Synthesis and Evaluation of Hydrogen Peroxide Sensitive Prodrugs of Methotrexate and Aminopterin for the Treatment of Rheumatoid Arthritis

Article abstract of DOI:10.1021/acs.jmedchem.7b01775

A series of novel hydrogen peroxide sensitive prodrugs of methotrexate (MTX) and aminopterin (AMT) were synthesized and evaluated for therapeutic efficacy in mice with collagen induced arthritis (CIA) as a model of chronic rheumatoid arthritis (RA). The prodrug strategy selected is based on ROS-labile 4-methylphenylboronic acid promoieties linked to the drugs via a carbamate linkage or a direct C-N bond. Activation under pathophysiological concentrations of H₂O₂ proved to be effective, and prodrug candidates were selected in agreement with relevant in vitro physicochemical and pharmacokinetic assays. Selected candidates showed moderate to good solubility, high chemical and enzymatic stability, and therapeutic efficacy comparable to the parent drugs in the CIA model. Importantly, the prodrugs displayed the expected safer toxicity profile and increased therapeutic window compared to MTX and AMT while maintaining a comparable therapeutic efficacy, which is highly encouraging for future use in RA patients.

Full text of DOI:10.1021/acs.jmedchem.7b01775

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Synthesis and Evaluation of Hydrogen Peroxide Sensitive Prodrugs of
Methotrexate and Aminopterin for the Treatment of Rheumatoid Arthritis
Jorge Peiró Cadahía, Jon Bondebjerg, Christian Abilgaard Hansen, Viola
Previtali, Anders E. Hansen, Thomas L. Andresen, and Mads Hartvig Clausen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01775 • Publication Date (Web): 31 Mar 2018
Downloaded from http://pubs.acs.org on April 1, 2018
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Synthesis and Evaluation of Hydrogen Peroxide
Sensitive Prodrugs of Methotrexate and Aminopterin
for the Treatment of Rheumatoid Arthritis
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Jorge Peiró Cadahía†, Jon Bondebjerg§, Christian A. Hansen#, Viola Previtali†, Anders E.
Hansen‡, Thomas L. Andresen‡, and Mads H. Clausen†,*
† Center for Nanomedicine & Theranostics, Department of Chemistry, Technical University of
Denmark, Kemitorvet 207, DKꢀ2800 Kongens Lyngby, Denmark. § MC2 Therapeutics, Ager
Alle 24ꢀ26, 2970 Hørsholm, Denmark, # Capdelta Group Aps, C/O Kavsbjerglund 30, DKꢀ2740
Skovlunde, Denmark, ‡ Center for Nanomedicine & Theranostics, Department of Microꢀ and
Nanotechnology, Technical University of Denmark, Ørsteds Plads, Building 345, DKꢀ2800
Kongens Lyngby, Denmark.
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ABSTRACT. A series of novel hydrogen peroxide sensitive prodrugs of methotrexate (MTX)
and aminopterin (AMT) were synthesized and evaluated for therapeutic efficacy in mice with
collagen induced arthritis (CIA) as a model of chronic rheumatoid arthritis (RA). The prodrug
strategy selected is based on ROSꢀlabile 4ꢀmethylphenylboronic acid promoieties linked to the
drugs via a carbamate linkage or a direct C‒N bond. Activation under pathoꢀphysiological
concentrations of H2O2 proved to be effective and prodrug candidates were selected in agreement
with relevant in vitro physicochemical and pharmacokinetic assays. Selected candidates showed
moderate to good solubility, high chemical and enzymatic stability, and therapeutic efficacy
comparable to the parent drugs in the CIA model. Importantly, the prodrugs displayed the
expected safer toxicity profile and increased therapeutic window compared to MTX and AMT
while maintaining a comparable therapeutic efficacy, which is highly encouraging for future use
in RA patients.
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KEYWORDS: aminopterin, collagenꢀinduced arthritis, hydrogen peroxide, methotrexate,
phenylboronic acid, prodrug, rheumatoid arthritis.
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INTRODUCTION
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Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic destructive
synovitis and is associated with progressive disability, systemic difficulties, premature death, and
socioeconomic costs.1 The cause of RA remains unknown and currently no cure for the disease
exists. Several studies have estimated the prevalence of RA to range between 0.5% to 1% of the
adult population in developed countries, with a 3 to 1 female to male ratio.2
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Aminopterin (1, AMT, Figure 1), an antiꢀfolate drug initially developed for treatment of
leukemia, was tested for RA treatment in 1951.3,4 Complex manufacturing,5 unpredictable
toxicities,6–9 and low therapeutic index8,10 led scientist to develop methotrexate (2, MTX,
Figure 1), as an alternative antifolate for cancer therapy which later showed antiꢀarthritic
properties as a disease modifying antiꢀrheumatic drug (DMARD). In 1988, lowꢀdose MTX
(LDꢀMTX, <30 mg in weekly pulses) was approved by the FDA for the treatment of RA and has
remained the standard of care since.11,12 Although efficacious, the mechanisms that govern the
antiꢀinflammatory and immunosuppressive effects of MTX in rheumatic diseases are still not
completely understood.13 Furthermore, the population of patients that will be responsive to an
MTX regiment cannot be reliably predicted before initiating treatment.14 Adverse effects and low
efficacy of LDꢀMTX in RA are the predominant reasons for discontinuation of treatment. Side
effects are usually mild, selfꢀlimiting, and/or preventable, but a subset of patients suffers from
severe sideꢀeffects. Toxicities including anemia, neutropenia, stomatitis, oral ulcers, lethargy,
fatigue, nodulosis, hepatic and pulmonary fibrosis, as well as renal insufficiency are associated to
15–18
this drug.
Additionally, progressive drug tolerance of disease, high patient variability, and
pharmacokinetic inadequacies of LDꢀMTX suggest the need of more efficient and safer RA
therapies.
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Figure 1. Chemical structure of aminopterin (AMT, 1) and methotrexate (MTX, 2)
Prodrugs are chemically modified drugs that have either no or greatly decreased activity
compared to the parent drug. Upon administration, prodrugs are activated in the body through
chemical and/or enzymatic transformation into the pharmaceutically active moiety.19
Approximately 20% of drugs approved since 2000 are prodrugs,20 mostly designed to minimize
and/or overcome unacceptable absorption, distribution, metabolism, excretion, and toxicity
(ADMET) properties of the native drug.21 Several prodrugs, drug conjugates, and drug delivery
systems for methotrexate have been reported in the literature but none of them have been
approved for clinical use yet. 22–33
Reactive oxygen species (ROS), like H2O2, HO•, O2 , produced by phagocytes and
ꢀ
neutrophils,34 have important physiological roles in priming the immune system.35,36 Increasing
evidence from different studies supports the relation between oxidative stress and the
pathogenesis of inflammation and rheumatoid arthritis.37,38 In RA, oxidative stress has been
described as an important mechanism in the pathogenesis of destructive proliferative synovitis.
Under pathological inflammatory conditions, the extracellular concentration of H2O2, the most
stable ROS, has been determined to be up to a 100ꢀfold higher compared to healthy tissue,
reaching concentrations as high as 1.0 mM.39–44 The presence of increased concentrations of
H2O2 in the inflammatory environment can serve as the stimulus for prodrug activation in siteꢀ
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selective drug delivery systems. Phenylboronic acids and esters,45–48 phenylsulfonate esters,49
thiazolidinones,44 Nꢀ(2,5ꢀdihydroxyphenyl)acetamides,50 and αꢀboryl ethers, carbonates, and
acetals51 have been reported as useful structural motifs for hydrogen peroxide activatable
prodrugs and imaging, but studies have mainly focused on their applications in oncology.
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Here we propose a new approach to RA therapy based on the use of phenylboronic acidꢀ
containing prodrugs that are inactive until activated locally in the inflammatory tissue. This
mechanism secures that the biological activity is limited to regions of inflammation to facilitate a
safer toxicity profile compared to AMT and MTX, and a more effective treatment due to the
expected localization and accumulation in the target tissue. We have designed and synthesized a
series of MTX and AMT prodrugs that are activated by diseaseꢀ and tissueꢀspecific factors like
H2O2. Their physicochemical, pharmacokinetic, and drugꢀlike properties have been evaluated in
in vitro assays. Additionally, their in vivo efficacy and preliminary toxicity in the murine
collagenꢀinduced arthritis model (CIA) was assessed.
RESULTS AND DISCUSSION
Synthesis of prodrugs. The pinacolates 4b and 4c were synthesized from the corresponding
4ꢀhydroxymethylphenyl boronic acids 3b and 3c via condensation with pinacol (Scheme 1).
