Total synthesis of methotrexate-γ-TRIS-fatty acid conjugates

Article abstract of DOI:10.1071/CH02125

Several routes for the regiospecific synthesis of lipophilic γ-conjugates of methotrexate are described. Coupling of methotrexate-α-tert-butyl ester with glycyl-TRIS-(mono-/di-/tri-)palmitate followed by trifluoroacetic acid cleavage of the α-protection a

Full text of DOI:10.1071/CH02125

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Aust. J. Chem. 2002, 55, 635–645
C
H
02152
C
a
L
.
F
a
nic
s
Total Synthesis of Methotrexate-γ-TRIS-Fatty Acid Conjugates
Craig L. Francis,A,B Qi Yang,A Noel K. Hart,A Fred Widmer,C
Michael K. MantheyC and H. Ming He-WilliamsC
A CSIRO Molecular Science, Ian Wark Laboratory, Bag 10, Clayton South
Clayton, Vic. 3169, Australia.
B Author to whom correspondence should be addressed (E-mail: craig.francis@csiro.au).
C CSIRO Molecular Science, Sydney laboratory, P.O. Box 184, North Ryde,
N.S.W. 1670, Australia.
Several routes for the regiospecific synthesis of lipophilic γ-conjugates of methotrexate are described. Coupling of
methotrexate-α-tert-butyl ester with glycyl-TRIS-(mono-/di-/tri-)palmitate followed by trifluoroacetic acid
cleavage of the α-protection afforded the target methotrexate-γ-glycyl-TRIS-(mono-/di-/tri-)palmitate derivatives.
Methotrexate-α-benzyl-γ-glycyl-TRIS-tripalmitate was prepared but no method was found to selectively cleave the
α-ester. A method where the diaminopteridinylmethyl moiety was attached last was successful, but was low yielding
in the final step. Coupling of 4-amino-4-deoxy-N10-methylpteroic acid with glutamoyl-γ-glycyl-TRIS-palmitate
derivatives efficiently afforded the desired conjugates.
Manuscript received: 19 June 2002.
Final version: 6 August 2002.
Introduction
in organs such as the liver and kidney. Therefore,
γ-substituted derivatives are less cytotoxic as they are
unable to form polyglutamates.
Methotrexate(MTX)[(1),seeDiagram1],awidelyuseddrug,
has cytotoxic and anti-inflammatory activities and is used to
treat a range of diseases including cancer,[1] rheumatoid
arthritis,[1] and psoriasis.[2] Whilst its anti-inflammatory
mechanism is not well understood, the drug inhibits cell
replicationthroughitsactionasafolateantagonist,preventing
theconversionoffolateintotetrahydrofolateviadihydrofolate
by dihydrofolate reductase (DHFR). MTX also inhibits other
folate-dependent enzymes.[3]
The carboxyl groups of the glutamyl moiety of MTX
have important biological roles.[3] Derivatization of the
carboxyl groups, particularly the α-carboxyl, impairs
transport into the cells and binding to target enzymes such
as DHFR. The addition of two to five glutamyl groups to
the γ-carboxyl group (polyglutamylation) in vivo increases
the binding of MTX to appropriate enzymes and prevents
MTX efflux via transport systems. Polyglutamate formation
and accumulation has unwanted effects in vivo where
prolonged intracellular retention leads to long-term toxicity
Studies in our laboratories have developed a method of
conjugating fatty acids to compounds using 2-amino-2-
hydroxymethylpropane-1,3-diol (TRIS).[3–10] We applied
this method to methotrexate and prepared fatty acid
conjugates (2)–(5) (Diagram 2) where the γ-carboxyl group
of MTX is selectively connected by an amide bond to a
conjugate group consisting of a glycyl-TRIS-palmitate
ester(s) linkage.[3,6,8,9]
These conjugates were biologically evaluated in the areas
of in vitro DHFR inhibition,[3,9] in vitro cytotoxicity,[3,9] and
with rats or mice for anti-tumour activity,[8,9] delayed type
hypersensitivity (immune) response,[9] and adjuvant-induced
arthritis inhibition.[9] The tripalmitate (2) was also evaluated
as a potential topical treatment for psoriasis in a proof-of-
concept clinical trial in humans.[9]
α
O
CO2H
γ
NH2
N
N
CO2H
H
N
N
N
N
H2N
(1) MTX
Diagram 1
© CSIRO 2002
10.1071/CH02125
0004-9425/02/100635

636
C. L. Francis et al.
This paper details improved synthetic methodology (over
(1)
that originally described[3,6,8]) for preparation of the
methotrexate conjugates (2)–(5).
