Total Synthesis and Biological Evaluation of Tiancimycins A and B, Yangpumicin A, and Related Anthraquinone-Fused Enediyne Antitumor Antibiotics

Article abstract of DOI:10.1021/jacs.9b12522

The family of anthraquinone-fused enediyne antitumor antibiotics was established by the discovery of dynemicin A and deoxy-dynemicin A. It was then expanded, first by the isolation of uncialamycin, and then by the addition to the family of tiancimycins A-

Full text of DOI:10.1021/jacs.9b12522

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Total Synthesis and Biological Evaluation of Tiancimycins A and B,
Yangpumicin A, and Related Anthraquinone-Fused Enediyne
Antitumor Antibiotics
K. C. Nicolaou,* Dipendu Das,∥ Yong Lu,∥ Subhrajit Rout,∥ Emmanuel N. Pitsinos, Joseph Lyssikatos,
Alexander Schammel, Joseph Sandoval, Mikhail Hammond, Monette Aujay, and Julia Gavrilyuk
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ABSTRACT: The family of anthraquinone-fused enediyne antitumor
antibiotics was established by the discovery of dynemicin A and deoxy-
dynemicin A. It was then expanded, first by the isolation of
uncialamycin, and then by the addition to the family of tiancimycins
A−F and yangpumicin A. This family of natural products provides
opportunities in total synthesis, biology, and medicine due to their
novel and challenging molecular structures, intriguing biological
properties and mechanism of action, and potential in targeted cancer
therapies. Herein, the total syntheses of tiancimycins A and B,
yangpumicin A, and a number of related anthraquinone-fused
enediynes are described. Biological evaluation of the synthesized
compounds revealed extremely potent cytotoxicities against a number
of cell lines, thus enriching the structure−activity relationships within
this class of compounds. The findings of these studies may facilitate future investigations directed toward antibody−drug conjugates
for targeted cancer therapies and provide inspiration for further advances in total synthesis and chemical biology.
of new members of this class of natural products. These efforts
led to the discovery of several new congeners (6−15, Figure
1), thus enlarging significantly this family of bioactive
molecules.10a,b,d It is interesting to note that, apart from
yangpumicin A (6),10b tiancimycin A (7),10a and tiancimycin
D (10),10d which were isolated from wild-type bacterial strains,
the remaining tiancimycins [i.e., B (8), C (9), E (11), and F
(12)] were produced through genetic manipulation of
Streptomyces sp. CB03234,10a,d the tiancimycin A (7)-
producing strain. It should also be noted that the existence
of three of these new anthraquinone-fused enediyne antibiotics
(i.e., 13−15, Figure 1, in brackets) could only be inferred from
isolation of the corresponding Bergman cycloaromatization11
products.10b,d Interestingly, tiancimycin A (7)10a,d and yangpu-
micin A (6)10b were found to be even more cytotoxic than
uncialamycin (3)10a,b against various human cancer cell
lines.10a,b,d
1. INTRODUCTION
The enediyne family of antitumor antibiotics attracted
considerable attention, initially due to their unprecedented
structural features, biological potency, and novel mode of
action1 and subsequently because of their promise as potential
payloads for antibody−drug conjugates (ADCs) as targeted
cancer therapies.2
Dynemicin A (1, Figure 1)3 and the closely related deoxy-
dynemicin A (2, Figure 1)4 were the first, and for more than a
decade the sole, examples of anthraquinone-fused enediyne
antitumor antibiotics. To this subclass of 10-membered
enediynes was added, in 2005, uncialamycin (3, Figure 1),5 a
rather simpler member of the growing class. Despite its potent
antibacterial activity and DNA-cleaving properties, the
extremely small amounts (∼300 μg) available from its natural
source hindered uncialamycin’s complete structural elucidation
and detailed biological evaluation,5 thus elevating it to an
attractive total synthesis target. This challenge was later fully
met by our group6 and partially by others.7 Recently, seeking
to identify the genes involved in the biosynthesis of dynemicin
A, genetic manipulation of the producing organism by C. A.
Townsend et al. led to the identification of additional, partially
oxygenated dynemicin A precursors (4, 5, Figure 1).8
Furthermore, motivated by the potential of anthraquinone-
fused enediyne antibiotics as payloads for ADCs,6c,9 B. Shen et
al.10 have surveyed a large number of actinomycetes in search
A plethora of both experimental3c,12 and theoretical13
studies have shed light on the mode of cleavage of double-
stranded DNA by dynemicin A (1). Based on its structural
Received: November 20, 2019
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Figure 1. Naturally occurring anthraquinone-fused enediyne antitumor antibiotics. Bracketed structures (13−15) were inferred from their
corresponding Bergman cycloaromatization products.
similarity with the most recent congeners (i.e., 3−12, Figure 1)
and the concurrent isolation of related cycloaromatized
products,6a,b,10b,d the latter are anticipated to cleave double-
stranded DNA by a mechanism similar to the one that has
been proposed for the former.12a,c Thus, reduction of the
anthraquinone core (II → III, Figure 2) would lead to opening
of the epoxide ring. The strain release caused by the latter
event would then trigger the Bergman cycloaromatization11 of
the enediyne moiety to form a reactive 1,4-benzenoid diradical
species (III → IV, Figure 2), which could abstract a hydrogen
atom from the DNA backbone, thus leading to the latter’s
cleavage (IV → V, Figure 2). Although the anthraquinone
moiety of the molecule is capable of intercalation into double-
stranded DNA,12b,13a studies on dynemicin A and related
analogues by A. G. Myers et al. indicated that the A ring and
C26 substituents that favor strong intercalative binding lead to
diminished DNA cleavage.12j,k Thus, reduction and subsequent
H-atom abstraction from DNA most likely occur at a minor-
groove-inserted state.13c,d According to this proposed transient
edgewise insertion of the anthraquinone moiety into the DNA
minor groove, the N-substituted site of the anthraquinone
moiety and C26 are expected to point toward the rim of
DNA’s minor groove (blue region in Figure 2), while the
Figure 2. Presumed mechanism of DNA binding and cleavage by
hydroxy-substituted site of the anthraquinone moiety should
anthraquinone-fused enediynes. Only structural features shared by all
face toward the floor of the minor groove (green region in
anthraquinone-fused enediynes are depicted. The blue colored region
of the molecule is presumed to face toward the rim of the DNA minor
groove, while the green colored region is anticipated to face toward
the floor of the minor groove during binding and cleavage.
