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8 Aeruginosins: From Natural Products to Achiral Analogs

8 Aeruginosins: From Natural Products to Achiral Analogs

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16.8 Aeruginosins: From Natural Products to Achiral Analogs

H



HO

HO



N



H



Me



H



O

NH



HO



H



O

Ph

O



NH



Me

O



N



MeO



OSO3 H N

2



NH2



HO



H

HN



N



O



HO



NH



HO

Me

NH

N

H



59



NH

O



NH

N

Ph



NH



H2N



Oscillarin, 58

(IC50 = 28 nM)



O

O



HO



N



HO



Dysinosin A, 57

(IC50 = 46 nM)

H



O



NH



H



Cl H

Me

O

MeO



O

N



NH

O



NH

N

OSO3 H N

2



NH2



Chlorodysinosin A, 60

(IC50 = 6 nM)



Figure 16.15 Structures of naturally occurring aeruginosins, dysinosin A, oscillarin, and

chlorodysinosin A.



Figure 16.16 (a) Cocrystal structure of synthetic oscillarin with thrombin. (b) Space-filling

structural comparison of dysinosin A (green) with chlorodysinosin A (purple) within the active

site from X-ray data.



587



588



16 Hybrids, Congeners, Mimics, and Constrained Variants



reported in another patent [108], whose structure and stereochemistry was

confirmed by total synthesis in our group [109]. The unusual (3R)-D-lysine moiety

was also found in aeruginosins 205A and 205B. Here too, the originally misplaced

chlorine atom in previous reports [110] was rectified through total synthesis [111].

Interestingly, oscillarin had improved activity against thrombin compared to

dysinosin A (IC50 ¼ 28 nM).

The remarkable improvement in in vitro thrombin inhibition in going from

dysinosin A to chlorodysinosin A (IC50 ¼ 6 nM) can be rationalized based on the Xray cocrystal structure with thrombin. Thus, it can be seen that the hydrophobic S2

pocket is better filled with the more hydrophobic chlorine atom in chlorodysinosin

A (purple contours) compared to dysinosin A (green contours). Molecular

mechanics calculations showed a more restricted x-angle around the carbonÀÀ

chlorine bond. Based on the hydrophobic interactions observed in chlorodysinosin

A, we reported the synthesis and excellent activities of analogs in which the

hydroxyl group(s) were removed from the octahydroindole subunit, the amidine

moiety was replaced by a benzamidine group, and the chlorine atom replaced with

other groups as shown in oscillarin analogs 61–64 (Figure 16.17) [112]. It was also

apparent that the hydroxyl groups in the octahydroindole core unit were not

necessary for activity since they did not make any contact with the enzyme.

Truncated analogs of oscillarin in which the appended phenyl lactic acid group was



H



Me



X H



H



O

N



NH

O



Me

NH



NH



Me

O



H

HN



H



NH



O

O

S O



NH

NH2



HO

NH



H

HN



N



NH



O

O

S O



O

N



NH

O

NH



NH



O



NH2



OH



64, (IC50 = 2 nM)

H



O



NH2



65, (IC50 = 2.9 µM)



H



63, (IC50 = 2 nM)



O

N



NH

O



OH



61, X = Cl (IC50 = 1 nM)

62, X = H (IC50 = 97 nM)



HO



N



NH



O



NH2



OH



H



Me H



H



O



MeO

NH



H

HN



O

N



NH



O

O

S O



NH2



NH2



66, (IC50 = 13.1 µM)



NH



67, (IC50 = 3.25 µM)



Figure 16.17 Structures of chemically modified and truncated analogs of oscillarin.



16.8 Aeruginosins: From Natural Products to Achiral Analogs



589



replaced by a sulfonamide or amide as in compounds 65 and 67 resulted in loss of

activity [113].

16.8.2

Constrained Peptidomimetics



The unique interactions of the bound aeruginosins with thrombin in the crystal

structures led us to design smaller molecules that would simulate the relevant

contacts. We were aided in our design by the cocrystal structure of the D-Phe–L-Pro–

L-Arg tripeptide as the chloromethyl ketone (PPACK, 68) [114]. Its interactions at

three sites in thrombin are shown in Figure 16.18. Based on this observation, we

conceived of a series of indolizidinones representing constrained peptidomimetic

analogs of PPACK. Modeling these molecules in thrombin provided excellent

congruence with the PPACK crystal structure. The synthesis of the first analog (69)

placed a tertiary hydroxyl group at C6 instead of an amino group [115]. We were

pleased to find an in vitro activity of IC50 ¼ 20 nM, which was corroborated by a

cocrystal structure with thrombin. When the corresponding C6 amino analog (70)

was synthesized and tested, the activity improved dramatically to IC50 ¼ 3 nM [116].

