Novel Hypoxia-Inducible Factor 1α (HIF-1α) Inhibitors for Angiogenesis-Related Ocular Diseases: Discovery of a Novel Scaffold via Ring-Truncation Strategy

Article abstract of DOI:10.1021/acs.jmedchem.8b00971

Ocular diseases featuring pathologic neovascularization are the leading cause of blindness, and anti-VEGF agents have been conventionally used to treat these diseases. Recently, regulating factors upstream of VEGF, such as HIF-1α, have emerged as a desirable therapeutic approach because the use of anti-VEGF agents is currently being reconsidered due to the VEGF action as a trophic factor. Here, we report a novel scaffold discovered through the complete structure-activity relationship of ring-truncated deguelin analogs in HIF-1α inhibition. Interestingly, analog 6i possessing a 2-fluorobenzene moiety instead of a dimethoxybenzene moiety exhibited excellent HIF-1α inhibitory activity, with an IC₅₀ value of 100 nM. In particular, the further ring-truncated analog 34f, which showed enhanced HIF-1α inhibitory activity compared to analog 2 previously reported by us, inhibited in vitro angiogenesis and effectively suppressed hypoxia-mediated retinal neovascularization. Importantly, the heteroatom-substituted benzene ring as a key structural feature of analog 34f was identified as a novel scaffold for HIF-1α inhibitors that can be used in lieu of a chromene ring.

Full text of DOI:10.1021/acs.jmedchem.8b00971

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Article
Novel Hypoxia-Inducible Factor 1# (HIF-1#) Inhibitors
for Angiogenesis-Related Ocular Diseases: Discovery
of a Novel Scaffold via Ring-Truncation Strategy
Hongchan An, Seungbeom Lee, Jung Min Lee, Dong Hyun Jo, Joohwan Kim, Yoo-Seong Jeong,
Mi Jeong Heo, Chang Sik Cho, Hoon Choi, Ji Hae Seo, Seyeon Hwang, Jihye Lim, Taewoo
Kim, Hyoung Oh Jun, Jaehoon Sim, Changjin Lim, Joonseong Hur, Jungmin Ahn, Hyun Su Kim,
Seung-Yong Seo, Younghwa Na, Seok-Ho Kim, Jeewoo Lee, Jeeyeon Lee, Suk-Jae Chung,
Young-Myeong Kim, Kyuwon Kim, Sang Geon Kim, Jeong Hun Kim, and Young-Ger Suh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00971 • Publication Date (Web): 25 Sep 2018
Downloaded from http://pubs.acs.org on September 26, 2018
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of their duties.

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Novel HypoxiaꢀInducible Factor 1α (HIFꢀ1α)
Inhibitors for AngiogenesisꢀRelated Ocular
Diseases: Discovery of a Novel Scaffold via Ringꢀ
Truncation Strategy
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†
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Hongchan An, Seungbeom Lee, Jung Min Lee, Dong Hyun Jo, Joohwan Kim, YooꢀSeong
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†
‡
†
†
†
Jeong, Mi Jeong Heo, Chang Sik Cho, Hoon Choi, Ji Hae Seo, Seyeon Hwang, Jihye
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‡
†,∥
†,∥
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Lim, Taewoo Kim, Hyoung Oh Jun, Jaehoon Sim, Changjin Lim, Joonseong Hur,
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†,∥
⊥
∥
∥
Jungmin Ahn, Hyun Su Kim,
SeungꢀYong Seo, Younghwa Na, SeokꢀHo Kim,
†
†
†
§
†
Jeewoo Lee, Jeeyeon Lee, SukꢀJae Chung, YoungꢀMyeong Kim, KyuꢀWon Kim, Sang
†
*,‡, #
*,†,∥
Geon Kim, Jeong Hun Kim,
and YoungꢀGer Suh
†
College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
‡
Fight against AngiogenesisꢀRelated Blindness (FARB) Laboratory, Clinical Research
Institute, Seoul National University Hospital, Seoul 03080, Republic of Korea
§
Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon
National University, Gangwonꢀdo 24341, Republic of Korea
∥
College of Pharmacy, CHA University, Gyeonggiꢀdo 11160, Republic of Korea
⊥
College of Pharmacy, Gachon University, Incheon 21936, Republic of Korea
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Department of Ophthalmology, College of Medicine, Seoul National University, Seoul
3080, Republic of Korea
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ABSTRACT
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Ocular diseases featuring pathologic neovascularization are the leading cause of blindness,
and antiꢀVEGF agents have been conventionally used to treat these diseases. Recently,
regulating factors upstream of VEGF, such as HIFꢀ1α, has emerged as a desirable therapeutic
approach because the use of antiꢀVEGF agents is currently being reconsidered due to the
VEGF action as a trophic factor. Here, we report a novel scaffold discovered through the
complete structureꢀactivity relationship of ringꢀtruncated deguelin analogs in HIFꢀ1α
inhibition. Interestingly, analog 6i possessing a 2ꢀfluorobenzene moiety instead of a
dimethoxybenzene moiety exhibited excellent HIFꢀ1α inhibitory activity, with an IC50 value
of 100 nM. In particular, the further ringꢀtruncated analog 34f, which showed enhanced HIFꢀ
1
α inhibitory activity compared to analog 2 previously reported by us, inhibited in vitro
angiogenesis and effectively suppressed hypoxiaꢀmediated retinal neovascularization.
Importantly, the heteroatomꢀsubstituted benzene ring as a key structural feature of analog 34f,
was identified as a novel scaffold for HIFꢀ1α inhibitors that can be used in lieu of a chromene
ring.
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INTRODUCTION
Ocular diseases featuring pathologic neovascularization are the leading cause of vision loss
worldwide. These ocular diseases include diabetic retinopathy (DR), wet ageꢀrelated macular
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degeneration (wet AMD), and retinopathy of prematurity (ROP). Vascular endothelial
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growth factor (VEGF) is known to play key roles in the pathogenesis of these diseases; thus,
the administration of antiꢀVEGF biologics, including bevacizumab and ranibizumab, has
been one of the standard therapies for angiogenesisꢀrelated ocular diseases. Despite the
convincing therapeutic effects of antiꢀVEGF agents, this treatment is being reevaluated in
view of the function of VEGF in maintaining neurons as well as its action as a trophic
3, 4
factor. Recently, VEGFA knockout in adult mice was reported to lead to vision loss and
downregulation of multiple angiogenic genes, highlighting the necessity of a longꢀterm safety
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study of VEGF antagonism in the eye. Accordingly, regulating factors upstream of VEGF
has received attention as a desirable therapeutic approach to eliminate offꢀtarget effects.