Subsequently, the chloroformates 5a‒c were achieved by reaction of 3a‒c with phosgene. In
parallel, the MTX core structure was synthesized starting with bromination of 2,4ꢀdiaminoꢀ6ꢀ
(hydroxymethyl)pteridine hydrochloride (6·HCl) and alkylation of 4ꢀmethylaminobenzoic acid
to form the pteoric acid 7, following the procedure described by Kralovej et al.52 Amide coupling
of 7 with dimethylglutamate hydrochloride 9·HCl, synthesized from glutamic acid 8, provided
methotrexate dimethylester 10. Finally, coupling between 10 and 5aꢀc formed the desired
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prodrugs 11‒13 after hydrolysis of the pinacolate with HCl in a mixture of H2O/MeCN. The use
of DMAP in the coupling step was crucial for the progression of the reaction. Compounds 11‒13
represent a set of MTX prodrugs in which the promoiety is linked to MTX via a carbamate
linkage. The promoiety contains different orthoꢀsubstituents (H, F or Me) in order to investigate
the effect of modifying the steric and electronic environment around the boronic acid on
reactivity towards H2O2 . Furthermore, the carboxylic acids of MTX were also masked as methyl
esters.
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Scheme 1. Synthesis of MTX prodrugs 11‒13a
aReagents and conditions: (a) pinacol, THF, reflux, 16 h (>94%). (b) COCl2 (20% in toluene),
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dioxane, 21 C, 20 h (>95%). (c) NaOH, H2O. (d) PPh3Br2, DMA, 21 C, 20 h, then
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4ꢀmethylaminobenzoic acid, DIPEA, 3 days (77%). (e) PyBOP, Et3N, DMF, 21 oC, 30 min, then
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9·HCl (86%). (f) MeOH, SOCl2, 21 C, 4 days (>95%). (g) 5a‒c, DIPEA, DMAP, CH2Cl2, 21
oC, 5 h. (h) HCl in H2O/MeCN, 21 oC, 16 h (27‒32%).
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Efforts aiming for hydrolysis of the methyl esters in 11‒13 using standard conditions based on
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alkaline hydrolysis with NaOH53 or LiOH,54 LiI in pyridine,55 or enzymatic hydrolysis using a
polymer supported lipase from Candida antarctica56 were unsuccessful. Therefore a new
synthetic strategy was proposed (see Scheme 2).
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Starting from MTX, the synthesis of the corresponding PMB protected intermediate 14 was
achieved. Following the same procedure described for the synthesis of 11‒13, coupling between
5a and 14 afforded 15, which was hydrolyzed to form the corresponding boronic acid 16. Lastly,
PMB deprotection under acidic conditions afforded the prodrug 17.
Scheme 2. Synthesis of MTX prodrug 17a
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aReagents and conditions: (a) PMBCl, 1,1,3,3ꢀtetramethylguanidine, CH2Cl2, 21 oC, 16 h (35%).
(b) 5a, DIPEA, DMAP, CH2Cl2, 21 oC, 5 h (73%). (c) HCl in H2O/MeCN, 21 oC, 20 h (31%). (d)
5% TFA in CH2Cl2, 21 oC, 30 min (54%).
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Cbz protection of 4ꢀaminobenzoic acid (18) formed 19 and twoꢀstep coupling with 9·HCl
produced 20. Hydrogenolysis of 20 afforded 21 in high yields, which was subjected to reductive
alkylation with 4ꢀformylphenylboronic acid to give 22. Alkylation of 22 with 6ꢀ(bromomethyl)ꢀ
2,4ꢀpteridinediamine hydrobromide (23·HBr) formed the first AMT prodrug 24. The
concentration of the reaction was critical to obtain a reasonable yield, as previously published by
Montgomery et al.57 for similar substrates. The last step consisted of alkaline hydrolysis of 24 to
form the second AMT prodrug 25. These compounds represent the proposed set of
phenylboronic acidꢀbased AMT prodrugs in which the promoiety is attached to the secondary
aniline group, bearing methylester protected (24) or free carboxylic acids (25).
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Scheme 3. Synthesis of AMT prodrugs 24 and 25a
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aReagents and conditions: (a) CbzCl, NaHCO3, H2O/dioxane, 21 oC, 2 h (89%). (b) SOCl2, DMF,
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CH2Cl2, reflux, 24 h. (c) 9·HCl, Et3N, CH2Cl2, 21 C, 2 h (>95%). (d) H2, Pd/C, MeOH, 21oC,
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12 h. (e) HCl in Et2O (>95%). (f) NaBH4, MeOH, 21 C, 24 h (67%). (g) DMA, 55 C, 3 days
(43%). (h) NaOH, H2O, 21oC, 5 min (40%).
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Activation of prodrugs. For a proofꢀofꢀprinciple of the prodrug strategy, a study on the
activation of 11‒13, 17, 24, and 25 under pathoꢀphysiological concentrations of H2O2 was
performed. The experiment consisted on the incubation of test compounds in a 30% DMSO in
PBS mixture at a concentration of 50 µM and H2O2 concentration found in inflammatory tissue
(0.5 mM, 10 equiv.) at 37 oC. The activation was followed by RPꢀUPLCꢀMS (see Figure S1 and
Figure S2 for examples on prodrug 17 and 25 chromatograms, respectively). Negative controls
(no H2O2 addition) were performed in parallel (Figure S3). The rationale behind the substitution
of a hydrogen atom in the orthoꢀposition of the phenylboronic acid promoiety by a methyl group
or a fluorine atom was the reactivity towards oxidative deboronation. Interestingly, prodrugs
11‒13 were activated and released 10 within less than 3 h (>95% conversion by UPLCꢀMS),
therefore no effects on the reaction rate by orthoꢀsubstituents was observed under the conditions
tested (Figure 2, left). Furthermore, 17 was activated within the same time frame, releasing
exclusively MTX, with no significant difference compared to 11‒13. The plot of the formation of
prodrug activation products versus time can be found in the SI (Figure S4). Moreover, a
concentration dependent release of drug was observed as determined in an activation assay using
different concentrations of H2O2 (see Figure S5).
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Figure 2. Activation of MTX prodrugs 11‒13 and 17 (left), and AMTꢀprodrugs 24 and 25, as
well as formation and disappearance of intermediates 26 and 27 (right). The experiment was
carried out at a compound concentration of 50 µM and 0.5 mM H2O2 (10 equiv.) in a mixture of
30% DMSO in PBS at 37 oC. Hidden error bars are smaller than symbols.
The activation of AMT prodrugs 24 and 25 was also examined. Full consumption of prodrugs
was observed in a similar timeframe compared to the MTX prodrugs (Figure 2, right). AMT
prodrugs did not release AMT nor 28 within the first two hours of incubation, but instead
afforded the corresponding phenol intermediates 26 and 27 (Scheme 4). After 2 h of assay
initiation, full disappearance of the prodrugs was observed and 26 and 27 started to slowly
release AMT and 28, in a H2O2 concentration independent manner as studied in a separate assay
(see Figure S5). Full formation of products was not observed even after 48 h of incubation. The
formation of the relatively stable intermediates 26 and 27 could potentially allow them to
disperse from the inflammatory environment and exert nonꢀselective drug actions upon AMT
release, unlike for the MTXꢀprodrugs 11‒13 and 17, where phenol intermediates were not
observed due to the faster selfꢀimmolation. This will have to be further evaluated in a future
study of the AMT prodrugs.
Scheme 4. Activation of AMT prodrugs 24 and 25 with H2O2 to form the intermediates 26 and
27, respectively, which slowly release 28 and AMT.
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Kinetic solubility. The low solubility of 11‒13 and 24 in the H2O2 activation assay required the
use of high concentrations of DMSO (30%) in PBS. Therefore, the evaluation of the prodrug
solubility was prioritized in the development program. Kinetic solubility in PBS was measured at
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37 C and the results were compared to those determined for the parent drugs, showing low
solubility (<10 µg/mL) for the methyl ester prodrugs 11‒13 and 24, according to the guidelines
for oral administration in humans and animal dosing formulations (Table 1). On the other hand,
prodrugs 17 and 25 showed moderate to good solubility for predicted oral dosing in humans (55
and 48 µg/mL, respectively), although still low for animal dosing applications. The low
solubility of 11‒13 and 24 prompted the decision to select 17 and 25 for further in vitro studies.
Table 1. Kinetic solubility of 11, 12, 13, 17, 24, and 25.
