SUMMARY OF SELECTED RESEARCH

John M. Mihelcic, Ph.D.2004 - 2010

CONTENTS

Progress Toward Hsp90 InhibitorsSlides 3-15

Probing Interactions Between Hsp90

and the Estradiol Receptor Slides 16-20

Total Synthesis of Opiate Analgesics Slides 21-27

PROGRESS TOWARD HSP90 INHIBITORS

Conducted with Dr. Brian S. J. BlaggDepartment of Medicinal ChemistryUniversity of Kansas2008 - 2010

Hsp90 (90 kDa heat shock protein) is a molecular chaperone responsible for the conformational maturation of numerous client proteins.1 Recent studies have demonstrated that Hsp90 multi-protein complexes from tumor cells have higher affinity for ligands than Hsp90 in normal cells; making the Hsp90 multi-protein complex a viable target for cancer therapy.2

Geldanamycin (GDA) and radicicol (RDC) are known inhibitors of Hsp90 and manifest their activity by binding and preventing Hsp90-catalyzed hydrolysis of ATP.3 The IC50 values for GDA and RDC are 49 nM and 23 nM, respectively, when determined in MCF-7 breast cancer cell lines.4 RDC is rapidly metabolized and displays no activity in vivo and GDA displays toxicity issues that are unassociated with Hsp90 inhibition.5

Naturally-Occurring Inhibitors of Hsp90

While derivatives of GDA have entered clinical trials for the treatment of several cancers,6,7 the quinone ring is redox- active. In cells GDA has been shown to generate superoxide radicals which can lead to cell death without interfering directly with Hsp90.8 Consequently, researchers have pursued the development of new Hsp90 inhibitors without these detrimental properties.3

Co-crystal structures of the natural products revealed numerous interactions with the Hsp90 architecture.9 Analysis of the crystal structures revealed that, in addition to other key interactions: 1) the resorcinol moiety of RDC binds in the same location as the adenine ring of ADP and 2) the quinone ring of GDA binds toward the exterior of the pocket and participates in hydrogen bond interactions with the amino acids that normally bind to the diphosphate region of ADP. 10

Interactions with Key Residues of Hsp90

Blagg and co-workers reasoned that chimeric molecules, such as Radester and Radamide, which contain the resorcinol ring of RDC and the quinone found in GDA might provide a molecule with high affinity for Hsp90.10,11

Chimeric Inhibitors of Hsp90

Radamide

OHHO

Cl

O

NH

O

OMe

O

O

O

OHHO

Cl

HO

NH

OH

OMe

O

O

O

OH

HN

OH

OMe

CO2Me

OH

OH

Cl

OO

HN

O

OMe

CO2Me

OH

OH

Cl

O

Blagg and co-workers synthesized several chimera that displayed activities in the low mM range in anti-proliferation assays against breast cancer cells.10,11 An unexpected and highly promising result was the enhanced activity that resulted upon replacement of the redox-active quinone ring with a dihydroquinone (1 vs. 2 and 3 vs. 4). The dihydroquinone of radester displayed the highest activity among the four.

Activities of Initial Chimera and Derivatives

3: Radester1: Radamide 2

# IC50 (mM) MCF-71 18.6 +/- 0.912

2 14.0 +/- 1.412

3 13.9 +/- 1.411

4 7.1 +/- 0.311

4

NH

O

HO OH

ClO

RO

OR

X

O

R=H, Me

NH

O

HO OH

ClO

OR

X

O

NH

O

HO OH

ClO

RO

NH2

X

O

NH

O

HO OH

ClO

NH2

X

O

X=CN, Cl

R=H, Me

X=CN, Cl

Proposed Modifications

The dihydroquinone can be metabolized in vivo to the redox-active quinone which displays unwanted toxicity. We sought to remove the dihydroquinone motif through the removal of one oxygen atom or replacement with a nitrogen atom. The phenol was available by default and the aniline was chosen for its excellent hydrogen-bonding characteristics.