DCC
We have reported syntheses of N-protected precursors of
the necessary amines [i.e. (6), (7), (8), and (9), see
Diagram 3].[10]
α
γ
O
O
O
O
NH2
N
N
H
N
N
O
H
OR1
OR2
N
N
N
N
R
H
H2N
(12)
OR3
(6) R = BnOCO, R1 = R2 = R3 = palmitoyl
(7) R = BnOCO, R1 = R2 = palmitoyl, R3 = H
(8) R = BnOCO, R1 = palmitoyl, R2 = R3 = H
(9) R = BnOCO, R1 = R2 = R3 = H
O
OCO(CH2)14CH3
OH
H
O
N
N
O
H
NH2
N
N
OH
CO2H
(10) R = H, R1 = R2 = R3 = palmitoyl
H
N
N
N
N
(11) R = H, R1 = R2 = palmitoyl, R3 = H
(12) R = H, R1 = palmitoyl, R2 = R3 = H
(13) R = H, R1 = R2 = R3 = H
(14) α-isomer
+ (4) γ-isomer
H2N
Diagram 3
Scheme 1
Results and Discussion
ester of 5-aminoisophthalic acid in the presence of one
equivalent of 4-dimethylaminopyridine (DMAP) to give the
γ-amide product. Other bases did not allow such high
selectivity (if any at all) and in the absence of base, an 85 : 15
α : γ ratio was observed.[11]
We briefly revisited our MTX-anhydride opening with
(10) employing one equivalent of DMAP in an ice-cooled
reaction. Unfortunately, as before, a 7 : 3 α : γ ratio was
observed. The greater γ-regioselectivity of the aniline
compared with our primary amine may be due to the reduced
nucleophilicity and greater steric bulk.[12]
Initially, direct coupling of glycyl-TRIS-palmitate moieties
to (1) was investigated. This was expected to produce a
mixture of the α- and γ-isomers, from which the desired γ-
derivative could be isolated.
The required amines (10), (11), (12), and (13) (see
Diagram 3) were liberated from their N-protected
precursors[10] (6), (7), (8), and (9), respectively, by standard
hydrogenolysis* over palladium-on-carbon catalyst as they
were required.
Treatment of (1) with 1.2 equivalents of 1,3-
dicyclohexylcarbodiimide
(DCC)
and
N-hydroxy-
Since selective attachment of the conjugate groups to the
γ-carboxyl moiety of the intact MTX molecule was not
achieved, nor separation of the α- and γ-isomer mixtures on
preparative scale, investigations into total syntheses to
unambiguously produce the MTX derivatives (2)–(5) were
undertaken.
The first method centred on the selectively protected
MTX-α-tert-butyl ester (15), initially prepared using
reported procedures.[13–15] A more efficient synthetic
method for (15), which avoided chromatography, is outlined
below (Scheme 2).
succinimide (HOSu) to provide a mix of active esters,
followed by reaction with amine (12) afforded an
approximately 1 : 1 mixture of α- and γ-isomers which was
practically chromatographically homogeneous.
We briefly studied reaction of (12) with the anhydride of
the glutamic acid moiety of (1) to determine if an amine
nucleophile would attack preferentially at the sterically less
hindered γ-carbonyl group of the anhydride, leading to the
γ-conjugate (see Scheme 1). The MTX anhydride
intermediate, formed with DCC in N,N-dimethylformamide
(DMF), was treated with monopalmitate (12) providing a
mixture of α- and γ-isomers (14) and (4), respectively, in a
ratio of 7 : 3.
The N-protected amino acid (16)[16,17] was converted into
its acid chloride (17) and reacted with α-tert-butyl-L-
glutamate (18) to give (19). The benzyloxycarbonyl group
was cleaved by standard hydrogenolysis to afford amine (20).