Kinetic solubility
Prodrug
µM
µg/mL
3.0 ± 0.5
2.0 ± 0.3
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12
13
17
3.1 ± 1.4
4.6 ± 0.9
87 ± 2.3
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83 ± 0.6
2.1 ± 0.9
3.1 ± 0.6
55 ± 1.5
2.5 ± 0.1
48 ± 0.4
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25
2137 ± 0.0 970 ± 0.0
>1000 ± 0.0 >440 ± 0.0
MTX
AMT
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Chemical (pH 7.4), SGF, SIF, plasma, and metabolic stability. The stability of 17 and 25
was evaluated in different relevant physiological environments including chemical stability at
blood pH 7.4 as well as in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), human
and mouse plasma and liver microsomes. Firstly, the halfꢀlives t1/2 of 17 and 25 in PBS (pH 7.4)
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at 37 C were determined to 238 and 577 h, respectively, with stabilities higher than 90% after
24 h in both cases (Table 2 and Figure S6). Secondly, the stability in SGF and SIF was studied
and halfꢀlives calculated to 8 and 770 h in SGF, and 239 and 866 h in SIF for 17 and 25,
respectively (Table 2 and Figure S7). The shorter halfꢀlife of 17 in SGF was found to be pHꢀ
dependent rather than a result of enzymatic hydrolysis (Figure S7). Thirdly, the stability in
human and mouse plasma proved to be high, with 70% and 82% of 17 and 25 remaining after 24
h in human plasma, respectively, and 62% and 91% after 8 h in mouse plasma (see Figure S8).
Their halfꢀlives were calculated using first order kinetics and the results are shown in Table 2.
Lastly, the metabolic stability was evaluated by the determination of intrinsic clearance (CLint) in
human and mouse pooled liver microsomes. Intrinsic clearance of 17 and 25 was calculated to 22
and 1.2 µL/min/mg respectively in human microsomes, while 6.4 and 2.6 µL/min/mg in mouse
microsomes (Table 2 and Figure S9), indicating their high metabolic stability.
Table 2. Chemical (pH 7.4), SGF, SIF, plasma, and metabolic stabilities of prodrug candidates
17 and 25.
SGF
SIF
Chemical
Stability
(pH 7.4)
Human
Plasma
Stability Stability
Mouse
Plasma
Human
Microsomal Microsomal
Stability
CLint
(µL/min/mg) (µL/min/mg)
Mouse
Stability Stability
Stability
CLint
Prodrug
t1/2 (h)
t1/2 (h)
t1/2 (h)
t1/2 (h)
t1/2 (h)
238
577
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239
866
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1.2
6.4
2.6
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25
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Antiꢀarthritic efficacy and preliminary toxicity. The efficacy of the prodrug candidates 17
and 25 was evaluated in the collagen typeꢀII induced arthritis (CIA) mouse model. This model
has been found to be characterised by oxidative stress58 and increased concentrations of H2O2 in
the joint tissue, paws and limbs,59,60 hence it represents a good model for the evaluation of the
prodrug concept. Prior to the in vivo analysis, the prodrug concept was evaluated in vitro for 17
and 25. To this end, the cell viability inhibition of the prodrugs against two MTXꢀ and AMTꢀ
sensitive cancer cell lines, NCIꢀH460 (lung cancer) and MCFꢀ7 (breast cancer),61 was tested. The
prodrugs showed significantly reduced efficacy in the two cell lines when applied directly to the
cells as compared to when they were activated by preꢀincubation with nonꢀcytotoxic
concentration of H2O2 (Figures S10–S12). Additionally, the IC50 values of the preꢀactivated
prodrugs 17 and 25 showed comparable biological activity to MTX and AMT (Figure S13 and
S14) in the two selected cancer cell lines. A detailed description of the assays can be found in the
SI.
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The in vivo antiꢀarthritic efficacy and preliminary toxicity study was performed using a
therapeutic intervention set up in the CIA model and the parent drugs MTX and AMT were
included as treatment controls. Vehicle control (2% DMSO in PBS), MTX (7.0 mg/kg), and
equimolar doses of AMT, 17, and 25 were i.p. administered on a daily basis for two weeks,
starting at onset of disease (day 27 after first immunization). Mean arthritis severity score and
average animal body weight were measured three times per week. Reduced incidence of arthritis
and reduction of macroscopic arthritis score was observed both for the drugs and prodrugs in
comparison to the control group (vehicle) (Figure 3, left). Additionally, remission of arthritis
started to be detected at the end of the study for MTX, 17, and 25, with no statistically significant
difference in efficacy between them. Interestingly, the AMTꢀgroup had to be sacrificed at day 33
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due to significant toxicity measured as decreased average body weight in the group, while the
AMT prodrug 25 did not show any sign of sideꢀeffects (Figure 3, right). Progressive weight loss
was observed in the MTXꢀgroup towards the end of the study but not in the group treated with
17. These results indicate the great potential of 17 and 25 as prodrugs of MTX and AMT,
respectively, showing comparable efficacy in managing arthritis development and control of
disease activity, and reducing sideꢀeffects compared to the parent drugs.
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Vehicle
MTX
17
**
†
**
†
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25
20
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10
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*
AMT
25
0
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Days after CII/CFA
Figure 3. Suppression of CIA development in DBA/1J mice (left) after treatment with MTX (7
mg/kg) and equimolar amounts of AMT (6.8 mg/kg), 17 (9.7 mg/kg), and 25 (8.8 mg/kg) (n = 8
per group). Mice were given the indicated dose of test compound once per day, starting on day
27, and arthritis scoring was performed three times per week. Data represents mean values of
arthritic score ± SEM. Statistics were calculated using a oneꢀtailed non parametric Mannꢀ
Whitney. * p < 0.05 and ** p < 0.01 between MTX and vehicle and † p < 0.05 between 17 and
vehicle. General health of mice (right) evaluated three times per week as the average body
weight in groups of animals (n = 8) tested with vehicle, MTX, AMT, 17, and 25. Data represents
mean values of bodyweight ± SEM. * p < 0.05 between AMT and vehicle. One animal in the
vehicle and the 25 groups were sacrificed preꢀtermination due to high arthritis scores. The AMT
group was removed at day 33 due to decline in health. AMT, aminopterin; CIA, collagen
inducedꢀarthritis; CII, collagen typeꢀII; MTX, methotrexate.
Although no toxicity associated to prodrugs 17 and 25 has been observed in the animal assays
performed, it is of relevance for future studies of the potential toxicities to also account for the
release of quinone methides as prodrug activation endꢀproduct. Their reactivity and toxicity has
been linked and reviewed elsewhere,62 and prior studies have demonstrated that it is possible to
modulate these factors through judicious choice of the substitution pattern on the quinone
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methides released. Noteworthy, quinone moieties are known in longꢀterm use marketed drugs,62
and newly developed ROSꢀsensitive drug delivery systems which employ phenyl boronates as
the responsive chemical entity have shown good safety profiles after longꢀterm administration in
mice.63
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CONCLUSION
In this report, we have described the synthesis of new arylboronic acidꢀbased hydrogen
peroxideꢀsensitive prodrugs of MTX and AMT for siteꢀselective delivery of drugs to
inflammatory tissues associated to RA with the aim of reducing side effects in MTX and AMT
RA therapy. Among the set of prodrugs synthesized, 17 and 25 showed moderate to good
solubility, high chemical stability, high stability in simulated gastrointestinal fluids, and high
plasma and metabolic stabilities both in human and mouse plasma and liver microsomes. Their
comparable efficacy and lower toxicity in comparison to parental drugs was identified using a
CIA mouse model of RA. Future work will focus on additional in vivo studies to establish
optimal dosing and to map toxicity in more detail to hopefully advance these promising prodrugs
into clinical evaluations.
EXPERIMENTAL SECTION
General considerations
Unless otherwise stated, commercially available reagents were used without further
purification and all solvents were of HPLC quality. Reactions under nitrogen atmosphere were
performed in ovenꢀ or flameꢀdried glassware and anhydrous solvents. Anhydrous CH2Cl2,
CH3CN, THF, DMF, and toluene were obtained from Innovative Technology PSꢀMDꢀ7 Pure
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solve solvent purification system. All reactions were monitored by thinꢀlayer chromatography
(TLC) and/or reversedꢀphased ultraꢀperformance liquid chromatography mass spectrometry
(RPꢀUPLCꢀMS). Analytical TLC was conducted on Merck aluminium sheets covered with silica
(C60). The plates were either visualized under UVꢀlight or stained by dipping in a developing
agent followed by heating. KMnO4 (3 g in water (300 mL) along with K2CO3 (20 g) and 5%
aqueous NaOH (5 mL)) or Ninhydrin (3 g in a mixture of nꢀbutanol (200 mL) and AcOH (6
mL)) were used as developing agents. Flash column chromatography was performed using
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1
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Matrex 60 Å, 35‒70 µ silica gel. All new compounds were characterized by IR, H NMR, C
NMR, HRMS (ESI) and melting point (m.p.) when applicable. For recording of 1H NMR and 13C
NMR a Bruker Ascend with a Prodigy cryoprobe (operating at 400 MHz for proton and 101
o
MHz for carbon) was used. Unless otherwise stated, all NMR spectra were recorded at 25 C.