We sought to enhance the ability to bind with the Hsp90 architecture by introducing new substituents onto the dihydroquinone ring. Co-crystal structures indicated that replacing the 17-methoxy of GDA with small groups might be well tolerated.13 Chloride and nitrile groups were chosen for their size and hydrogen-bonding characteristics.

The necessity of both hydroxyl or alkoxyl groups of the dihydroquinone would also be investigated.

MeO

HO

NO2H2

Pd/C

MeOH

rt 12 h

MeI

K2CO3

DMF

rt 12 h

tBuC(O)Cl

DMAP pyr.

DCM

0°C 2 hMeO

MeO

NHPv

78% over 3 steps

a) nBuLi, THF

0°C 2 h

b)

THF 0°C to rt

O

85%

MeO

MeO

NHPv

OH

BCl3DCM

-40°C to rtMeO

HO

NHPv

OH80%

conc. HCl

1,4-dioxane

80°C 16 h

30%

a)NaNO2

HCl AcOH

H2O 0°C

b) CuCN

PhH 0°C

HO OH

Cl CO2H

DCC DMAP

THF DMF

50°C 15 hHO OH

ClO

MeO

HO

CN

O

6% over 2 steps

5

6

5

Synthesis of the New Analogs Compounds lacking the formamide were pursued first. A substantial amount of

experimentation was performed on an unsuccessful synthetic approach before establishing the robust route to pivalanalide 5 described below.

Mono-demethylation of 5 was achieved via the directing ability of the amide. Acidic hydrolysis of the amide was complicated by formation of a 7-membered ring by the

proximal alcohol and a low yield was obtained. Sandmeyer reaction to install the nitrile was low-yielding and the coupling reaction to

produced compound 6 suffered from the presence of the free phenol.

MeO

NHPv

OH

TFAA

NH4NO3

MeCN -10°C

35%

a) NaNO2 HCl

AcOH H2O 0°C

b) CuCN PhH 0°C

9%

H2 Pd/C

MeOH NH3

3 h

90%MeO

HO

NHPv

OH

Tf2NPh

Hunig's base

DMF

75%

HCl

1,4-dioxane

80°C 20 h

80%

33%

HO OH

Cl CO2H

DCC DMAP

THF DMF

50°C 15 h 35%

H2, Pd/C

MeOH 2 h

HO OH

ClO

MeO

NH2

CN

O

MeO

NH2

OH

NO2

MeO

NH2

OH

NO2

7

IC50 (mM) MCF-7

6 28.0 +/- 3.57 45.2 +/- 6.7

The free phenol was replaced with a nitro group in three steps. Yield for the hydrolysis was greatly enhanced in the case of the deactivated aromatic ring.

The sequence just described was used to provide a nitroester that was hydrogenated to give compound 7.

Activity of the new compounds was lower than compound 4 by 4-6 times. The observation that

aniline 7 was less active the phenol 6 suggested that the NH2 was not making significant

interactions with the Hsp90 architecture.

Synthesis and Activity of the New Analogs

HO OH

Cl CO2H

DCC DMAP

THF DMF

50°C 15 h

HO OH

ClO

MeO

OH

CN

O

Isolation of product from either sequence was unsuccessful.

O2N

MeO

HO

CN

OH

O2N

MeO

HO

NH2

OH

O2NHO OH

Cl CO2H

DCC DMAP

THF DMF

50°C

12% HO OH

ClO

MeO

OH

CN

O

O2Na) NaNO2 HCl

AcOH H2O 0°C

b) CuCN PhH 0°C

9%

ca. 25%

Synthesis of analogs containing the formamide commenced with the monomethylnitrile. The coupling reaction gave a mixture which proved impossible to separate. Reversing the sequence did not facilitate the purification.

