Streamlined Total Synthesis of Uncialamycin and Its Application to the Synthesis of Designed Analogues for Biological Investigations

Article abstract of DOI:10.1021/jacs.6b04339

From the enediyne class of antitumor antibiotics, uncialamycin is among the rarest and most potent, yet one of the structurally simpler, making it attractive for chemical synthesis and potential applications in biology and medicine. In this article we describe a streamlined and practical enantioselective total synthesis of uncialamycin that is amenable to the synthesis of novel analogues and renders the natural product readily available for biological and drug development studies. Starting from hydroxy- or methoxyisatin, the synthesis features a Noyori enantioselective reduction, a Yamaguchi acetylide-pyridinium coupling, a stereoselective acetylide-aldehyde cyclization, and a newly developed annulation reaction that allows efficient coupling of a cyanophthalide and a p-methoxy semiquinone aminal to forge the anthraquinone moiety of the molecule. Overall, the developed streamlined synthesis proceeds in 22 linear steps (14 chromatographic separations) and 11% overall yield. The developed synthetic strategies and technologies were applied to the synthesis of a series of designed uncialamycin analogues equipped with suitable functional groups for conjugation to antibodies and other delivery systems. Biological evaluation of a select number of these analogues led to the identification of compounds with low picomolar potencies against certain cancer cell lines. These compounds and others like them may serve as powerful payloads for the development of antibody drug conjugates (ADCs) intended for personalized targeted cancer therapy.

Full text of DOI:10.1021/jacs.6b04339

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Article
Streamlined Total Synthesis of Uncialamycin and Its Application to
the Synthesis of Designed Analogues for Biological Investigations
K. C. Nicolaou, Yanping Wang, Min Lu, Debashis Mandal, Manas R. Pattanayak, Ruocheng Yu, Akshay
A Shah, Jason S. Chen, Hongjun Zhang, James J. Crawford, Laxman Pasunoori, Yam B. Poudel, Naidu
S. Chowdari, Chin Pan, Ayesha Nazeer, Sanjeev Gangwar, Gregory Vite, and Emmanuel N. Pitsinos
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b04339 • Publication Date (Web): 07 Jun 2016
Downloaded from http://pubs.acs.org on June 8, 2016
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Journal of the American Chemical Society
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Streamlined Total Synthesis of Uncialamycin and Its
Application to the Synthesis of Designed Analogues for
Biological Investigations
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K. C. Nicolaou,*,†,‡ Yanping Wang,† Min Lu,†,‡, Debashis Mandal,†,‡ Manas R. Pattanayak,†,‡
Ruocheng Yu,† Akshay A. Shah,† Jason S. Chen,‡ Hongjun Zhang,‡ James J. Crawford,‡ Laxman
Pasunoori,‡ Yam B. Poudel,§ Naidu S. Chowdari,§ Chin Pan,§ Ayesha Nazeer,§ Sanjeev Gangwar,§
Gregory Vite,# and Emmanuel N. Pitsinos†,◊
,∥
∥
†Department of Chemistry, BioScience Research Collaborative, Rice University, 6100 Main St., Houston, Texas
77005, United States
‡Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California
92037, United States
§Bristol-Myers Squibb Research & Development, 700 Bay Road, Redwood City, California 94063, United States
#Bristol-Myers Squibb Research & Development, Princeton, New Jersey 08543-4000, United States
◊Laboratory of Natural Products Synthesis & Bioorganic Chemistry, Institute of Nanoscience & Nanotechnology,
National Centre of Scientific Research “Demokritos”, Agia Paraskevi GR-15310, Greece
Supporting Information Placeholder
ABSTRACT: From the enediyne class of antitumor antibiotics, uncialamycin is among the rarest and most potent, yet one
of the structurally simpler, making it attractive for chemical synthesis and potential applications in biology and medicine.
In this article we describe a streamlined and practical enantioselective total synthesis of uncialamycin that is amenable to
the synthesis of novel analogues and renders the natural product readily available for biological and drug development
studies. Starting from hydroxy- or methoxyisatin, the synthesis features a Noyori enantioselective reduction, a Yamaguchi
acetylide-pyridinium coupling, a stereoselective acetylide-aldehyde cyclization, and a newly developed annulation reaction
that allows efficient coupling of a cyanophthalide and a p-methoxy semiquinone aminal to forge the anthraquinone moiety
of the molecule. Overall, the developed streamlined synthesis proceeds in 22 linear steps (14 chromatographic separations)
and 11% overall yield. The developed synthetic strategies and technologies were applied to the synthesis of a series of
designed uncialamycin analogues equipped with suitable functional groups for conjugation to antibodies and other delivery
systems. Biological evaluation of a select number of these analogues led to the identification of compounds with low
picomolar potencies against certain cancer cell lines. These compounds and others like them may serve as powerful
payloads for the development of antibody-drug conjugates (ADCs) intended for personalized targeted therapy.
appropriate delivery systems, have found applications as
anticancer clinical agents. Neocarzinostatin was the first
1. INTRODUCTION
enediyne to be approved as a polymer drug conjugate
(styrene maleic acid neocarzinostatin, SMANCS;
zinostatin stimalamer) for the treatment of certain types
of leukemia and cancers of the liver and the brain, while
calicheamicin γ1I (2) as an antibody drug conjugate (ADC,
gemtuzumab ozogamicin; Mylotarg®) was approved for
the treatment of certain types of acute myeloid leukemia
(later with-
The naturally occurring enediyne antitumor antibiotics
represent some of the most potent cytotoxic agents
known to date.1 Their structures are characterized by 9-
or 10-membered ring conjugated enediyne structural
motifs which play a principal role in their Bergman
cycloaromatization-based double strand DNA-cleaving
mechanism of action. Figure 1 depicts a number of
enediynes, including the stable 10-membered ring
enediynes uncialamycin (1),2-4 calicheamicin γ1I (2),5,6
dynemicin A (3),7,8 shishijimicin A (4)9,10 and the labile 9-
membered ring enediynes neocarzinostatin (5)11,12 and
presporolide (6),13 the latter being only a proposed
naturally biosynthesized substance based on the isolation
of its expected Bergman cycloaromatization products,14
sporolides A and B.15,16 Because of their high potencies,
these compounds are not suitable for chemotherapy by
themselves. Two of them however, as conjugates to
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O
Scheme 1. First Generation Retrosynthetic Analysis of
26-epi-Uncialamycina
HO
MeSSS
H
NHCO2Me
1
2
3
4
5
6
7
8
Me
OH
OH
Me
Me
O
Me
H
O
I
O
Me
O
O
O
HN
21
S
O
O
N
O
HO
O
OMe
OH
19
O
Hauser–Kraus
CN
O
23
Me
26
Me
OMe
annulation
O
H
N
MeO
Me
HO
MeO
25
OH
O
H1N
N
O
OTES
OH
O
OH
OH
O
3
5
I
7
1: uncialamycin
2: calicheamicin γ1
O
15
O
17
7
OH
11
O
HO
MeSSS
N MeS
NHCO2Me
13
OH
acetylide-
9
8
aldehyde
Me
H
O
cyclization
Me
1' : 26-epi-uncialamycin
CO2H
OMe
O
OH
OH
O
O
HN
O
HO
9
O
HO
HN
NH
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
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O
H
Me
O
Yamaguchi
coupling
N
MeO
Friendländer–
Pfitzinger
OH
Me
OMe
12
H
3: dynemicin A
4: shishijimicin A
quinoline
Me
synthesis
Me
O
O
OMe
O
N
AllocN
O
Me
OTES
OH
OH
OH
O
OTES
H
O
O
O
Me
O
O
OH
O
ODMB
O
TMS
OH
ODMB
H
O
O
11
10
9
Me
O
MeO
HN
OH
aTES = triethylsilyl, DMB = 3,4-dimethoxy-benzyl, Alloc =
allyloxycarbonyl.
HO
O
Me
HO
6: presporolide [unstable]
5: neocarzinostatin
In a preliminary communication in 2007,3a we reported
the total synthesis of racemic uncialamycin [26(R)] and
its C26 epimer [26-epi-uncialamycin, 26(S)] and assigned
the relative stereochemistry of the natural product as
C26-(R). The strategy employed in this synthesis is shown
in retrosynthetic format in Scheme 1. In addition to
lacking enantioselectivity, this synthesis suffered from
the intermediacy of iminoquinone 8 (Scheme 1) whose
low yielding preparation, chemical instability and
modest performance as a substrate in the Hauser−Kraus
annulation18a,b with cyanophthalide 7 left much to be
desired. In a subsequent communication3b we disclosed
an enantioselective total synthesis of both (+)-
uncialamycin [(+)-1, Figure 1)] and 26-epi-uncialamycin
[(+)-1′, Scheme 1)], their DNA-cleaving activities, and
antibiotic and cytotoxic properties. In this article we
report the details of the evolution of a streamlined
asymmetric and scalable total synthesis of uncialamycin
that renders it readily available for further biological
investigations. We also report the application of our
latter synthesis to the construction of a series of designed
analogues as well as the biological evaluation of a select
number of them.