The chemical shifts (δ) are reported in parts per million (ppm) and the coupling constants (J) in
Hz. For spectra recorded in CDCl3, signal positions were measured relative to the signal for
CHCl3 (7.26 ppm for 1H NMR and 77.16 ppm for 13C NMR). For spectra recorded in DMSOꢀd6
1
signal positions were measured relative to the signal for DMSO (δ 2.50 ppm for H NMR and
39.52 ppm for 13C NMR). For spectra recorded in D2O signal positions were measured relative to
the signal for H2O (δ 4.79 ppm for 1H NMR at 25 oC).64 The 13C NMR signal of the ipso carbon
to the boron atom was not observed in any characterized compound, aulthough they were
identified by the characteristic HMBC correlation signals. Infrared (IR) spectra were recorded on
a Bruker AlphaꢀP FTꢀIR (Bruker) instrument. Neat application into the apparatus was used for
all compounds. Transmittance units were plotted in the y axis and wavenumber (cmꢀ1) on the x
axis. Optical rotations were measured on a Perkin Elmer Model 241 Polarimeter (cuvette 1.0 mL,
o
100 mm) using a sodium source lamp (589 nm, 20 C). Melting points (m.p.) were recorded
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using a Stuart melting point SMP30, and reported in C, uncorrected. Analytical RPꢀUPLCꢀMS
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(ESI) analysis was performed on a Waters AQUITY RPꢀUPLC system equipped with a diode
array detector using a Thermo accucore C18 column (d 2.6 µm, 2.1 x 50 mm; column temp: 50
°C; flow: 0.6 mL/min). Four different methods were use. Method A: eluents A (0.1% HCO2H in
milliꢀQ water) and B (0.1% HCO2H in CH3CN) were used in a linear gradient (5% B to 100% B)
in a total run time of 2.6 min. Method B: eluents A (0.1% HCO2H in H2O) and B (0.1% HCO2H
in CH3CN) were used in a linear gradient (5% B to 100% B) in a total run time of 5.0 min.
Method C: eluents A (10 mM NH4OAc in milliꢀQ water) and B (0.1% NH4OAc in milliꢀQ
water/MeCN, 90/10, v/v) were used in a linear gradient (5% B to 100% B) in a total run time of
2.6 min. Method D: eluents A (0.1% NH4OAc in H2O) and B (0.1% NH4OAc in CH3CN) were
used in a linear gradient (5% B to 100% B) in a total run time of 5.0 min. The LC system was
coupled to a SQD mass spectrometer operating in both positive and negative electrospray modes.
The temperature for all recordings was 20 °C. Analytical LCꢀHRMS (ESI) analysis was
performed on an Agilent 1100 RPꢀLC system equipped with a diode array detector using a
Phenomenex Luna C18 column (d 3 µm, 2.1 x 50 mm; column temp: 40 °C; flow: 0.4 mL/min).
Eluents A (0.1% HCO2H in H2O) and B (0.1% HCO2H in CH3CN) were used in a linear gradient
(20% B to 100% B) in a total run time of 15 min. The LC system was coupled to a Micromass
LCT orthogonal timeꢀofꢀflight mass spectrometer equipped with a Lock Mass probe operating in
positive or negative electrospray mode. Purification of reactions by preparative RPꢀHPLC was
performed on a Waters Alliance reverseꢀphase HPLC system consisting of a Waters 2545 Binary
Gradient Module equipped with either an xBridge BEH C18 OBD Prep Column (130 Å, 5 µm,
30 x 150 mm) or an xBridge Peptide BEH C18 OBD Prep Column (130 Å, 5 ꢀm, 19 mm x 100
mm) both operating at 20 °C and a flow rate of 20 mL/min, a Waters Photodiode Array Detector
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(detecting at 210‒600 nm), a Waters UV Fraction Manager, and a Waters 2767 Sample Manager.
Elution was carried out in a reversedꢀphase gradient fashion combining A1 (0.1% HCO2H in
milliꢀQ water) and B1 (0.1% HCO2H in CH3CN) or A2 (5 mM NH4OAc in H2O) and B2 (5 mM
NH4OAc in CH3CN): 5% B to 70 % B in 10 min, hold for 3.5 min, then 70% B to 100% B in 1.5
min, and hold 3 minutes. Total run time: 20 min. The purity of the compounds was assessed by
RPꢀUPLCꢀUV and NMR, and purities ≥95% were considered acceptable for evaluation purposes
both in vitro and in vivo assays.
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Synthesis
3ꢀMethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)benzyl carbonochloridate (4b).
General procedure A: a suspension of 3b (0.9 g, 5.4 mmol) and pinacol (0.7 g, 6.0 mmol) in
anhydrous THF (25 mL) was refluxed for 16 h under an N2 atmosphere. The solids dissolved
during the reaction time. Then, the mixture was concentrated in vacuo and the residue purified
by flash column chromatography on silica gel using a mixture of CH2Cl2/EtOAc (9/1, v/v) as the
eluent to give the title compound 4b as a clear oil (1.27 g, 95%) which solidified upon storage in
the refrigerator. Rf = 0.84 (silica, CH2Cl2/EtOAc, 1/1, v/v); m.p.: 38.5 – 41.3 oC; IR (neat, cmꢀ1):
1
3390.6, 2978.1, 2925.3, 2867.7, 1609.0, 1347.1, 335.9; H NMR (400 MHz, CDCl3) δ 7.76 (d,
13
J = 7.4 Hz, 1H), 7.17 – 7.09 (m, 2H), 4.67 (s, 2H), 2.54 (s, 3H), 1.34 (s, 12H); C NMR (101
MHz, CDCl3) δ 145.5, 143.6, 136.4, 128.3, 123.2, 83.6, 65.4, 25.0, 22.3; HRMS (ESI) m/z: calcd
for C14H22BO3 [M+Na]+ 271.1476, found 271.1512.
(3ꢀFluoroꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)methanol (4c). Following
general procedure A using 3c (0.90 g, 5.30 mmol), the title compound 4c was afforded as a clear
oil (1.26 g, 94%) after purification by flash column chromatography on silica gel using a mixture
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of CH2Cl2/EtOAc (9/1, v/v) as the eluent. The oil became a white solid upon storage in the
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refrigerator. Rf = 0.71 (silica, eluent CH2Cl2/EtOAc, 1/1, v/v); m.p.: 52.5 – 56.4 C (Litt.65 35‒
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38); IR (neat, cmꢀ1): 3424.6, 2978.6, 2932.3, 2864.3, 1624.1, 1351.8; H NMR (400 MHz,
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CDCl3) δ 7.77 – 7.67 (m, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 10.1 Hz, 1H), 4.72 (s, 2H),
1.36 (s, 12H);65 13C NMR (101 MHz, CDCl3) δ 167.6 (d, J = 251.4 Hz), 147.2 (d, J = 8.0 Hz),
137.2 (d, J = 8.4 Hz), 121.7 (d, J = 2.9 Hz), 113.4 (d, J = 24.7 Hz), 84.0, 64.6, 25.0.65
4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)benzyl carbonochloridate (5a). General
procedure B: a solution of 4a (2.0 g, 8.54 mmol) in anhydrous dioxane (25 mL) was treated with
phosgene (20% in toluene, 22.6 mL, 42.7 mmol) and the reaction was stirred for 20 h under an
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N2 atmosphere at 21 C. The mixture was concentrated in vacuo, redissolved and concentrated
with toluene (2x) to afford the crude title compound 5a as a clear oil (2.5 g, > 95%). The crude
1
chloroformate was used in subsequent reactions within few hours after its isolation. H NMR
(400 MHz, CDCl3) δ 7.84 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 5.31 (s, 2H), 1.35 (s,
12H);66 13C NMR (101 MHz, CDCl3) δ 150.8, 136.2, 135.4, 128.0, 84.2, 73.4, 25.0.66
3ꢀMethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)benzyl carbonochloridate (5b).