Synthesis of the New Analogs

MeO

MeO

NH2

OH

O2N

HO OH

Cl CO2H

DCC DMAP

THF DMF

50°C

HO OH

ClO

MeO

MeO

NH2

O

O2N

65%

1.) tBuONO,CuCl22.) H2,Pd/C,MeOH

3.) PhOCHO

40% over 3 steps

HO OH

ClO

MeO

MeO

Cl

O

OHCHN

8

Coupling of the dimethylhydroquinone proceeded in good yield to provide nitroaniline 8 that was used to make the remaining analogs. Formation of the dimethylchloride is shown.

Mono-demethylation was highly problematic because, as anticipated, the amide directing group was no longer present. After significant experimentation only the chloride was selectively demethylated.

Isolation and subsequent testing of the dihydroquinones was accomplished.

Synthesis of the New Analogs

HO OH

ClO

MeO

Cl

O

OHCHN

HO OH

ClO

HO

Cl

O

OHCHN

11 12HO OH

ClO

MeO

CN

O

OHCHN

HO OH

ClO

HO

CN

O

OHCHN

9 10

MCF-7 SKBR3 >100 >100 >100 >100 >100 >100 >100 >100

9101112

IC50 (mM) No activity was observed for compounds 9-12; indicating that the presence only one oxygen atom of the hydroquinone ring is not sufficient to maintain effective binding with Hsp90.

Results of Anti-Proliferation Assays

HO OH

ClO

HO

MeO

Cl

O

OHCHN

HO OH

ClO

MeO

OH

CN

O

HO OH

ClO

MeO

NH2

CN

O

HO OH

ClO

HO

HO

CN

O

OHCHN

HO OH

ClO

HO

HO

Cl

O

OHCHN

6 7 13 14 15

MCF-7 SKBR322.3+/- 2.4 58.3+/- 7.231.8+/- 4.5 65.7+/- 6.4 47.4+/- 6.2 >1008.4 +/- 0.7 22.4 +/- 3.216.4+/- 3.4 29.2 +/- 6.3

67

131415

IC50 (mM)

Results and Conclusions

Further testing indicated that aniline 7 was a less effective inhibitor than phenol 6.

Chloride 14 was the most active among the new compounds tested; suggesting that a chlorine atom interacts more effectively with the Hsp90 architecture than a nitrile group (i.e., 13 vs. 14).

The diminished activity of 15 would indicate that the methyl group might be disrupting a key hydrogen bonding interaction.

Improved binding interactions that would have allowed removal of the dihydroquinone motif were not established.

Further work is needed to develop non-hydroquinone-based inhibitors.

This work was supported by funding from NIH/NCI grant CA109265.

1. Zhang, H.; Burrows, F. J. Mol. Med. 2004, 82, 488.2. Kamal, A.; Thao, L.; Sensintaffar, J.; Zhang, L.; Boehm, M. F.; Fritz, L. C.; Burrows, F. J. Nature 2003, 425, 4073. Chiosis, G.; Vilenchik, M.; Kim, J.; Solit, D. Drug Discovery Today 2004, 9, 881.4. Yamamoto, K.; Garbaccio, R. M.; Stachel, S. J.; Solit, D. B.; Chiosis, G.; Rosen, N.; Danishefsky, S. J. Angew. Chem.,

Int. Ed. 2003, 42,1280.5. Geng, X.; Yang, Z.-Q.; Danishefsky, S. J. Synlett 2004, 8, 1325.6. Sausville, E. A.; Tomaszewski, J. E.; Ivy, P. Curr. Cancer Drug Targets 2003, 3, 377.7. (a) Adams, J.; Elliott, P. J. Oncogene 2000, 19, 6687. (b) Neckers, L. Trends Mol. Med. 2002, 8, S55.8. Dikalov, S.; Landmesser, U.; Harrison, D. G. J. Biol. Chem. 2002, 277, 25480.9. Roe, M.S.; Prodromou, C.; O’Brien, R.; Ladbury, J.; Piper, P.; Pearl, L. J. Med Chem. 1999, 26010.Clevenger, R.S.; and Blagg, B.S.J. Org.Lett. 2004, 24, 445911.Shen, G.; Blagg, B.S.J. Org.Lett. 2005, 25, 215712.Hadden, K.; Blagg, B.S.J. J.Org.Chem. 2009, 74, 469713.Immormino, R.; Metzger, L.; Reardon, P.; Dollins, D.E.; Blagg, B.S.J; Gewirth, D. J. Mol. Biol. 2009, 388, 1033