Figure 2).13c,d
Based on the above scenario, several notable structure−
activity relationships (SARs) might be anticipated for this class
of compounds: (a) The electronic nature of substituents on
ring A could influence the redox potential of the anthraquinone
moiety, and thus affect the initiation of the cascade that leads
to DNA damage. (b) In parallel, substituents at C6/C7 and
C8/C9 could potentially alter the equilibrium between the
intercalated and triggerable (i.e., minor groove inserted) states
of the enediyne molecule through, for example, favorable
interactions with the DNA deoxyribose phosphate backbone,
or by exploiting the hydrogen-bonding potential of the DNA’s
B
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Figure 3. Designed and synthesized tiancimycin B and uncialamycin analogues. Targeted analogues 25, 37, and 38 were not reached; instead,
analogues 24, 37′, and 38′ were obtained. Fmoc = 9-fluorenylmethyloxycarbonyl; Boc = tert-butoxycarbonyl.
minor groove floor, respectively. (c) As previously indicated by
studies on dynemicin A models,14 the oxidation state/
substitution of the carbons adjacent to the epoxide ring (i.e.,
C17 and C26) might influence the biological activity since they
could favor or hinder the consequential opening of this moiety.
(d) In either the intercalation9,13a or insertion−intercalation13c
mode of binding, uncharged substituents at C26 are not
anticipated to interfere with the molecule’s interaction with
DNA, and thus might offer an alternative favorable site for
linking to antibodies or other delivery systems.
naturally occurring yangpumicin A (6, Figure 1) and
tiancimycins A and B (7 and 8, Figure 1); (b) the syntheses
of related anthraquinone-fused enediyne antibiotics possessing
the core framework of either tiancimycin B (i.e., analogues
16−25, Figure 3) or uncialamycin (i.e., analogues 26−38′,
Figure 3) and different substituents on the ring A and positions
C26 or C17, including fluorine residues, with interesting
chemical and biological consequences; and (c) the results of
the biological evaluation of a select number of the synthesized
compounds against a series of cancer and related cell lines.
Our prior endeavors toward the total synthesis of unciala-
mycin (3)6 resulted in the development of an efficient and
flexible synthetic route that provided access not only to
uncialamycin itself but also to related analogues.6c Aiming to
enrich the SARs within this class of compounds, and in order
to facilitate further exploration of the potential of anthra-
quinone-fused enediyne antibiotics as payloads for ADCs, we
initiated further studies within this field of investigation.
Herein we report the application of our developed synthetic
strategies and technologies to (a) the total syntheses of the
2. RESULTS AND DISCUSSION
2.1. Total Synthesis of Yangpumicin A (6), Tiancimy-
cins A (7) and B (8), and Analogues 16−38′. According to
our established approach toward uncialamycin,6 the synthesis
of the targeted anthraquinone-fused enediyne antibiotics
bearing varying A ring substituents (i.e., 23−25, and 29−37
and 38, Figure 3; general structure VII, Figure 4), including
the naturally occurring yangpumicin A (6, Figure 1) and
tiancimycins A and B (7 and 8, Figure 1), was projected to rely
C
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[(COCl)2] to excess diethylamine. Subsequent protection of
the phenol moiety as methoxymethyl (MOM) ether
compounds 43a (93% yield) and 43b (98% yield),
respectively, set the stage for the regioselective ortho-lithiation
of the aromatic ring (n-BuLi) of the resulting intermediates,
which, upon quenching with dimethylformamide, provided the
corresponding formyl derivatives 44a (81% yield) and 44b
(85% yield), respectively. Their exposure to TMSCN, in the
presence of catalytic amounts of KCN and 18-crown-6, and
then to AcOH secured the targeted substituted cyano-
phthalides 45a (93% yield) and 45b (97% yield), respectively,
as shown in Scheme 1.
Annulation of MOM-protected cyanophthalides 45a and
45b with the readily obtainable6a,c p-methoxy-semiquinone
aminal (+)-39 (Scheme 2) with KHMDS led to the desired
Figure 4. General retrosynthetic analysis of targeted anthraquinone
antitumor antibiotics and analogues thereof. TES = triethylsilyl, Alloc
= allyloxycarbonyl.
Scheme 2. Total Synthesis of Yangpumicin A (6) and
a
Tiancimycin A (7)
on the availability of the appropriately substituted 3-cyano-
phthalides IX (Figure 4) and their successful fusion via a
Hauser−Kraus-type annulation,15 with the Alloc-protected p-
methoxy semiquinone aminal [(+)-39,6a,c Figure 4]. This key
building block can be secured on large scale and in
enantiomerically pure form from hydroxyisatin (40, Figure
4) as previously described.6a,c The synthesis of tiancimycin B
(8, Figure 1) and related targeted analogues (i.e., 16−25,
Figure 3; general structure VII, Figure 4) was expected to rely
on selective oxidation of the C26 hydroxyl group of the
corresponding anthraquinone-fused enediynes (VIII, Figure 4)
and subsequent Horner−Wadsworth−Emmons (HWE) olefi-
nation16 of the resulting ketone.
Guided by the above retrosynthetic analysis, synthetic efforts
were initially focused on the preparation of the appropriately
substituted 3-cyanophthalides required for the synthesis of
yangpumicin A (6) and tiancimycin A (7, Figure 1). Thus,
commercially available 3-hydroxybenzoic acid (41a) and
isovanillic acid (41b, Scheme 1) were converted to the
corresponding diethyl amides 42a (93% yield) and 42b (93%
yield), respectively, through exposure of their acid chlorides
a
a
Reagents and conditions: (a) 45a or 45b (3.0 equiv), KHMDS (4.0
Scheme 1. Synthesis of Cyanophthalides 45a and 45b
equiv), THF, −78 °C, 20 min; then (+)-39 (1.0 equiv), −78 to 0 °C,
1.5 h; then 25 °C, 2.5 h (for 46a), or (+)-39 (1.0 equiv), −78 °C, 15
min; then 0 °C, 2 h (for 46b); (b) Pd(PPh3)4 (0.07 equiv),
morpholine (2.6 equiv), THF, 0 to 25 °C, 2.3−2.5 h, 47a or 47b
(49% or 59% yield over two steps, respectively); (c) MgBr2·Et2O (3.0
equiv), THF, 0 °C, 3 h; then 25 °C, 15 min, 48a (89%) or 0 to 25 °C,
1.75 h, 48b (88%); (d) 3HF·Et3N (150 equiv), THF, 25 °C, 1 h, 6
(86%) or 7 (97%). KHMDS = potassium hexamethyldisilazide.
