treating diabetes requires the development of inhibitors with high selectivity for PTP IB over several highly related tyrosine phosphatases. So far, most of the reported PTP IB inhibitors are derived from synthetic compound library by HTS. The PTP IB inhibitors from plants especially from folklore medicinal plants are not reported yet. In this paper we report our effort in searching of PTP IB inhibitors from Traditional Chinese Medicine.

Discovery of PTP1B inhibitors from Traditional Chinese Medicine National Center for Drug Screening has established an extract bank based on Traditional Chinese Medicine ( T C M ) since 2000. To date, about 2,000 medicinal plants around China have been collected and 15,000 fractions prepared. Each plant was extracted with 95% ethanol, and the residues partitioned between water and chloroform. The water phase was subjected to AB-8 macro porous resin, eluted with water-ethanol (0:100, 15:85, 30:70, 50:50, 75:25, 95:5) to yield six fractions. In our research work for natural products with anti-diabetes activity from our extract bank since 2001, we have screened about 10,000 fractions for inhibitors against PTP IB enzyme by High-Throughput


145

50

= 384 nM


IC

Takeda

Abbot

Pyrrol phenoxy propionic acid derivatives 1C50 = 90 nM

A-366901 IC50 = 7 nM

Ο 7

Figure I A. Companies involved in protein tyrosine phosphatases IB (PTP IB) inhibitor discovery and representative compounds.



146

Merk-Frosst

Albert Einstein College of Medicine

Aryldifluoromethylphosphonic

4'-phosphony!difIuoromethyl

acid derivatives

phenylanaline derivatives

>90% inhibition at 10 uM

IC

Pharmacia 3'-carboxy-4'-((;-cai boxy methyl) tyrosine derivatives K\ = 870 nM

K

o

r

e

= 14 |Èvl

50

a

R

e

s

e

a

r

c

n

Institute of Chemical Technology 1,2-naphtoquinone derivatives 1C = 650 nM 50

Figure IB. Companies involved in protein tyrosine phosphatases IB (PTPIB) inhibitor discovery and representative compounds (continued).


147 Screening technique, and found that several fractions from Ardisia japonia, Hypericum erectum, Broussonetia papyrifera, Ginkgo biloba, Eobotrya japonica showed strong inhibitory activity.


p-Benzoquinonoid Inhibitors from Ardisia japonica and Hypericum erectum The chloroform fraction from A. japonica showed 69% inhibition against PTP IB at the concentration of 5 μg/mL. Under bioassay-guided purification, four active benzoquinonoids, 5-ethoxy-2-hydroxy-3-[pentadec-10'(Z)-enyl]-l,4benzoquinone (1), 5-ethoxy-2-hydroxy-3-[tridec-8'(Z)-enyl]-1,4-benzo-quinones (2), maesanin (3), and 2,5-dihydroxy-3-[(Z)-10'-pentadecenyl]-l,4-benzoquinone (4) were isolated (see Figure 2). We found compounds 1-4 have the same 2,5dihydroxy-l,4-benzoquinones skeleton, and more nonpolar substituents of this skeleton increases their inhibitory activities [from 4 (a hydroxyl group at C-6) to 3 (a methoxyl group at C-6), to 2 (an ethoxyl group at C-6)] (see Table I). Two other 1,4-benzoquinone derivatives, erecquinone A (5) and Β (6) were isolated from the active CHC1 fraction of H. erectum (see Figure 2) (4). Compound 5, with two free hydroxyl groups, showed stronger activity than compound 6, with two masked hydroxyl groups. L i u et al. reported that several 3,6-diaryl-2,5dihydroxy benzoquinones showed significant reduction in hyperinsultnemia in ob/ob mice (6). In order to study the relationships between the structure of 2,5dihydroxy-1,4-benzoquinone derivatives and the activity against PTP IB, we have synthesized several aromatic substituted 3,6-diaryl-2,5-dihydroxy benzoquinone derivatives using the same synthetic method reported by Liu et al. 3

(5) . Unfortunately, all of these aromatic substituted benzoquinones showed no inhibitory activity against PTP IB enzyme. These results indicated the long aliphatic chain on the 1,4-benzoquinone was helpful for the inhibitory activity. Further structural modification of 2,5-dihydroxy-l,4-benzoquinonoids will be carried out in our lab.