Hypoxiaꢀinducible factor 1 (HIFꢀ1) is a transcription factor consisting of an oxygenꢀ
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regulated αꢀsubunit and a constitutively expressed βꢀsubunit. Under normal oxygen
concentrations, HIFꢀ1α is hydroxylated by prolyl hydroxylases (PHDs), using molecular
oxygen and 2ꢀoxoglutarate as coꢀsubstrates. Subsequently, hydroxylated HIFꢀ1α is
recognized by the tumor suppressing protein von HippelꢀLindau (VHL) and then
polyubiquitinated for degradation by proteasomes. Under hypoxic conditions, however, the
function of PHD is limited, and the stabilized HIFꢀ1α dimerizes with HIFꢀ1β for translocation
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into the nucleus to activate the transcription of target genes, including VEGF. The molecular
biology of HIFꢀ1 has been well studied in solid tumors due to their hypoxic conditions, which
are caused by high proliferation. Additionally, the roles of the HIFꢀVEGF axis in the eye
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have been studied over the last ten years, and it has become clear that HIF plays an important
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ꢀ10
role in the pathogenesis of many ocular diseases.
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Figure 1. Structures of (−)ꢀdeguelin (1), SHꢀ1242 (2), and SHꢀ1280 (3). The turquoise color
indicates a part eliminated from the parent compound.
We recently reported that (−)ꢀdeguelin (1) interferes with ATP binding to heat shock protein
90 (HSP90), a molecular chaperone associated with the translocation and stabilization of
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HIFꢀ1α. We also identified a series of ringꢀtruncated deguelin analogs using a synthetic
1
2
strategy for the synthesis of (−)ꢀdeguelin. The two representative analogs, SHꢀ1242 (2) and
SHꢀ1280 (3) exhibited potent cell growth inhibition and inhibitory activities of HIFꢀ1α, a
client protein of HSP90, and concentrationꢀdependent antiangiogenic activities in zebrafish
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embryos. In particular, analogs 2 and 3 showed the ability to suppress hypoxiaꢀmediated
retinal neovascularization and vascular leakage in the diabetic retina by destabilizing HIFꢀ1α,
implying that these compounds can be used as alternatives to direct VEGF inhibition for the
1
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treatment of angiogenesisꢀrelated ocular diseases. Additional studies on the mode of action
of 2 also revealed its therapeutic potential as a potent antitumor agent with minimal
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toxicity.
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Figure 2. Strategy for structureꢀactivity relationship studies of 2 and 3.
Despite the therapeutic potential of ringꢀtruncated deguelin analogs, structureꢀactivity
13, 16, 17
relationship (SAR) studies of 2 and 3 have been limited in terms of structural diversity.
In addition, we have been interested in structural features of analogs related to their
physicochemical properties, especially aqueous solubility and their biological activities, since
intravitreal injection is the most commonly used administrative method for the ocular
diseases. In this article, we fully describe the design, synthesis, and biological evaluation of
ringꢀtruncated deguelin analogs to establish a complete SAR and identify novel and advanced
HIFꢀ1α inhibitors having therapeutic potential for angiogenesisꢀrelated ocular diseases. We
first investigated the role of the three pharmacophoric parts of 2 and 3 shown in Figure 2. For
part A, the substituent effects of the dimethoxy benzene ring were examined. Part B was
investigated through replacement with various functional moiety that may play an important
role for HIFꢀ1α inhibition. Particularly, the role of substituents on the benzylic position was
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intensively studied and built on our earlier work. The C partꢀmodified analogs were
designed on the basis of a chromeneꢀtruncation strategy to discover a novel scaffold for
potent HIFꢀ1α inhibitors. Truncation of the chromene unit could be more interesting as
hydrophobic chromene ring was replaced by diverse hydrophilic moieties to increase water
solubilities of the analogs. The synthesized analogs were evaluated for HIFꢀ1α inhibitory
activity by performing luciferaseꢀreporter assays with HREꢀA549 cells. The selected analogs
based on the primary in vitro assay were further evaluated by in vitro angiogenesis assays,
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including proliferation, migration, and tube formation assays, and solubility for intravitreal
injection. Finally, the suppressing effect of hypoxiaꢀmediated retinal neovascularization was
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assessed using a mouse oxygenꢀinduced retinopathy (OIR) model.
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RESULTS AND DISCUSSION
Chemistry Two synthetic methods for coupling the A and C parts, which concomitantly
produce the B part, are outlined in Figure 3. We previously reported the addition of aryl
anions to aldehydes to afford secondary alcohols that were transformed into the
12, 13
corresponding ketones by oxidation (method A).
However, the chromeneꢀcontaining aryl
bromide 5 was prepared via a relatively long sixꢀstep sequence from resorcinol. To overcome
this drawback of method A, we adapted reverse anionic coupling between the phenyl sulfonyl
16, 19
intermediates 7 and the chromeneꢀcontaining aldehyde 8 (method B).
The chromene unit
was efficiently prepared from 2,4ꢀdihydroxybenzaldehyde in two steps.
Figure 3. Synthetic methods for coupling parts A and C.
Syntheses of the analogs modified at part A by method A are summarized in Scheme 1.
Phenethyl alcohol 10 was oxidized to afford the corresponding aldehyde 4, which was
13
directly subjected to addition of an aryl anion generated by treatment of aryl bromide 5 with
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nꢀbutyllithium to produce secondary alcohol 11. DessꢀMartin periodinane oxidation of 11
afforded the ketone analog 6.
a
Scheme 1. Synthesis of the part A-modified analogs.
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a
Reagents and conditions: (a) DMP, NaHCO
3
, CH
2
2
Cl , 0 °C to rt; (b) 5, nꢀBuLi, THF, −78 °C
to rt, 17−60% (over 2 steps); (c) DMP, NaHCO
3
, CH
2
Cl , 0 °C to rt, 20−83%.