This accounts for a more effective interaction with Gly216. The C6 epimeric

analogs were inactive, which emphasizes the importance of stereochemistry at that

site, correctly orienting the hydrophobic benzyl group into the S2 site. Finally, the

cyclic sulfonamide 71 analog was synthesized, and found to be significantly less

active compared to 69 and 70 [117].

16.8.3

Achiral Inhibitors



Focusing on the interactions of the natural products and the synthetic inhibitors

with the Gly216 antiparallel bridge and the basic amidine moiety at the P1 site,

we conceived of a core unit comprising an o-aminophenol onto which

requisite functionality would be appended as pictorially illustrated in expression 72

S2



S3

Ph

H



N

H

O



O



N

O



O



HN



X

H



Cl



O



H

N



Gly216

68, PPACK



H



Ph



NH

HN

O



NH2

O



Ph



N

O

H

N



O



H2N



NH

NH



H

N

S

O O



O



NH



NH2



NH2



S1

69, X = O (IC50 = 20 nM)

70, X = NH (IC50 = 3 nM)



NH



71, (IC50 = 494 nM)



Asp189



Figure 16.18 Constrained indolizidinone and sultam analogs inspired by the structure of PPACK.



590



16 Hybrids, Congeners, Mimics, and Constrained Variants

S2

P2



S3

X

H



P3



O



R



H2N



N

H P

1



OH



S1



H

N



O O

S

N

H



72



Gly216



NH



O

Me

O

N

H



OH

73

(IC50 = 17 nM)



Figure 16.19 Design and synthesis of achiral thrombin inhibitors based on a phenolic core.



(Figure 16.19) [118]. Consequently, we synthesized a small library of achiral phenolbased molecules and assessed their inhibitory activity against thrombin. A

representative inhibitor (73) showed excellent activity (IC50 ¼ 17 nM), which was

corroborated by a cocrystal structure with thrombin [118].

A refinement of this model, strongly guided by molecular modeling, led to the

design and synthesis of an achiral motif based on an N-amino-2-pyridone core

unit (Figure 16.20) [119]. A small library of such molecules was synthesized and

tested, showing good inhibitory activity as exemplified by compound 74

(IC50 ¼ 23 nM). A remarkable improvement in inhibitory activity was observed

with the N-amino-2-dihydropyridones exemplified by analog 75 (IC50 ¼ 11 nM)

[120] compared to the pyridone counterpart 76 [120]. Excellent selectivity against

trypsin was also observed for 75.

The remarkable difference can be explained by the fact that in the pyridone

series, the amino group is ionized at physiological pH (as in the assay protocol),

whereas in the dihydropyridone series, it is not, thus being able to effectively

engage in the antiparallel H-bonding interaction with Gly216. It can be seen that in

the X-ray cocrystal structure of an inactive pyridone analog 77, the H-bonding

lengths with Gly216 are quite long (Figure 16.21). The corresponding N-2dihydropyridone analog showed IC50 ¼ 26 nM, which is comparatively less active

than 75, but much more active than 77 (IC50 ¼ 17 100 nM).



H2N



Me

O

Me



N

H



N

O



N

H



74, (IC50 = 23 nM)



NH



H2N



O O

N

S

N

H

OMe



Me

O

O



N

H



75, (IC50 = 11 nM)



NH



H2N



O O

N

S

N

H

OMe



NH



Me

O

O



N

H



76, (IC50 = 1050 nM)



Figure 16.20 Design and synthesis of achiral thrombin inhibitors based on aminopyridone and

aminodihydropyridone cores.



16.9 Avermectin B1a and Bafilomycin A1



3.87 Å

4.36 Å

Gly 216

2.87 Å

2.64 Å



Asp 189



O O

N

S

N

H

O



Me

O

N

H



X X

O



H

N



N

HN



O



O

Me

Asp 189



77, (IC50 = 1240 nM)

Figure 16.21 Cocrystal structure of a prototypical weak thrombin inhibitor based on an

aminopyridone core.