Figure 2. The structures of benzoquinonoids from A. japonica and H. erectum.


148


Flavanoid Inhibitors from Broussonetia papyrifera and Ginkgo biloba B. papyrifera (L.) (Moraceae) is a deciduous tree, and its fruits have been used for impotency and to treat ophthalmic disorders in China. Extracts of B. papyrifera have shown antifungal, antihepatotoxic, antioxidant and lens aldose reductase inhibitory activities. Also, several flavonoid constituents of this plant have been shown to inhibit lipid peroxidation, aromatase activity, and to exhibit antiplatelet effects (6). The chloroform fraction from the bark of B. papyrifera (L.) Vent, was found to significantly inhibit PTP IB enzyme (93% inhibition at 20 //g/mL). Bioassay-guided fractionation of the chloroform fraction led to the isolation of four flavonoids that were found to be active. They were characterized as quercetin (7), uralenol (8), 8-(l,l-dimethylallyl)-5'-(3methyibut-2-enyl)-3',4',5,7-tetrahydroxyflanvonol (9) and 5',8-di(3-methyIbut-2enyl)-3',4',5,7-tetrahydroxyflanvonol (10) (see Figure 3 and Table 1) (7). Analyzing their structures and activity, we found that with the introduction of unsaturated aliphatic substituents into the flavanol skeleton, the activity against PTP IB was improved . G. biloba leaf extract is being widely used for memory improvement, mental alertness, vertigo and tinnitus, allergies-through ginkgo's ability to antagonize, or inhibit platelet activating factor (PAF), altitude sickness-through improved blood flow to the brain to compensate for low oxygen levels at high altitude, early stage Alzheimer's disease, asthma, impotence, intermittent claudication (lameness), macular degeneration, migraines, to deter aging overall-through antioxidant-nature of chemical compounds in ginkgo, as a mood enhancer, and more. It was also used in the prevention of complications of diabetes mellitus (8). The chloroform fraction of G biloba leaf showed strong inhibitory activity with an I C of 8.6 //g/mL. Its chemical study yielded two biflavonoid compounds: ginkgetin(ll, IC 4.0//M)andsciadopitysin(12, I C 6.0 μΜ) (Figure 3). 50

50

50

Triterpenoid Inhibitors from Eriobotrya japonica The leaves of E. japonica has been documented for the treatment of various skin disease and diabetes mellitus in folk medicine (P). 3,6,19-trihydroxy-urs-12en-28-oic acid and corosolic acid, isolated from this plant, were reported to reduce blood glucose levels in normoglycemic acid. We found the crude triterpene acids, prepared from this plant, showed high activity against PTP IB enzyme with an I C of 2.5 //g/mL. Chemical analyses afforded four main active triterpenoids, oleanolic acid (13), ursolic acid (14), 2oc-hydroxyoleanolic acid (15), and corosolic acid (16) (see Figure 4 and Table I). The quantitative analysis of the crude terpene acids from this plant by H P L C - E L S D indicated the 50



149

Figure 3. The structures offlavonoids from B. papyrifera and G. biloba.


150 content of oleanolic acid, 2a-hydroxyoIeanolic acid, corosolic acid, and ursolic acid was about 1%, 5%, 10%, and 10% (w/w), respectively.

13 R,=H, R =H, R = C H 14R,=H,R =CH , R =H 2

3

2

3

3

3

15 R,=OH, R =H, R = C H 16 R,=OH, R =CH , R =H 2


2

3

3

3

3

Figure 4. The structures of triterpenoids from E. japonica.