Reaction of amine (20) with 6-bromomethyl-2,4-
diaminopteridine hydrobromide (21)[18] provided (15).
Ester (15) was also synthesized from (22) (see
Diagram 4),[14,15] and (18) using 2-(2-oxo-1(2H)-pyridyl)-
An analogous experiment with tripalmitate (10) provided
a near identical isomer ratio. We were unable to separate the
isomers on a preparative scale.
During the preparation of this manuscript, a report[11]
appeared describing regioselective opening of N-
benzyloxycarbonyl-glutamic anhydride with the dibenzyl
* ‘Standard hydrogenolysis’ refers to use of 10% palladium-on-carbon (Pd/C) catalyst with ethanol as solvent and a hydrogen atmosphere from a
hydrogen-filled balloon. In the tripalmitate case (conversion of (6) into (10)), ethyl acetate was added as co-solvent.

Total Synthesis of Methotrexate-γ-TRIS-Fatty Acid Conjugates
637
α
O
CO2tBu
O
H
OR1
OR2
γ
N
N
NH2
N
N
H
H
N
N
O
OR3
N
N
(23) R1 = R2 = R3 = palmitoyl
(24) R1 = R2 = palmitoyl, R3 = H
(25) R1 = palmitoyl, R2 = R3 = H
(26) R1 = R2 = R3 = H
H2N
Diagram 5
O
O
CO2tBu
OR1
H
N
N
H
N
OR2
H
R
O
N
OR3
(27) R = BnOCO, R1 = R2 = R3 = palmitoyl
(28) R = H, R1 = R2 = R3 = palmitoyl
Diagram 6
Synthesis of the desired conjugates using the
corresponding α-tert-butyl esters required two consecutive
and inefficient (particularly in solvent volume)
chromatographic purification procedures that were not
amenable to scale-up. Multi-gram quantities of the desired
conjugates were required to enable comprehensive
investigations of their therapeutic potential and scale-up of
two inefficient procedures was not desired. Therefore, we
investigated another route utilizing benzyl ester protection
for the α-carboxyl group instead of tert-butyl ester; a
modification we hoped would result in a milder and cleaner
α-deprotection step.
CO2H
NH2
N
N
N
N
N
H2N
The glutamate (29) was coupled with amine (10) using
DCC and catalytic DMAP to give (30). The tert-
butyloxycarbonyl group was removed using TFA in DCM
providing (31) (Scheme 3). Coupling of (31) to (22), using
either DCC or diethyl cyanophosphonate (DECP), produced
(32) in moderate yield (max. 50% with DECP).
Cleavage of the α-benzyl ester of (32) to give the
tripalmitate conjugate (2) proved to be problematic. Standard
hydrogenolysis of (32) in ethanol, or ethyl acetate,
containing a small amount of acetic acid, resulted in minimal
reaction. Treatment with either palladium-on-carbon or
platinum oxide catalyst in acetic acid resulted in a complex
mixture of products.
(22)
Diagram 4
1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU) as the
coupling agent.
Coupling of (15) with amine (10) was accomplished with
DCC and catalytic DMAP in dichloromethane (DCM)/DMF
to give the α-protected conjugate (23). Cleavage of the α-
tert-butyl ester moiety of (23), see Diagram 5, was achieved
with trifluoroacetic acid (TFA) in DCM to afford the
tripalmitate conjugate (2).
Analogous syntheses from (15) with amines (11), (12),
and (13) provided conjugates (3), (4), and (5), respectively,
via the corresponding α-tert-butyl esters (24), (25), and (26),
see Diagram 5.
The tripalmitate (23) was also prepared in moderate yield
from the bromomethylpteridine (21)[18] and amine (28), see
Diagram 6. Amine (28) was prepared by DCC-mediated
coupling of (10) and (19) to give (27), followed by
hydrogenolysis.
A literature search suggested the hydrogenolysis would
have problems. Hydrogenations (of double bonds) in folate-
related molecules have been achieved[19–21] but with
difficulty. Reduction of the heterocyclic ring occurred as a
side reaction.[19,20] Interestingly, none of these substrates had
a nitrogen atom at the 10-position (unlike our case).
We briefly investigated hydrolytic cleavage of (32) to (2).