Figure 1. Selected enediyne antitumor antibiotics.
drawn because of side effects). Kadcyla® (ado-
trastuzumab emtansine; a conjugate of the maytansinoid
DM1) and Adcertris® (brentuximab vedotin; a conjugate
of monomethyl auristatin E) are two recently approved
ADCs, the former being used against HER-2-positive
breast cancer and the latter against Hodgkin lymphoma.
Furthermore, several other ADC’s of calicheamicin γ1I
(and other potent cytotoxic agents) are currently in
advanced clinical trials.17
Not surprisingly, the enediyne natural products have
continued to attract considerable interest from biologists
and chemists, both from academia and industry, due to
their important biological properties, potential in
medicine, and intriguing molecular structures and
mechanisms of action.
In 2005, while screening for antibiotics effective against
Burkholderia cepacia, a major cause of lung infections in
cystic fibrosis patients, Davies, Andersen, and coworkers
isolated uncialamycin (1, Figure 1), a 10-membered ring
enediyne antibiotic from an unreported strain of
streptomycete related to Streptomyces cyanogenus.2 In
addition to its extremely high potency against B. cepacia
(minimum inhibitory concentration MIC = 0.001 μg/mL),
uncialamycin was reported to possess equally impressive
antibacterial potency against Staphylococcus aureus
(MIC = 0.0000064 μg/mL) and Escherichia coli (MIC =
0.002 μg/mL) as well as potent DNA-cleaving properties.2
Unfortunately, the extreme scarcity of uncialamycin
(only 300 μg were isolated)2 precluded its complete
structural elucidation and thorough biological
investigation. Thus, its relative stereochemistry at C26
(for numbering see structure 1', Scheme 1) was not
assigned and its absolute stereochemistry was presumed,
but not proven, to be the same as that of the related
natural product dynemicin A (3, Figure 1).2
2. RESULTS AND DISCUSSION
2.1. Enantioselective, Streamlined and Practical
Total Synthesis of (+)-Uncialamycin. Our experiences
during our campaign toward racemic and enantiopure
uncialamycin and other intelligence gathered during
these endeavors positioned us well to develop a
streamlined process applicable for larger scale synthesis
of uncialamycin in its naturally occurring enantiomeric
form. Below we describe the evolution of the process that
led to this development.
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Scheme 2. Streamlined Retrosynthetic Analysis
pure key building block (+)-22. Thus, BBr3-induced
Through p-Methoxy Semiquinone Aminal 13a
1
2
3
4
5
6
7
8
demethylation of 12 at room temperature led to
hydroxyisatin (18) in 96% yield. To our delight, this
substrate exhibited exceptional reactivity under
Friedländer–Pfitzinger conditions (aq. NaOH, 25 °C,
15 min) in the presence of methyl ketone 17, leading
directly to quinoline hydroxyketoacid 16 in 91% yield,
sparing the troublesome aldol-type condensation (i.e. 19
→16, see Scheme 3) that was required in the original
route.3a,b The presumed intermediate aryl vinylogous
amide 19 (Scheme 3) was not detected in this reaction.
The subsequent bis-alkylation with 3,4-dimethoxy-
benzyl bromide (DMBBr) also required modification due
to the lability of the substrate under the original
conditions [DMBBr (3.0 equiv), K2CO3 (8.0 equiv), n-
Bu4NI (0.15 equiv), DMF, 25 °C].3a,b Thus, by switching to
the more effective and yet mild Cs2CO3 and feeding the
resulting bis-DMB derivative 20 directly into the
previously employed3b Noyori asymmetric reduction
[HCO2H, Et3N, (S,S)-21 cat.]22 allowed the formation of
(+)-22 in 82% overall yield and 96% ee (≥99% ee after one
recrystallization from EtOAc). All compounds in this
series proved crystalline and only one column
chromatographic purification was required [at the stage
of (+)-22]. Note that the seemingly wrong absolute
configuration of (+)-22 [26(S), uncialamycin numbering]
was intentional, its aim being to direct
Improved
CN
Hauser–Kraus
annulation
Me
Me
OTES
OH
O
O
HN
AllocN
MeO
O
OH
OH
O
O
7
acetylide-
aldehyde
cyclization
O
OH
O
1: uncialamycin
13
9
Noyori asymmetric
reduction
Yamaguchi
coupling
10
11
12
13
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15
16
17
18
19
20
21
22
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25
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TIPS
H
Me
H
15
N
Me
O
AllocN
O
OTES
OTES
inversion
O
ODMB
11
ODMB
14
Friedländer–Pfitzinger
quinoline synthesis
O
O
O
MeO
Me
HO
N
Me
17
O
CO2H
N
H
18: hydroxyisatin
16
OH
aTIPS = triisopropylsilyl
As shown retrosynthetically in Scheme 2, a major
innovation in this strategy was the utilization of the p-
methoxy semiquinone aminal 13 (together with
cyanophthalide 7) in the Hauser–Kraus-type fusion,18
instead of the customary p-iminoquinones (e.g. see 8,
Scheme 1).8c.d Sequential disconnections of the 10-
membered enediyne ring within 13 through acetylide–
aldehyde ring closure and Yamaguchi quinoline–
acetylide coupling19 revealed first acetylene aldehyde 14,
and then key intermediates 11 and 15,20 the latter being
this time the TIPS-monoprotected enediyne building
block. This alternative intermediate was deemed more
practical due to its relative non-volatility and other
reasons that will be discussed below. Another important
development in the new process was the discovery that
hydroxyisatin (18, Scheme 2) could be used without
protection in the Friedländer–Pfitzinger quinoline
synthesis21 of hydroxy carboxylic acid methyl ketone 16,
circumventing the cumbersome cleavage of the methoxy
group under acidic conditions.3a,b The motivation and
rationale for this and other improvements will be further
discussed below as we traverse through the conduits of
the developing strategy.
Scheme 3. Streamlined Process for the Preparation of
Quinoline Derivative (+)-22a
O
O
O
Me
HN
HN
a) BBr3
b) NaOH
O
O
O
HN
O
CO2H
MeO
Me
12
18
17
OMe
OH
19
OH
Ts
N
Ph
Ph
Me
O
Ru
O
N
N
O
N
Me
CO2H
Cl
H2
N
Me
d) (S,S)-21
c) DMBBr
O
CO2DMB
HCO2H, Et3N
ODMB
(+)-22
16
OH
20
ODMB
aReagents and conditions: (a) BBr3 (2.6 equiv), CH2Cl2, 0 to
25 °C, 2 h, 96%; (b) NaOH (2.0 equiv), 17 (2.0 equiv), H2O,
25 °C, 15 min, 91%; (c) Cs2CO3 (5.0 equiv), n-Bu4NI
(0.15 equiv), DMBBr (4.0 equiv), DMF, 0 to 25 °C, 5 h; (d)
(S,S)-21 (0.05 equiv), HCO2H (4.3 equiv), Et3N (2.5 equiv),
CH2Cl2, 0 °C, 24 h, 82% over the two steps, 96% ee;
recrystallization from EtOAc, ≥99% ee.
the incoming acetylide attack from the desired side (anti)
in the pending Yamaguchi coupling (see below).
The original preparation of hydroxyketoacid 16 from
methoxyisatin (12) suffered heavily from prolonged
reaction times and drastic conditions, especially at the
demethylation step (48% aq. HBr, 110 °C, 40 h).3a,b In
pondering how to render this process practical, we
wondered whether the Friedländer–Pfitzinger quinoline
synthesis would proceed from the free hydroxyisatin (18)
which, in addition to being commercially available, could
be prepared in principle from the less costly
methoxyisatin (12). Scheme 3 shows the solution to this
problem and the efficient synthesis of enentiomerically
With a practical process for the synthesis of the
quinoline fragment, we next turned our attention to the
enediyne system of uncialamycin. The required enediyne
fragment 10 was originally prepared as shown in Scheme
4. The readily available enyne alcohol 2323 was oxidized
to aldehyde 24 (TPAP cat., NMO) and then converted to
10 through the action of TMSCHN2 and LDA (≤30%
overall yield). The volatile nature of 24 and 10 made it
impractical to improve the yield of this sequence,
compelling us to devise the alternative route shown in
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Scheme 4. Thus, MnO2 oxidation of eneyne alcohol 2323
acetylene 30 in 96% yield. The latter was smoothly and
1
2
3
4
5
6
7
8
followed by reaction of the resulting crude aldehyde (i.e.
24) with CBr4/PPh3 furnished dibromodieneyne 25 in 75%
overall yield as a colorless solid. The Grignard reagent 10a
required for the coupling with quinoline 11 could be
prepared from 25 in THF by sequential treatment with
NaHMDS and EtMgBr or from 10 directly by treatment
with EtMgBr, as shown in Scheme 4 [for more details on
the generation and quantitation of reagent 10a from 10 or
25, see the Supporting Information (SI)].
selectively mono-desilylated (–TMS) with K2CO3 in
MeOH to give desired TIPS-monoprotected cis-enediyne
15 in 96% yield (≥98% geometrical purity, as determined
by 1H NMR spectroscopy).
