Total syntheses of (-)-pyrimidoblamic acid and P-3A

Article abstract of DOI:10.1021/ja412298c

Total syntheses of (-)-pyrimidoblamic acid and P-3A are disclosed. Central to the convergent approach is a powerful inverse electron demand Diels-Alder reaction between substituted electron-deficient 1,2,3-triazines and a highly functionalized and chiral primary amidine, which forms the pyrimidine cores and introduces all necessary stereochemistry in a single step. Intrinsic in the convergent approach is the potential it provides for the late stage divergent synthesis of modified analogs bearing deep-seated changes in either the pyrimidine cores or the highly functionalized C2 side chain common to both natural products. The examination of the key cycloaddition reaction revealed that the inherent 1,2,3-triazine mode of cycloaddition (C4/N1 vs C5/N2) as well as the amidine regioselectivity were unaffected by introduction of two electron-withdrawing groups (-CO₂R) at C4 and C6 of the 1,2,3-triazine even if C5 is unsubstituted (Me or H), highlighting the synthetic potential of the powerful pyrimidine synthesis.

Full text of DOI:10.1021/ja412298c

Article
pubs.acs.org/JACS
Total Syntheses of (−)-Pyrimidoblamic Acid and P‑3A
Adam S. Duerfeldt and Dale L. Boger*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: Total syntheses of (−)-pyrimidoblamic acid and P-
3A are disclosed. Central to the convergent approach is a powerful
inverse electron demand Diels−Alder reaction between substituted
electron-deficient 1,2,3-triazines and a highly functionalized and
chiral primary amidine, which forms the pyrimidine cores and
introduces all necessary stereochemistry in a single step. Intrinsic
in the convergent approach is the potential it provides for the late
stage divergent synthesis of modified analogs bearing deep-seated
changes in either the pyrimidine cores or the highly functionalized
C2 side chain common to both natural products. The examination of the key cycloaddition reaction revealed that the inherent
1,2,3-triazine mode of cycloaddition (C4/N1 vs C5/N2) as well as the amidine regioselectivity were unaffected by introduction
of two electron-withdrawing groups (−CO2R) at C4 and C6 of the 1,2,3-triazine even if C5 is unsubstituted (Me or H),
highlighting the synthetic potential of the powerful pyrimidine synthesis.
toxicity and maintain efficacy, which can be used as single
component oncology drugs.
INTRODUCTION
Bleomycin A2 (BLM, 1, Figure 1) is the major component
(∼70%) of the clinical anticancer drug Blenoxane, which is
■
Each structural unit of BLM contributes an important role in
the DNA binding and cleavage cascade. Systematic evaluation
of its structure through single site deep-seated modifications
prepared by total synthesis has delineated the function of each
subunit and the role of their individual substituents.1,21,29−74
These studies, in conjunction with NMR-derived structural
models of DNA bound BLM,75−83 continue to define the
remarkable combination of functional, structural, and con-
formational properties integrated into the natural product. The
pyrimidoblamic acid subunit not only participates in the metal
chelation and O2 activation but also is responsible for the DNA
cleavage selectivity via triplex-like hydrogen bonding to guanine
at the 5′-GC and 5′-GT cleavage sites.76 In such studies, we
have additionally shown that removal of the pyrimidine C5
methyl group, enlisting the functionalized pyrimidine found in
P-3A, has no impact in the functional activity of the resulting
BLM analog.56 From these studies, (−)-pyrimidoblamic acid
(2, Figure 2) and the related peptide-derived natural product,
P-3A (3, Figure 2), have been identified as key subunits for the
continued preparation of improved or simplified bleomycin
analogs. Total syntheses of (−)-pyrimidoblamic acid were
originally described by Umezawa,32,42 Hecht,30,50 and later by
us.60,64 Although elegant and pioneering in their own regards,
these previous efforts have suffered from the inability to fully
control the stereochemistry at the benzylic pyrimidine C2
tertiary center (Scheme 1). Similarly, our initial total synthesis
of P-3A, whose pyrimidine core differs from that of
pyrimidoblamic acid by only the lack of the C5 methyl
Figure 1. Structure of bleomycin A2.