2
The chromeneꢀcontaining moiety 8 was prepared from 2,4ꢀdihydroxybenzaldehyde 12,
which possesses an electronꢀwithdrawing group intrinsically required for regioselective
20ꢀ22
13
electrocyclization.
The previously reported procedure using aryl bromide 5 to
synthesize the chromeneꢀcontaining analogs was inefficient in terms of chemical yields and
the number of steps. Thus, resorcinol 12 was condensed with 3ꢀmethylꢀ2ꢀbutenal to give the
cyclized products, which were Oꢀmethylated to afford aldehyde 8.
a
Scheme 2. Preparation of a chromene unit.
a
Reagents and conditions: (a) 3ꢀmethylꢀ2ꢀbutenal, CaCl
CO , acetone, reflux, 83%.
2 2 3
·2H O, Et N, EtOH, reflux, 65%; (b)
MeI, K
2
3
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The syntheses of the analogs with modifications to part A via a phenyl sulfonyl intermediate
(method B) are shown in Scheme 3. Hydroxyphenyl methanol 14 was treated with allyl
bromide to afford allyloxyphenyl methanol 15. Bromination of 15 with phosphorus
tribromide followed by substitution using sodium pꢀtoluenesulfinate gave phenyl sulfonyl
intermediates 7. The anion of 7, which was generated by treatment with nꢀbutyllithium, was
coupled with aldehyde 8 to yield the corresponding secondary alcohol, which was converted
to ketone intermediate 16 by DessꢀMartin oxidation. Finally, Pdꢀcatalyzed allyl deprotection
of 16, followed by desulfonylation, produced ketone 9.
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a
Scheme 3. Synthesis of the part A-modified analogs.
a
Reagents and conditions: (a) allyl bromide, K CO , acetone, reflux, 89−97%; (b) PBr ,
2
3
3
CH Cl , 0 °C to rt; (c) PhSO Na, DMF, rt to 80 °C, 88−92% over 2 steps; (d) 8, nꢀBuLi,
2
2
2
THF, −78 °C; (e) DMP, NaHCO , CH Cl , 0 °C to rt, 76−86% over 2 steps; (f) Pd(PPh ) ,
3
2
2
3 4
MeNHPh, THF; (g) Zn, NH Cl, THF, reflux, 91−100% over 2 steps.
4
The analogs modified at the benzylic position in part B were synthesized from ketone 2. The
treatment of ketone 2 with trimethylsilyl trifluoromethanesulfonate afforded the
corresponding silyl enol, which was subjected to electrophilic fluorination to afford
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fluorination product 17. Allylated analog 18 and benzylated analog 19 were prepared by
alkylation of ketone 2 with allyl bromide or benzyl bromide, respectively (Scheme 4).
a
Scheme 4. Synthesis of part B-modified analogs having benzylic substituents.
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a
Reagents and conditions: (a) TMSOTf, Et
3
N, CH
2
Cl
2
;
NꢀChloromethylꢀN’ꢀ
fluorotriethylenediammonium bis(tetrafluoroborate), TBAF, DMF, 67% over 2 steps; (b)
allyl bromide, NaH, THF, 0 °C to RT, 61%; (c) benzyl bromide, NaH, THF, 0 °C to RT,
70%.
13
To synthesize the B partꢀmodified analogs as outlined in Scheme 5, known alcohol 20 was
acetylated using acetic anhydride in the presence of DMAP and trimethylamine to yield
acetate 21. Amide 23 was prepared by oxidation of aldehyde 8 using silver nitrate and
coupling the resulting acid 22 with 3,4ꢀdimethoxyaniline in the presence of PyBOP and
diisopropylethylamine. Tertiary amide analogs 24−26 were prepared by alkylation using the
corresponding electrophiles and sodium hydride as a base.
Scheme 5. Synthesis of the part B-modified analogs containing alcohol, ester, and
a
amide moieties.
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0
a
Reagents and conditions: (a) Ac
2 3 2 2 3
O, DMAP, Et N, CH Cl , 63%; (b) AgNO , NaOH, EtOH,
H O, reflux, 35%; (c) 3,4ꢀdimethoxyaniline, PyBOP, iPr NEt, CH Cl , 75%; (d) MeI, NaH,
2
2
2
2
THF, 0 °C to rt, 85%; (e) EtI, NaH, THF, 0 °C to rt, 97%; (f) BnBr, NaH, THF, 0 °C to rt,
87%.
The syntheses of the chromeneꢀtruncated analogs are shown in Scheme 6. MOM protection
of the hydroxyl group of 4ꢀbromoꢀ3ꢀmethoxyphenol (27) followed by treatment with nꢀ
13
butyllithium gave the bromobenzene anion, which was coupled with the known aldehyde 35
to provide corresponding alcohol 29. Oxidation of the alcohol using DessꢀMartin periodinane
produced ketone 30. MOM deprotection with hydrochloric acid afforded phenol intermediate
31, which could be transformed into both oxygenꢀsubstituted chromeneꢀtruncated analogs
32a to 32c and a precursor for nitrogenꢀsubstituted chromeneꢀtruncated analogs 34a to 34d.
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2
2
2
2
2
2
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6
Ether analogs 32a and 32b were synthesized by alkylation of 31 using iodomethane and
iodoethane, respectively. Cyclohexyl ether 32c was prepared via a Mitsunobu reaction with
cyclohexanol. To prepare the nitrogenꢀcontaining chromeneꢀtruncated analogs, phenol 31
was treated with phenyl triflimide to yield triflate 33. Finally, amine analogs 34a to 34f were
synthesized by palladiumꢀcatalyzed BuchwaldꢀHartwig reactions of triflate 33 with the
corresponding amines in the presence of palladium acetate.