Following general procedure B using 4b (1.2 g, 4.8 mmol), the title compound 5b was afforded
as a clear oil (1.5 g, >95%). The crude chloroformate was used in subsequent reactions within
few hours after its isolation. 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 8.0 Hz, 1H), 7.20 – 7.13
13
(m, 2H), 5.26 (s, 2H), 2.55 (s, 3H), 1.34 (s, 12H); C NMR (101 MHz, CDCl3) δ 150.8, 145.7,
136.5, 135.7, 130.1, 125.0, 83.8, 73.5, 25.0, 22.3.
3ꢀFluoroꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)benzyl carbonochloridate (5c).
Following general procedure B using 4c (1.2 g, 4.8 mmol), the crude product 5c was afforded as
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a white solid (1.5 g, >95%). The crude chloroformate was used in subsequent reactions within
few hours after isolation. 1H NMR (400 MHz, CDCl3) δ 7.77 (dd, J = 7.5, 6.1 Hz, 1H), 7.15 (dd,
J = 7.5, 1.3 Hz, 1H), 7.07 (dd, J = 9.6, 1.1 Hz, 1H), 5.29 (s, 2H), 1.36 (s, 12H);65 13C NMR (101
MHz, CDCl3) δ 167.7 (d, J = 251 Hz), 150.8, 138.92 (d, J = 8.3 Hz), 137.6 (d, J = 8.5 Hz), 123.5
(d, J = 3.2 Hz), 115.3 (d, J = 25.4 Hz), 84.3 (s), 72.2 (s), 25.0 (s).65
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4ꢀ(((2,4ꢀDiaminopteridinꢀ6ꢀyl)methyl)(methyl)amino)benzoic acid (7). 2,4ꢀDiaminoꢀ6ꢀ
(hydroxymethyl)pteridine hydrochloride 6·HCl (4.4 g, 19 mmol) was dissolved in hot water (150
mL) and after cooling to 21 oC the solution was neutralized with 1 M aq. NaOH to pH ~ 7 (ca. 20
mL). The formed precipitates were collected by filtration, washed with water, and dried in vacuo
over P2O5 to afford an orangeꢀbeige solid corresponding to 2,4ꢀdiaminoꢀ6ꢀ
(hydroxymethyl)pteridine. The solid was suspended in anhydrous DMA (25 mL) and
triphenylphosphine dibromide (18 g, 43 mmol) was added to the suspension. The turbid and dark
mixture was stirred for 24 h under an N2 atmosphere at 20 oC. Then, 4ꢀaminobenzoic acid (3.0 g,
20 mmol) was added to the reaction and stirred for an additional three days. The reaction mixture
was poured into 250 mL of 0.33N NaOH and the precipitate was filtered off. The filtrate was
neutralized with 10% aq. acetic acid (ca. 20 mL) and the precipitate formed upon neutralization
was filtered off, washed with water, triturated over MeOH, filtered, and dried in vacuo to afford
o
the title compound 7 as an orangeꢀbeige solid (5.70 g, 91%). m.p.: > 240 C (decomp.) (lit.52
o
242 C); IR (neat, cmꢀ1): 3378.7, 3279.5, 2233.3, 1663.6, 1598.6, 1288.4, 1186.0;67 1H NMR
(400 MHz, DMSOꢀd6) δ 12.15 (br s, 1H), 8.63 (s, 1H), 8.17 (br s, 1H), 7.94 (br s, 1H), 7.73 (d,
J = 9.0 Hz, 2H), 7.04 (br s, 2H), 6.83 (d, J = 9.0 Hz, 2H), 4.81 (s, 2H), 3.23 (s, 3H);26 13C NMR
(101 MHz, DMSOꢀd6) δ 167.8, 163.2, 161.1, 152.3, 149.5, 147.8, 131.5, 122.2, 118.1, 111.7,
100.0, 55.2, 39.6.
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Dimethyl
L
ꢀglutamate hydrochloride (9·HCl). Thionyl chloride (5.0 mL, 68 mmol) was
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added dropwise to 50 mL of anhydrous MeOH at 0 oC. The mixture was stirred for 30 min at the
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same temperature followed by addition of Lꢀglutamic acid 8 (5.0 g, 34 mmol). Then, the reaction
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was stirred under an N2 atmosphere for 3 days at 21 C and concentrated in vacuo to afford the
title compound 9·HCl as a colourless solid (7.19 g, >95%). The crude product was used without
further purification in the next reactions. m.p.: 63.2 – 64.0 oC (lit.68 77‒79); [ꢀ]ꢁꢂꢃ = +25.0 (c 5.0,
H2O)69; IR (neat, cmꢀ1): 2952.45, 2902.73, 2852.33, 1728.6770; 1H NMR (400 MHz, D2O) δ 4.24
– 4.19 (m, 1H), 3.85 (s, 3H), 3.73 (s, 3H), 2.65 (td, J = 7.3, 2.2 Hz, 2H), 2.37 – 2.13 (m, 2H);70
13C NMR (101 MHz, D2O) δ 174.8, 170.2, 53.6, 52.4, 52.0, 29.2, 24.7.70
Dimethyl
(4ꢀ(((2,4ꢀdiaminopteridinꢀ6ꢀyl)methyl)(methyl)amino)benzoyl)ꢀ ꢀglutamate
L
(10). To a solution of 7 (3.0 g, 9.2 mmol) in anhydrous DMF (80 mL) was added Et3N (6.4 mL,
46.1 mmol) followed by PyBOP (6.5 g, 12.4 mmol). The mixture was stirred for 30 min under an
N2 atmosphere at 21 oC. Then, 9·HCl (2.1 g, 9.9 mmol) was added and the reaction mixture was
stirred for 5 h at the same temperature. Then, the reaction was filtered through a path of Celite®
and the filtrate concentrated in vacuo. Next, the residue was dissolved in a mixture of
o
EtOAc/CHCl3 (150 mL, 1/1) and poured into 750 mL of Et2O at 0 C under strong stirring. The
resultant suspension was filtered off, washed with Et2O and cold water, triturated in hot MeOH,
and filtered off again. The resultant orange solid wash purified by flash column chromatography
on silica gel using a mixture of CH2Cl2/MeOH (92.5/7.5, v/v) as the eluent to give the title
compound 10 as a yellow solid (2.39 g, 54%). Rf = 0.48 (silica, MeOH/CH2Cl2, 1/9, v/v); m.p.:
>168.6 (decomp.) (lit.52 130‒134); α
[ ]ꢂꢃ
= +2.6 (c 0.5, DMSO); IR (neat, cmꢀ1): 3306.96,
ꢁ
1
3128.91, 2950.99, 2923.52, 1731.36, 1626.09, 1604.36, 1503.14, 1435.41; H NMR (400 MHz,
DMSOꢀd6) δ 8.56 (s, 1H), 8.34 (d, J = 7.4 Hz, 1H), 7.77 – 7.69 (m, 3H), 7.45 (br s, 1H), 6.82 (d,
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8
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J = 8.8 Hz, 2H), 6.61 (br s, 2H), 4.78 (s, 2H), 4.39 (ddd, J = 9.4, 7.4, 5.6 Hz, 1H), 3.61 (s, 3H),
3.57 (s, 3H), 3.21 (s, 3H), 2.41 (t, J = 7.4 Hz, 2H), 2.17 –1.72 (m, 2H);71 13C NMR (101 MHz,
DMSOꢀd6) δ 172.7, 172.6, 166.4, 162.9, 162.7, 155.2, 151.0, 149.2, 145.9, 129.0, 121.4, 120.7,
111.0, 54.9, 51.8, 51.7, 51.4, 38.7, 30.0, 25.8;71 HRMS (ESI) m/z: calcd for C22H27N8O5 [M+H]+
483.2099, found 483.2119.