currently used for the treatment of Hodgkin’s lymphoma,
melanomas, head and neck carcinomas, and testicular
cancers.1−4 BLM exerts its biological effects through a metal
ion and oxygen dependent5−19 DNA cleavage that occurs
selectively at 5′-GC or 5′-GT sites.4,7,12,15,20−25 Although
capable of producing single-strand and double-strand breaks,
the latter is believed to be the most biologically relevant
event.26 Despite being an effective therapy for numerous
malignancies, BLM treatment is often limited by dose-
dependent pneumonitis, lung fibrosis, and skin toxicities,
some of which have been attributed to the C-terminal
bithiazole appendage.2,3,27,28 Thus, there remains an interest
in identifying synthetic BLM-based analogs that lack off-target
Received: December 9, 2013
Published: January 12, 2014
© 2014 American Chemical Society
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Scheme 2. Retrosynthetic Analysis
Figure 2. Structures of (−)-pyrimidoblamic acid and P-3A.
Scheme 1. Prior Late Stage Installations of the Benzylic C2-
Stereocenter of (−)-Pyrimidoblamic Acid
would produce the pyrimidine core found in P-3A.This late
stage convergent assemblage of the pyrimidine cores not only
allows the early stage synthesis of the stereochemically rich C2
side chain common to both pyrimidoblamic acid and P-3A but
also permits the late stage divergent synthesis of the natural
products utilizing two different 1,2,3-triazines. Amidine 4 was
expected to arise from the reductive alkylation product of N-
(triphenylmethyl)-L-asparagine (6) with aldehyde 7. Key to the
construction of 4 is the manipulation of intermediates,
especially the desired free-based amidine, without sacrificing
the stereochemical integrity of the α-stereocenter, as similar
amidines have demonstrated a propensity to epimerize.86,87
This concern dictated the late stage amidine introduction in
order to minimize the potential impact of amidine epimeriza-
tion. 1,2,3-Triazines 5a and 5b, the electron-deficient
heterocyclic azadienes for use in the key cycloaddition reaction,
were anticipated to arise from an oxidative ring expansion of N-
aminopyrazoles 9a and 9b.88−91 A key caveat to the approach is
that although the two electron-withdrawing substitutents on the
1,2,3-triazines would be expected to enhance their cyclo-
addition reactivity, it was unknown whether their placement at
the C4 and C6 positions would sterically slow the reaction and
electronically redirect the mode of cycloaddition (C4/N1 vs
C5/N2) with amidines.
group, relied on a late stage diastereoselective introduction of
the benzylic pyrimidine C2 tertiary center that proceeded with
modest diastereoselectivity (87:13).59,66
Recently, we defined the cycloaddition scope and productive
reactivity of substituted electron-deficient 1,2,3-triazines with
various dienophiles, including the first report of their ability to
participate in previously unexplored [4 + 2] cycloaddition
reactions with amidine or imidate heterodienophiles to provide
highly substituted pyrimidines.84,85 These latter studies
provided the basis for the potential development of second
generation asymmetric total syntheses of (−)-pyrimidoblamic
acid and P-3A, in which all necessary stereochemistry is
installed prior to a late stage [4 + 2] cycloaddition introduction
of the pyrimidine, addressing the common limitation of
previously reported routes, permitting the late stage divergent
modification of their structures, and further expanding the
scope of the 1,2,3-triazine inverse electron demand Diels−Alder
reaction to encompass the use of chiral amidines.
RESULTS AND DISCUSSION
■
Synthesis of 1,2,3-Triazines 5a and 5b. The requisite N-
aminopyrazole 9a for oxidative ring expansion to the 1,2,3-
triazine 5a was prepared from 8a that was accessed by a
palladium-mediated tandem cross-coupling/electrocyclization
reaction of commercially available ethyl diazoacetate and the
vinyl triflate derived from t-butyl acetoacetate (Scheme 3).92 N-
Amination of 8a was accomplished upon treatment with O-4-
nitrobenzoylhydroxylamine in the presence of potassium t-
We envisioned that the pyrimidine core of (−)-pyrimido-
blamic acid would arise from an inverse electron demand
Diels−Alder reaction between the highly functionalized
amidine 4 and 1,2,3-triazine 5a (Scheme 2). Additionally, the
same amidine 4 and its cycloaddition with 1,2,3-triazine 5b
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Scheme 3. Synthesis of Requisite N-Aminopyrazoles
Figure 3. Oxidative ring expansion survey.