0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 6. Synthesis of chromene-truncated analogs modified at part C.
a
Reagents and conditions: (a) MOMCl, NaH, DMF, 0 °C to rt, 96%; (b) 35, nꢀBuLi, THF,
−
78 °C, 72%; (c) DMP, CH
iodide, Cs CO , CH CN, 0 °C to rt. 32a: MeI, 71%. 32b: EtI, 91%; (f) 32c: cyclohexanol,
DEAD, PPh , THF, 42%; (g) PhNTf , K CO , DMF, quant.; (h) amines, Pd(OAc) , BINAP,
Cs CO , toluene, 100 °C. 34a: dimethylamine, 41%. 34b: pyrrolidine, 90%. 34c: piperidine,
2 2
Cl , 0 °C to rt, 82%; (d) 2NꢀHCl, MeOH, 60 °C, 94%; (e) alkyl
2
3
3
3
2
2
3
2
2
3
45%. 34d: morpholine, 53%. 34e: benzylamine, 88%. 34f: 4ꢀcyanobenzylamine, 91%.
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9
Syntheses of the chromeneꢀtruncated analogs without a methoxy group on the original
chromene moiety are described in Scheme 7. Treatment of 1,4ꢀdibromobenzene with nꢀ
butyllithium gave the 4ꢀbromobenzene anion, which was then coupled with the known
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
3
3
4
4
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6
0
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9
0
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0
13
aldehyde 35 to provide the corresponding alcohol. The alcohol was then oxidized with
DessꢀMartin periodinane to afford ketone 36. Palladiumꢀcatalyzed amination of 36 produced
the desired chromeneꢀtruncated analogs 37a and 37b.
Scheme 7. Synthesis of the chromene-truncated analogs without a methoxy group on
a
the original chromene moiety.
a
Reagents and conditions: (a) 1,4ꢀdibromobenzene, nꢀBuLi, THF, −78 °C; (b) DMP, CH Cl ,
2
2
0
°C to rt, 71% over 2 steps. (c) amines, Pd(OAc)
2 2 3
, BINAP, Cs CO , toluene, 100 °C. 37a:
pyrrolidine, 57%. 37b: morpholine, 53%.
We also synthesized the analogs possessing the key structural features of parts A, B, and C
responsible for high HIFꢀ1α inhibition as shown in Scheme 8. Starting from 2ꢀfluoro
benzeneacetaldehyde 38, we prepared intermediates 40 and 43 for Pdꢀcatalyzed reaction
which gave analogs 41aꢀb and 44aꢀb.
Scheme 8. Synthesis of the chromene-truncated analogs combined with the
a
pharmacophoric structural features of part A and B.
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0
a
Reagents and conditions: (a) 1,4ꢀdibromobenzene (for 39) or 28 (for 42), nꢀBuLi, THF, −78
°
C; (b) DMP, CH
rt, 40: 93%. 43: 64%; (d) amines, Pd(OAc)
1b: 71%. 44a: 67%. 44b: 58%; (e) 2NꢀHCl, MeOH, 60 °C, 97%; (f) PhNTf
quant.
2
Cl
2
, 0 °C to rt, 39: 55%. 42: 38% over 2 steps; (c) MeI, NaH, THF, 0 °C to
, BINAP, Cs CO , toluene, 100 °C. 41a: 68%.
, K CO , DMF,
2
2
3
4
2
2
3
Finally, we prepared (S)ꢀ and (R)ꢀenantiomers of racemate 34f as outlined in Scheme 9.
13
Syntheses commenced with active aldehydes (S)ꢀ35 and (R)ꢀ35 and followed the same
procedure for the racemic analog except for the last step to avoid racemization at high
temperature and long reaction time. (Supporting Information Figure 1)
Scheme 9. Synthesis of optically active analogs (S)-34f and (R)-34f
a
2 2
Reagents and conditions: (a) 28, nꢀBuLi, THF, −78 °C; (b) DMP, CH Cl , 0 °C to rt; (c) 2Nꢀ
HCl, MeOH, 60 °C; (d) PhNTf
2
, K CO , DMF, (S)-33: 34%, (R)-33: 42% over 4 steps; (e) 4ꢀ
2
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cyanobenzylamine, Pd(OAc)
4f: 92%.
2 2 3
, BINAP, Cs CO , toluene, 60 °C to 100 °C, (S)-34f: 83%, (R)-
3
1
1
1
1
1
1
1
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Structure-activity relationship (SAR) study based on an HRE-luciferase reporter assay
13ꢀ15
Our previous studies
to elucidate the structural features of deguelin mainly focused on
the Bꢀ and Cꢀring truncation of deguelin although analog 2 not only showed antiangiogenic
15
effects and antitumor effect with minimal toxicity, but also suppressed hypoxiaꢀmediated
1
4
retinal neovascularization. Specifically, investigation of a distinctive moiety, such as the
dimethoxy benzene ring in part A and the terminal chromene moiety in part C, were quite
limited. Thus, we have initially established the complete SAR focusing on analog 2. The
HREꢀluciferase assay in the A549 cell line was used for the primary evaluation of the newly
synthesized analogs. We also examined calculated molecular descriptors such as log P and
log S to predict aqueous solubilities of the analogs.
We first investigated substituent effects in part A of 2 on HIFꢀ1α inhibitory activities. The
two methoxy groups in part A were originally considered essential for HIFꢀ1α inhibition
because we observed a loss of antiproliferative activity upon demethylation of one or both
1
3
methoxy groups of deguelin. Van Meir and coworkers reported a similar observation about
the dimethoxybezene moiety, which played a crucial role in HIFꢀ1α inhibitory activities in
2
4, 25
their sulfonamide analogs.
Thus, we prepared analogs devoid of one or both methoxy
groups or analogs possessing alkoxyꢀreplacing substituents at the 3ꢀ and 4ꢀpositions.
Interestingly, analog 6a, with no substituent on the benzene ring in Aꢀpart, exhibited
considerably increased inhibition, with an IC value of 880 nM, which implied the necessity
5
0
of investigation into the substituent effects on HIFꢀ1α inhibition. Analog 6b, which has a
dioxolane ring instead of two methoxy substituents, showed a lower inhibitory activity than
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analog 2, while analog 6c, which has a dioxane ring, exhibited slightly enhanced activity. The
3,4ꢀdifluoro analog 6d exhibited enhanced activity compared to analog 2, whereas the
dichloro analog 6e showed lower activity. All the monofluoro analogs showed improved
inhibitory activities compared to analog 2 and the most potent 2ꢀfluoro analog 6i exhibited
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20ꢀfold higher activity than analog 2, with an IC50 value of 100 nM. The monomethoxy
analogs 6f and 6h exhibited lower activity than analog 2 and the 3ꢀmethoxy analog 6g
showed the improved activity. Curiously, 2ꢀhydroxy analog 9a and 4ꢀhydroxy analog 9c
showed significantly increased activity compared to the corresponding methoxy analogs,
13
which contradicted the previous SAR study of deguelin using antiproliferative assays.