With both building blocks quinoline (+)-22 and
enediyne 15 available in deca-gram quantities, we next
proceeded to construct the advanced intermediate Alloc-
protected p-aminophenol (+)-38, as shown in Schemes 6
and 7. Thus, reduction of the lactone moiety of (+)-22
with DIBAL-H followed by silylation of the resulting
lactol (TESCl) gave TES–ether 11 in 91% yield (ca. 1:1 dr).
Addition of the latter mixture to a preformed solution of
the Grignard reagent derived from enediyne 15 and i-
PrMgCl, (THF, 0 to 25 °C) at 0 °C followed by addition of
AllocCl furnished exclusively anti addition product (+)-
30 in a pleasing 90% yield (as compared to 72% yield, plus
20% recovered starting material in the case of TMS-
acetylene 10 that was used in the original approach3a,b).
The improved yield in this Yamaguchi coupling is
attributed to the lower volatility of the enediyne 15 that
allows the formation of its Grignard reagent without loss
of substrate. The anti-selectivity observed in this reaction
is most likely due to the controlling effect of the methyl
group on substrate 11 as opposed to other substrates we
tested (such as the open-chain bis-TES ether of the diol
derived from 22 that led to ca. 1:1 mixture of C-24 epimers)
in our attempts to develop this highly efficient and
stereoselective process.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. Construction of Enediyne Key Building
Block 10a
b) TMSCHN2,
LDA
a) TPAP,
NMO
OHC
H
TMS
10
TMS
24
c) EtMgBr
HO
23
d) MnO2
e) CBr4, PPh3
f) NaHMDS
g) EtMgBr
TMS
Br
Br
25
BrMg
TMS
TMS
10a
aReagents and conditions: (a) NMO (1.5 equiv), TPAP
(0.1 equiv), 4 Å molecular sieves, CH2Cl2, 25 °C, 5 min; (b)
TMSCHN2 (1.2 equiv), LDA (1.2 equiv), THF, –78 °C, 20 min;
then 24 (1.0 equiv), –78 to 25 °C, 1 h, 30% over the two steps;
(c) EtMgBr (1.1 equiv), THF, 25 °C, 40 min; (d) MnO2
(10.0 equiv), CH2Cl2, 0 to 25 °C, 5 h; (e) CBr4 (1.5 equiv), PPh3,
CH2Cl2, 0 °C, 1 h, 75% over the two steps; (f) NaHMDS
(1.0 equiv), THF, –78 to –50 °C, 3 h; (g) EtMgBr (2.2 equiv),
THF, 0 to 25 °C, 30 min. NMO = N-methylmorpholine-N-
oxide, TPAP = tetrapropylammoniumperruthenate, LDA =
lithium diisopropylamide, HMDS = hexamethyldisilazane.
Scheme 6. Yamaguchi Coupling and Synthesis of
Monoprotected Diol (−)-33a
Although the originally employed TMS-enediyne 10
could be routinely prepared on gram-scale through the
above described procedures, the overall routes (see
Scheme 4) and properties of this building block left much
to be desired (i.e. high cost of reagents, chromatographic
purification, and volatility of final product). To remedy
this situation, we adopted TIPS-enediyne 1520 (Scheme 5)
as a superior building block by virtue of its expected
lower volatility. Scheme 5 summarizes a streamlined
synthesis of TIPS-enediyne 15 from readily available cis-
dichloroethene (26), TIPS-acetylene (27), and TMS-
acetylene
Me
O
Me
TIPS
a) DIBAL-H;
then TESCl
b) 15, i-PrMgCl;
Me
O
N
N
O
26
c) AcOH
then AllocCl
AllocN
O
OTES
OTES
TIPS
ODMB
ODMB
H
15
11
(+)-22
ODMB
(+)-30
TIPS
TIPS
Me
TIPS
Me
Me
AllocN
AllocN
AllocN
d) NaBH4;
e) AcCl
O
OH
O
OH
OH
O
Scheme 5. Streamlined Synthesis of Enediyne 15a
then
m-CPBA
OAc (see Table 1)
OH
ODMB
ODMB
ODMB
(+)-31
b) Pd(PPh3)4,
a) Pd(PPh3)4,
TIPS
Cl
CuI,
CuI,
(–)-33
(+)-32
Cl
Cl
TIPS
H
TMS
H
TIPS
aReagents and conditions: (a) DIBAL-H (2.4 equiv), CH2Cl2,
−78 °C, 3 h; then TESCl (1.3 equiv), imidazole (2.6 equiv),
28
R
27
29
26
30: R = TMS
15: R = H
c) K2CO3
DMF,
0 to 25 °C, 20 min, 91%, ca. 1:1 mixture of
aReagents and conditions: (a) Pd(PPh3)4 (0.03 equiv), CuI
(0.03 equiv), n-BuNH2 (2.0 equiv), 27 (1.0 equiv), 26 (1.9
equiv), Et2O, 25 °C, 10 h, 93%; (b) Pd(PPh3)4 (0.03 equiv), CuI
(0.03 equiv), n-BuNH2 (2.0 equiv), 29 (2.0 equiv), Et2O,
25 °C, 12 h, 96%; (c) K2CO3 (1.1 equiv), benzene, MeOH,
25 °C, 2 h, 96%.
diastereoisomers; (b) 15 (2.0 equiv), i-PrMgCl (2.0 equiv),
THF, 0 to 25 °C, 2 h; then (+)-11 (1.0 equiv), 0 °C, 1 h; then
AllocCl
(2.0 equiv),
0 °C,
30 min,
90%;
(c)
CH3CN/H2O/AcOH (4:1:2), 25 °C, 2 h, 91%; (d) NaBH4
(1.3 equiv), MeOH, 0 °C, 20 min; then m-CPBA (1.0 equiv),
NaHCO3 (2.0 equiv), CH2Cl2, 0 °C, 3 h, 81% over the two
steps; (e) AcCl (1.0 equiv), i-Pr2NEt (2.0 equiv), CH2Cl2,
−78 °C, 12 h, 86%. DIBAL-H = diisobutylaluminum hydride,
m-CPBA = 3-chloro-perbenzoic acid.
(29).20 Thus, Sonogashira coupling24 of 26 with 27
proceeded in the presence of Pd(PPh3)4 and CuI catalysts
to afford chloroenyne 28 (93% yield), which was coupled
[once again in the presence of Pd(PPh3)4 and CuI cat.] this
time with TMS-acetylene (29) furnishing bis-silyl
As good as the yield and stereoselectivity of this
process was, it left behind the undesired stereochemical
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Journal of the American Chemical Society
configuration of C-26(S) [see (+)-30, Scheme 6]. Our next latter was treated with DMP to give the coveted
1
2
3
4
5
6
7
8
objective therefore was the advancement of intermediate
(+)-30 to the cyclization precursor (+)-37 (Scheme 7) with
the corrected C26 configuration. To this end, the TES
group from (+)-30 was removed (AcOH, 91% yield) and
the resulting lactol was treated with NaBH4 in MeOH to
afford the corresponding diol, whose epoxidation with m-
CPBA furnished epoxide diol (+)-32 in 81% overall yield
for the two steps. At this stage, we decided to attempt to
optimize the monoacetylation of (+)-32, which we
previously achieved only partially.3a,b Table 1 summarizes
the results of this optimization study. Our previously
achieved selectivity (82% yield, contaminated with 15–20%
of the corresponding bis-acetate, 100 mg scale) with AcCl
and collidine (CH2Cl2, 0 to 25 °C)3a,b was surpassed only
through the use of AcCl and Hünig’s base (i-Pr2NEt,
CH2Cl2, −78 °C, 86% yield, deca-gram scale, Table 1, entry
5). The latter procedure had the advantage of being
reproducible on a large scale, while the former suffered
upon scale up in terms of yield, selectivity and
reproducibility.
cyclization substrate aldehyde (+)-14 (90% yield).
Preliminary attempts to induce cyclization on this
substrate were plagued by instability of the DMB ether
moiety protecting the phenolic group, forcing us to
remove the latter (DDQ, 94% yield), an operation that
led to aldehyde (+)-37 possessing a free phenolic group.