16.9

Avermectin B1a and Bafilomycin A1



The macrolide natural product avermectin B1a (78) is a well-known marketed

anthelmintic agent (Figure 16.22) [121]. Subsequent to our first total synthesis of

avermectin B1a in 1986 [122], we became interested in the synthesis of analogs to

probe the relevance of its functional and structural features. In the course of our

synthetic studies, we prepared the 19-epi-analog 79, in which the original lactone

oxygen atom in avermectin A1a [123] had been inverted via an intramolecular

Mitsunobu macrocyclization reaction to assume a pseudoaxial orientation [124].

The inactivity of this analog compared to avermectin B1a pointed to the importance

of stereochemistry at that site, possibly resulting in the altered conformation of the

macrocyclic lactone.

In another instance, we wished to study the effect of lactone ring enlargement in

avermectin B1a. Controlled ozonolysis of the natural product [125], followed by

functional group manipulation and an intramolecular Julia coupling, led to the ring



591



592



16 Hybrids, Congeners, Mimics, and Constrained Variants

OMe

HO

OMe

Me



O



O

Me



O



Me



Me

O



O



Me



O

Me



O

OH

O



H



Me



O



Me

OH



Avermectin B1a, 78



Me



Me

Me



Me

RO



O

O



Me



O



Me



RO



O

O



Me



Me

O



Me



O



OH

O



H



OH

Me



OMe



19-epi-Avermectin A1a, 79



Me



O



H



OMe

HO



O



OMe

R=

Me



OH



Me



O



O

Me



O



bis-Homo-avermectin B1a, 80



Figure 16.22 Structures of avermectin B1a and semisynthetic variants.



expanded analog, bis-homo avermectin B1a, 80, now harboring an extended triene

unit [126] (Figure 16.22). Unfortunately, this ring-expanded macrolactone did not

show activity in the brine shrimp assay relative to avermectin B1a.



16.10

Bafilomycin A1



Bafilomycin A1 (81) is among a group of 16-membered macrolides belonging to the

hygrolides [127] (Figure 16.23). It has shown promising activity against Grampositive bacteria and as a selective inhibitor of enzymes involved in membrane

ATPases [128]. Following our total synthesis of bafilomycin A1 [129], we studied the

chemical reactivity of various hydroxyl groups toward substitution reactions.

Treatment of the partially protected methyl glycoside of bafilomycin A1 with an

organocopper reagent followed by deprotection led to a ring-expanded 18-membred

macrolactone 82, whose structure was ascertained by single-crystal X-ray crystal-



16.10 Bafilomycin A1

OMe Me



Me



O

HO

Me

Me



OH

O



OH



Me



OH

O



Me



Me



HO

Me



Me



OMe



OMe Me



O



Me



Me



O



Me



Me

OH



O



OH



593



OH

Me



Me

Me



OMe



Me

iso-Bafilomycin A1, 82



Bafilomycin A1, 81



Figure 16.23 Structure of bafilomycin A1 and a semisynthetic ring-expanded analog.



lography [130]. This ring expansion resulted from the initial formation of an

alkoxide at C17, which attacked the lactone carbonyl, leading to an orthoacid salt,

then collapsed to the enlarged lactone. The process could be reversed in the

presence of n-Bu4NF, while attempting to deprotect a TMS ether.

In another attempt to invert the C7 hydroxyl group by a Mitsunobu reaction, we

discovered that a new product (83) was formed in the presence of triphenylphosphine and diethylazodicarboxylate alone and in high yield [131] (Figure 16.24).

This product resulted from a Grob-type fragmentation of the sugar-like ring, and

had remarkably maintained its macrocyclic ring topology as evidenced from a

single-crystal X-ray structure, which was virtually superimposable on the structure

of bafilomycin A1 itself, including the presence of an intramolecular H-bond with

the lactone carbonyl. Contrary to the natural product, the fragmentation product 83

was highly stable to strong acids and strong bases at room temperature, possibly

due to the shielding of the lactone carbonyl group by two hydrophobic side chains.



OMe Me



HO

Me

Me



OH

O



OH



OMe Me



Me



O

O



PPh3



Me



Me



DEAD

Me



Me



Me



OMe



Me



Me



O



OH



86%



Me

O



OH



OH

O



Me



O

Me



Me



Bafilomycin A1, 81



Figure 16.24 X-ray structures of bafilomycin A1 and a macrocyclic analog.