Table 1). Overall, the function of the methoxy groups of part A in analog 2 for HIFꢀ1α
(
inhibitory activities was not clearly revealed. However, a small substituent or no substituent
at the 2ꢀ, 3ꢀ, or 4ꢀposition seems beneficial for improved inhibitory activity regardless of
electronic effect. The excellent HIFꢀ1α inhibition of 2ꢀfluoro analog 6i is currently not
explained although the small 2ꢀfluoro substituent seems specifically optimized for an
interaction with the corresponding binding site and not associated with electronic interaction
or hydrogen bonding. Calculated hydrophilicity descriptor (log S) and lipophilicity descriptor
(log P) were not dramatically changed with substituent variation except two chlorines at para
and meta positions (6e) and hydroxy substituent at ortho (9a), meta (9b), and para (9c)
2
6
position. Lipophilicity parameter π of substituents seems significant as hydrophilic
substituent could be beneficial for intravitreal drugs to have better aqueous solubility.
Table 1. HIF-1α inhibitory activities of the part A-modified analogs.
a
b
c
c
Compound
R
IC50 (µM)
S.E.
log S
log P
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2
3,4ꢀdimethoxy
2.15
0.88
3.41
1.33
1.27
2.88
3.21
0.82
6.21
0.10
0.36
1.02
0.94
1.44
0.79
0.52
0.32
0.71
0.34
0.40
0.54
0.74
0.20
2.39
0.01
0.15
0.29
0.41
0.88
ꢀ 5.36
ꢀ 5.27
ꢀ 5.15
ꢀ 5.23
ꢀ 5.38
ꢀ 6.49
ꢀ 5.34
ꢀ 5.38
ꢀ 5.38
ꢀ 5.36
ꢀ 5.39
ꢀ 5.39
ꢀ 4.69
ꢀ 4.82
ꢀ 4.85
4.68
4.53
4.30
4.61
4.31
5.43
4.50
4.52
4.53
4.21
4.34
4.47
4.45
4.45
4.47
6a
none
3,4ꢀmethylenedioxy
3,4ꢀethylenedioxy
3,4ꢀdiꢀF
3,4ꢀdiꢀCl
2ꢀOMe
6b
6c
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6d
6e
6f
6g
3ꢀOMe
6h
4ꢀOMe
6i
2ꢀF
6j
3ꢀF
6k
4ꢀF
9a
2ꢀOH
9b
3ꢀOH
9c
4ꢀOH
0.49
b
a
c
Data are the mean values of three independent experiments. Standard error. The log S
and log P values of the analogs were calculated with the software ALOGPS 2.1 (VCCLAB
Virtual Computational Chemistry Laboratory)
27
We next examined the effects of variations to part B on HIFꢀ1α inhibitory activity. We first
examined substitution effects at the benzylic position by incorporating diverse substituents. In
13
accordance with a previous study, bulky substituents generally decreased HIFꢀ1α inhibitory
activities apart from methylꢀsubstituted rac-3, which seemed to have beneficial conformation
induced by a structural constraint. We also examined alcohol analog 20 since deguelol, a
naturally occurring reduced form of deguelin, exhibited 10ꢀfold higher antiproliferative
activity than deguelin. Both alcohol 20 and acetate 21 exhibited lower inhibitory activity
compared to 2. The amide analogs 23ꢀ25 showed increased log S value although their
inhibitory potencies were not satisfactory. The inhibitory activity of the Nꢀalkylated amides
2
4–26 decreased as the size of the alkyl group increased (Table 2). The size effect of the Nꢀ
alkyl group was quite similar to the trend shown in the corresponding ketone series.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. HIF-1α inhibitory activities of analogs with part B modified.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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2
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4
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6
7
8
9
0
a
IC50
b
c
c
Compound
A
R
1
R
2
S.E.
log S
log P
(µM)
2
C
C
C
C
C
C
C
N
N
N
N
H
Me
F
O
O
2.15
1.36
6.35
8.45
12.03
8.88
3.04
2.04
3.15
5.25
10.78
0.52
0.50
6.49
5.58
6.91
2.75
0.96
0.78
2.75
2.53
2.35
ꢀ 5.36
ꢀ 5.55
ꢀ 5.38
ꢀ 5.99
ꢀ 6.61
ꢀ 5.01
ꢀ 5.83
ꢀ 4.89
ꢀ 4.91
ꢀ 4.94
4.68
4.87
3.86
5.34
5.87
4.18
4.79
3.73
3.49
3.72
4.67
rac-3
17
O
18
Allyl
Bn
H
O
1
2
2
2
9
0
1
3
O
OH
OAc
O
H
H
24
Me
Et
O
25
O
26
Bn
O
ꢀ 5.80
b
a
c
Data are the mean values of three independent experiments. Standard error.
The log S and log P values of the analogs were calculated with the software
2
7
ALOGPS 2.1 (VCCLAB Virtual Computational Chemistry Laboratory)
The modification of part C of 2 included truncation of the terminal chromene unit, one of
the key parts of deguelin. The chromene unit was traditionally known as a ‘privileged
28
structure’ with versatile binding properties, and many natural products containing chromene
29
units were reported along with their various biological activities. For this reason, various
attempts to incorporate chromene units into therapeutic agents have been made. Nicolaou et
al. constructed natural productꢀlike chemical libraries via the solidꢀphase synthesis of
3
0ꢀ32
chromene units.
Van Meir et al. recently developed a group of HIFꢀ1α inhibitors
24, 25, 33, 34
possessing chromene units based on a hit compound from Nicolaou’s library.
