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2 Phlorizin: A Drug Lead from Apple Trees

2 Phlorizin: A Drug Lead from Apple Trees

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9.2 Phlorizin: A Drug Lead from Apple Trees



from the root bark of apple trees. First characterized and named by De Koninck [10]

in 1835 using the Greek words for “root” and “bark,” phlorizin is now known to

occur in other parts of apple trees, especially the leaves [9]. Whereas it is the

predominant phenolic compound found in domestic apple trees (M. Â domestica),

only small amounts have been detected in other apple (genus Malus) species and

plant families.

Soon after its first isolation, phlorizin was characterized by acid hydrolysis as

being a sugar derivative of phloretin (5, Scheme 9.1), a dihydrochalcone

derivative also isolated from apple bark. The empirical formula

(C21H24O10Á2H2O) was first established in 1887, at which time glucose was

recognized as being the component sugar [11]. Although the structure of

phloretin was definitively established by synthesis in 1928, the structure of

phlorizin remained ambiguous with regard to the point of glucose attachment

and the stereochemistry of the glucoside linkage. In 1930, building on earlier

work that had established the point of glucose attachment to be on the



HO



OH



OH O

Stas

(1836)



OH



phloretin (5)

aq. H2SO4



HO



OH



OH



HO



OH

OH



HO



O



HO



O



O



warm aq. Ba(OH)2

Cremer, Seufelt

(1912)



OH

OH



HO



O



O



HO



HO

O

phloretic acid



OH

OH



phloroglucinol β-glycoside



phlorizin (4)

Johnson,

Robertson MeI, K2CO3, acetone

(1930)

MeO



OMe



OMe

MeO



HO



O



HO



O



O



H2SO4, MeOH, H2O



OMe



OMe



Glucose

OH



OH

OH



O

Ac2O, AcOH, 180oC



MeO



OMe

O

O

6



Scheme 9.1 Phlorizin structure determination.



OMe



303



304



9 From Natural Product to New Diabetes Therapy: Phlorizin and the Discovery



phloroglucinol (1,3,5-trihydroxybenzene) ring, Johnson and Robertson carried

out a series of reactions starting with phlorizin that afforded the benzopyrone

6 (Scheme 9.1) [12]. This material could only be formed if the point of

attachment to phloretin was on one of the two equivalent phenolic hydroxyl

groups ortho to the ketone.

Finally, after some confusion as to the stereochemistry of the glycosidic linkage

based on rates of hydrolysis catalyzed by glycosidases [13], the complete structure of

phlorizin was unambiguously assigned by total synthesis in 1942 (Scheme 9.2) [14].

Although not appreciated at the time, this work constituted the first synthesis of an

SGLT inhibitor and remained the general approach to preparing analogs of

phlorizin for over six decades. An X-ray crystal structure of phlorizin was published

in 1986 [15].



Ph

Ph



O



OH



O



AcO



O



Br

aq. KOH, acetone, 0oC



AcO

OH



OAc



O



O



OH



O

AcO



OAc



O



AcO



O



O



OAc

OAc



OH

HO



HO



CHO



O

H2, Pd, C, EtOH



Phlorizin (4)



aq. KOH, EtOH

HO



O



O



O

naringenin-2'-glucoside



HO



OH

OH



Scheme 9.2 Phlorizin — first total synthesis, 1942.



9.3

Phlorizin: Mechanism of Action



Long before its chemical structure was fully established, the ability of phlorizin to

induce glucosuria in humans and animals was well known and had been well

characterized. Indeed, following the first report of its biological activity by von

Mering in 1886 [16], phlorizin became a valuable tool that was used for many years

in physiological studies, especially with regard to renal function. For example, in

the 1930s, using intravenously administered phlorizin, methods were developed

that allowed the noninvasive measurement of glomerular filtration rate and renal

blood flow [17].

Later in the twentieth century, researchers began to zero-in on the mechanism of

action. By the 1960s it was recognized that phlorizin was an inhibitor of active

glucose transport across cell membranes, not only in the kidney (leading to

blockade of filtered glucose reabsorption), but also in erythrocytes and the small



9.3 Phlorizin: Mechanism of Action



Figure 9.2 Representation of SGLT-mediated glucose reabsorption in the kidney.



intestine [18]. The phlorizin-sensitive active transport system responsible for the

renal reabsorption of glucose was shown to be Naỵ dependent and localized in the

brush border of proximal tubule cells in the kidney. With the advent of expression

cloning techniques in the 1980s, the proteins making up this active transport

system, termed “sodium-glucose cotransporters,” were finally isolated and characterized [19].

Several members of the SGLT transporter family are now known [1] with SGLT1

and SGLT2 being the major isoforms; both are found in the kidney (Figure 9.2).

The first, SGLT1, has a high affinity (Km $ 0.4 mM) and low capacity for glucose.

Found primarily in the small intestine but also in the kidney and heart, SGLT1

transports two sodium ions with every molecule of glucose. Although glucose is

transported against its concentration gradient from the lumen (the “pro-urine”)

into renal tubule cells, this “uphill” transport is coupled to the simultaneous

“downhill” transport of sodium ions along a concentration gradient maintained by

ATP-driven Kỵ/Naỵ exchange. The second transporter, SGLT2, transports glucose

in a similar way to SGLT1 except that the affinity for glucose is lower (Km $ 2 mM)

and only one glucose molecule is transported along with each sodium ion.