Scheme 7. Streamlined Synthesis of Alloc-Protected
p-Aminophenol (+)-38a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
TIPS
H
TIPS
Me
Me
OR1
OR2
Me
O
b) TBAF, HOAc;
then NaBH4
a) DMP
AllocN
AllocN
AllocN
O
OH
O
O
OAc
OAc
ODMB
(–)-33
ODMB
ODMB
(–)-34
c) TESCl;
then K2CO3
(+)-35: R1 = H, R2 = Ac
(+)-36: R1 = TES, R2 = H
d) DMP
H
Me
OTES
Me
Me
AllocN
AllocN
AllocN
f) CeCl3, KHMDS
(see Table 2)
O
O
OTES
OH
O
OTES
+
OH
Table 1. Optimization Study of Monoacetylationa
C17
C17
O
OH
(+)-38: 17(R)
OH
17-epi-38: 17(S)
OR
(+)-14: R = DMB
(+)-37: R = H
e) DDQ
TIPS
TIPS
aReagents and conditions: (a) DMP (2.0 equiv), NaHCO3
(4.0 equiv), CH2Cl2, 0 to 25 °C, 2 h, 94%; (b) TBAF (2.5 equiv),
AcOH (2.5 equiv), THF, 0 to 25 °C, 2 h; NaBH4 (2.0 equiv),
MeOH, 0 °C, 30 min, 93% over the two steps, ≥ 25:1 dr; (c)
TESCl (1.5 equiv), imidazole (2.0 equiv), DMF, 0 °C, 15 min;
then saturated K2CO3 in MeOH, THF, −10 °C, 20 min, 85%
for the two steps; (d) DMP (2.0 equiv), NaHCO3 (4.0 equiv),
CH2Cl2, 0 to 25 °C, 90 min, 90%; (e) DDQ (3.0 equiv), pH 6.8
phosphate buffer, CH2Cl2, 25 °C, 12 h, 94%; (f) CeCl3
(4.0 equiv), THF, 25 °C, sonication, 30 min; then KHMDS
(6.0 equiv), −78 to −40 °C, 1 h, 79% (+)-38 [17(R)-isomer],
plus 13% 17(S)-isomer, (+)-17-epi-38. DMP = Dess–Martin
Me
Me
conditions
AllocN
AllocN
O
OH
OH
O
OR
OAc
S
O
ODMB
(+)-32
ODMB
(–)-33: R = H
(–)-33a: R = Ac
S
Me
N
ATT
yieldb
product(s)b
entry
1
conditions
Ac2O, Et3N, DMAP,
≥95%
only 33a
THF, −78 °C
n-Bu2SnO, Ac2O,
2
3
4
10%
80%
82%
only 33
DMF, 25 °C
periodinane,
DDQ
=
2,3-dichloro-5,6-dicyano-1,4-
benzoquinone.
ATT, NaH, THF, 25 °C
33:33a (ca. 95:5)
At this stage, and in order to develop a high-yielding
and reproducible procedure for the ring closure to the
AcCl, collidine,
CH2Cl2, 0 to 25 °C
33:33a (ca. 5–
20:1)
desired cyclic enediyne system, we undertook
a
AcCl, i-Pr2NEt,
CH2Cl2, −78 °C
5
86%
only 33
systematic optimization study. As seen in Table 2, this
exploration included changes of the base metal, additives,
and sonication, the latter proving itself as a crucial
parameter. Thus, while NaHMDS and LiHMDS proved
inferior to KHMDS in the presence of various additives,
the latter base performed even better upon addition to a
pre-sonicated mixture of CeCl3 and substrate (+)-37. It
was delightful to realize in the end that the phenol served
well as a substrate, leading to good selectivity in favor of
the desired 17(R)-epimer (ca. 6:1 dr, see entry 7, Table 2).
It was also interesting to observe that the use of LiHMDS
resulted in reversal of the selectivity of the acetylide
addition to the aldehyde, furnishing the 17-epimer of (+)-
38 as the major product, (+)-17-epi-38 (single isomer, see
entry 3, Table 2, not optimized). Thus, a stereo-divergent
cyclization was discovered that potentially could be
employed for the selective synthesis of designed
analogues with differing C17-configurations.
aReactions were carried out on 0.1–1.0 mmol scale. Yields
and selectivities were determined by 1H NMR spectroscopic
analysis. ATT = 3-acetylthiazolidine-2-thione.
b
Hydroxyacetate (−)-33 was then oxidized with Dess–
Martin periodinane25 (DMP) to afford ketoacetate (−)-34
(94% yield) as shown in Scheme 7. Removal of the TIPS
group from the enediyne terminus of the latter (TBAF,
AcOH) followed by NaBH4 reduction led smoothly to the
C26-inverted hydroxy compound (+)-35 in 93% overall
yield and ≥ 25:1 diastereoselectivity (as determined by 1H
NMR spectroscopic analysis).
In preparation for the pending ring closure, the
hydroxyl group within (+)-35 (Scheme 7) was protected
as a TES ether (TESCl) and the acetate group was cleaved
(K2CO3, MeOH, 85% overall yield), releasing the primary
hydroxyl group and furnishing compound (+)-36. The
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The next challenge to be addressed in the synthesis was
the annulation intended to form the amino-
anthraquinone system of uncialamycin. In our first3a and
second3b syntheses of uncialamycin we achieved this goal
by joining cyanophthalide 7 with p-iminoquinone (+)-39
(i.e. 26-epi-8, Scheme 1), the latter obtained from Alloc-
protected p-aminophenol (+)-38 through oxidation and
Alloc cleavage (Scheme 8). In our initial attempts to
optimize this process, we achieved some improvements
but also encountered persistent intransigence as shown
in Schemes 8 and 9. Mindful of the reports from Myers8d
and Danishefsky,8c we were not surprised to observe by-
product 40 (43% yield), with LDA as a base, or by-product
41 (30% yield) plus desired product 42 (60% yield), with
LiHMDS as a base (Scheme 9A).
as partner in the annulation reaction with
a
1
2
3
4
5
6
7
8
cyanophthalide 7. Substrate (+)-13 had the advantage of
offering only a single viable site of attack from the
cyanophthalide anion (see Scheme 9B), as compared to
the two presented (and observed)4d,e by p-iminoquinone
39 (see Scheme 9A). Indeed, when p-methoxy
semiquinone aminal (+)-13 was exposed to the anion
generated from cyanophthalide 7 and LiHMDS, either as
the limiting reagent [3.0 equiv of 7, 3.0 equiv of LiHMDS,
1.0 equiv of (+)-13, THF, −78 to 25 °C, 84% yield] or in
excess [1.0 equiv of 7, 4.0 equiv of LiHMDS, 3.0 equiv of
(+)-13, THF, −78 to 25 °C, 81% yield based on 62%
recovery of 13], protected uncialamycin (+)-43 (Scheme
9B) was obtained cleanly and in high yield. Cleavage of
the Alloc protecting group from the latter led smoothly
to the desired protected uncialamycin (+)-42 as a single
product and in excellent yield. As it turned out, this mode
of annulation became the method of choice for preparing
amino-anthraquinones of wide ranging complexity and
diversity.18c
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Optimization Study for Acetylene-Aldehyde
Cyclizationa
H
Me
OTES
Me
Me
conditions
AllocN
AllocN
Finally, a three-step sequence from p-methoxy-semi-
quinone aminal (+)-13 to (+)-uncialamycin [(+)-1]
without purification of intermediates was developed as
shown in Scheme 9B. Thus, reaction of cyanophthalide 7
(3.0 equiv) with LiHMDS (4.0 equiv) in THF at −78 °C for
20 min followed by addition of semiquinone (+)-13
(1.0 equiv, −78 to 25 °C, 1 h) afforded bis-protected
uncialamycin 43 in high yield, whose Alloc and TES
protecting groups were sequentially removed without
purification by exposure to Pd(PPh3)4 cat. and 3HF•Et3N
to furnish, after chromatographic purification, pure
(+)-uncialamycin [(+)-1] in 73% overall yield (see Scheme
9B). This improved and streamlined 22-step (14
chromatographic separation) synthesis of (+)-
uncialamycin from 5-hydroxyisatin (18, Scheme 3)
proceeds in 11% overall yield.
O
O
OTES
OH
AllocN
+
OH
O
OTES
–78 to –40 °C
C17
C17
O
OH
(+)-38: 17(R)
OH
(+)-17-epi-38: 17(S)
(+)-37
OH
yieldb
90%
90%
33%
38:17-epi-38b
entry
conditions
1
CeCl3, LiHMDS, THF
2:3
1:3
2
3
CeCl3•2LiCl, LiHMDS, THF
LiCl, LiHMDS, THF
17-epi-38 only
CeCl3, KHMDS,
toluene/THF
4
80%
5:1
5
CeCl3, KHMDS, THF
85%
70%
6:1
1:3
6
CeCl3•2LiCl, KHMDS, THF
CeCl3, KHMDS,THF,
pre-sonication
7
92%
6:1
8
Yb(OTf)3, LiHMDS, THF
decomposition
aReactions were carried out on 0.1–1.0 mmol scale. bYields
and ratios determined by 1H NMR spectroscopic analysis.