anthraquinones 46a and 46b, respectively, as shown in Scheme
2. The so-obtained fused products were more conveniently
purified and characterized upon cleavage of the Alloc
protecting group, by treatment of the corresponding crude
annulation products (46a, 46b) with catalytic amounts of
Pd(PPh3)4 in the presence of morpholine. Thus, products 47a
and 47b were obtained in good overall yield for the two steps
[49% and 59%, respectively, based on (+)-39]. Finally,
sequential removal of the MOM and TES protecting groups,
by exposure to, initially, MgBr·Et2O and then 3HF·Et3N,
furnished yangpumicin A (6) and tiancimycin A (7) in 77%
and 85% overall yield from 47a and 47b, respectively, as
summarized in Scheme 2.
a
Reagents and conditions: (a) (COCl)2 (2.6 equiv), DMF (cat.),
CH2Cl2, 0 to 25 °C, 12 h; then Et2NH (4.6 equiv), THF, 0 to 25 °C,
12 h, 42a (93%), 42b (93%); (b) i-Pr2NEt (3.0 equiv), MOMCl (3.0
equiv), CH2Cl2, 25 °C, 2−5 h, 43a (93%), 43b (98%); (c) TMEDA
(1.1 equiv), n-BuLi (1.1 equiv), THF, −78 °C, 40 min; then DMF
(1.2 equiv), −78 °C, 40−60 min, 44a (81%), 44b (85%); (d)
TMSCN (1.3 equiv), KCN (0.2 equiv), 18-crown-6 (0.2 equiv),
CH2Cl2, 0 °C, 4.5−5 h; then AcOH, 25 °C, 13−16 h, 45a (93%), 45b
(97%). MOMCl = methoxymethyl chloride; TMEDA = tetramethyl-
ethylenediamine; TMSCN = trimethylsilyl cyanide.
D
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Seeking to secure a synthetic route to tiancimycin B (8)
requiring the minimum protecting group manipulations, the
TES-protected uncialamycin 496c (Scheme 3) was exploited as
Further oxidation of analogue 27 with Dess−Martin period-
inane led to the diketo analogue 28 (89% yield), as shown in
Scheme 3.
Advanced synthetic intermediate 536c (Scheme 4) was
employed as starting material for the synthesis of tiancimycin B
analogues 17−22 and uncialamycin carbamate derivative 29 as
summarized in Scheme 4. Thus, for the synthesis of analogue
29, the phenolic group and C17 hydroxyl moiety of 53 were
sequentially and selectively protected as methoxymethyl
(MOM) ether and acetate, respectively, through treatment,
first with MOMCl in the presence of i-Pr2NEt, and then with
Ac2O and catalytic amounts of DMAP, to afford the
corresponding fully protected Alloc derivative (54, 75% yield
over two steps). The latter was subjected to TES-ether
deprotection (3HF·Et3N) to yield the expected hydroxyl
intermediate and subsequent reaction of this alcohol with
excess trichloroacetylisocyanate (Cl3CCONCO), followed by
concurrent quenching of excess reagent and methanolysis of
the acetate protecting group with K2CO3 in MeOH, furnished
carbamate 55 (72% yield over two steps). Uncialamycin
analogue 29 was obtained from the latter upon sequential
removal of the Alloc [Pd(PPh3)4, 96% yield] and MOM
(MgBr2·Et2O, 87% yield) protecting groups under standard
conditions, as shown in Scheme 4.
a
Scheme 3. Total Synthesis of Tiancimycin B (8)
For the synthesis of tiancimycin B analogues 17−22, the
same intermediate 53 (see Scheme 4) was subjected, as above,
to MOM and acetate protection steps, followed this time by
cleavage of the Alloc protecting group to provide intermediate
56, in 74% overall yield. Subsequent deprotection of the C26
alcohol within the latter (3HF·Et3N), followed by oxidation
with Dess−Martin periodinane delivered ketone 57 in 86%
overall yield, setting the stage for the pending HWE
olefination. Gratifyingly, the latter transformation proceeded,
in this case, with exquisite stereoselectivity (compare 27 → 8 +
16, Scheme 3 where the HWE reaction was not as exclusive),
furnishing the targeted tiancimycin B analogue 17 as the sole
product in 81% yield, as shown in Scheme 4. Treatment of the
latter with LiOH in THF/H2O (3:1, v/v) cleaved both the
acetate protecting group and the methyl ester moiety,
furnishing the MOM-protected tiancimycin B acid derivative
18 in 87% yield. The targeted free tiancimycin B acid (19) was
obtained, in quantitative yield, from 18 upon treatment with
MgBr2·Et2O. Its primary amide analogue (20) was secured by
treatment with a 0.4 M solution of NH3 in THF in the
presence of EDCI·HCl, HOAt and i-Pr2NEt (19 → 20, 74%
yield) as shown in Scheme 4. To explore the feasibility of
employing its carboxylic acid moiety as a linking functionality
to construct linker-drugs and ADCs, analogue 19 was coupled
with Fmoc-monoprotected 1,2-diaminoethane to yield amide
21 (74% yield), from which the targeted primary amine
derivative 22 was liberated upon treatment with Et2NH in
DMSO in quantitative yield as depicted in Scheme 4.
a
Reagents and conditions: (a) i-Pr2NEt (2.0 equiv), DMAP (0.1
equiv), Ac2O (2.0 equiv), CH2Cl2, 0 °C, 10 min, quant.; (b) 3HF·
Et3N (150 equiv), THF, 25 °C, 2 h, 85%; (c) DMP (2.5 equiv),
CH2Cl2, 0 to 25 °C, 3 h, 89%; (d) K2CO3 (1.0 equiv), MeOH, 0 °C,
1.5 h, 96%; (e) DMP (2.5 equiv), CH2Cl2, 0 to 25 °C, 3 h, 89% (f)
(EtO)2POCH2COOMe (11 equiv), NaH (10.0 equiv), THF, −78
°C, 15 min; then 27, −78 to 0 °C, 5 h, 84% (plus 10% of 16). DMAP
= 4-dimethylaminopyridine, DMP = Dess−Martin periodinane.
starting material. Thus, starting with 49, a sequence involving
selective acetylation of the propargylic hydroxyl group (i-
Pr2NEt, DMAP, Ac2O, 50, quant.) of the molecule, followed
by cleavage of the TES protecting group (3HF·Et3N, 85%
yield) provided the targeted uncialamycin analogue 26.