Me

83



OMe



Me



594



16 Hybrids, Congeners, Mimics, and Constrained Variants



16.11

3-N,N-Dimethylamino Lincomycin



Elegant crystallographic studies by Schl€

unzen et al. [15] have shown that there are

overlapping regions when erythromycin (84) and clindamycin (85) are bound to the

peptide exit tunnel region near the P- and A-sites of the ribosomal-tRNA complex.

There is a virtual overlap of the methylthio lincosaminide unit of clindamycin (85)

on the desosamine sugar of erythromycin (84) (Figure 16.25). The interactions of

the 2–4 hydroxyl groups in clindamycin (and lincomycin) with specific nucleic acid

residues are matched with the 3-dimethylamino and 4-hydroxyl groups in

desosamine (Figure 16.22). It was hypothesized that replacing the C3 hydroxyl

group in lincomycin by a dimethylamino group as in the proposed analog 86, could

provide a better interaction with the nucleic acid residues [132]. Unfortunately, the

hybrid analog of lincomycin (86) did not inhibit the growth of S. aureus or E. coli at

concentrations of 10 mM. The corresponding 3-azido and 3-amino analogs were



NMe2

HO



O



OH

Me

O



HO



Me



O



HO

O



O

O



N

Me



O



Cl

H

N

HO O

O

OH



OH

OMe



SMe

OH



Clindamycin, 85



Erythromycin, 84

SMe

O



HO



A-2058



OH OH

G-2505

PO4-



-2505



O

HO



PO4- -2053

A-2058

A-2059



Methylthio lincosaminide, A



G-2505

A-2059



A-2058

A-2059



G-2505



PO4-



C



OH

-2505



PO4- -2053

A-2059



Me

NMe2

PO4- -2505



Desosamine, B



SMe

O

HO

Me2N



O



N

Me



Me

HO

H

N

HO O

O

NR2

SMe

OH



Proposed analog (R = Me), 86

Figure 16.25 Structures of erythromycinA, clindamycin, and a semisynthetic 3-N,N-dimethylamino

lincomycin analog.



16.12 Oxazolidinone Ketolide Mimetics



595



also inactive. However, lincomycin was only moderately active against the two

microorganisms at concentrations of 10 and 2.5 mg/ml, respectively.



16.12

Oxazolidinone Ketolide Mimetics



Chemically modified erythromycins such as the ketolides telithromycin (87) [20] and

cethromycin (88) [133] contain an oxazolidinone ring spanning C10/C11. It has been

known for many years that erythromycin adopts a rigid crown-shaped structure in

solution and in the solid state based on classical X-ray [134] and NMR studies [135].

We therefore hypothesized that bicyclic cis- and trans-oxazolidinone lactones,

represented by structures 89 and 90 and bearing aromatic appendages fixed on the

nitrogen atom, as well as various heterocycles could possibly act as miniaturized

mimetics of the “western” segment of the ketolides [136] (Figure 16.26).



N



N

N



O



O



O



N



O



N



O

OMe



O



O



O



NMe2



HO

O



O



H

N



HO

O



O



O



O



O



87 telithromycin



NMe2

O



O



88, cethromycin

O



O O N



O



R



O



O

Me



ketolide

substructure



O



O



O



O



Me

O



O

O



Me



Me



NR



O



N

R



bicyclic

lactone



O



89, cis



O



O

O



N



N



R

90, cis



O



N



O

NHAc

91, linezolid



N



N



R=

N



N



Figure 16.26 Structures of telithromycin, cethromycin, linezolid, and synthetic oxazolidinones.



596



16 Hybrids, Congeners, Mimics, and Constrained Variants



Unfortunately, no inhibition of S. aureus was observed at concentrations of >50 mg/

ml, which may not be unexpected in view of the drastic simplification of structure

compared to the exquisitely functionalized ketolides. Nevertheless, it was the hope

that the deployment of aromatic and heterocyclic appendages as shown for

structures A and B would give a hint of activity, compared with linezolid (91) [137]

in view of the nominal functional similarity. Clearly, simplification and truncation

of complex structures found in natural and semisynthetic macrolides to prototypes

such as A and B were detrimental to the recognition elements at the ribosomal

level, assuming that cell penetration and efflux were not gatekeepers in preventing

these compounds to reach their target.