butoxide (t-BuOK) in N-methylpyrrolidone (NMP), which
yielded N-aminopyrazole 9a in 73% yield in a near 1:1 mixture
of inconsequential regioisomers.93 In an analogous fashion and
with similar yield (72%), the requisite N-aminopyrazole 9b
used to access 1,2,3-triazine 5b was prepared from pyrazole 8b,
which was generated from a [3 + 2] dipolar cycloaddition
between ethyl diazoacetate and t-butyl propiolate.94
and aldehyde 7 (Scheme 4). Reductive alkylation of 6 with 7 in
the presence of excess NaBH(OAc)3 provided 10 in 84% yield.
Scheme 4. Synthesis of Chiral Amidine 4
Although a range of oxidants have been reported for
transforming N-aminopyrazoles into 1,2,3-triazines, we have
found that the optimal reagent for this oxidative ring expansion
reaction is substrate dependent. The ring expansion is often
suggested to occur through a nitrene intermediate, which
initiates N−N bond migration and inserts the central nitrogen
of the 1,2,3-triazine.90 Adaptation of conditions developed by
Ohsawa and colleagues, in which an N-aminopyrazole is treated
with iodine (I2) in the presence of aqueous potassium
bicarbonate (KHCO3), provided the 1,2,3-triazine 5a in
excellent yield (75%).91 These conditions also proved effective
for the production of 5b, yielding the 1,2,3-triazine in 68%
yield. Notably, 1,2,3-triazines 5a and 5b proved stable to
extended storage at room temperature and purification by flash
chromatography using silica gel. As summarized in Figure 3, the
most commonly used oxidants for this transformation (i.e.,
Pb(OAc)4, Ni2O3, and MnO2)88−91 proved unsuccessful in
converting 9a to the desired 1,2,3-triazine 5a, instead resulting
in N−N bond cleavage and reversion to the starting pyrazole
8a.
Silver(I) nitrate, a commonly utilized nitrene transfer reagent
in aziridination,95,96 alone failed to provide 5a but produced 5a
in modest yield (34%) when used in the presence of
phenyliodine diacetate (PIDA) as a co-oxidant. Similarly,
sodium periodate (NaIO4),84 which promotes the oxidative
ring expansion efficiently for 4-substituted N-aminopyrazoles,
afforded the 1,2,3-triazine 5a in moderate yield (28%) with a
majority (70%) of the material isolated being a mixture of
unreacted starting material (9a) and free pyrazole (8a).
Addition of a phase transfer catalyst improved the conversions
to as high as 70%, but was less consistent, and the use of
tetrabutylammonium periodate failed to generate any desired
compound.
Treatment of 10 with 1-hydroxybenzotriazole (HOBt) and 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) fol-
lowed by NH3/THF, provided the primary amide 11.
Dehydration of 11 to provide the nitrile 12 was accomplished
by treatment with N-propanephosphonic acid anhydride (T3P)
in the presence of Hunig’s base (i-Pr2NEt). Treatment of 12
with 50% aqueous NH2OH cleanly afforded amidoxime 13. All
intermediates up to this stage, including 13, exhibited a robust
stability and no epimerization was observed throughout the
reaction sequence. Treatment of 13 with Raney Nickel (Ra−
Ni) in the presence of acetic acid (AcOH) in MeOH generated
the acetate salt of the desired amidine, which could be filtered
through Celite, redissolved in CH2Cl2, and treated with 20%
aqueous NaOH to yield the free-based amidine 4. Chromato-
graphic purification of the amidine 4, as either the acetate salt
or the free base, resulted in partial epimerization. As a result,
the crude amidine was used without purification and produced
immediately prior to its use. Notably, the free-based amidine
epimerized much more readily than the protonated amidine in
solution, and if the adjacent secondary amine was protected as
the t-butylcarbamate (Boc), even more significant slow
epimerization of the α-stereocenter occurred upon amidine
Synthesis of Amidine 4. Amidine 4 was prepared from
commercially available N-(triphenylmethyl)-L-asparagine (6)
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formation. Therefore, the free secondary amine is essential to
the observed stereochemical stability of the adjacent amidine
chiral center.