Selective oxidation of the secondary alcohol of the latter in
the presence of the free phenolic group to the corresponding
ketone (51, 89% yield) by Dess−Martin periodinane, and
finally careful methanolysis of the propargylic acetate (K2CO3,
96% yield) secured the targeted methyl ketone 27 as depicted
in Scheme 3. HWE olefination of ketone 27 with 52
[(EtO)2POCH2COOMe, NaH] provided, after chromato-
graphic purification, not only tiancimycin B [8, (E)-isomer,
major product, 84% yield] but also the (Z)-isomer (16, minor
product, ∼10% yield) as an analogue of the natural product.
Our experience with uncialamycin analogues revealed that
introduction of a methylaminomethyl substitution at C8 (ring
A, see structures 30, 23, and 24, Scheme 5) not only provides
an anchoring point for the construction of ADCs but also leads
to derivatives of increased potency.6c In order to investigate
whether such substitution might have similar effects on
tiancimycin B-related analogues, derivatives 23−25 and 30
(see Figure 2) were targeted for synthesis as shown in Scheme
5. Their pursuit started from the known annulation product6c
(structure not shown) of cyanophthalide 596c and p-
methoxysemiquinone aminal (+)-39.6a,c Thus, and as shown
E
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a
Scheme 4. Synthesis of Analogues 29 and 17−22
Scheme 4. continued
i-Pr2NEt (5.0 equiv), CH2Cl2, 0 to 25 °C, 1 h, 74%; (p) EDCI·HCl
(1.5 equiv), HOAt (1.5 equiv), NH2CH2CH2NHFmoc (1.3 equiv), i-
Pr2NEt (5.0 equiv), CH2Cl2, 0 to 25 °C, 1 h, 74%; (q) Et2NH,
DMSO, 25 °C, 15 min, quant. EDCI·HCl = N3-(ethylcarbonimidoyl)-
N1,N1-dimethyl-1,3-propanediamine hydrochloride, HOAt = 1-
hydroxy-7-azabenzotriazole.
in Scheme 5, the resulting annulation product was first
acetylated under the standard conditions (66% overall yield for
the two steps) and then desilylated (3HF·Et3N, 83% yield) to
afford hydroxy acetate 61, via intermediate 60, as summarized
in Scheme 5. Selective oxidation of alcohol 61 with DMP led
to the triprotected analogue 62 in 86% yield. The latter
precursor was converted to the targeted analogue 30 by
sequential removal of the acetate (K2CO3, 81% yield) and
Alloc [Pd(PPh3)4, morpholine, 71% yield] protecting groups.
Alternatively, precursor 62 was employed as a starting material
in an HWE olefination with 52 [(EtO)2POCH2COOMe,
NaH] to produce, exclusively, the desired (E)-α,β-unsaturated
methyl ester 64 in 82% yield. Exposure of 64 to LiOH induced
concurrent cleavage of both the methyl ester and the acetyl
group, leading to Alloc-protected hydroxy acid 65 (89% yield),
from which the targeted tiancimycin analogue 23 was
generated, in 78% yield, by removal of the Alloc protecting
group through the action of Pd(PPh3)4 and morpholine.
Carboxylic acid 65 was also successfully coupled with Fmoc-
monoprotected 1,2-diaminoethane (58) in the presence of
EDCI·HCl and HOAt to afford bis-protected amide 24 (80%
yield). Interestingly, attempts to generate the targeted free
primary amine analogue of 24 (i.e., 25 in Figure 2) under the
standard Fmoc-deprotection conditions failed, in contrast to
the results obtained in Scheme 4 (cf. 21 → 22).
Substitution of a hydrogen atom with a fluorine residue
within a molecule, especially in the vicinity of a basic functional
group, can lead to potential drug candidates with improved
pharmacokinetic profiles.17 Furthermore, polar interactions
between a fluoro-substituted ligand and its biological receptor
can contribute to increased binding affinity, and thus potency,
as compared to the affinity of its unsubstituted version.18
These potentially beneficial effects prompted us to design and
target of a series of fluoro-substituted uncialamycin analogues
(31−36, Figure 2). The corresponding prerequisite fluoro-3-
cyanophthalides were synthesized as shown in Schemes 6 (69,
70, 75a, and 75b) and 7 (82, 83, 88a, and 88b). Thus, the
carbonyl moiety of commercially available 4-bromo-3-
fluorobenzaldehyde (66, Scheme 6) was protected as an acetal
with ethylene glycol (99% yield) and the resulting product was
lithiated (LDA) regioselectively and reacted with methyl
chloroformate to give aryl methyl ester bromide 67 in 79%
yield. This versatile intermediate was utilized as precursor to