16.13

Epilogue



For nearly a century, humanity has relied on the pharmaceutical industry to develop

medicines that have contributed to the well-being of hundreds of millions of

people. We have come a long way from the use of quinine and sulfa drugs, which,

in spite of their poorly understood pharmacology at the time, have paved the way to

what we benefit from today as modern medicinal agents. Through periods of

success and turmoil alike, the drug discovery enterprise has become a multibillion

dollar business, with a complex structure, encompassing many subdisciplines of

the physical, biological, and medical sciences.

Unraveling the genetic code, leading to the discovery of a multitude of new

proteins, has necessitated the development of sophisticated assays and techniques

that allow the testing of millions of molecules for their potential activities. Synthetic

chemists have risen to the challenge of “feeding” the high-throughput assay

machines by inventing ingenious technologies for the mass production of new

chemical entities, albeit in milligram amounts. However, experience has shown

that the main issue in drug discovery today is not the ability to test millions of

compounds but to ensure that any “hit-to-lead” compounds can eventually endure

the rigors of pharmacological testing before moving forward. Even in the most

favorable cases, the many hurdles that lie in the path of a seemingly promising

preclinical candidate may eventually lead to disappointment. Recent cases of

discontinuing the use of some marketed drugs due to toxicity and other

contraindications have not been without severe financial consequences. As a result,

many pharmaceutical and biotech companies continue to undergo paradigm and

organizational shifts in an effort to deal with the prohibitive costs of research,

development, and clinical trials. In addition, rigorous safety guidelines required by

the regulatory agencies must be followed before a drug is approved. There are also

socioeconomic challenges in being able to maintain a competitive edge of the share

of the marketplace, at times arousing public criticism. Outsourcing projects to

contract research organizations(CROs) has become the norm for many companies

in an effort to secure services at a lower cost. While this practice may be convenient

for the time being, its long-term prospects for sustaining creativity and innovation



16.13 Epilogue



may be jeopardized. Nevertheless, the noble objective of providing the most

beneficial drugs to alleviate human suffering is still at the heart of the industry. It is

hoped that despite many of the challenges, unmet medical needs such as stroke,

diabetes, cancer, a host of autoimmune diseases, and Alzheimer’s to mention a few,

will continue to be at the forefront of priorities within the pharmaceutical

companies for some time to come [138].

Drug discovery is a biology-based, chemistry-driven endeavor spanning many

years of dedicated effort by large teams of scientists. It is labor intensive and

unpredictable with regard to outcome. The role of basic academic research cannot

be understated in the context of drug discovery. This can be accomplished by

providing deeper insights into the molecular basis of drug action, by developing

practical methods that facilitate the synthesis of novel entities more efficiently, and

by seeking solutions to unanswered fundamental questions at the interface of

biology and chemistry. In this regard, collaborations between academia and the

pharmaceutical industry are of paramount importance. In this chapter, I highlighted a number of projects dealing with natural products chemistry that were the

direct result of such sustained collaborations, and leading to relevant discoveries of

interest to the companies in guiding their own research efforts in a particular area.

Even negative results produced in our academic research during such collaborations may have been of some utility to our collaborators in knowing what paths not

to follow when time and efficiency are measured on a different scale.

Over the years, our collaborative projects have led to over 130 scientific

publications in peer-reviewed journals and a number of patents. In this regard, it

should be acknowledged that highly significant practical knowledge can be gained

from such collaborations. In turn, academic research can offer innovative and

potentially useful technology to our industrial partners. Not surprisingly, intellectual property issues and legal matters may cause delays in publishing the results of

such collaborative projects. However, such problems can be resolved at the outset

by a reasonable compromise without penalizing efforts given by dedicated

coworkers who wish to see their work published. We hope that the potentially

beneficial collaborative partnerships between academia and the pharmaceutical

industry will continue to provide opportunities for the coming generation of

brilliant young scientists to fulfill their ambitions of helping humanity through the

noble cause of drug discovery and development.



Acknowledgments



I wish to thank NSERC and FQRNT for financial support. The generous support

from many companies over the years is gratefully acknowledged. They are cited in

alphabetical order: Abbott, Achaogen, AstraZeneca (Sweden, UK), Biomira,

Boehringer-Ingelheim, Ciba-Agro, Ciba-Central Research, Ciba-Geigy, Ciba-Vision,

Dupont (Agro), Farmitalia (Milan), Genextra (Milan), GSK (Verona), Hassle

(Sweden), Isis Pharmaceuticals, Lederle, Lilly, Medicure, Merck, Merck-Frosst,



597



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