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Our early work on deguelin, a parent natural product of 2, revealed that the terminal
chromene unit is crucial for cell growth inhibition via its interaction with HSP90. However,
studies on the truncation of the chromene unit were not included. To establish a complete
SAR study of ringꢀtruncated analog 2, a series of analogs with part Cꢀmodification was
evaluated on the basis of a chromene truncation strategy. Specifically, chromene ring
truncation was anticipated to provide improved physicochemical properties, which would be
beneficial for intravitreal injection. We introduced a methyl substituent at the benzylic
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
position for the chromeneꢀtruncated analogs because our early work confirmed that the B,
Cꢀringꢀtruncated analog 3 with a methyl substituent at the benzylic position exhibited better
activity than analog 2 without a methyl substituent. Additionally, the methyl substituent
might limit the flexibility of the chromeneꢀtruncated analogs via conformational rigidity. We
examined the HIFꢀ1α inhibitory activities of the chromeneꢀtruncated analogs in racemic form
because we focused on the effect of the chromeneꢀtruncation at this stage, although our
13
previous work revealed that the βꢀmethyl substituted chromene analog was twice as potent
as the αꢀmethyl chromene analog for cell growth inhibition. However, we investigated
stereochemical effects on the in vitro and in vivo activities via chiral switching for the finally
selected analog. HIFꢀ1α inhibitory activities of the chromeneꢀtruncated analogs are
summarized in Table 3. Generally, the methoxy substituent at the 2ꢀposition corresponding to
the methoxy group of the chromene unit was kept intact. The alkoxy (31, 32a–32c) and
amine (34a–34f) substituents at the 4ꢀposition correspond to the alkoxy moiety of the
chromene ring. HIFꢀ1α inhibitory activities of the alkoxyꢀsubstituted analogs increased as the
size of the alkyl group increased. The same pattern was observed in all alkyl/cyclic amine
analogs (34a–34d), except for the morpholine analog which showed possible advantage with
regard to aqueous solubility. The introduction of benzylamine (34e) at the 4ꢀposition
exhibited enhanced inhibitory activities compared to the alkyl/cyclic amine substituted
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2
2
2
2
2
2
2
2
2
3
3
3
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3
4
4
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4
4
4
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6
analogs although its log S value decreased. Interestingly, analog 34f, which possesses a 4ꢀ
cyanobenzylamine substituent, showed higher activity than analog 2, with an IC value of
50
600 nM. The calculated molecular descriptors predicted better aqueous solubility for analog
0
1
2
3
4
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8
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34f compared to 34e, which is likely due to a negative lipophilicity parameter π of the cyanoꢀ
26
substituent. Those results also suggested that a heteroatomꢀsubstituted benzene ring could
be used as a scaffold for HIFꢀ1α inhibitors instead of a chromene unit. Next, we investigated
the role of the methoxy substituent at the 2ꢀposition by comparing analogs 34b and 37a as
well as analogs 34d and 37b. Unlike the chromene ring case, the 2ꢀmethoxy group of the
chromeneꢀtruncated analogs did not exert any significant role in HIFꢀ1α inhibitory activity.
Table 3. HIF-1α inhibitory activities of analogs modified in part C
a
b
c
c
Compound
R
1
R
2
IC50 (µM)
S.E.
log S
log P
3.23
3.41
3.89
5.29
3.93
4.66
5.15
3.73
5.12
4.38
4.49
3.73
d
31
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OH
OMe
OEt
N.A.
ꢀ
ꢀ
ꢀ 4.11
ꢀ 4.44
ꢀ 4.69
ꢀ 5.87
ꢀ 3.94
ꢀ 4.13
ꢀ 4.50
ꢀ 4.08
ꢀ 5.95
ꢀ 5.21
ꢀ 4.20
ꢀ 3.80
3
2a
2b
2c
4a
4b
4c
34d
4e
34f
7a
7b
N.A.
11.68
2.59
N.A.
2.82
1.85
8.74
1.43
0.60
2.61
6.85
3
9.45
2.45
ꢀ
3
OCy
3
N(CH
N(CH
3
2
2
)
)
)
2
4
2
3
1.66
0.57
8.00
0.76
0.23
1.27
3
N(CH
N(CH
NHBn
NH(4ꢀCN)ꢀBn
N(CH
N(CH CH
2
CH
CH
2
CH
)
O
2
2
2
3
3
2 4
)
3
H
2
2
)
2
O
2.74
a
b
c
Data are the mean values of three independent experiments. Standard error. The log S
and log P values of the analogs were calculated with the software ALOGPS 2.1 (VCCLAB
2
7 d
Virtual Computational Chemistry Laboratory)
No activity (N.A.) indicates that the
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analog exhibits activity with an IC50 higher than 15 µM.
We explored additive effects by combining structural features of each part, which were
considered important for high HIFꢀ1α inhibitory activity. Unfortunately, analogs 41a/44a,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
41b, and 44b, which consist of a 2ꢀfluoro benzene ring in part A, did not exhibited improved
activities compared to the corresponding analogs (34c, 34e, and 34f) possessing a
dimethoxybenzene unit possibly due to a conformational change by chromeneꢀring truncation
(
Table 4).
Table 4. HIF-1α inhibitory activities of analogs modified in parts Α−C
a
b
c
c
Compound
1a
41b
4a
4b
R
1
R
2
IC50 (µM)
S.E.
log S
log P
4.98
5.13
4.86
4.90
4
H
N(CH
NHBn
2
N(CH CH )
2
CH
2
)
2
CH
CH
2
2
2.06
7.35
6.52
5.28
0.72
4.15
2.00
ꢀ 4.76
ꢀ 5.92
ꢀ 4.63
ꢀ 5.35
H
4
OMe
OMe
2
2
4
NH(4ꢀCN)ꢀBn
2.13
a
b
c
Data are the mean values of three independent experiments. Standard error. The log S
and log P values of the analogs were calculated with the software ALOGPS 2.1 (VCCLAB
2
7
Virtual Computational Chemistry Laboratory)
We finally examined stereochemical effects on the HIFꢀ1α inhibitory activity for racemate
34f. As shown in Figure 4, (R)ꢀ34f exhibited more potent HIFꢀ1α inhibitory activity than its
antipode (S)ꢀ34f. It is interesting that absolute configuration of the stereogenic center of (R)ꢀ
13
3
4f is opposite to that of analog 3 , which was previously reported as a B, Cꢀring truncation
analog. This change is likely due to a conformational change induced by the chromeneꢀ
truncation.
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0
1
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8
9
0
1
2
3
4
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8
9
0
Figure 4. Relative HREꢀluciferase activities of analog 34f and its enantiomers at 1 µM.