required cyanophthalides (69, 70, 75a, and 75b), as depicted
in Scheme 6. Thus, Buchwald−Hartwig coupling19 [Pd-
(OAc)2, Xphos cat., Cs2CO3] of aryl bromide 67 with tert-
butyl carbamate (BocNH2, 97% yield), followed by acid-
induced hydrolysis (PTSA, 78% yield) of the acetal moiety
furnished o-formyl methyl benzoate derivative 68 as shown in
Scheme 6 (center). Conversion of the latter to cyanophthalide
69 was achieved by exposure, first to TMSCN, KCN cat., 18-
crown-6 cat., and then AcOH, in 60% overall yield as depicted
in Scheme 6. Treatment of 69 with montmorillonite K 10
(MK10),20 a mild reagent to remove the Boc group, allowed
a
Reagents and conditions: (a) i-Pr2NEt (3.0 equiv), MOMCl (3.0
equiv), CH2Cl2, 25 °C, 12 h; (b) i-Pr2NEt (2.0 equiv), DMAP (0.1
equiv), Ac2O (2.0 equiv), CH2Cl2, 0 °C, 0.5 h, 75% (over two steps);
(c) 3HF·Et3N (150 equiv), THF, 25 °C, 2 h; (d) Cl3CCONCO (2.5
equiv), ClCH2CH2Cl, 0 °C, 5 h; then K2CO3 (1.3 equiv), MeOH, 0
to 25 °C, 12 h, 72% (over two steps); (e) Pd(PPh3)4 (0.08 equiv),
morpholine (2.6 equiv), THF, 0 to 25 °C, 2.5 h, 96%; (f) MgBr2·Et2O
(5.0 equiv), THF, 0 to 25 °C, 12 h, 87%; (g) i-Pr2NEt (3.0 equiv),
MOMCl (3.0 equiv), CH2Cl2, 25 °C, 12 h; (h) i-Pr2NEt (2.0 equiv),
DMAP (0.1 equiv), Ac2O (2.0 equiv), CH2Cl2, 0 °C, 0.5 h; (i)
Pd(PPh3)4 (0.08 equiv), morpholine (2.6 equiv), THF, 0 to 25 °C,
2.5 h, 74% (over three steps); (j) 3HF·Et3N (150 equiv), THF, 25
°C, 2 h; (k) DMP (2.5 equiv), CH2Cl2, 0 to 25 °C, 12 h, 86% for the
two steps; (l) (EtO)2POCH2COOMe (6.0 equiv), NaH (5.0 equiv),
THF, −78 °C, 15 min; then 57, −78 to 0 °C, 5 h, 81%; (m) LiOH
(10.0 equiv), THF/H2O (3:1, v/v), 0 to 25 °C, 12 h, 87%; (n)
MgBr2·Et2O (5.0 equiv), THF, 0 to 25 °C, 12 h, quant; (o) EDCI·
HCl (1.5 equiv), HOAt (1.5 equiv), 0.4 M NH3 in THF (1.3 equiv),
F
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a
Scheme 5. Synthesis of Analogues 23, 24, and 30
Scheme 6. Synthesis of 7-Fluoro-3-Cyanophthalides 69, 70,
75a, and 75b
a
a
Reagents and conditions: (a) ethylene glycol (excess), PTSA (0.5
equiv), toluene, reflux, 12 h, 99%; (b) LDA (1.1 equiv), THF, −78
°C, 1 h; then ClCO2Me (1.1 equiv), −78 °C, 1 h, 79%; (c) BocNH2
(1.2 equiv), Pd(OAc)2 (3 mol%), XPhos (9 mol%), Cs2CO3 (1.4
equiv), dioxane, 100 °C, 8 h, 97%; (d) PTSA (0.5 equiv), acetone, 25
°C, 1 h, 78%; (e) TMSCN (2.0 equiv), KCN (0.1 equiv), 18-crown-6
(0.1 equiv), CH2Cl2, 0 to 25 °C, 1 h; then AcOH, 80 °C, 24 h, 60%;
(f) MK10, DCE, reflux, 3 h, 98%; (g) pinacol vinylboronate (71, 1.1
equiv), Pd(OAc)2 (5 mol%), Cs2CO3 (2.0 equiv), PPh3 (0.2 equiv),
1,4-dioxane, 70 °C, 12 h, 91%; (h) O3, CH2Cl2, −78 °C; then Me2S
(5.0 equiv), 25 °C, 1 h, 81%; (i) MeNH2 (1.0 equiv), CF3CH2OH, 25
°C, 5 min; then NaBH4 (1.2 equiv), 25 °C, 5 min; then NaHCO3 (2.0
equiv), AllocCl (1.5 equiv), THF/H2O (1:1, v/v), 25 °C, 40 min; (j)
HCl (4 N, 15 equiv), THF, 25 °C, 4 h, 74a (64%) or 74b (44% over
two steps, respectively); (k) TMSCN (2.0 equiv), KCN (0.1 equiv),
18-crown-6 (0.1 equiv), CH2Cl2, 0 to 25 °C, 4 h; then PTSA (0.5
equiv), AcOH, 80 °C, 12 h, 75a (56%) or 75b (66%); (l) allyl alcohol
(1.1 equiv), Pd(dba)2 (2 mol%), 2-(di-tert-butylphosphino)-1-
phenylindole (76, 6 mol%), Cy2NMe (1.1 equiv), DMF, 100 °C, 1
h, 78%. PTSA = p-toluenesulfonic acid, LDA = lithium diisopropy-
lamide, XPhos = 2-dicyclohexyl-phosphino-2′,4′,6′-triisopropylbi-
phenyl, MK10 = montmorillonite K 10, DCE = 1,2-dichloroethane.
a
Reagents and conditions: (a) 59 (3.0 equiv), LiHMDS (4.0 equiv),
THF, −78 °C, 20 min; then (+)-39 (1.0 equiv), −78 to 25 °C, 1 h;
(b) i-Pr2NEt (2.0 equiv), DMAP(0.1 equiv), Ac2O (2.0 equiv),
CH2Cl2, 0 °C, 10 min, 66% (over two steps); (c) 3HF·Et3N (150
equiv), THF, 25 °C, 2 h, 83%; (d) DMP (2.5 equiv), CH2Cl2, 0 to 25
°C, 3 h, 86%; (e) K2CO3 (1.0 equiv), MeOH, 0 °C, 1.5 h, 81%; (f)
Pd(PPh3)4 (0.2 equiv), morpholine (5.2 equiv), DMF, 0 to 25 °C, 2.5
h, 71%; (g) (EtO)2POCH2COOMe (6.0 equiv), NaH (5.0 equiv),
THF, −78 to 0 °C, 5 h, 82%; (h) LiOH (10.0 equiv), THF/H2O
(3:1, v/v), 0 to 25 °C, 12 h, 89%; (i) Pd(PPh3)4 (0.2 equiv),
morpholine (5.2 equiv), DMF, 0 to 25 °C, 12 h, 78%; (j) EDCI·HCl
(1.5 equiv), HOAt (1.5 equiv), 58 (1.3 equiv), i-Pr2NEt (5.0 equiv),
CH2Cl2, 0 to 25 °C, 1 h, 80%. LiHMDS = lithium hexamethyldi-
silazide.