The α-benzyl ester group in glutamoyl moieties in similar
molecules has been hydrolysed with barium hydroxide in

638
C. L. Francis et al.
O
CO2Bn
O
CO2Bn
(10)
+
O
N
CO2H
O
N
H
CO2H
H
BnO
N
(29)
Pm = palmitoyl
(35)
(i) DCC
HOSu
(ii) (10) or
(12) or (13)
O
CO2Bn
OPm
OPm
H
N
R
N
N
H
H
O
O
CO2R'
OR1
OR2
H
O
N
N
H
N
OPm
(30) R = ButOCO
(31) R = H
H
R
TFA
O
N
OR3
DECP, Et2NPri
(33) R = BnOCO, R' = Bn, R1 = R2 = R3 = palmitoyl
(34) R = R' = H, R1 = R2 = R3 = palmitoyl
(22)
a
or DCC
(21)
(21)
(2)
(4)
O
CO2Bn
O
(36) R = BnOCO, R' = Bn, R1 = palmitoyl, R2 = R3 = H
(37) R = R' = H, R1 = palmitoyl, R2 = R3 = H
H
N
OPm
OPm
a
a
N
H
NH2
N
N
H
N
O
OPm
N
N
(38) R = BnOCO, R' = Bn, R1 = R2 = R3 = H
(39) R = R' = R1 = R2 = R3 = H
(21)
N
H2N
(32)
(5)
a = H2/Pd-C
Scheme 3
Scheme 4
aqueous ethanol[13] or sodium hydroxide in aqueous
methanol.[22] However, we were unable to find conditions to
hydrolyse the benzyl ester whilst leaving the palmitate esters
intact.
from the corresponding step was very poor (less than 10%).
Hence another synthetic method was required.
A more efficient synthetic route involved the pteroic acid
(22),[14,15] which we had in plentiful supply from earlier work.
Treatment of α-benzyl-N-benzyloxycarbonyl-L-glutamate
(43) with DCC/HOSu and then aminotriol (13) provided (44).
Analogous reactions with monopalmitate (12), dipalmitate
(11), and tripalmitate (10) afforded (45), (46), and (47),
respectively (Scheme 5). Hydrogenolysis of (44) provided
free amino acid (48). TPTU-mediated coupling of (48) to
(22)[14,15] produced (5). Analogous chemistry with (45)
afforded monopalmitate (4) via (49), and with tripalmitate
(45) gave (2) via (50) (Schemes 5 and 6).
Another route, utilizing chemistry developed by Piper and
co-workers,[17] also involves benzyl protection, but avoids the
need to carry out hydrogenolysis in the presence of the
(pteridinylmethyl)methylamino moiety. Treatment of amine
(31) with acid chloride (17) provided (33). Standard
hydrogenolysis of this compound cleaved both the
benzyloxycarbonyl protecting group and the benzyl ester,
providing (34). Reaction of this compound with the pteridine
bromide (21)[18] afforded the tripalmitate conjugate (2) in
moderate yield (Scheme 4). Compound (33) was prepared
more efficiently by coupling of amine (10) to the γ-carboxyl
groupof(35).[17] Thechemistrydescribedabovewas repeated
with monopalmitate (12) to provide (4) via intermediates (36)
and (37). Use of aminotriol(13) afforded(5)via(38)and(39).
Theyieldof(36)fromcouplingof(12)to(35)[17] was lower
than desired (ca. 40%) and we suspected side reactions were
occurring at the two free hydroxyl groups of (12) The process
was improved by the coupling of (35)[17] with the
monopalmitate/acetonide (41) (liberated from (40),[10] see
Diagram7),whichhasthenon-palmitoylatedhydroxylgroups
blocked.
We were unable to isolate dipalmitate (51) from
hydrogenolysis of (46). Thin-layer chromatography (TLC)
O
O
H
N
O
N
R
H
OCO(CH2)14CH3
(40) R = BnOCO
(41) R = H
O
O
CO2Bn
O
H
N
This modification resulted in a 73% yield of (42). The
acetonide group was cleaved by acid-catalysed hydrolysis
affording (36) and the sequence continued as before.