Table I. The Inhibitory Activity of Natural Compounds Against P T P 1 B Enzyme

No 1 2 3 4 5 6 7 8

ICso^M) 3.01±1.25 3.63±0.97 4.62±2.41 19.18±3.77 4.40±1.32 12.74±5.37 25.78±2.53 21.55±6.48

No 9 10 11 12 13 14 15 16

ICJO^M)

4.38±1.05 5.20±2.02 4.03±0.62 6.08±0.39 21.96^1.47 8.11±3.64 4.57 ±0.56 13.43±4.77

Structural Modification of Oleanolic Acid Compounds 13-16 are four very common triterpene acids existng in many higher plants. Corosolic acid, an anti-diabetes ingredient from Lagerstroemia speciosa leaf, showed significant glucose transport-stimulating activity in Ehrlich ascites tumor cells at a concentration of 1.0 μΜ (10). In a randomized clinical trial type II diabetics, the anti-diabetic activity of an L speciosa leaf extract standardized to 1% corosolic acid (Glucosol™) demonstrated a


151 statistically significant reduction in blood glucose level at 48 mg per day dose for 2 weeks and clinical use suggests it is very safe (//). Some oleanolic acid glycosides were reported to have the activity of lowering blood glucose level in oral glucose-loaded rats (72). Oleanolic acid has been shown to protect against some hepatotoxicants and it has been used to treat hepatitis as a drug for decades in China (13). Oleanolic acid is very cheap and commercial available in China. In order to improve the activity and selectivity of oleanolic acid, structureactivity relationship studies were performed by our group. Wrobel reported in the docking study of compound 17, a strong inhibitor of PTP IB ( I C = 61 nM), with the X-ray crystal structure of PTP IB that the carboxylic acid group of 17 binds to the side chain of active site Arg 221 via a charge-charge interaction (14). Comparing oleanolic acid with compound 17, we found they had the similar stereo-structure. Their difference was that the distance between the carboxylic acid and five-ring skeleton in oleanolic acid is shorter than the distance between the carboxylic acid and tetracyclic ring in 17. If we lengthen the distance between the carboxylic acid and the five-ring skeleton, perhaps its potency would increase. Therefore, we synthesiezed two series of oleanolic acid derivatives: the C-28 long-chain peptide derivatives and the C-28 long-chain acid derivatives. Their structures and activities are listed in Tables II and III, respectively.


50

When the carboxylic acid of oleanolic acid was masked by methyl group (18), it was inactive. We hypothesized that the carboxylic acid of oleanolic acid might bind to the side chain of active site Arg 221 via a charge-charge interaction. Then we linked some types of amino acid to find out which type of amino acid residue will improve the activity. In this experiment, we have found that the activities of oleanolic acid C-28 long-chain peptide derivatives were related to the length of amino acid chain. When the length of carbon chain were


152 4 or 6, such as compounds 19 and 20, no activity was observed; and the length of the carbon chain was increased to 11 carbons, the active compound 21 (with the IC so of 2.5 μΜ) was obtained, whose activity was 10-fold greater than the parent compound 5. According to the docking study of compound 17 with the X-ray crystal structure PTP IB (14), there is a large hydrophobic pocket beside the active site Arg 221. So we inserted a (/?)-benzyl moiety on the α-carbon and obtained compound 22 (IC : 0.89 μΜ), which is 3-fold more potent than 21. 50


Table 11. Structures and Inhibitory Activity of Oleanolic Acid C-28 Longchain Peptide Derivatives Against P T P 1 B Enzyme

Ο HO*

No

R

IC Q 5

CH

19

NH(CH ) COOH

>20

20

NH(CH ) COOH

>20

21

N H ( C H ) , QCOOH

22

O H

(μΜ)