Scheme 6 (right). The latter was transformed to the desired
product through a sequence involving (i) ozonolysis to afford
aldehyde 73a (81% yield); (ii) reductive amination with of 73a
MeNH2 and NaBH4; (iii) Alloc protection of the resulting
amine, followed by HCl-induced hydrolysis of the acetal
protecting group leading to aldehyde methyl ester 74a (64%
yield for the two steps); and finally (iv) TMSCN/PTSA-
facilitated cyclization (TMSCN, PPTS, 56% yield) to generate
the targeted cyanophthalide 75a. Required cyanophathalide
75b was constructed from intermediate aryl bromide 67 (see
Scheme 6, top center) through a sequence involving: (i) Heck
coupling22 with allyl alcohol in the presence of Pd(dba)2 and
phosphine ligand 76 to give aldehyde 73b (78% yield; note
the generation of the required free amino cyanophthalide 70,
in 98% yield. For the preparation of cyanophthlide 75a,
common precursor aryl bromide 67 (Scheme 6) was subjected
to Suzuki coupling21 [Pd(OAc)2] with pinacol vinylborane
(71) to give aryl vinyl compound 72 (91% yield) as shown in
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(iii) ring closure to form the cyanolactone ring as facilitated by
TMSCN and PTSA (66% yield) to afford the targeted
cyanophathalide building block (75b) as shown in Scheme 6.
For the preparation of the remaining required cyano-
phthalides (82, 83, 88a, and 88b), commercially available 2-
amino-4-fluoro-5-bromobenzoic acid (77) was employed as
starting material as depicted in Scheme 7. Thus, aryl amine 77
was converted to its iodo counterpart by sequential treatment
with NaNO2, HCl and KI, followed by methyl ester formation
(H2SO4, MeOH) to afford iodo-bromo-fluoro methyl ester 78
(82% yield over two steps). The latter was reacted with pinacol
vinylboronate (71) in a Suzuki coupling21 [Pd(OAc)2, 92%
yield] to give vinyl bromoaryl derivative 79, whose Buchwald−
Hartwig coupling19 with BocNH2 under standard conditions
furnished Boc-protected aniline derivative 80 (95% yield) as
shown in Scheme 7. Ozonolysis of the vinyl group within the
latter (O3; Me2S, 79% yield), followed by ring closure of the
resulting aldehyde methyl ester (TMSCN, KCN cat., 18-
crown-6; AcOH) led to the desired fluoro cyanophthalide 82
(65% yield) and, upon removal of the Boc group from the
latter (MK10, 98% yield), cyanophthalide 83, as summarized
in Scheme 7 (left). Through a second pathway, intermediate
78 was converted to common intermediate 85, via 84
[ozonolysis (78% yield); followed by acetal formation, 85
(95% yield), Scheme 7, right], which was diverted through
different routes to targeted cyanophthalides 88a (left) and 88b
(right), as summarized in Scheme 7. Thus, the sequence
toward cyanophthalide 88a proceeded from 85 through Suzuki
coupling with pinacol vinylboronate (71) under the standard
conditions [Pd(PPh3)4; 94% yield], followed by ozonolysis to
afford fluoro aldehyde 86a (82% yield), whose reductive
amination with MeNH2, Alloc protection, and acetal hydrolysis
led to aryl aldehyde methyl ester 87a (81% overall yield from
86a), as shown in Scheme 7. Finally, cyanophthalide 88a was
obtained from aldehyde methyl ester 87a through the standard
TMSCN/PTSA protocol, in 72% overall yield as summarized
in Scheme 7. The two-carbon extended homologue of the
latter, cyanophthalide 88b, was synthesized in a similar manner
from common intermediate 85 as depicted in Scheme 7. Thus,
reaction of 85 with allyl alcohol in the presence of Pd(dba)2
and phosphine ligand 76 furnished aldehyde 86b (84% yield),
whose conversion to the targeted cyanophthalide 88b
proceeded through intermediate 87b under the same
conditions, and in similar yields, as for the conversion of 86a
to 88a via 87a, as shown in Scheme 7.
With the prerequisite fluorocyanophthalides fragments in
hand, their Hauser−Kraus-type annulation15 onto the Alloc-
protected p-methoxy semiquinone aminal (+)-39 under the
conditions previously established [i.e., LiHMDS, THF; then
(+)-39, −78 to 25 °C],6,15e followed by either sequential
removal of first the Alloc and then TES protecting groups
(Procedure a, Scheme 8) or first removal of the TES protecting
group and then concurrent global deprotection of both the
aniline and amino functional groups (Procedure b, Scheme 8),
provided fluorinated uncialamycin analogues 31−36, respec-
tively, as summarized in Scheme 8.
Interestingly, in the cases of targeted analogues 37 and 38,
our attempts to cleave the Alloc group and liberate the
methylamino group in the last step (Procedure b, Scheme 8)
resulted in exclusive formation of compounds 37′ and 38′,
respectively, apparently through an intramolecular displace-
ment of the fluorine residue by the generated methylamino
group under the reaction conditions. This reaction is
Scheme 7. Synthesis of 5-Fluoro-3-cyanophthalides 82, 83,
88a, and 88b
a
a
Reagents and conditions: (a) NaNO2 (1.2 equiv), HCl (conc., 10.0
equiv), H2O, 0 °C, 0.5 h; then KI (1.5 equiv), 0 °C, 1 h; then 90 °C,
0.5 h; (b) H2SO4 (2.0 equiv), MeOH, reflux, 12 h, 82% (over two
steps); (c) pinacol vinylboronate (71, 1.1 equiv), Pd(OAc)2 (5 mol
%), Cs2CO3 (2.0 equiv), PPh3 (0.2 equiv), 1,4-dioxane, 70 °C, 12 h,
92%; (d) BocNH2 (1.2 equiv), Pd(OAc)2 (3 mol%), XPhos (9 mol
%), Cs2CO3 (1.4 equiv), dioxane, 100 °C, 8 h, 95%; (e) O3, CH2Cl2,
−78 °C; then Me2S (5.0 equiv), 25 °C, 1 h, 81 (79%), 84 (78%) or
86a (82%); (f) TMSCN (2.0 equiv), KCN (0.1 equiv), 18-crown-6
(0.1 equiv), CH2Cl2, 0 to 25 °C, 1 h; then AcOH, 80 °C, 24 h, 65%;
(g) MK10, DCE, reflux, 3 h, 98%; (h) 1,2-bis(trimethylsilyloxy)-
ethane [(TMSOCH2)2, 2.0 equiv], TMSOTf (0.1 equiv), CH2Cl2, 0
to 25 °C, 4 h, 95%; (i) pinacol vinylboronate (71, 1.1 equiv),
Pd(OAc)2 (5 mol%), Cs2CO3 (2.0 equiv), PPh3 (0.2 equiv), 1,4-
dioxane, 80 °C, 18 h, 94%; (j) MeNH2 (1.0 equiv), CF3CH2OH, 25
°C, 5 min; then NaBH4 (1.2 equiv), 25 °C, 5 min; then NaHCO3 (2.0
equiv), AllocCl (1.5 equiv), THF/H2O (1:1, v/v), 25 °C, 40 min; (k)
HCl (4 N, 15 equiv), THF, 25 °C, 4 h, 87a (81%) or 87b (46% yield
over two steps, respectively); (l) TMSCN (2.0 equiv), KCN (0.1
equiv), 18-crown-6 (0.1 equiv), CH2Cl2, 0 to 25 °C, 4 h; then PTSA
(0.5 equiv), AcOH, 80 °C, 12 h, 88a (72%) or 88b (74%); (m) allyl
alcohol (1.1 equiv), Pd(dba)2 (2 mol%), 2-(di-tert-butylphosphino)-
1-phenylindole (76, 6 mol%), Cy2NMe (1.1 equiv), DMF, 100 °C, 1
h, 84%.