References and Acknowledgement

PROBING PROXIMAL INTERACTIONS BETWEEN HSP90 AND THE ESTRADIOL RECEPTORConducted with Dr. Brian S. J. BlaggDepartment of Medicinal ChemistryUniversity of Kansas2008 - 2010

O

NH

HO

O

OMeOMe

O

OH2N

O

OOH

HO

Linker

The aim of this novel application is to determine the proximity of the estradiol receptor to the Hsp90 chaperone complex while the two are bound in an activated state.

A molecule of GDA would be tethered to one component of a “click chemistry” partner and an estradiol derivative would contain the complementary “click” appendage.

Probes with various length tethers would first be allowed to bind to their respective targets in vivo. The hypothesis is that tethers of appropriate length would meet and enter into a click reaction to form a covalently bound macromolecule.

Isolation of the macromolecule and determination of the tether length would provide an estimate of the distance between Hsp 90 and the estradiol receptor.

Background

N3

CO2R

F

F

Bertozzi Cycloalkyne CO2R

F

F+

NN

N

The Bertozzi cycloalkyne could be used as one of the probes in conjugation with an azide tether.

“Click reaction” would provide a fused bicyclic triazine macromolecule.

Background

Bertozzi, et. al., JACS, 2008, 11486–11493

TMSClNa

toluenereflux

CO2Et

CO2Et

8 g scale 85-90%

OTMS

OTMS

O

O

Et2ZnCH2I2

toluene0°C to rt, 12 h

95%

SelectfluorCs2CO3MeCN

0°C to rt, 1.5 h40%

HIO4EtOH

0°C to rt, 3 h42%

O

O

DBU THF0°C to rt, 12 h

60%

CO2Me

Br,PPh3Pd/C, H2MeOH

0°C to rt, 3 h95%

O

CO2Me

F

F

Tf2NPhKHMDS

THF -78°C to rt, 15 h

58%

OTf

CO2Me

F

F

CO2H

F

FLDA THF-20°C to rt

35%

O

CO2Me

F

F LiOHH2O dioxane

55°C95%

The published synthesis was reproduced with minor modifications to provide gram quantities of the cycloalkyne.

Synthesis of the Bertozzi Cycloalkyne

Proof of Concept for the GDA-EA Tether

O

NH

HO

O

OMeOMe

O

OH2N

O

HN

HN

O

F

FO

NH

HO

O

OMeOMe

O

OH2N

O

OMe H2N NH21.

DCM 2.

C(O)Cl

F

F

Geldanamycin

AH 7-30

N3O

OH

HO

+

O

NH

HO

O

OMeOMe

O

OH2N

O

HN

HN

O

FF

N

NNO

OH

HO

IC50=15.2 +/- 3.0 mM MCF-7

Initial proof of concept was achieved by first coupling GDA with 1,5-diaminopentane. The Bertozzi alkyne was then converted to an acid chloride and used to form an amide bond with the GDA amino tether.

Click reaction of the tethered alkyne was conducted in the lab of Dr. Robert Hanson at Northeastern University in Boston.

The GDA-estradiol AH 7-30 displayed moderate activity in the anti-proliferation assay against MCF-7 breast cancer cells.

The ability of the macromolecule to permeate the cell walls and exhibit a cytotoxic effect has been demonstrated. The next phase will be to determine if AH 7-30 is binding to the HSP90 chaperone complex or if cell death is occurring by another pathway.