5 18

2

2

21.90±10.11 >20

3

4

6

2

H

HN(H C), 2

0 v

Τ

Ο

.N XOOH

2.52± 1.61

0.89±0.32

v

μ

When the carbon chain of acid was prolonged by fatty acid instead of amino acid, a series of oleanolic acid C-28 long-chain aliphatic acid derivatives were synthesized. Their structure-activity relationship study results are listed in Table III. When the C-28 long-chain aliphatic acids are saturated acid, they are inactive (compounds 23-25). Comparing to oleanolic acid C-28 long-chain peptide derivatives, we found the existence of amide bond very important for inhibitory activity against PTP IB enzyme. When the C-28 long-chain residues contain olefinic bonds, their activity was improved. For 3-OH and 3-OMe derivatives,


153 compounds 27 and 32 were the most potent inhibitors, respectively. From above data, we hypothesized that different substituent at C-3 might bind to the different site of enzyme, and the distance between the carboxylic acid and the five-ring skeleton of oleanolic acid is also different.


Table III. Structures and Inhibitory Activity of Oleanolic Acid C-28 Longchain Acid Derivatives Against PTP1B Enzyme

R-,0

No 23

Ri H

R

2

13.45±7.23 COOH

(CH ) COOH (CH ) COOH

>20 >20 7.80±1.33

24 25 26

H H H

27

H

0.98±0.34

28

H

1.41±0.95

29

H

2

2

8

12

^ ^ ^ C O O H

•

/

^

i

\ ^ .

>20 C

O

O

H

30

CH

3

7.80±2.45

31

CH

3

5.72±1.65

32

CH

3

2.60±1.14

33

CH

3

>20

The PTPase domains of receptor and nonreceptor are highly conserved with -35% mean sequence identity among known phosphatases (75). Therefore it is critical that inhibitors of PTPases used for therapeutic purpose show requisite


154 selectivity. Several potent oleanolic acid derivatives were tested on inhibitory activity against CDC25A, CDC25B and P T P - L A R (Table IV). They all have good selectivity over PTP-LAR, and 32 has good selectivity over CDC25A and CDC25B. So we surmised that the hydrophobic substituted at C-3 might interact at a special site on the PTP1B and this site is not existent in CDC25A, CDC25B and P T P - L A R . (See Table IV).

Table IV. The Inhibitory Activity ( I C (μΜ)) of Compounds 21,22, 27, 28, 31 and 32 Against P T P 1 B , C D C 2 5 A , C D C 2 5 B , and P T P - L A R .


50

No

21 22 27 28 31 32

PTP1B 2.52±1.61 0.89±0.32 0.98±0.34 1.41±0.95 5.72±1.65 2.60±1.14

CDC25A 6.55 5.50 1.56 >100 >100 >100

CDC25B 1.5±0.74 l.liO.ll 2.52±0.21 0.85±0.12 4.50±1.3 >100

PTP-LAR >100 >100 >100 >100 >100 >100

Biological Activity Evaluation A l l PTPases are C-terminal truncated, soluble form of recombinant human PTPases. Their catalytic activities were routinely measured as rates of hydrolysis of /?-nitrophenyl phosphate (pNPP) in a 96-well microtiter plate format (16). N a V 0 acts as positive control ( I C = 2 μΜ). Three independent measurements were performed for I C determinations. Similar results were obtained in multiple measurements. The reported values are the average of all experiments and the errors are standard deviations. 3

4

50

50

Conclusions Several natural PTP IB inhibitors,/^-benzoquinonoids, isoprenylflavanoids, biflavanois, and triterpenoic acids, were discovered from T C M . Modifying the structure of oleanolic acid, we have gotten more potent PTP IB inhibitors with high selectivity over CDC25A, CDC25B and P T P - L A R .


155 Acknowledgements This work was supported by the National Natural Science Fund of China (30100229 and 30371679) and the Science and Technology Development Fund of Shanghai, China (01QB14051).

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Protein Tyrosine Phosphatases 1B Inhibitors from Traditional Chinese

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