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(S)ꢀ(4ꢀ((((2ꢀAminoꢀ6ꢀ(((4ꢀ((1,5ꢀdimethoxyꢀ1,5ꢀdioxopentanꢀ2ꢀ
yl)carbamoyl)phenyl)(methyl)amino)methyl)pteridinꢀ4ꢀyl)carbamoyl)oxy)methyl)
phenyl)boronic acid (11). General procedure C: to a suspension of 10 (313 mg, 0.68 mmol) in
anhydrous CH2Cl2 (25 mL) was added DMAP (417 mg, 3.42 mmol) followed by DIPEA (0.60
o
mL, 3.42 mmol). The mixture was cooled to 0 C and a solution of 5a (1.01 g, 3.42 mmol) in
CH2Cl2 (10 mL) was added dropwise. The reaction was allowed to warm to 21 oC and stirred for
6 h under an N2 atmosphere. Then, the mixture was diluted with CH2Cl2 (100 mL), washed with
0.1 M aq. HCl (2 x 75 mL), sat. aq. NaHCO3 (2 x 75 mL), and brine (75 mL). The organic layer
was dried over Na2SO4, filtered, and concentrated in vacuo to afford a yellow solid that was
purified by reversedꢀphase preparative HPLC. The CH3CN/H2O collected fractions containing
the pinacolate intermediate were combined into a 250 mL round bottom flask and HCl cc. (0.3
o
mL, pH ~ 2) was added. The reaction mixture was stirred for 16 h at 21 C and quenched with
sat. aq. NaHCO3 (ca. 50 mL). After removal of the CH3CN under rotatory evaporation the
formed precipitate was filtered off, washed with H2O and dried in vacuo to afford the title
compound 11 as a yellow solid (122 mg, 27%). Additional preparative HPLC purification was
o
sometimes needed in specific substrates. m.p.: >153.0 C (decomp.); [ꢀ]ꢂꢁꢃ = +4.7 (c 0.32,
DMSO); IR (neat, cmꢀ1): 3354.4, 3233.1, 2954.7, 1732.5, 1627.5, 1605.2, 1573.9, 1536.9,
1
1198.5, 1173.2; H NMR (400 MHz, DMSOꢀd6) δ 9.88 (br s, 1H), 8.68 (s, 1H), 8.34 (d, J = 7.5
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Hz, 1H), 8.08 (s, 2H), 7.82 (d, J = 7.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 7.8 Hz, 2H),
7.27 (br s, 2H), 6.82 (d, J = 8.8 Hz, 2H), 5.24 (s, 2H), 4.84 (s, 2H), 4.39 (ddd, J = 9.3, 7.5, 5.7
Hz, 1H), 3.60 (s, 3H), 3.56 (s, 3H), 3.20 (s, 3H), 2.40 (t, J = 7.4 Hz, 2H), 2.15 – 1.90 (m, 2H);
13C NMR (101 MHz, DMSOꢀd6) δ 172.7, 172.6, 166.4, 162.0, 157.8, 155.1, 151.1, 150.7, 150.3,
148.0, 137.7, 134.2, 129.0, 127.0, 120.9, 120.9, 111.1, 66.7, 54.9, 51.8, 51.7, 51.4, 38.9, 30.0,
25.8. HRMS (ESI) m/z: calcd for C30H34BN8O9 [M+H]+ 661.2536, found 661.2566.
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(S)ꢀ(4ꢀ((((2ꢀAminoꢀ6ꢀ(((4ꢀ((1,5ꢀdimethoxyꢀ1,5ꢀdioxopentanꢀ2ꢀ
yl)carbamoyl)phenyl)(methyl)amino)methyl)pteridinꢀ4ꢀyl)carbamoyl)oxy)methyl)ꢀ2ꢀ
methylphenyl)boronic acid (12). Following general procedure C using 5b (1.45 g, 4.66 mmol),
o
the title compound 12 was afforded as a yellow solid (179 mg, 22%). m.p.: >166.0 C
(decomp.); [ꢀ]ꢂꢁꢃ = +4.0 (c 0.7, DMSO); IR (neat, cmꢀ1): 3340.8, 3230.2, 2951.6, 1741.8, 1604.5,
1
1510.3, 1197.7, 1166.6, 1028.4; H NMR (400 MHz, DMSOꢀd6) δ 9.82 (s, 1H), 8.69 (s, 1H),
8.34 (d, J = 7.5 Hz, 1H), 8.02 (s, 2H), 7.72 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 1H), 7.28 (br
s, 2H), 7.23 – 7.16 (m, 2H), 6.83 (d, J = 9.0 Hz, 2H), 5.18 (s, 2H), 4.84 (s, 2H), 4.39 (ddd,
J = 9.5, 7.5, 5.4 Hz, 1H), 3.61 (s, 3H), 3.56 (s, 3H), 3.20 (s, 3H), 2.44 – 2.37 (m, 5H), 2.13 – 1.88
13
(m, 2H).; C NMR (101 MHz, DMSOꢀd6) δ 172.7, 172.6, 166.4, 162.0, 157.8, 155.1, 151.0,
150.7, 150.2, 147.9, 141.4, 136.2, 133.3, 129.0, 128.9, 124.3, 120.9, 120.8, 111.1, 66.7, 54.9,
51.8, 51.7, 51.3, 38.9, 29.9, 25.8, 22.1; HRMS (ESI) m/z: calcd for C31H36BN8O9 [M+H]+
675.2693, found 675.2720.
(S)ꢀ(4ꢀ((((2ꢀAminoꢀ6ꢀ(((4ꢀ((1,5ꢀdimethoxyꢀ1,5ꢀdioxopentanꢀ2
yl)carbamoyl)phenyl)(methyl)amino)methyl)pteridinꢀ4ꢀyl)carbamoyl)oxy)methyl)ꢀ2ꢀ
fluorophenyl)boronic acid (13). Following general procedure C using 5c (1.30 g, 4.19 mmol),
o
the title compound 13 was afforded as a yellow solid (237 mg, 34%). m.p.: >158.1 C
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(decomp.); [ꢀ]ꢂꢁꢃ = +4.3 (c 0.6, DMSO); IR (neat, cmꢀ1): 3499.4, 3448.3, 3360.5, 3326.1, 3218.7,
5
6
1
2952.9, 1760.0, 1733.0, 1625.6, 1490.3, 1196.3, 1171.6; H NMR (400 MHz, DMSOꢀd6) δ 9.95
7
8
9
(s, 1H), 8.69 (s, 1H), 8.34 (d, J = 7.5 Hz, 1H), 8.22 (s, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.59 (t,
J = 7.0 Hz, 1H), 7.40 – 7.20 (m, 4H), 6.83 (d, J = 8.9 Hz, 2H), 5.25 (s, 2H), 4.85 (s, 2H), 4.39
(ddd, J = 9.4, 7.5, 5.7 Hz, 1H), 3.61 (s, 3H), 3.56 (s, 3H), 3.20 (s, 3H), 2.41 (t, J = 7.4 Hz, 2H),
2.16 – 1.86 (m, 2H); 13C NMR (100 MHz, DMSOꢀd6) δ 173.2, 173.04, 166.9, 165.9 (d, J = 244.5
Hz), 162.5, 158.3, 155.6, 151.5, 151.1, 150.7, 148.4, 140.6 (d, J = 8.3 Hz), 136.0(d, J = 9.8 Hz),
129.5, 123.4 (d, J = 2.7 Hz), 121.4, 121.3, 114.5 (d, J = 25.5 Hz), 111.6, 66.2, 55.4, 52.3, 52.2,
51.8, 39.1, 30.4, 26.2; HRMS (ESI) m/z: calcd for C30H33BFN8O9 [M+H]+ 679.2442, found
679.2455.
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Bis(4ꢀmethoxybenzyl) (4ꢀ(((2,4ꢀdiaminopteridinꢀ6ꢀyl)methyl)(methyl)amino)benzoyl)ꢀ ꢀ
L
glutamate (14). A solution of methotrexate (1.0 g, 2.20 mmol) in anhydrous DMF (50 mL) was
o
treated with 1,1,3,3ꢀtetramethylguanidine (0.55 mL, 4.4 mmol) at 0 C and the mixture was
stirred for 30 min under an N2 atmosphere. Then, 4ꢀmethoxybenzyl chloride (0.59 mL, 4.4
o
mmol) was added dropwise and the mixture was allowed to warm to 21 C and stirred for 24 h.
Next, volatiles were removed in vacuo and the crude was purified by flash column
chromatography on silica gel using a mixture of CH2Cl2/MeOH (gradient elution from 97/3 to
94/6, v/v) as the eluent to give the title compound 154as a yellow solid (915 mg, 60%). Rf = 0.24
o
(silica, CH2Cl2/MeOH, 95/5, v/v); m.p.: 96.5 – 99.2 C; [ꢀ]ꢂꢁꢃ = +8.0 (c 1.0, DMSO); IR (neat,
cmꢀ1): 3325.0, 3181.8, 2936.0, 2835.6, 1730.6, 1603.3, 1511.2, 1442.8; 1H NMR (400 MHz,
DMSOꢀd6) δ 8.56 (s, 1H), 8.34 (d, J = 7.5 Hz, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.65 (br s, 1H), 7.43
(br s, 1H), 7.27 (d, J = 6.9 Hz, 2H), 7.25 (d, J = 6.9 Hz, 2H), 6.90 – 6.86 (m, 4H), 6.82 (d, J = 8.9
Hz, 2H), 6.60 (br s, 2H), 5.10 – 4.99 (m, 2H), 4.97 (s, 2H), 4.78 (s, 2H), 4.42 (ddd, J = 9.6, 7.5,
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5.4 Hz, 1H), 3.73 (s, 6H), 3.21 (s, 3H), 2.42 (t, J = 7.6 Hz, 2H), 2.13 – 1.90 (m, 2H); 13C NMR
(101 MHz, DMSOꢀd6) δ 172.1, 172.0, 166.5, 162.9, 162.7, 159.1, 155.2, 151.0, 149.2, 145.9,
129.8, 129.7, 129.0, 128.0, 127.9, 121.4, 120.8, 113.8, 111.0, 65.7, 65.3, 55.1, 54.8, 51.9, 38.8,
30.2, 25.8; HRMS (ESI) m/z: calcd for C36H39N8O7 [M+H]+ 695.2936, found 695.2954.