Aqueous solubility of the representative analogs Through this intensive SAR study of
ringꢀtruncated deguelin analogs, we successfully identified potent HIFꢀ1α inhibitors with IC50
of submicromolar level. With several novel and potent HIFꢀ1α inhibitors in hand, we
examined their physicochemical properties to select a suitable candidate for additional
biological evaluation and determined their therapeutic potential for the treatment of ocular
diseases via intravitreal injection. We compared the water solubility of analog 2 (IC50 = 2.15
µM) with those of analogs 6i (IC = 0.10 µM), 34c (IC = 1.85 µM), and 34f (IC = 0.60
50
50
50
µM), which were selected based on their HIFꢀ1α inhibitory activities and structural novelties
Figure 5). The water solubilities of analogs 34c and 34f were 93ꢀfold and 17ꢀfold higher,
(
respectively, than that of parent compound 2. The improved solubility is likely due to a result
of the increase in polarity induced by the amine moiety and the increase in flexibility induced
by the truncation of the fused ring system (i.e., the chromene ring). Unfortunately, the most
potent analog, 6i, was much less water soluble than parent compound 2. For these reasons,
analogs 34c and 34f were chosen for further biological evaluation associated with
angiogenesisꢀrelated ocular diseases. Both analogs also have a structural advantage over
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analog 2 in terms of structural novelty since analog 2, which was developed from widely used
natural products, consists of a popular chromene moiety.
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Water solubility of the representative analogs.
In vitro validation of analogs 34c and 34f We initially examined the inhibitory effects of
analog 34c and 34f on hypoxiaꢀmediated angiogenic processes because angiogenesis is an
important process involving extensive interaction between cells, soluble factors and
3
5
extracellular matrix components. Construction of a vascular network involves consecutive
36
steps, including cell proliferation, migration, and tube formation. Thus, we used conditioned
media (CM) extracted from human colon cancer HCT116 cells or HeLa cells treated with or
without analogs 2, 34c, and 34f under hypoxia. The antiangiogenic effects of the analogs
were evaluated by observing changes in angiogenic processes following the treatment of
human umbilical vein endothelial cells (HUVECs) with CM. Analog 34c effectively
suppressed the hypoxiaꢀinduced proliferation, migration, and tube formation of HUVECs and
14
was nearly equipotent to analog 2 (Supporting Information Figure 2). More interestingly,
analog 34f exhibited better antiꢀangiogenic effects than analog 2. Analog 34f suppressed the
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2
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6
angiogenic processes of human retinal microvascular endothelial cells (HRMECs) by
treatment of CM extracted from retinal pigment epithelial (RPE) cells (Figure 6).
0
1
2
3
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8
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0
1
2
3
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9
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1
2
3
4
5
6
7
8
9
0
Figure 6. In vitro antiangiogenic effect of analog 34f in HUVECs (A−C) and HRMECs
3
(
D−F) at 100 nM. Effect of analog 34f on cell proliferation determined by [ H]ꢀthymidine
incorporation assay (A, D). Analog 34f inhibited cell migration (B, E) and suppressed tube
formation (C, F). Error bars indicate the mean ± SEM. Control (Con).
We also compared HIFꢀ1α regulations by analog 2 and 34f using Western blot analysis.
Analog 34f effectively induced the destabilization of HIFꢀ1α on the HCT116 cell line at 100
nM (Figure 7a). With the destabilization of HIFꢀ1α, analog 34f effectively reduced the
production of VEGF (Figure 7b) and inhibited the expression of VEGFA, an angiogenesisꢀ
related target gene of HIFꢀ1α (Figure 7c).
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2
2
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0
Figure 7. Destabilization of HIFꢀ1α by analogs 2 and 34f suppresses hypoxiaꢀinduced
expression of VEGF in HCT116 cells at 100 nM. (A) Destabilization of HIFꢀ1α (upregulated
by hypoxia) by analogs 2 and 34f. (B) Inhibition of VEGF secretion by analogs 2 and 34f in
enzymeꢀlinked immunosorbent assay. (C) Inhibition of VEGFA expression, a target gene of
HIFꢀ1α, by analogs 2 and 34f.
Finally, we confirmed an association of HSP90 in the HIFꢀ1α inhibition by analog 34f via
immunoprecipitation and immunoblotting assays using A549 cell lysates. As anticipated,
analog 34f interrupted the interaction between HIFꢀ1α and HSP90 in vitro as shown in the
previous ringꢀtruncation analogs (Supporting Information Figure 3).
Suppression of hypoxia-mediated retinal neovascularization Following examination of
the in vitro antiangiogenic activities of 34c and 34f, we confirmed their in vivo efficacy using
18, 37, 38
a mouse OIR model.
Analogs 2, 34c, and 34f were injected intravitreally at a
concentration of 100 nM or 1 µM. Staining of wholeꢀmounted retinal tissues and their
quantitative analysis revealed that analog 34c suppressed retinal neovascularization
comparable to analog 2 (Supporting Information Figure 4). In particular, 34f at a
concentration of 100 nM effectively reduced retinal neovascular tufts at an enhanced level
compared to analog 2 (Figure 8). We also confirmed that analogs (S)ꢀ34f and (R)ꢀ34f, the
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stereoisomers of analog 34f, did not show significant difference in vivo antiangiogenic
activities at a concentration of 100 nM (Supporting Information Figure 5).
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0
Figure 8. Suppression of retinal neovascularization by analog 34f at 100 nM. (A) Reduction
of retinal neovascular tufts by analogs 2 and 34f. Scale bar = 1 mm (B) Quantitative analysis
was performed using the ImageJ image processing program by measuring the neovascular
area per retina (n = 6). Error bars indicate the mean ± SEM.