concomitant isomerization/tautomerization of the allylic
alcohol to the aldehyde moiety)23 as shown in Scheme 6
(left); (ii) reductive amination of 73b with MeNH2 (NaBH4)
followed by Alloc protection (AllocCl) and acetal cleavage (aq.
HCl) to form aldehyde 74b (44% yield over two steps); and
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a
Scheme 8. Synthesis of Fluorinated Analogues 31−36, 37′, and 38′
a
Reagents and conditions: A. General scheme for the preparation of analogues 31−36, 37′ and 38′ from building blocks 69, 70, 82, 83, 75a, 75b,
88a, and 88b. B. Individual syntheses of fluorinated uncialamycin analogues 31−36, 37′ and 38′ through procedures a and b. Procedure a: (a)
LiHMDS (4.0 equiv), THF, −78 to 25 °C, 12 h; (b) Pd(PPh3)4 (0.1 equiv), morpholine (3.0 equiv), THF, 0 to 25 °C, 2 h; (c) 3HF·Et3N (30.0
equiv), THF, 0 to 25 °C, 1.5 h. Procedure b: (a) LiHMDS (4.0 equiv), THF, −78 to 25 °C, 24 h; (b) 3HF·Et3N (30.0 equiv), DMF, 0 to 25 °C, 4
h; (c) Pd(PPh3)4 (0.1 equiv), morpholine (3.0 equiv), DMF, 0 to 25 °C, 12 h.
presumably facilitated by activation of the fluoride residue
through the electron-withdrawing effect of the conjugated
carbonyl group on one hand, and the proximity effect of the
amino group to the fluoride residue coupled with the
formation of the six-membered ring of 37′ and 38′ on the
other hand, as depicted in Scheme 8. The presumed
mechanism for the conversion of 37 and 38 to 37′ and 38′,
respectively, is shown in Scheme 8B (bottom center). Needless
to say, these serendipitous reactions leading to the unexpected
and novel analogues 36′ and 37′ were of interest with regards
to their biological profiles due to their unprecedented
structures.
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2.2. Biological Evaluation of Synthesized Com-
pounds. The synthesized anthraquinone-fused enediynes
(natural products and designed analogues) were evaluated
for their ability to inhibit the proliferation of the following cell
lines: human embryonic kidney (HEK 293T), multidrug-
resistant uterus sarcoma (MES SA/DX), and multidrug-
resistant uterine sarcoma cell line with PGP inhibitor elacridar
(MES SA/DXE) (Table 1), breast cancer cell line SK-BR-3,
Table 2. In Vitro Cytotoxicity of Select Synthesized
Anthraquinone-Fused Enediynes
a
Table 1. In Vitro Cytotoxicity of Synthesized
a
Anthraquinone-Fused Enediynes
a
For details of the biological assays, see Supporting Information.
b
c
d
Breast cancer cell line. Ovarian cancer cell line. Cervical cancer cell
e
line. N-Acetyl calicheamicin γI1.
(MeNHCH2)-3: 6 pM (HEK293T); 15 pM (MES SA/DX); 4
pM (MES SA/DXE); 25 pM (SKBR3), 10 pM (SKOV3), 1.2
nM (HeLa)]. Furthermore, introduction of a single fluorine
atom in the ortho position to the secondary benzylamine
handle (cf. 35 and 36) led to single digit picomolar cytotoxic
activities in five out of the six cell lines evaluated. In
comparison, fluoro aniline analogues 32 and 34 (Figure 3)
were found to be 20−100-fold less cytotoxic as compared to
their corresponding analogues 35 and 36. These results also
demonstrate that the combination of the potency enhancing
effects of the C8-methylaminomethyl moiety and the fluorine
residue at C7 or C9 of the molecule within the structures of 35
and 36 was apparently responsible for the extreme potencies
exhibited by these analogues.
Interestingly, introduction of the potential linker attachment
handles on the opposite side of the molecule [compounds 16
(carboxylic acid moiety derived from methyl ester) and 22
(primary amino group), Figure 3] resulted in a significant
decrease in cytotoxicity (low nanomolar IC50 values) and
significant increase in PGP efflux susceptibility (see Table 1).
Compound 19 (Figure 3) has shown no apparent
cytotoxicity up to the highest concentration tested (100
nM), most likely due to the presence of the free carboxylic acid
moiety and poor cell permeability. Similarly, compound 21
(Figure 3), that still contained an Fmoc protecting group, has
shown no activity.
a
For details of the biological assays, see Supporting Information.
b
c
Immortalized human embryonic kidney cell line. Multidrug-
d
resistant uterine sarcoma cell line. Multidrug-resistant uterine
e
sarcoma sell line with PGP inhibitor elacridar. N-Acetyl calichea-
micin γI1.
ovarian cancer cell line SKOV3, and cervical cancer cell line
HeLa (Table 2). Uncialamycin (3) and N-acetyl calicheamicin
γI1 were tested in parallel for direct comparison. Unlike N-
acetyl calicheamicin γI1, uncialamycin (3) is not a good
substrate for PGP and is equally cytotoxic to MES SA/DX and
MES SA/DXE cells. This multidrug pump resistance activity
has been preserved among the other most active analogues
[i.e., yangpumicin A (6), tiancimycin A (7), and analogues 27,
30, 35 and 36, Table 1 and Figure 3]. The majority of the
other designed analogues have demonstrated low nanomolar to
sub-nanomolar IC50 values.