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Bis(4ꢀmethoxybenzyl)
(4ꢀ(((2ꢀaminoꢀ4ꢀ((((4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
ꢀglutamate
yl)benzyl)oxy)carbonyl)amino)pteridinꢀ6ꢀyl)methyl)(methyl)amino)benzoyl)ꢀ
L
(15). To a suspension of 15 (800 mg, 1.15 mmol) in anhydrous CH2Cl2 (25 mL) was added
DMAP (703 mg, 5.76 mmol) followed by DIPEA (1.0 mL, 5.76 mmol). The mixture was cooled
o
to 0 C and a solution of 5a (1.71 g, 5.76 mmol) in CH2Cl2 (10 mL) was added dropwise. The
o
reaction was allowed to warm to 21 C and stirred for 5 h under an N2 atmosphere. The mixture
was then diluted with CH2Cl2 (100 mL), washed with 1 M aq. HCl (2 x 75 mL), sat. aq. NaHCO3
(2 x 75 mL), and brine (75 mL). The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. Purification of the residue by reversedꢀphase preparative HPLC afforded
the title compound 15 as a yellow solid (800 mg, 73%). m.p.: >105.0 (decomp.); [ꢀ]ꢂꢁꢃ = +8.8 (c
0.42, DMSO); IR (neat, cmꢀ1): 3351.5, 3230.0, 2975.5, 2836.5, 1731.3, 1605.6, 1512.6, 1356.9;
1H NMR (400 MHz, DMSOꢀd6) 9.87 (s, 1H), 8.69 (s, 1H), 8.34 (d, J = 7.5 Hz, 1H), 7.72 (d, J =
7.9 Hz, 4H), 7.47 (d, J = 7.9 Hz, 2H), 7.33 – 7.17 (m, 6H), 6.91 – 6.85 (m, 4H), 6.82 (d, J = 9.0
Hz, 2H), 5.26 (s, 2H), 5.06 – 4.98 (m, 2H), 4.96 (s, 2H), 4.84 (s, 2H), 4.43 (ddd, J = 9.5, 7.4, 5.4
Hz, 1H), 3.72 (s, 6H), 3.19 (s, 3H), 2.41 (t, J = 7.4 Hz, 2H), 2.13 – 1.91 (m, 2H), 1.29 (s, 12H);
13C NMR (101 MHz, DMSOꢀd6) δ 172.1, 172.0, 166.5, 162.0, 159.1 (2C), 157.8, 155.1, 151.1,
150.7, 150.3, 148.0, 139.3, 134.6, 129.8, 129.7, 129.0, 128.0, 127.9, 127.3, 121.0, 120.8, 113.8
(2C), 111.1, 83.7, 66.5, 65.7, 65.3, 55.1 (2C), 54.9, 51.9, 39.5, 30.2, 25.8, 24.7; HRMS (ESI)
m/z: calcd for C50H56BN8O11 [M+H]+ 955.4156, found 955.4191.
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(S)ꢀ(4ꢀ((((2ꢀAminoꢀ6ꢀ(((4ꢀ((1,5ꢀbis((4ꢀmethoxybenzyl)oxy)ꢀ1,5ꢀdioxopentanꢀ2ꢀ
yl)carbamoyl)phenyl)(methyl)amino)methyl)pteridinꢀ4ꢀ
7
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yl)carbamoyl)oxy)methyl)phenyl)boronic acid (16). To a solution of 15 (0.70 g, 0.73 mmol) in
a mixture of CH3CN/THF/H2O (200 mL, 2/1/1, v/v) was added cc. HCl until pH ~ 2. The
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reaction mixture was stirred at 21 C for 6 h followed by neutralization with sat. aq. NaHCO3
(approx. 50 mL). After removal of organic solvents in vacuo, the formed precipitate was filtered
off, washed with H2O, and purified by reversedꢀphase preparative HPLC to afford the title
o
compound 16 as a yellow solid (199 mg, 31%). m.p.: >147.8 C; [ꢀ]ꢂꢁꢃ = +8.3 (c 0.99, DMSO);
IR (neat, cmꢀ1): 3362.3, 3230.6, 2954.1, 2836.7, 1732.0, 1628.9, 1605.5, 1512.4, 1198.7, 1170.9;
1H NMR (400 MHz, DMSOꢀd6) δ 9.10 (br s, 1H), 8.64 (br s, 1H), 8.49 (s, 2H), 8.35 (d, J = 7.5
Hz, 1H), 7.78 (d, J = 7.9 Hz, 2H), 7.71 (d, J = 8.9 Hz, 2H), 7.40 (d, J = 7.9 Hz, 2H), 7.37 – 7.10
(m, 6H), 6.87 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 9.0 Hz, 1H), 5.21 (s, 2H),
5.10 – 4.98 (m, 2H), 4.96 (s, 2H), 4.83 (s, 2H), 4.42 (ddd, J = 9.6, 7.4, 5.4 Hz, 1H), 3.72 (s, 6H),
13
3.19 (s, 3H), 2.41 (t, J = 7.6 Hz, 2H), 2.15 – 1.79 (m, 2H); C NMR (101 MHz, DMSOꢀd6) δ
172.2, 172.0, 166.6, 162.1, 159.1 (2C), 157.8, 155.2, 151.1, 150.7, 150.3, 148.0, 132.7, 134.3,
129.9, 129.7, 129.1, 128.0, 127.9, 127.1, 121.0, 120.9, 113.8 (2C), 111.1, 66.7, 65.8, 65.4, 55.1
(2C), 54.9, 51.9, 39.35, 30.2, 25.8; HRMS (ESI) m/z: calcd for C44H46BN8O11 [M+H]+ 873.3374,
found 873.3405.
(4ꢀ(((2ꢀaminoꢀ4ꢀ((((4ꢀBoronobenzyl)oxy)carbonyl)amino)pteridinꢀ6ꢀ
yl)methyl)(methyl)amino)benzoyl)ꢀ ꢀglutamic acid (17). A solution of 16 (150 mg, 0.17mmol)
L
in a mixture of 5% TFA in anhydrous CH2Cl2 (16 mL) was stirred for 25 min under an N2
atmosphere at 21 oC. The crude mixture was then concentrated in vacuo and the residue purified
by reversedꢀphase preparative HPLC to afford the title compound 17 as an yellow/orange solid
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(59 mg, 54%). m.p.: >200 oC (decomp.); [ꢀ]ꢁꢂꢃ = +10.0 (c 0.50, DMSO); IR (neat, cmꢀ1): 3336.8,
5
6
1
3203.3, 2929.5, 1728.8, 1604.5, 1505.5, 1199.4, 1171.1; H NMR (400 MHz, DMSOꢀd6) δ 12.29
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8
9
(br s, 2H), 9.89 (s, 1H), 8.68 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.07 (s, 2H), 7.82 (d, J = 7.9 Hz,
2H), 7.73 (d, J = 8.9 Hz, 2H), 7.43 (d, J = 7.9 Hz, 2H), 7.28 (br s, 2H), 6.82 (d, J = 8.9 Hz, 2H),
5.24 (s, 2H), 4.84 (s, 2H), 4.34 (ddd, J = 9.7, 7.8, 5.0 Hz, 1H), 3.19 (s, 3H), 2.31 (t, J = 7.5 Hz,
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2H), 2.11 – 1.99 (m, 1H), 1.99 – 1.79 (m, 1H); C NMR (101 MHz, DMSOꢀd6) δ 173.9, 173.7,
166.3, 162.0, 157.8, 155.1, 151.0, 150.7, 150.3, 148.0, 137.7, 134.2, 129.0, 127.0, 121.3, 120.9,
111.1, 66.7, 54.9, 51.7, 39.0, 30.4, 26.0; HRMS (ESI) m/z: calcd for C28H28BN8O9 [MꢀH]ꢀ
631.2078, found 631.2028.