For a related approach see: Danishefsky, et. al., BMCL, 1999, 1233

TOWARD A MORE EFFICIENT SYNTHESIS OF OPIATE ANALGESICSConducted at Mallinckrodt – A Division of Covidien, formerly a division of Tyco Healthcare2003 - 2004

Background

MeO

ONCH3

Thebaine

MeO

O

O

NCH3

Hydrocodone

MeO

O

O

NCH3

Oxycodone

OH

MeO

O

HO

NCH3

CodeineMeO

TotalSynthesis

2 steps4 steps2 steps

H H

Mallinckrodt refines the morphine, codeine and thebaine that are present in poppy resin. The retrosynthetic analysis illustrates how thebaine is converted to the popular analgesic

oxycodone. Codeine is a commercial product and can also be converted to hydrocodone which is a precursor to thebaine.

Supply of oxycodone is limited by the naturally occurring raw materials. . Mallinckrodt licensed the synthesis developed by Kenner Rice and conducted extensive

process research to establish a scalable route to hydrocodone.

NR

O

MeO Br

HO Br

OMe

HO

O

NR

"H+"

Br

OMe

HO

O

NROH

OMe

O

O

NROH

4 steps

12 steps

H

Is an advanced intermediate available via oxidative cyclization?

Oxycodone16 17

18

Intramolecular Cyclization Strategies

Intermediate 16 was prepared in 10 steps and converted into tetracyclic intermediate 17 with anhydrous acidic catalysis. Oxycodone is then produced from 17 in 12 more steps. We envisioned that oxidative cyclization of 16 could produce intermediate 18 which could be quickly transformed into oxycodone.

Preparation of the Cyclization Substrate

MeO

MeO NH2

CO2H

NCHO

O

MeO Br

HO

N

MeO

MeO

HO

MeO

CO2HHO

NH

MeO

MeO

HO

1.Br2, AcOH, 95%2.NaOH, CuSO4 H2O, reflux then HCl, 90%

Xylene, DST reflux, 85%

1. Ru cat. NEt3-HCO2H ACN 95%, 90%ee2.Birch Reduction 90%

1. iPrCHO2. MeSO3H, CHCl3 ethylene glycol then NBA, -20°C 3. 88% HCO2H, DMF 70%

1. POCl3, ACN, reflux then NaOH/H2O reflux2. NH4OH, H2O/MeOH, 85%

16

This chemistry was performed on scales between 200 g and 1 kg.

NCHO

O

MeO Br

HOBr

OMe

HO

O

NCHO

"H+" NH4F-HF in TfOH: 60%TfOH: 30%

TfOH/TfO2: 70-85%

16 17

Anhydrous Acidic Catalysis

Trace amounts of water adversely affected the yield of the cyclization reaction conducted with triflic acid, TfOH.

Refluxing triflic acid with triflic anhydride prior to adding the substrate provided extremely anhydrous conditions that led to highly improved yield.

Proposed Oxidative Cyclization

NCHO

O

MeO Br

HO

BrMeO

HO

O

NCHONCHO

O

MeO Br

HO

-e- -H+

-e-

H

BrMeO

HO

O

NCHO

H

The proposed oxidative cyclization would be conducted in an electrolysis cell. Loss of one electron to the anode would produce a radical cation that could undergo

attack by the aromatic ring. Re-aromatization via deprotonation followed by loss of a second electron would produce

the tertiary cation shown. The cation could by trapped by methanol to give methyl-protected oxycodone.

NCHO

O

MeO Br

RO

NCHO

MeO

MeO Br

RO

R= H or Ac

NR

MeO

MeO Br

HO

Li/TMSClTHF

then aq. HCl

NR

O

OMe

Br

OH

TMS

Anodic Oxidation

Anodic Oxidation

Anodic Oxidation

R= H or Ac

Product not observed

Product not observed

Product not observed

Attempts at Oxidative Cyclization

The majority of effort was devoted to making 0.25 kg of intermediate 16.

A new electrolysis system was purchased, validated and calibrated.

The attempted cyclization reactions shown are representative of the numerous unsuccessful trials that were attempted.

Further study is needed.