Scheme 8. Optimized Preparation of p-Methoxy
Semiquinone Aminal (+)-13 and p-Iminoquinone (+)-
39a
Me
OTES
Me
Me
AllocN
b) Pd(PPh3)2Cl2,
n-Bu3SnH
AllocN
N
O
O
O
OTES
OH
a) PhI(OAc)
2 MeO
O
OTES
OH
OH
OH
(+)-38
O
(+)-13
(+)-39
aReagents and conditions: (a) PhI(OAc)2 (1.1 equiv), MeOH,
0 to 25 °C, 15 min, 83%; (b) n-Bu3SnH (1.6 equiv), H2O
(5.2 equiv), Pd(PPh3)2Cl2 (0.2 equiv), CH2Cl2, 25 °C, 20 min,
63% (based on 65% conversion).
The varying yields in this coupling reaction and the
difficulties associated with the preparation and stability
of p-iminoquinone (+)-39 led us to explore its precursor,
Alloc-protected p-methoxy semiquinone aminal (+)-13,
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Scheme 9. Completion of Uncialamycin [(+)-1] Total
acetylenic units that allows the obligatory orbital
Synthesisa
1
2
3
4
5
6
7
8
interactions for the cycloaromatization to take place. The
uncialamycin cascade shown in Scheme 10 can be rapidly
and cleanly induced by anhydrous HCl in CH2Cl2 to
afford the Bergman cycloaromatization product 47 (90%
A. Conventional Hauser–Kraus annulation using iminoquinone 39
O
path b
Me
Me
Me
yield, dark blue).3a Interestingly,
a
Bergman
CN
O
N
N
CN
N
O
O
OTES
OH
a) LiHMDS
O
OTES
OH
O
OTES
OH
cycloaromatization secondary metabolite related to
uncialamycin has recently been reported and shown to
exhibit anti-HIV activity,27 suggesting this type of
structures as potential lead compounds for further
optimization. The cd distance [see structure 1, Scheme 10]
between the two acetylenic carbons to be bonded during
the cycloaromatization of cyclic conjugated enediynes
has been correlated with the stability of these systems
toward benzenoid diradical formation.28 Computational
studies (AMBER* force field) placed the cd distance of
uncialamycin at 3.41 Å.29 X-Ray crystallographic analysis
of (±)-uncialamycin [(±)-1] revealed the cd to be 3.60 Å.3a
This value is consistent with the molecule’s stability as
opposed to that of the transient chlorohydrin 45, whose
calculated cd distance was found to be 3.00 Å,29 a value in
line with its rapid cycloaromatization observed. The
likely mechanism of action of uncialamycin is, therefore,
thought to involve binding to double-stranded DNA,
activation, and double strand cleavage of the genetic
material by the generated benzenoid diradical as
proposed for dynemycin7 and calicheamicin γ1I,5 and
confirmed through experimentation.3b
path
a
O
NC
path a
O
O
7
39
O
OLi
O
I
41: expected by-product
9
(based on ref 8c,d)
b) LDA
path b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
Me
Me
Me
CN
N
O
O
O
HN
NC
N
O
OTES
OH
O
OTES
OH
O
OTES
OH
O
ꢀCN
O
OH
OH
Li
II
42: Hauser–Kraus product
40: expected by-product
(based on ref. 8d)
B. Improved annulation using semiquinone aminal (+)-13
Me
Me
Alloc
O
CN
O
AllocN
MeO
N
O
OTES
OH
O
OTES
OH
c) LiHMDS
or
d) LiHMDS
7
O
(+)-13
O
O
OH
43
g) LiHMDS;
then Pd(PPh3)4,
morpholine;
e) Pd(PPh3)4
Et N
3
then 3HF•
Me
Me
O
HN
O
HN
O
OTES
OH
f) 3HF• Et3N
O
OH
OH
O
OH
O
OH
Scheme 10. Bergman Cycloaromatization Study of (±)-
Uncialamycina
(+)-1: uncialamycin
(+)-42: annulation product
aReagents and conditions: (a) 7 (3.0 equiv), LiHMDS (3.0
equiv), THF, −78 °C, 20 min; then 39 (1.0 equiv), −78 to 25 °C,
1 h, 60%; (b) 7 (3.0 equiv), LDA (3.0 equiv), THF, −78 °C, 20
min; then 39 (1.0 equiv), −78 to 25 °C, 1 h, 43%; (c) 7
(3.0 equiv), LiHMDS (3.0 equiv), THF, −78 °C, 20 min; then
(+)-13 (1.0 equiv), −78 to 25 °C, 1 h, 84%; (d) 7 (1.0 equiv),
LiHMDS (1.0 equiv), THF, −78 °C, 20 min; then (+)-37
(3.0 equiv), LiHMDS (3.0 equiv), THF, −78 to 25 °C, 1 h, 81%,
with 62% recovered (+)-13; (e) Pd(PPh3)4 (0.08 equiv),
morpholine (2.4 equiv), THF, 0 to 25 °C, 2.5 h, 85%; (f)
c
Me
Me
d
H
O
HN
O
O
N
O
OH
OH
OH
OH
HO
a) CH2Cl2
c) HCl
or
Cl
b) CDCl3
O
OH
OH
44
1: uncialamycin
c
Me
Me
d
Me
OH
OH
O
O
HN
HO
O
HN
HO
O
O
HN
HO
OH
OH
OH
OH
Cl
3HF•Et3N (100 equiv), THF, 25 °C, 1.5 h, 99%; (g)
7
Cl
Cl
(3.0 equiv), LiHMDS (4.0 equiv), THF, −78 °C, 20 min; then
(+)-13 (1.0 equiv), −78 to 25 °C, 1 h; then Pd(PPh3)4
(0.1 equiv), morpholine (2.4 equiv), THF, 0 °C, 2 h, then
25 °C, 30 min; then 3HF•Et3N (100 equiv), THF, 25 °C, 1.5 h,
73% overall for the three steps from (+)-13.
CH2Cl2
OH
47
O
OH
46
OH
45
aReagents and conditions: (a) CH2Cl2, 25 °C, 12 h; (b) CDCl3,
25 °C, 30 min; (c) 5 mM HCl in CH2Cl2, 25 °C, 5 min, 90%.
Synthetic uncialamycin [(+)-1] was found to be a deep
purple crystalline solid stable at ambient temperature,
both in the solid phase and in solution in a variety of
solvents (e.g. DMSO, MeOH, EtOAc, CH3CN). However,
upon dissolution in HPLC grade CH2Cl2 or NMR grade
CDCl3 that had been exposed to air, uncialamycin rapidly
turned dark bluish, indicating a chemical change. As
proven by NMR spectroscopic analysis,3a this change was
brought about by the Bergman cycloaromatization
reaction,14 induced by traces of HCl or DCl present in the
solvents used, and which led to the hexacyclic benzenoid
system 47 via the pentacyclic chlorohydrin enediyne 45
(as shown in Scheme 10).3a,26 The latter intermediate is a
fleeting species due to the induced closeness of its
2.2. Design, Synthesis and Biological Evaluation of
Uncialamycin Analogues. Having developed a practical
and efficient total synthesis of uncialamycin [(+)-1; see
general strategy in retrosynthetic format, Figure 2A], we
proceeded to apply it to the synthesis of a series of
designed analogues of this scarce natural product for the
purposes of biological evaluation as cytotoxic agents to
be used as payloads to ADCs (48–59, Figure 2B). The
masked amino group in some of these analogues was
intended as the handle through which the molecule
could be attached to a linker, and thence to an
appropriate antibody. According to Figure 2A the
required substituted cyanophthalides II were to be fused
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with semiquinone aminal (+)-13 to form the targeted Scheme 11) through a sequence involving: (a) Boc
1
2
3
4
5
6
7
8
analogues (I) following our standard protocol.
A
protection (Boc2O; quant.); (b) amidation (AlCl3, Et2NH;
82% yield); (c) oxidation of the resulting primary alcohol
(PDC; 62% yield); (d) cyanation and ring closing (TMSCN,
KCN cat., 18-crown-6 cat., followed by AcOH; 83% yield);
and (e) deprotection (montmorillonite K 10; 96% yield).
Anthraquinone
fusion
Me
Me
CN
O
AllocN
O
O
HN
O
OTES
OH
O
OH
OH
MeO
+
Scheme 11. Synthesis of Aminocyanophthalides 60b
and 60ca
O
substitution(s)
substitution(s)
O
OH
(+)-13
I
II
B
O
O
O
9
H2N
BocHN
BocHN
a) Boc2O
b) AlCl3, Et2NH
NEt2
OH
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Me
Me
Me
61
62
63
O
O
HN
O
OH
OH
O
O
HN
O
O
HN
O
OH
OH
O
OH
OH
c) PDC
O
R
O
O
N
H
d) TMSCN;
then AcOH
H2N
BocHN
BocHN
OH
e) MK10
NEt2
CHO
OH
OH
O
O
49a: R = Boc
49b: R = H
1: uncialamycin
48
CN
CN
60c
60b
64
aReagents and conditions: (a) Boc2O (1.0 equiv), THF, reflux,
16 h, 100% (based on 88% conversion); (b) AlCl3 (1.3 equiv),
Et2NH (2.5 equiv), CH2Cl2, 25 °C, 30 min, then 62, 0 °C,
15 min, 82%; (c) PDC (1.1 equiv), 3 Å MS, CH2Cl2, 25 °C, 2 h,
62%; (d) TMSCN (1.4 equiv), KCN (0.02 equiv), 18-C-6
(0.02 equiv), CH2Cl2, 0 to 25 °C, 2 h; then AcOH, 25 °C, 24 h,
83%; (e) MK10, ClCH2CH2Cl, reflux, 4 h, 96%. PDC =
pyridinium dichromate; MS = molecular sieves; TMSCN =
trimethylsilyl cyanide; MK10 = montmorillonite K 10.