Total Synthesis of (−)-Pyrimidoblamic Acid. With an
effective route to 14 in hand, the synthesis of (−)-pyrimido-
blamic acid was completed (Scheme 5). The secondary amine
[4 + 2] Cycloaddition Reaction of 1,2,3-Triazine 5a
with Amidine 4. Effective access to the requisite intermediates
4 and 5a set the stage for examination of the key inverse
electron demand Diels−Alder reaction. Consistent with the
electron-deficient character of the 1,2,3-triazine, the cyclo-
addition reaction was found to proceed at room temperature or
lower. Significantly, the intrinsic regioselectivity of the amidine
cycloaddition and 1,2,3-triazine cycloaddition mode were
unaltered by the C4/C6 substitution with electron-withdrawing
groups, and the amidine was found to add exclusively across
C4/N1 (C6/N3) with no evidence of a redirected C5/N2
mode of cycloaddition. By design and because of the pseudo-
C2-symmetric nature of the product pyrimidine, the cyclo-
addition across C4/N1 versus C6/N3 are indistinguishable,
permitting the differential ester protection. Although not all
conditions surveyed are included, Figure 4 summarizes
Scheme 5. Completion of the Total Synthesis of
(−)-Pyrimidoblamic Acid
of 14 was protected as the t-butylcarbamate to provide 15 to
eliminate oxidation risks later in the synthesis. Chemoselective
hydrolysis of 15 with 1 M aqueous NaOH in THF:MeOH
(3:1) was followed with a Curtius rearrangement to provide 16
(78% yield over two steps). Removal of the acetonide
protecting group with p-toluenesulfonic acid monohydrate (p-
TsOH·H2O) in MeOH yielded the Boc-protected amino
alcohol 17, which was subsequently treated with Jones’ reagent
to give the carboxylic acid 18. Conversion of 18 to amide 19
was accomplished upon treatment with HOBt and EDCI
followed by NH3/THF, which provided the fully functionalized
and protected (−)-pyrimidoblamic acid 19 in 96% yield.
Although attempts to promote a global deprotection with 4 M
HCl in EtOAc proved unsuccessful, as the tritylated
carboxamide exhibited an unusual stability to these traditional
reaction conditions, treatment of 19 with a 3:1 mixture of
trifluoroacetic acid (TFA) and CH2Cl2 followed by deliberate
counterion exchange with a 1 M aqueous HCl workup provided
(−)-pyrimidoblamic acid (2) in quantitative yield and identical
in all respects with authentic material.29,64
Generality of the 1,2,3-Triazine/Amidine [4 + 2]
Cycloaddition. Although the similarity in the pyrimidine
cores of (−)-pyrimidoblamic acid and P-3A is striking and may
suggest that extrapolation of the approach to P-3A is
straightforward, it was not clear what the impact of the C5
substituent might be. With electron-withdrawing groups at the
C4- and C6-positions of 5a and 5b, it was still unknown
whether the C5 methyl group affects the efficiency of the
cycloaddition reaction of 5a and if its absence in 5b would alter
the mode of cycloaddition. Thus, before embarking on the
synthesis of P-3A, we first examined the reactions of the two
1,2,3-triazines 5a and 5b in parallel. Remarkably, the less
substituted 1,2,3-triazine 5b was even more reactive than 5a,
providing the product pyrimidine at a faster rate and in a higher
yield in its reaction at room temperature with the aliphatic
amidine substrate, where good conversion to the product was
observed even within 5 min (Figure 5). Slower and a more
Figure 4. Optimization of the [4 + 2] cycloaddition reaction.
representative examples in the optimization of the reaction
between 4 and 5a to provide the desired pyrimidine 14. In
accordance with our previous studies,84,85 which utilized 2.0
equiv of amidine, the investigation began with this reaction
stoichiometry. As summarized in Figure 4, an increase in
reaction time and temperature significantly increases the overall
yield (entries 1−4). However, increasing the reaction temper-
ature diminished the diastereomeric purity of the product,
resulting from competitive amidine epimerization. Notably, we
found that the reaction produces a single diastereomer of 14
when conducted at 5 °C in acetonitrile (entry 5). Because the
1,2,3-triazine 5a is the simpler of the two reaction components,
we focused on identifying conditions that employed amidine 4
as the limiting reagent (entries 8 and 9 vs 6 and 7). When the
reaction was conducted with 1.0 equiv of amidine 4 and 2.0
equiv of the 1,2,3-triazine 5a, the yield increased to a
respectable 46%. Finally, if the reaction was allowed to stir at
5 °C for 14 h and then warmed to 25 °C for 6 h, 14 was
obtained as a single diastereomer in 54% yield.