Importantly, as opposed to SGLT1, SGLT2 is found exclusively in the kidney (in the

proximal tubules) and has a higher capacity for glucose transport – it is responsible

for approximately 90% of glucose reabsorption in the kidney. Phlorizin inhibits

both SGLT1 (Ki ¼ 151 nM) and SGLT2 (Ki ¼ 18.6 nM) [20].

Other transporter proteins are now known to transport glucose, most notably the

GLUT family of transporters [3], which facilitate passive diffusion of glucose across

cell membranes. Indeed, once glucose is reabsorbed from the urine into renal

tubule cells, it reenters the bloodstream passively via the GLUT1 and GLUT2

isoforms. In this way, SGLTs and GLUTs work together in a healthy individual to

achieve almost complete reuptake of glucose from the urine, thereby conserving a

vital energy source (Figure 9.2). Phlorizin does not appreciably inhibit transporters

in the GLUT family.



305



306



9 From Natural Product to New Diabetes Therapy: Phlorizin and the Discovery



Genetic information has shed valuable light on the roles of SGLTs and other

glucose transporters in vivo. In humans, mutations of SGLT2 lead to a rare

autosomal genetic condition known as familial renal glucosuria [21]. Interestingly,

subjects with this condition (sometimes referred to as a “nondisease”) can excrete

up to 140 g of glucose each day while otherwise being perfectly healthy and living

normal lives. Besides confirming phlorizin’s mechanism of action, these observations suggest that chronic inhibition of SGLT2 (e.g., via a SGLT2-selective inhibitor)

may not lead to serious mechanism-based side effects. On the other hand,

outcomes associated with mutations in the SGLT1 gene are less benign. Because

SGLT1 is a major sugar transporter in the gut, mutations in humans lead to

severe and potentially fatal diarrhea resulting from malabsorption of glucose and

galactose [22].



9.4

Phlorizin, SGLTs, and Diabetes



Experiments exploring the effects of phlorizin in diabetic animal models

followed logically from observations regarding its ability to cause urinary

glucose excretion. In these key experiments, phlorizin administration

reduced plasma glucose levels and improved insulin sensitivity, thereby

establishing hyperglycemia as a contributor to insulin resistance and the

development of diabetes [23]. However, despite its positive effects in animal

models and potential for therapeutic use, phlorizin was not developed as a

drug for T2DM therapy due to several shortcomings. First, the compound

exhibits very low oral bioavailability due to poor absorption in the small

intestine and its susceptibility to intestinal b-glycosidases that cleave the

molecule to glucose and phloretin. Second, as a nonselective SGLT

inhibitor, phlorizin acts on SGLT1, adding the potential for gastrointestinal

side effects. Exacerbating the potential for side effects, the aglycone cleavage

product (phloretin) is a micromolar inhibitor of GLUT facilitative glucose

transporters, which are expressed in various tissues including the brain and

small intestine.



9.5

Phlorizin Analogs: O-Glucosides



Prior to the 1990s, efforts to prepare analogs of phlorizin were associated with

research on sugar transport and phlorizin’s mechanism of action [24]. Following

the characterization of SGLTs as distinct therapeutic targets, several pharmaceutical

companies initiated research programs aimed at the discovery of phlorizin analogs

for the treatment of T2DM. Using or modifying the procedure developed by



9.5 Phlorizin Analogs: O-Glucosides



Zemplen and Bognar in the 1940s (Scheme 9.2), the early analog programs focused

on O-glucoside analogs with superior characteristics to phlorizin: increased

potency versus SGLT2, increased selectivity over SGLT1, improved absorption in

the small intestine, increased resistance to b-glycosidase cleavage, and, for the

aglycone, reduced inhibition of GLUT transporters.

One of the first papers describing structure–activity relationships (SARs) around

phlorizin was published by investigators at Tanabe who profiled analogs in a rat in

vivo model of urinary glucose excretion (UGE) [25]. Activity in this model was

reduced by structural changes in the linker between aryl groups (A- and B-rings)

and required one, but not both, phenolic hydroxyl group on the A-ring (Figure 9.1).

A few sugar modifications were also explored, replacing glucose with galactose,

xylose (pyranoside), and the disaccharides maltose and lactose. In all these cases,

activity was attenuated. On the other hand, changes on the B-ring were often

tolerated and sometimes improved potency, for example, by replacement of the

phenolic hydroxyl group with a small alkyl substituent or fusion to form a 6,5-fused

heterocycle (e.g., benzofuran). From this work, which did not directly measure

potency versus SGLT1 and SGLT2, compound 7 (Figure 9.3) emerged as a

promising candidate based on its superior activity in the rat UGE model compared

to phlorizin. However, realizing that 7 would likely remain a substrate for

b-glycosidases, various ester and carbonate derivatives linked to the sugar moiety

were explored as potential prodrugs. Ideally, these would not be recognized by

intestinal b-glycosidases and would be cleaved by esterases to the parent drug (7).



Me



OH



O

H

N



RO



O



HO



O



7: R = H (T-1095A)

8: R = CO2Me (T-1095)



OH

OH

H

N



CF3



SMe



CF3



N



O



9



HO



SMe



OMe



N

HO



O



HO



O



RO



10



OH



O



O



OH



OH 1: R = H (sergliflozin)

13: R = CO2Et (sergliflozin etabonate)



HO



OH



OMe



N



S



HO



O



HO



O

OH



OH



O



N

RO



12 (AVE-2268)



O



O



OH



OH 11: R = H (remogliflozin)

14: R = CO2Et (remogliflozin etabonate)



HO



Figure 9.3 O-Glucoside SGLT inhibitor candidates.



307



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