Me
Me
R1
R2
Me
N
O
O
HN
O
O
HN
O
OH
OH
O
OH
OH
O
O
HN
O
OH
OH
R1
N
R2
OH
OH
OH
50a: R1, R2 = Phth
51a: R1, R2 = Phth
PhthN
50b: R1 = R2 = H
51b: R1 = R2 = H
50c: R1 = Boc, R2 = H
51c: R1 = Boc, R2 = H
52
Me
Me
SO2Ph
Me
O
HN
O
HN
O
OH
OH
O
OH
OH
The phthalimidomethyl cyanophthalide 60d was
prepared from the commercially available 2,5-dimethyl
benzoic acid (65, Scheme 12). Thus, reaction of 65 with
MeOH in the presence of H2SO4 gave methyl ester 66 (86%
yield) which was selectively brominated [Br2, benzoyl
peroxide (BPO) cat.] and cyclized (150 °C) to afford
bromide 67 (68% yield).30 The latter was reacted with
phthalimide in the presence of K2CO3 and catalytic
amounts of n-Bu4NI to furnish phthalimide 68 (99%
yield), which was then converted into the targeted
phthalimidomethyl cyanophthalide 60d following
procedures analogous to the ones employed for the
construction of cyanophthalide 60b from phthalide 62
(see Scheme 11). Analogously and in similar yields,
MeO
O
O
HN
O
OH
OH
O
O
N
N
OMe O
OH
NHMe
O
OH
Me
R1
R2
OH
53a: R1, R2 = Phth
53b: R1 = R2 = H
54
55
Me
Me
O
O
HN
O
O
O
HN
OH
OH
O
OH
OH
CONHMe
H
N
H
N
R
O
OH
OH
56
57: R = PhCO
58: R = CH3CO
59: R = p-NH2C6H4CO
Figure 2. A: General retrosynthetic analysis of
uncialamycin analogues. B: Structures of uncialamycin (1)
and designed analogues (48–59). Boc = tert-butoxy-
carbonyl; Phth = phthaloyl.
Scheme 12. Synthesis of Phthalimidomethyl
Cyanophthalide 60da
CN
CN
CN
CN
O
O
O
O
R
NPhth
O
O
BocHN
H2N
Me
CO2R
Me
O
b) NBS, BPO
c) 150 °C
e) AlCl3, Et2NH
O
O
O
PhthN
NEt2
60a
60b
60c
60d
O
CN
CN
CN
CN
MeO
65: R = H
66: R = Me
a) MeOH,
H2SO4
67: R = Br
68: R = PhthN
69
f) PDC
OH
d) PhthNH
Me
O
PhthN
O
O
O
N
Alloc
O
O
O
PhthN
OMe
60g
O
PhthN
O
O
O
60e
60f
60h
g) TMSCN
then AcOH
PhthN
PhthN
NEt2
CHO
O
= Phth
Figure 3. Required cyanophthalides for this work (60a–h).
CN
60d
70
Figure 3 shows the individual cyanophthalides (60a18c
and 60b–h) required for the synthesis of the targeted
uncialamycin analogues (48–59, Figure 2B). Their
rendering readily available became our first task. The
synthesis of cyanophthalide 60a has been reported.18c
The construction of the remaining required cyano-
phthalides (60b–h) from simple building blocks is
summarized in Schemes 11–14.
O
aReagents and conditions: (a) H2SO4 (0.1 equiv), MeOH,
reflux, 4 h, 86%; (b) NBS (2.2 equiv), BPO (0.06 equiv), CCl4,
reflux, 1 h, 82%; (c) 150 °C, 1.5 h, 68%; (d) PhthNH (1.1 equiv),
K2CO3 (1.5 equiv), n-Bu4NI (0.1 equiv), DMF, 70 °C, 1.5 h,
99%; (e) AlCl3 (1.3 equiv), Et2NH (2.5 equiv), CH2Cl2, 0 to
25 °C, 30 min, then 68, 0 °C, 15 min, 78%; (f) PDC (1.5 equiv),
3 Å MS, CH2Cl2, 25 °C, 6.5 h, 63%; (g) TMSCN (2.0 equiv),
KCN (0.1 equiv), 18-C-6 (0.1 equiv), CH2Cl2 /THF (1 : 1), 25 °C,
2.5 h; then PTSA (0.05 equiv), AcOH, 25 to 40 °C, 24 h, 92%.
Aminocyanophthalides 60b and 60c were prepared
from commercially available 6-aminophthalide 61 (see
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Journal of the American Chemical Society
NBS = N-bromo succinimide; BPO = benzoylperoxide; PTSA
= p-toluenesulfonic acid.
25 °C, 2 h; then AcOH, PTSA (0.05 equiv), 40 °C, 24 h, 88%.
TFE = trifluoroethanol.
1
2
3
4
5
6
7
8
phthalimidomethyl cyanophthalides 60e and 60f (Figure
3) were prepared from the corresponding dimethyl
benzoic acids (see SI for further details).
The preparation of N-Alloc-N-methyl cyanophthalide
60h was accomplished from the readily available
bromide 67 (see Scheme 12) as shown in Scheme 14. Thus,
heating of 67 at 110 °C with hexamethylenetetramine in
the presence of AcOH in H2O produced aldehyde 77 in 81%
yield. Reductive amination of the latter with
MeNH2/NaBH4 in trifluoroethanol followed by Alloc
protection of the resulting secondary amine gave
intermediate lactone 78 (78% yield), whose sequential
aminolysis (AlCl3, Et2NH, 95% yield) and PDC oxidation
(79% yield) furnished aldehyde 80 via hydroxyl amide 79.
Exposure of 80 first to TMSCN in the presence of catalytic
amounts of KCN and 18-C-6 and then to AcOH led to the
targeted Alloc protected N-methyl cyanophthalide 60h
in 88% yield.
Scheme 13 depicts the synthesis of cyanophthalide 60g
from commercially available methyl 2,4-dihydroxy-3-
methylbenzoate 71. Thus sequential treatment of 71 with
K2CO3/MeI (93% yield) and AlMe3/Et2NH (85% yield) led
to amide 73 via methyl ester 72. Regioselective
formylation of the latter to aldehyde 74 was achieved
through lithiation (t-BuLi, TMEDA) followed by
quenching with DMF (96% yield). Benzylic bromination
of 74 with NBS in the presence of catalytic amounts of
BPO furnished benzyl bromide 75 which was converted
smoothly to its phthalimide derivative 76 (PhthNH, 60%
yield for the two steps). Finally, reaction of phthalimide
76 with TMSCN in the presence of catalytic quantities of
KCN and 18-C-6 followed by addition of AcOH led to the
desired bis-methoxy cyanophthalide 60g in 81% yield.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
With the required cyanophthalide partners (i.e. 60a–h)
in hand we turned our attention to their fusion with the
readily available semiquinone aminal (+)-13 as the key
step before reaching the targeted uncialamycin
analogues (48–59).
Scheme 13. Synthesis of Methoxy Cyanophthalide 60ga
OMe
OMe
OR
Me
CONEt2
Me
CONEt2
CHO
Me
RO
CO2Me
b) AlMe3, Et2NH
a) MeI
c) t-BuLi;
then DMF
MeO
MeO
71: R = H
72: R = Me
73
74
d) NBS, BPO
Br OMe
OMe
O
PhthN
OMe
CONEt2
CHO
CONEt2
CHO
PhthN
MeO
f) TMSCN;
then AcOH
e) PhthNH
MeO
O
MeO
CN
60g
76
75
aReagents and conditions: (a) MeI (1.2 equiv), K2CO3
(1.5 equiv), DMF, 50 °C, 9 h, 93%; (b) AlMe3 (2.1 equiv),
Et2NH (3.8 equiv), PhH, 0 to 120 °C, 7 h, 85%; (c) TMEDA
(2.0 equiv), t-BuLi (2.0 equiv), THF, −78 °C, 50 min; then
DMF (12 equiv), −78 to 25 °C, 3 h, 96%; (d) NBS (1.2 equiv),
BPO (0.06 equiv), CCl4, 85 °C, 2 h; (e) PhthNH (1.1 equiv),
K2CO3 (1.5 equiv), n-Bu4NI (0.1 equiv), DMF, 40 °C, 2 h, 60%
over the two steps; (f) TMSCN (2.0 equiv), KCN
(0.025 equiv), 18-C-6 (0.02 equiv), CH2Cl2, 0 to 25 °C, 2 h;
then AcOH, 25 °C, 80 h, 81%. TMEDA = tetramethyl-
ethylenediamine.