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Scheme 6. Total Synthesis of P-3A
Figure 5. Comparison of the cycloaddition reactions between 5a or 5b
and amidines.
comparable reactivity between 5a and 5b were observed with
the aryl amidines. Most significant in these studies is the fact
that 5b showed no evidence of a potentially competitive
cycloaddition across C5/N2 (vs C4/N1) despite the lack of a
C5 substituent. With this knowledge in hand, the total synthesis
of P-3A was pursued.
Total Synthesis of P-3A. In accordance with the model
substrates, the cycloaddition reaction between 4 and 5b (25 °C,
CH3CN, 12 h) yielded the desired pyrimidine 26 in 76% yield
as a single diastereomer (Scheme 6). In contrast to the reaction
between 4 and 5a, which requires 5 °C for 12 h prior to
warming to 25 °C to prevent competitive epimerization and
preserve the amidine stereochemical integrity, the faster
reaction between 4 and 5b can be conducted at 25 °C without
compromising the diastereomeric purity of the product and
provided the stereochemically pure pyrimidine 26 in even
higher yield (76%).
Following a synthetic route modeled on that used to
complete the synthesis of (−)-pyrimidoblamic acid, compound
26 was advanced (Scheme 6). Boc-protection of the secondary
amine provided compound 27, which was subjected to
saponification of the ethyl ester. However, attempts to
selectively hydrolyze the ethyl ester by treatment with 1 M
aqueous NaOH in THF/MeOH provided a mixture of
compounds, which included those derived from transester-
ification or hydrolysis of the t-butyl ester. By substituting t-
butanol (t-BuOH) for MeOH as the reaction cosolvent the
competitive reactions were avoided and the reaction cleanly
provided the requisite carboxylic acid, which was subjected to
Curtius rearrangement conditions to provide 28 in good yield
(82% over two steps). Removal of the acetonide protecting
group with p-TsOH·H2O in MeOH afforded the amino alcohol
29, which could be transformed to the primary carboxamide 31
in two steps. Global deprotection of 31 was effected by
treatment with TFA:CH2Cl2 (3:1) to provide 32, a key analog
of (−)-pyrimidoblamic acid lacking only the C5 methyl group,
in quantitative yield. Additionally, treatment of 31 with 1 M
aqueous NaOH in THF:MeOH (3:1) results in t-butyl ester
hydrolysis and provided carboxylic acid (33), which was
coupled with N(Im)-Boc-L-His-L-Ala-O(t-Bu) to provide the
fully assembled but protected P-3A (34). Notably, this coupling
reaction enlisted a more highly protected derivative of 32 than
our prior efforts60,64 and proceeded more smoothly, providing
higher yields of the product 34. Global deprotection of 34 with
TFA:CH2Cl2 (3:1) followed by counterion exchange with a 1
M aqueous HCl workup provided P-3A (3), in quantitative
yield and spectroscopically identical in all comparable respects
with authentic material.66,97
CONCLUSIONS
■
Convergent total syntheses of (−)-pyrimidoblamic acid and P-
3A were detailed based on the early stage preparation of the
chiral highly functionalized C2 side chain and subsequent late
stage, divergent construction of the pyrimidine cores through
use of a powerful inverse electron demand amidine/1,2,3-
triazine [4 + 2] cycloaddition reaction conducted at ≤25 °C. In
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ASSOCIATED CONTENT
* Supporting Information
Full experimental details including NMR results and graphs.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
boger@scripps.edu
■
(33) Umezawa, Y.; Morishima, H.; Saito, S.; Takita, T.; Umezawa, H.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support of the National
Institutes of Health (CA042056). A.S.D. is an American Cancer
Society postdoctoral fellow (PF-12-122-01-CDD).
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