4ꢀ(((Benzyloxy)carbonyl)amino)benzoic acid (19). To a solution of 4ꢀmethylaminobenzoic
acid 18 (4.11 g, 29.9 mmol) and NaHCO3 (22.6 g, 269 mmol) in a mixture of H2O/THF (100
o
mL, 1/1, v/v) was added benzyl chloroformate (4.26 mL, 29.9 mmol) dropwise at 0 C. The
o
reaction mixture was stirred for 4 h at 21 C followed by addition of 20 mL of water. Then, the
mixture was stirred for an additional 16 h followed by acidification with 1 M aq. HCl to pH ~ 3.
Next, the precipitate was filtered off and dried in vacuo to afford 19 as a white solid (7.2 g,
o
o
89%). m.p.: 213.0–217.0 C (lit.72 217 C, decomp.); IR (neat, cmꢀ1): 3324.4, 2951.7, 2677.4,
1
2557.2, 1702.1, 1672.4, 1528.6, 1512.8, 1225.8, 1062.6; H NMR (400 MHz, DMSOꢀd6) δ 12.66
(br s, 1H), 10.15 (s, 1H), 7.87 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.51 – 7.17 (m, 5H),
5.17 (s, 2H);73 13C NMR (101 MHz, DMSOꢀd6) δ 167.0, 153.2, 143.3, 136.3, 130.5, 128.5,
128.2, 128.2, 124.5, 117.3, 66.1.74
Dimethyl (4ꢀ(((benzyloxy)carbonyl)amino)benzoyl)ꢀ ꢀglutamate (20). To a suspension of
L
19 (2.90 g, 10.7 mmol) in anhydrous CH2Cl2 was added 1 drop of DMF followed by thionyl
chloride (3.88 mL, 53.4 mmol) and the reaction mixture was refluxed for 24 h under an N2
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atmosphere. Next, all volatiles were removed in vacuo to afford the acyl chloride as a pale
yellow solid (3.1 g, >95%) which was added to a stirred mixture of 9·HCl (2.40 g, 11.3 mmol)
and Et3N in anhydrous CH2Cl2 (100 mL) and an additional 2 h stirring was applied under an N2
atmosphere at 21 oC. The mixture was then diluted with EtOAc (250 mL), washed with 1 M aq.
HCl (2 x 150 mL), sat. aq. NaHCO3 (2 x 150 mL), and brine (200 mL), dried over Na2SO4,
filtered, and concentrated in vacuo to afford the title compound 20 as a white solid (4.3 g, 97%).
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m.p.: 121.7–124.0 C; [ꢀ]ꢂꢁꢃ = ꢀ16.2 (c 1.0, MeOH); IR (neat, cmꢀ1): 3378.3, 3297.8, 2952.4,
1730.5, 1627.7, 1526.8, 1227.4; 1H NMR (400 MHz, DMSOꢀd6) δ 10.07 (s, 1H), 8.60 (d, J = 7.4
Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.50 – 7.26 (m, 5H), 5.18 (s, 2H),
4.44 (ddd, J = 9.5, 7.4, 5.4 Hz, 1H), 3.64 (s, 3H), 3.59 (s, 3H), 2.45 (t, J = 7.5 Hz, 2H), 2.22 –
1.83 (m, 2H); 13C NMR (101 MHz, DMSOꢀd6) δ 172.7, 172.4, 166.1, 153.2, 142.1, 136.4, 128.5
(2C), 128.2, 128.1, 127.3, 117.2, 66.0, 51.9, 51.9, 51.4, 29.9, 25.7; HRMS (ESI) m/z: calcd for
C22H25N2O7 [M+H]+ 429.1656, found 429.1676.
Dimethyl (4ꢀaminobenzoyl)ꢀ ꢀglutamate hydrochloride (21·HCl). A solution of 20 (2.0 g,
L
4.8 mmol) in anhydrous MeOH (100 mL) was treated with Pd/C (200 mg, 10% w/w). The
o
mixture was stirred for 12 h under a H2 atmosphere at 21 C, then filtered through a path of
Celite®, concentrated in vacuo over silica gel and purified by flash column chromatography on
silica gel using a mixture of EtOAc/Hep (2/1, v/v) as the eluent to give a clear oil corresponding
to the free amine 21. Treatment with 2 M HCl in ether afforded the hydrochloride salt 21·HCl as
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a white solid (1.3 g, 95%). Rf = 0.38 (free amine; EtOAc/Hep, 4/1, v/v); m.p.: 65.2 – 70.0 C;
[ꢀ]ꢂꢁꢃ = ꢀ17.8 (c 1.26, H2O); IR (neat, cmꢀ1): 3270.9, 2952.3, 2834.2, 2577.2, 1731.4, 1641.3,
1609.8, 1538.9, 1501.4, 1436.4, 1206.7, 1171.7; 1H NMR (400 MHz, D2O) δ 7.78 (d, J = 8.5 Hz,
2H), 7.33 (d, J = 8.5 Hz, 2H), 4.58 (dd, J = 9.4, 5.2 Hz, 1H), 3.70 (s, 3H), 3.56 (s, 3H), 2.49 (t,
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Journal of Medicinal Chemistry
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J = 7.0 Hz, 2H), 2.35 – 2.20 (m, 1H), 2.10 (ddt, J = 14.2, 9.4, 6.9 Hz, 1H); C NMR (101 MHz,
D2O) δ 175.8, 173.7, 169.8, 136.8, 131.5, 129.2, 121.9, 53.0, 52.7, 52.3, 30.2, 25.5; HRMS (ESI)
m/z: calcd for C14H19N2O5 [M+H]+ 295.1288, found 295.1297.
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(S)ꢀ(4ꢀ(((4ꢀ((1,5ꢀDimethoxyꢀ1,5ꢀdioxopentanꢀ2ꢀ
yl)carbamoyl)phenyl)amino)methyl)phenyl) boronic acid (22). To a solution of 21⋅HCl (0.80
g, 2.42 mmol) in anhydrous MeOH was added (4ꢀformylphenyl)boronic acid (0.55 g, 3.67 mmol)
followed by portionꢀwise addition of NaBH3CN (0.80 g, 12.1 mmol). The reaction mixture was
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stirred for 12 h under an N2 atmosphere at 21 C. Extra (4ꢀformylphenyl)boronic acid (0.55 g,
3.67 mmol) was then added to the stirred mixture followed by an additional 24 h of stirring for
completion of the reaction. Next, the crude mixture was concentrated in vacuo, redissolved in
EtOAc (150mL), and washed with sat. aq. NaHCO3 (2 x 150 mL) and brine (150 mL). The
organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to afford a pale yellow
solid. Purification by reversedꢀphase preparative HPLC afforded the title compound 22 as a
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white solid (699 mg, 67%). Rf = 0.77 (silica, MeOH/CH2Cl2, 1/1, v/v); m.p.: 84.1‒88.2 C;
[ꢀ]ꢂꢁꢃ = ꢀ16.3 (c 1.04, DMSO); IR (neat, cmꢀ1): 3382.1, 2951.9, 1726.9, 1601.9, 1506.4, 1330.4;
1H NMR (400 MHz, DMSOꢀd6) δ 8.24 (d, J = 7.5 Hz, 1H), 7.98 (s, 2H), 7.73 (d, J = 7.8 Hz, 2H),
7.62 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 6.84 (t, J = 6.0 Hz, 1H), 6.58 (d, J = 8.6 Hz,
2H), 4.38 (ddd, J = 9.5, 7.5, 5.6 Hz, 1H), 4.33 (d, J = 6.0 Hz, 2H), 3.61 (s, 3H), 3.57 (s, 3H), 2.41
13
(t, J = 7.4 Hz, 2H), 2.14 – 2.03 (m, 1H), 2.02 – 1.86 (m, 1H); C NMR (101 MHz, DMSOꢀd6) δ
172.8, 172.7, 166.6, 151.4, 141.6, 134.2, 129.0, 126.2, 120.4, 111.1, 51.8, 51.7, 51.4, 46.0, 30.0,
25.8; HRMS (ESI) m/z: calcd for C21H26BN2O7 [M+H]+ 429.1828, found 429.1850.
(S)ꢀ(4ꢀ((((2,4ꢀDiaminopteridinꢀ6ꢀyl)methyl)(4ꢀ((1,5ꢀdimethoxyꢀ1,5ꢀdioxopentanꢀ2ꢀ
yl)carbamoyl)phenyl)amino)methyl)phenyl)boronic acid (24). A solution of 22 (461 mg, 1.08
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