Scheme 14. Synthesis of N-Methyl Cyanophthalide 60ha
Alloc
Me
N
N
O
Br
O
O
N
N OHC
a)
b) MeNH2, NaBH4;
then AllocCl
N
O
O
O
67
77
78
c) AlCl3,
Et2NH
Alloc
Me
Alloc
Me
N
O
N
O
O
NEt2
d) PDC
AllocN
Me
NEt2
e) TMSCN;
then AcOH
O
CHO
CN
60h
80
79
OH
aReagents and conditions: (a) hexamethylenetetramine
(2.0 equiv), AcOH (4.0 equiv), H2O, 110 °C, 2 h, 81%; (b)
MeNH2 (1.0 equiv), TFE, 25 °C, 5 min; NaBH4 (1.2 equiv),
25 °C, 5 min; AllocCl (1.5 equiv), NaHCO3 (2.0 equiv),
THF/H2O (1:1), 25 °C, 40 min, 78%; (c) AlCl3 (1.5 equiv),
Et2NH (3.0 equiv), CH2Cl2, 0 to 25 °C, 30 min, 95%; (d) PDC
(2.0 equiv), 3 Å MS, CH2Cl2, 25 °C, 3 h, 79%; (e) TMSCN
(2.0 equiv), KCN (0.1 equiv), 18-C-6 (0.1 equiv), CH2Cl2, 0 to
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Page 10 of 15
Scheme 15. Synthesis of Uncialamycin Analogues 48–
uncialamycin (50b) in high yield. Apparently the
54a
1
2
3
4
5
6
7
8
presence of the aminomethyl group within the
uncialamycin structure imparts considerable lability to
the molecule (presumably due to higher propensity for
A
Me
CN
O
Alloc
O
Bergman
cycloaromatization).
To
facilitate
N
a) LiHMDS, 13
O
OTES
OH
PhthN
characterization, immediate trapping of freshly
generated product 50b in the MeNH2-induced de-
phthaloylation of 50a with Boc2O in the presence of
NaHCO3 led to Boc-protected 8-aminomethyl
uncialamycin (50c) in 70% overall yield from 50a. The
latter compound was found to be quite stable under
neutral conditions, apparently due to the diminished
electron density on its aminomethyl substituent.
O
PhthN
60d
Me
O
OH
AllocN
MeO
81
O
OTES
OH
b) Pd(PPh3)4,
9
morpholine
13
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Me
Me
c) 3HF•Et3N
O
O
HN
O
O
HN
O
OH
OH
O
OTES
OH
PhthN
PhthN
In a similar fashion, uncialamycin analogues 48–49b
and 51a–54 were synthesized from the corresponding
cyanophthalides (60a−c and 60e–h) and semiquinone
aminal (+)-13 (see SI for details).
8
OH
OH
82
50a
d) MeNH2
Scheme 16 summarizes the synthesis of the
phenylsulfonyl uncialamycin analogue 55 from
cyanophthalide 60h and semiquinone aminal (+)-13.
Thus, reaction of 60h with LiHMDS and (+)-13 under the
standard conditions led to Alloc-protected uncialamycin
derivative 83, from which secondary amine derivative 84
was generated by treatment with Pd(PPh3)4 cat. and
morpholine. The latter compound was treated with
sulfone containing chloroformate 8531 in the presence of
pyridine to give carbamate 86. Then, removal of the TES
group with HF•Et3N secured the desired phenylsufonyl
uncialamycin analogue 55 (51% overall yield from (+)-13).
Me
Me
O
HN
O
O
HN
e) Boc2O
OH
OH
O
OH
OH
8
8
NH2
O
OH
NHBoc
O
OH
50c
50b
B
60a
60b
60c
60e
Me
Me
Me
O
HN
O
HN
R
O
O
HN
O
OH
OH
O
OH
OH
O
OH
OH
R
O
OH
O
OH
OH
48
49a: R = NHBoc
49b: R = NH2
51a: R = NPhth
51b: R = NH2
51c: R = NHBoc
Scheme 16. Synthesis of Phenylsulfonyl Uncialamycin
Analogue 55a
60f
60g
60h
Me
OH
Me
Me
O
HN
O
HN
O
HN
Me
O
O
OH
OH
O
OH
OH
CN
O
R
MeO
R
OH
O
O
N
a) LiHMDS, 13
Me
N
O
OTES
OH
R
N
Alloc
O
OH
OMe O
OH
NHMe
O
OH
O
PhthN
Me
O
60h
55
53a: R = NPhth
53b: R = NH2
54
Me
OH
AllocN
MeO
83: R = Alloc
84: R = H
b) Pd(PPh3)4,
morpholine
aReagents and conditions: (a) 13 (1.0 equiv), LiHMDS
(2.6 equiv), THF, −78 °C, 20 min; then 60d (1.3 equiv), −78 to
25 °C, 1 h; (b) Pd(PPh3)4 (0.1 equiv), morpholine (2.4 equiv),
THF, 0 °C, 2 h; then 25 °C, 30 min, 81% for the two steps
(based on 13); (c) 3HF•Et3N (100 equiv), THF, 25 °C, 1.5 h,
90%; (d) MeNH2 (200 equiv), THF/H2O (1 : 5), 0 to 25 °C, 2 h;
(e) Boc2O (1.2 equiv), sat. NaHCO3 (aq.), THF, 0 °C, 1.5 h,
70% over the two steps.
O
OTES
OH
13
c)
O
Cl
O(CH2)2SO2Ph
85
SO2Ph
SO2Ph
Me
Me
O
HN
O
HN
O
OH
OH
O
OTES
OH
O
O
N
O
O
d) 3HF•Et3N
N
Me
Me
O
OH
O
OH
55
86
As a representative example, the synthesis of 8-
aReagents and conditions: (a) 60h (2.0 equiv), LiHMDS
(2.6 equiv), THF, −78 °C, 20 min; then 13 (1.0 equiv), −78 to
25 °C, 1 h; (b) Pd(PPh3)4 (0.1 equiv), morpholine (2.4 equiv),
THF, 0 °C, 1 h; (c) 85 (5 equiv), pyridine (5 equiv), CH2Cl2,
0 °C, 10 min; (d) 3HF•Et3N (100 equiv), THF, 25 °C, 1.5 h, 51%
from 13.
aminomethyl
phthalimidomethyl
uncialamycin
cyanophthalide
(50b)
60d
from
and
semiquinone aminal (+)-13 is presented below. Thus, as
summarized in Scheme 15A, coupling of 60d (2.0 equiv)
with (+)-13 (1.0 equiv) under the developed conditions
(60d, LiHMDS, THF; then 13, −78 to 25 °C) gave
uncialamycin derivative 81. The latter was treated
without purification with catalytic amounts of Pd(PPh3)4
in the presence of morpholine to induce Alloc cleavage,
furnishing 82 in 81% overall yield for the two steps (based
on 13). Treatment of 82 with 3HF•Et3N afforded
desilylated product 50a (90% yield), whose exposure to
MeNH2 led to the rather labile 8-aminomethyl
Phthalimidomethyl-uncialamycin derivative 81 (see
Scheme 15A) served as a common precursor for the
syntheses of uncialamycin analogues 56–59 (Scheme 17).
Thus, treatment of 81 with mild basic conditions (MeNH2
in THF) at room temperature opened the phthalimide
moiety to give the corresponding bis-amide compound,
which was globally deprotected to furnish the bis-amide
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Journal of the American Chemical Society
uncialamycin analogue 56. Alternatively, by employing aromatic moiety of the molecule (uncialamycin ring A,
1
2
3
4
5
6
7
8
the optimized conditions for complete phtalimide
removal (aqueous MeNH2 in MeOH/THF), the free
amino derivative 87 was obtained. Derivatization of this
primary amine with acetic anhydride, benzoic acid or N-
Fmoc-4-aminobenzoic acid (88) furnished the
corresponding amide derivatives which were subjected to
global deprotection to afford the targeted uncialamycin
analogues (57−59), respectively, as shown in Scheme 17.
compound 50b, Table 3, entry 5). This compound
exhibited remarkably high potency against the tested cell
lines (H226: IC50 = 28 pM; N87: IC50 = 11 pM; OVCAR3:
IC50 = 316 pM; Adr: IC50 = 20 pM). Not surprisingly, the
regioisomeric
uncialamycin
analogue
51b
(C7
aminomethyl, Table 3, entry 7) exhibited practically the
same potency as its C8 sibling (H226: IC50 = 12 pM; N87:
IC50 = 10 pM; OVCAR3: IC50 = 66 pM; Adr: IC50 = 29 pM;
see Table 3, entry 7). The precise origin of the extreme
potencies of these compounds is not fully understood.
