bleomycin a2 ( blm , 1 , figure 1 ) is the major 
 component ( 70% ) of the clinical anticancer 
 drug blenoxane , which is currently used for the treatment of hodgkin s 
 lymphoma , melanomas , head and neck carcinomas , and testicular cancers .
blm exerts its biological effects through a metal ion and oxygen 
 dependent dna cleavage that occurs selectively at 5-gc or 5-gt 
 sites .
although capable of producing single - strand and double - strand breaks , 
 the latter is believed to be the most biologically relevant event . 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 .
thus , there remains 
 an interest in identifying synthetic blm - based analogs that lack off - target 
 toxicity and maintain efficacy , which can be used as single component 
 oncology drugs .
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 .
these studies , in conjunction with nmr - derived structural models 
 of dna bound blm , continue to define the remarkable combination of functional , structural , 
 and conformational 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 . 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 . 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 , hecht , and later 
 by us .
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 group , 
 relied on a late stage diastereoselective introduction of the benzylic 
 pyrimidine c2 tertiary center that proceeded with modest diastereoselectivity 
 ( 87:13 ) .
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 .
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
we envisioned that the pyrimidine core 
 of ( )-pyrimidoblamic 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 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 -stereocenter , as similar amidines have demonstrated 
 a propensity to epimerize .
this concern dictated 
 the late stage amidine introduction in order to minimize the potential 
 impact of amidine epimerization .
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 .
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 cycloaddition 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 .
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).n - amination of 8a was accomplished upon treatment with o-4-nitrobenzoylhydroxylamine 
 in the presence of potassium t - butoxide ( t - buok ) in n - methylpyrrolidone ( nmp ) , which 
 yielded n - aminopyrazole 9a in 73% yield 
 in a near 1:1 mixture of inconsequential regioisomers . 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 .
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 .
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% ) .
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 ) proved unsuccessful in converting 9a to the desired 1,2,3-triazine 5a , instead 
 resulting in n 
silver(i ) nitrate , a commonly utilized nitrene 
 transfer reagent 
 in aziridination , 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 ) , 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 .
amidine 4 was prepared from commercially 
 available n-(triphenylmethyl)-l - asparagine 
 ( 6 ) and aldehyde 7 ( scheme 4 ) .
reductive alkylation of 6 with 7 in the presence of excess nabh(oac)3 provided 10 in 84% yield .
treatment of 10 with 1-hydroxybenzotriazole 
 ( hobt ) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( edci ) followed 
 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 ) .
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 .
chromatographic 
 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 -stereocenter 
 occurred upon amidine formation .
therefore , the free secondary amine 
 is essential to the observed stereochemical stability of the adjacent 
 amidine chiral center .
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 cycloaddition 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 cycloaddition across c4/n1 versus c6/n3 are indistinguishable , 
 permitting the differential ester protection .
although not all conditions 
 surveyed are included , figure 4 summarizes 
 representative examples in the optimization of the reaction between 4 and 5a to provide the desired pyrimidine 14 . in accordance with our previous studies , which utilized 2.0 equiv of amidine ,
as summarized in figure 4 , an increase in reaction time and temperature significantly 
 increases the overall yield ( entries 14 ) .
however , increasing 
 the reaction temperature 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 .
optimization of the [ 4 + 2 ] cycloaddition reaction . with 
 an effective route to 14 in hand ,
the secondary 
 amine 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 - tsohh2o ) 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 .
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 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 ,
comparison of the cycloaddition 
 reactions between 5a or 5b and amidines . 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 transesterification 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 - tsohh2o 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 - 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 efforts 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 .
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 addition to permitting full control 
 of the natural product stereochemistry , the approach provides the 
 opportunity for the late stage divergent synthesis of modified analogs 
 bearing deep - seated changes in either the pyrimidine core ( c4 and 
 c5 ) or the highly functionalized c2 side chain .
such investigations 
 are in progress and will be reported in due course . the examination 
 of the key 1,2,3-triazine cycloaddition reaction with amidines defined 
 nonobvious 1,2,3-triazine substituent effects that maintain or enhance 
 the heterocyclic azadiene reactivity without altering the intrinsic 
 regioselectivity or mode of cycloaddition .
these observations with 
 1,2,3-triazines extend the utility of the inverse electron demand 
 cycloaddition reactions of electron - deficient heterocyclic azadienes 
 that includes the complementary 1,2,4- and 1,3,5-triazines , 1,2,4,5-tetrazines , and 1,2-diazines in the synthesis of highly substituted 
 and functionalized heterocycles found in complex natural products .
 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 . 