Notably, introduction of a secondary benzylic amine [i.e., 8-
(MeNHCH2)-3] as a potential handle for ADC linker
attachment resulted in a compound with cytotoxic properties
very similar or even improved as compared to the parent
natural product in the tested cell lines [IC50 values for 8-
Novel analogues 37′ and 38′ showed differentiated cytotoxic
activities, with compound 38′ being more potent and closely
resembling the cytotoxic activity of uncialamycin (3), albeit
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being more susceptible to PGP efflux, while compound 37′
displayed low nanomolar potencies in five out of six cell lines
evaluated.
Overall, cytotoxicity evaluation of the synthetic anthra-
quinone-fused enediynes have shown distinct SARs. Chemical
modifications of the uncialamycin core allow for selective
modulation of cytotoxic activity from low picomolar to high
nanomolar IC50 values and also tune the analogue’s
susceptibility to PGP efflux.
evaluating payloads, sometimes it might be desirable to
sacrifice potency in order to avoid unwanted side-effects
caused by leakage of extremely potent payloads. The scalable
reported synthetic strategies and technologies are destined to
facilitate further studies in this relatively new oncology
paradigm.2e
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b12522.
3. CONCLUSION
Applying our streamlined synthetic strategies and methods, we
synthesized the newly discovered uncialamycin congeners,
tiancimycins A (7) and B (8), and yangpumicin A (6) and a
series of novel designed analogues of these anthraquinone-type
enediyne antitumor antibiotics. Biological evaluation of the
synthesized natural and designed compounds revealed
interesting and useful structure−activity relationships (see
Figure 5B) and led to the discovery of extremely potent
Experimental procedures and characterization data for
all compounds; cytotoxicity data (HEK 293T, MES SA
DX, and MES SA DXE) (PDF)
AUTHOR INFORMATION
Corresponding Author
■
K. C. Nicolaou − Department of Chemistry, BioScience Research
Collaborative, Rice University, Houston, Texas 77005, United
States; orcid.org/0000-0001-5332-2511; Email: kcn@
rice.edu
Authors
Dipendu Das − Department of Chemistry, BioScience Research
Collaborative, Rice University, Houston, Texas 77005, United
States
Yong Lu − Department of Chemistry, BioScience Research
Collaborative, Rice University, Houston, Texas 77005, United
States
Subhrajit Rout − Department of Chemistry, BioScience Research
Collaborative, Rice University, Houston, Texas 77005, United
States
Emmanuel N. Pitsinos − Department of Chemistry, BioScience
Research Collaborative, Rice University, Houston, Texas 77005,
United States; Laboratory of Natural Products Synthesis &
Bioorganic Chemistry, Institute of Nanoscience and
Nanotechnology, National Centre for Scientific Research
“Demokritos”, 153 10 Agia Paraskevi, Greece; orcid.org/
0000-0003-0626-0578
Joseph Lyssikatos − Abbvie Stemcentrx, LLC, South San
Francisco, California 94080, United States
Alexander Schammel − Abbvie Stemcentrx, LLC, South San
Francisco, California 94080, United States
Joseph Sandoval − Abbvie Stemcentrx, LLC, South San
Francisco, California 94080, United States
Mikhail Hammond − Abbvie Stemcentrx, LLC, South San
Francisco, California 94080, United States
Monette Aujay − Abbvie Stemcentrx, LLC, South San Francisco,
California 94080, United States
Figure 5. (A) Molecular structures of most potent uncialamycin
analogues 8-(MeNHCH2)-3,6c 30, 35, and 36. (B) Summary of
derived structure−activity relationships (SARs).
Julia Gavrilyuk − Abbvie Stemcentrx, LLC, South San Francisco,
California 94080, United States
compounds against a number of cell lines, including the
multidrug-resistant MES SA/DX cell line. Specifically, the 26-
keto-8-methylamino methyl analogue 30, 9-fluoro-8-methyl-
amino methyl analogue 35, and 7-fluoro-8-methylamino
methyl analogue 36 (see Figure 5A for structures) were
found to be more potent than their parent naturally occurring
molecules possessing potencies down to low single digit
picomolar IC50 values against the tested cell lines (see Table
1). These readily available analogues and others like them, and
their derivatives, may serve as suitable payloads for antibody−
drug conjugates and other possible delivery systems for
personalized targeted cancer therapies. It should, however, be
noted that potency is not necessarily the only criterium when
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.9b12522
Author Contributions
∥D.D., Y.L., and S.R. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Drs. Lawrence B. Alemany and Quinn Kleerekoper
(Rice University) for NMR spectroscopic assistance, and Drs.
■
K
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(9) Chowdari, N. S.; Pan, C.; Rao, C.; Langley, D. R.; Sivaprakasam,
P.; Sufi, B.; Derwin, D.; Wang, Y.; Kwok, E.; Passmore, D.; Rangan, V.
S.; Deshpande, S.; Cardarelli, P.; Vite, G.; Gangwar, S. Uncialamycin
as a novel payload for antibody drug conjugate (ADC) based targeted
cancer therapy. Bioorg. Med. Chem. Lett. 2019, 29, 466−470.
Christopher L. Pennington (Rice University) and Ian
Riddington (University of Texas at Austin) for mass
spectrometric assistance. This work was supported by AbbVie
Stemcentrx, the Cancer Prevention & Research Institute of
Texas (CPRIT), and The Welch Foundation (grant C-1819).
K.C.N. is a CPRIT scholar in cancer research.
(10) (a) Yan, X.; Ge, H.; Huang, T.; Hindra; Yang, D.; Teng, Q.;
̌
́
Crnovcic, I.; Li, X.; Rudolf, J. D.; Lohman, J. R.; Gansemans, Y.; Zhu,
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