However, the relatively low chemical stability of these
analogues (i.e. compounds 50b and 51b) suggests that the
aminomethyl group renders them more prone to the
Bergman cycloaromatization14 and/or increases their
binding affinity to DNA through polar interactions of
their protonated forms with the phosphate backbone,
and consequently more potent against their targeted
biological system. The bis-dimethoxy analogue 53b was
proven to be only slightly less potent than 50b (Table 3,
entry 8). On the other hand, phthalimido-protected
analogue 50a (Table 3, entry 4) was found to be
significantly less potent than the free amine 50b,
underscoring the importance of the free aminomethyl
group for the high potency observed with these
analogues. Compound 56 (Table 3, entry 10), an
intermediate isolated from an incomplete cleavage of the
phthalimido group, proved to be almost as potent as the
free amine (i.e. 50b). It was speculated, and later
confirmed (by HPLC analysis), that this bis-amide
uncialamycin derivative (i.e. 56, for structure see Figure
2B) acts as a prodrug undergoing internal cyclization
under the biological testing conditions, releasing free
amine 50b. Not surprisingly, the Boc-protected
derivative 50c (for structure see Figure 2B) also exhibited
relatively low potency (Table 3, entry 6). The more stable
4-aminobenzoic derivative 59 proved quite potent (Table
3, entry 13) in contrast to its benzoic acid counterpart,
derivative 57, which was found to be considerably less
potent (Table 3, entry 11). Once again, these differences
in potencies between 50b and 50c highlight the role of a
free amine as an enhancing structural feature within the
uncialamycin class. It is interesting to note that
acetamide 58 (Table 3, entry 12) was considerably more
potent than benzamide 57 (Table 3, entry 11) and that the
N-methyl C8-aminomethyl uncialamycin 54 (Table 3,
entry 9) proved essentially equipotent (H226: IC50 = 10
pM; N87: IC50 = 16 pM; OVCAR3: IC50 = 311 pM; Adr: IC50
= 15 pM) to the most potent analogues C8- (i.e. 50b Table
3, entry 5) and C7-aminomethyl (i.e. 51b, Table 3, entry 7)
analogues. These results provided a set of useful structure
activity relationship (SARs) within the uncialamycin
structural type. It should be
9
Scheme 17. Synthesis of Uncialamycin Analogues 56–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
59a
Me
OTES
Me
Alloc
O
N
O
O
HN
O
O
OH
OH
a) MeNH2, THF
OH
b) Pd(PPh3)4,
morpholine
c) 3HF•Et3N
H
N
NPhth
O
OH
O
OH
MeHN
O
81
56
Me
O
O
HN
d) MeNH2,
THF/H2O/MeOH
(2:3:10)
O
OH
OH
e) PhCO2H, HATU
H
N
b) Pd(PPh3)4,
morpholine
c) 3HF•Et3N
O
OH
57
Me
Me
Alloc
O
f) Ac2O
OTES
OH
N
O
HN
O
O
OH
OH
b) Pd(PPh3)4,
morpholine
c) 3HF•Et3N
H
N
Me
NH2
O
OH
O
O
OH
58
87
COOH
Me
g) 88, HATU
b) Pd(PPh3)4,
morpholine
O
O
HN
O
OH
OH
H2N
H
N
NHFmoc
88
c) 3HF•Et3N
O
OH
59
aReagents and conditions: (a) MeNH2 (36 equiv), THF, 0 to
25 °C; (b) Pd(PPh3)4 (0.1 equiv), morpholine (2.4 equiv), THF,
0 °C, 2 h; then 25 °C, 30 min; (c) 3HF•Et3N (100 equiv), THF,
25 °C, 1.5 h, 56 (77% overall yield) or 57 (33% overall yield) or
58 (35% overall yield) or 59 (48% overall yield); (d) MeNH2
(124 equiv), THF/H2O/MeOH (2:3:10), 0 to 10 °C; (e) benzoic
acid (1.5 equiv), HATU (2 equiv), DIPEA (3 equiv), DMF, 0 °C
to rt; (f) Ac2O (2.0 equiv), DIPEA (3.0 equiv), CH2Cl2, 0 °C to
rt, 1.5 h; (g) 88 (2.0 equiv), HATU (1.5 equiv), DIPEA (3 equiv),
MeCN, rt, 1.5 h. DIPEA = diisopropylethylamine; HATU= 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate.
A number of the synthesized uncialamycin analogues
were tested for their ability to inhibit the proliferation of
four different types of tumor cell lines: lung (H226),
gastric (N87), ovarian (OVCAR3) and multidrug resistant
(Adr) (see Table 3). Synthetic uncialamycin (1, Table 3,
entry 1) was found to exhibit similar activity against these
cell lines as that of natural uncialamycin,2 while its
extended anthraquinone moiety counterpart 48 (Table 3,
entry 2) lost considerable potency. Introduction of an
amino group at C8 (compound 49b, Table 3, entry 3) did
not make a significant difference in the potency.
However, a real breakthrough was observed when the
amino group was moved one carbon away from the
Table 3. In vitro Cytotoxicity of Uncialamycin
Analogues
Cell lines
Compoun
d
H226a
IC50 (pM)
N87b
IC50 (pM)
OVCAR3c
IC50 (pM)
Adrd
IC50 (pM)
Entry
1
2
1
48
1770
180200
1764
37670
4686
26130
388
9050
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Page 12 of 15
3
4
5
6
7
8
9
10
11
12
13
49b
50a
50b
50c
51b
53b
54
56
57
58
59
2043
6946
28
3763
12
77
10
67
2683
3014
11
3067
10
148
16
31
7638
15500
316
8552
66
401
311
1245
10550
2051
368
666
1007
20
1021
29
335
15
39
under further refinement and development as potential
targeted cancer therapies.4b
1
2
3
4
5
6
7
8
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and
characterization data for key compounds (pdf files). This
material is available free of charge via the Internet at
http://pubs.acs.org.
3164
855
706
5600
892
210
5171
1146
300
AUTHOR INFORMATION
9
a
b
For details of the biological assays, see SI. lung; gastric;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Corresponding Author
covarian; dmultidrug resistant.
*kcn@rice.edu
noted that although labile as free amines, the most
potent compounds 50b, 51b and 53b can be generated
from their stable protected precursors and trapped by
appropriate linkers, leading to stable molecular entities
for further conjugation to antibodies and other delivery
systems.
Author Contributions
∥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
3. CONCLUSION
This work was partially carried out at The Scripps Research
Institute. We thank Drs. D. H. Huang (TSRI), L. Pasternack
(TSRI), L. B. Alemany (Rice) and Q. Kleerekoper (Rice) for
NMR spectroscopic assistance, Drs. G. Siuzdak (TSRI) and C.
Pennington (Rice) for mass spectrometric, and Drs. R. K.
Chadha (TSRI) and C. Moore (UCSD) for X-ray
crystallographic assistance. This work was supported by the
National Institutes of Health (USA) (grant AI055475), the
Skaggs Institute for Research (TSRI), the Cancer Prevention
We have described the evolution of a streamlined
synthetic strategy for the total synthesis of the rare
marine cytotoxic agent uncialamycin [(+)-1]. Starting
from hydroxyisatin, the present strategy proceeds in 22
steps (14 chromatographic separations) and delivers the
natural product in 11% overall yield on multi-100 mg
scales (within the confines of academic laboratories;
projected to be scalable to multi-gram scales in
appropriate industrial laboratories) in stark contrast to
the 300 µg isolated from its natural source.2 Of particular
interest in this endeavor is the discovery, development
and application of a new type of annulation reaction to
forge the p-amino-anthraquinone structural motif of the
molecule, a method proven to be of general scope and
applicability and which can also be extended to o-amino-
anthraquinones and related systems.18c Thus, employing
our developed streamlined process for the total synthesis
of uncialamycin (1) we synthesized an array of designed
uncialamycin analogues equipped with appropriate
protecting groups and handles for further manipulation
and attachment to cancer cell associated antibodies and
other suitable drug delivery systems. Biological
evaluation of a number of the synthesized compounds
led to the discovery of a number of cytotoxic agents with
low picomolar potencies (e.g. compounds 50b, 51b, 53b,
54, 56). The chemistry described in this article sets the
foundation for further developments to occur toward
drug discovery and development starting with
uncialamycin, a rare naturally occurring substance, as a
lead compound that by itself proved too toxic to be useful
as a therapeutic agent. These investigations demonstrate
the power of modern organic synthesis to facilitate
biology and medicine by rendering readily available,
otherwise scarce complex molecules of natural or design
origins. Thus, in addition to providing useful structure
activity relationships (SARs) within the uncialamycin
class of antitumor agents, these efforts enabled
conjugation studies that led to the preparation of
antibody drug conjugates (ADCs) that are currently
&
Research Institute of Texas (CPRIT), The Welch
Foundation (grant C-1819), and Bristol–Myers Squibb (BMS).
J.J.C. acknowledges a post-doctoral fellowship award from
the US–UK Fulbright Commission.
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