CONCLUSION
In summary, we fully elucidated structural features by establishing the SAR of the ringꢀ
truncated deguelin analog 2 for potent HIFꢀ1α inhibitory activity using an HREꢀluciferase
reporter assay. Analog 6i, in which part A contains a 2ꢀfluorobenzene system, was identified
as a highly potent HIFꢀ1α inhibitor, with an IC50 of 100 nM. Analog 6i is currently under
investigation for its anticancer activities, as well as the distinctive physicochemical properties
required for intravitreal drugs. The modification of part B, including the benzylic position
and the ketone moiety, confirmed the benefits of structural constraints to induce proper
conformation of the ringꢀtruncated deguelin analogs. Noticeably, the Cꢀpart modified analog
34f, which possesses a 4ꢀcyanobenzylamineꢀsubstituted benzene moiety in place of the
original chromene unit, exhibited enhanced HIFꢀ1α inhibitory activity and in vitro
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antiangiogenic activity compared to analog 2. Particularly, analog 34f exhibited highly
improved solubility and suppression of retinal neovascularization in the OIR model compared
to parent analog 2. These results support the therapeutic potential of the ringꢀtruncation
analogs with the heteroatomꢀsubstituted benzene ring as a novel scaffold replacing the
chromene moiety, which is known as a privileged structure for natural productꢀbased HIFꢀ1α
inhibitors targeting angiogenesis, for the development of novel HIFꢀ1α inhibitors targeting
pathological angiogenesis in ocular diseases.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
EXPERIMENTAL SECTION
Chemistry. General Methods. Unless otherwise described, all commercial reagents and
solvents were purchased from commercial suppliers and used without further purification.
Tetrahydrofuran was distilled from sodium benzophenone ketyl and dichloromethane,
acetonitrile, triethylamine and pyridine were freshly distilled with calcium hydride. Flash
column chromatography was carried out using silicaꢀgel 60 (230ꢀ400 mesh, Merck) and
preparative thin layer chromatography was used with glassꢀbacked silica gel plates (1mm,
Merck). Thin layer chromatography was performed to monitor reactions. All reactions were
1
13
performed under dry argon atmosphere in flameꢀdried glassware. H NMR and C NMR
spectra were recorded on a JEOL JNMꢀLA 300 (300MHz), JEOL JNMꢀGCX (400MHz),
BRUKERAMXꢀ500 (500MHz), JEOL (600MHz) or Bruker Avance III HD (800MHz, with a
5ꢀmm CPTCI CryoProbe) spectrometers. Chemical shifts are provided in parts per million
(ppm,δ) downfield from tetramethylsilane (internal standard) with coupling constant in hertz
(Hz). Multiplicity is indicated by the following abbreviations: singlet (s), doublet (d), doublet
of doublet (dd), triplet (t), quartet (q), quintet (quin) multiplet (m) and broad (br). Mass
spectra and HRMS were recorded on VG Trioꢀ2 GCꢀMS instrument and JEOL JMSꢀAX,
respectively. All the final compounds were purified up to more than 95% purity. The purity
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2
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2
2
2
2
2
2
3
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3
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3
4
4
4
4
4
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4
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5
6
of the compounds was determined by normal phase high performance liquid chromatography
®
(HPLC), (Waters, CHIRALPAK ADꢀH (4.6 × 250 mm)). The detailed analytical conditions
are available in Supporting Information
0
1
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0
General procedure A for Dess-Martin oxidation of alcohols 10a-h and 11a-h.
2 2 3
To a solution of alcohol (1 equiv.) in CH Cl were added NaHCO (3 equiv) and DessꢀMartin
periodinane (1.5 equiv) at 0 °C. The reaction mixture was stirred for 1 h at room temperature,
quenched with 10% sodium thiosulfate solution, and extracted with CH Cl . The organic
2
2
4
layer was washed with brine, dried over MgSO , and concentrated under reduced pressure.
The residue was purified by flash column chromatography on silica gel to afford the
corresponding carbonyl products.
General procedure B for anionic coupling of parts A and C.
To a solution of aryl bromide or phenylsulfone (1.8 equiv) in THF was added dropwise nꢀ
BuLi solution (1.6 M solution in nꢀhexane, 1.7 equiv) at ꢀ78 °C. The resulting solution was
stirred for 30 min at ꢀ78 °C, and aldehyde (1.0 equiv) in THF was added. The reaction
mixture was stirred for 1 h at ꢀ78 °C, washed with saturated NH
4
Cl solution, and extracted
, and concentrated
with EtOAc. The organic layer was washed with brine, dried over MgSO
4
under reduced pressure. The residue was purified by flash column chromatography on silica
gel to afford the corresponding coupled product.
General procedure C for O-alkylation of phenols 14a-c.
To a solution of phenol (1.0 equiv) in acetone were added K
3.0 equiv) at room temperature. The resulting mixture was refluxed for 12 h and extracted
with EtOAc. The combined organic layer was washed with brine, dried over anhydrous
2 3
CO (3.0 equiv) and alkyl halide
(
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MgSO
4
, and concentrated under reduced pressure. The crude residue was purified by flash
column chromatography on silica gel to afford the corresponding aryl ethers.
General procedure D for conversion of alcohols 15a-c to bromides.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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9
0
2 2 3
To a solution of alcohol (1.0 equiv) in dried CH Cl was added dropwise PBr (1.4 equiv) at
o
0
C. The reaction mixture was stirred for 3 h at room temperature, quenched with absolute
CO powder was added, the mixture was
stirred for 30 min. and extracted with CH Cl . The organic layer was dried over anhydrous
EtOH, and stirred for additional 30 min. After Na
2
3
2
2
4
MgSO and concentrated under reduced pressure. The crude residue was purified by flash
column chromatography on silica gel to afford the corresponding bromides.
General procedure E for conversion of bromides to sulfones 7a-c.
To a solution of bromide (1.0 equiv) in DMF were added benzenesulfinic acid sodium salt
o
(1.1 equiv) at room temperature. The resulting solution was heated to 80 C and stirred for 1
h at the same temperature. The reaction mixture was cooled to room temperature and
extracted with EtOAc. The organic layer was washed with water, dried over anhydrous
4
MgSO , and concentrated under reduced pressure. The residue was purified by flash column
chromatography on silica gel to afford the corresponding sulfone.
General procedure F for deallylation of allyl ethers 16a-c.
3 4
To a solution of allyl ether (1.0 equiv) in THF were added Pd(PPh ) (0.1 equiv) and
MeNHPh (0.2 equiv) at room temperature. The reaction mixture was stirred for 4h, quenched
with water and 2NꢀHCl solution, and extracted with EtOAc. The organic layer was dried over
4
anhydrous MgSO and concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel to afford the corresponding phenol.
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