The last step of coupling amine (37) with pteridine
bromide (21)[18] was low yielding (20–40%). When
aminotriol (13) was used as the conjugate group, the yield
O
O
N
H
N
H
O
OCO(CH2)14CH3
O
N
(42)
Diagram 7

Total Synthesis of Methotrexate-γ-TRIS-Fatty Acid Conjugates
639
Experimental
O
CO2Bn
(43)
General
BnO
N
CO2H
H
General experimental conditions have been described previously.[10]
For high-pressure liquid chromatography (HPLC) analysis of di- and
tri-palmitate derivatives, a gradient system was used commencing with
50%acetonitrile/37.5% tetrahydrofuran/12.5%water(containing0.05%
TFA) for 3 min followed by a 22 min gradient to 50% acetonitrile/50%
tetrahydrofuran (containing 0.05% TFA). For the mono-palmitate
derivatives, an isocratic mobile phase of 80% methanol/water
(containing 0.05% TFA) was used. For triol (5), a gradient system was
used commencing with 20% acetonitrile/80% aqueous ammonium
formate solution (0.05 M, adjusted to pH 3.5with formic acid) for 2 min
followed by a 13 min gradient to 80% acetonitrile/20% aqueous
ammonium formate solution (0.05 M, adjusted to pH 3.5with formic
acid). For MTX-α/γ-tripalmitate mixtures, a mobile phase of 50%
acetonitrile/35.5% tetrahydrofuran/14.5% water (containing 0.05%
TFA) was used on a 250 × 4.6 mm Alltima C18 column. For MTX-α/γ-
monopalmitate mixtures, a mobile phase of 50% acetonitrile/50% water
(containing 0.05% TFA) on a 250 × 10 mm Alltima C18 column with a
flow rate of 4 mL/min was used. Preparative, reversed-phase HPLC of
(5) was performed with a Waters µBondapak C18, 40 × 100 mm column,
using a flow rate of 40 mL/min. A gradient system was used starting with
water for 10 min, followed by a 25 min gradient to 8% methanol/water.
(i) DCC
HOSu
(ii) (13) or (12)
or (11) or (10)
O
CO2Bn
O
OR1
OR2
H
N
BnO
N
N
H
H
O
OR3
(44) R1 = R2 = R3 = H
(45) R1 = palmitoyl, R2 = R3 = H
(46) R1 = R2 = palmitoyl, R3 = H
(47) R1 = R2 = R3 = palmitoyl
H2
Pd/C
1
In the H NMR spectra of compounds prepared in this work, NH2
signals usually appeared as very broad signals, which were difficult to
discern. They are quoted only when clearly observed.
O
CO2H
OR1
OR2
H
N
H2N
N
Synthesis
H
O
N-[2-[(1-Oxohexadecyl)oxy]-1,1-bis[[(1-
oxohexadecyl)oxy]methyl]ethyl]glycinamide (10)
OR3
(48) R1 = R2 = R3 = H
The N-protected tripalmitate (6)[10] (13.38 g, 13.02 mmol) was
dissolved with warming in a mixture of ethyl acetate (80 mL) and
ethanol (200 mL). Palladium-on-carbon (10%, 1.00 g) was added. The
reaction mixture was vigorously stirred under hydrogen gas (balloon) at
40°C for 20 h, cooled, and then filtered. The filtrate was evaporated to
yield the title compound (10) (11.16 g, 96%) as a white, waxy solid,
(49) R1 = palmitoyl, R2 = R3 = H
(50) R1 = R2 = R3 = palmitoyl
(51) R1 = R2 = palmitoyl, R3 = H
Scheme 5
1
which was used without further purification. H NMR δ ((CD3)2SO+
and some CDCl3) 7.56, s, NH; 4.43, br, s, 3 × CH2O; 3.15, br s, CH2N;
2.32, t, J 7.2 Hz, 3 × CH2CO; 1.69–1.50, m, 3 × CH2CH2CO; 1.28, br,
36 × CH2; 0.88, t, J 6.5 Hz, CH3. Mass spectrum (ESI+) m/z 894 (M + H,
100%), 916 (M + Na, 17).
The following compounds were prepared by employing the
appropriate N-protected substrate in the above procedure, but using
ethanol as sole solvent (this procedure in 100% ethanol is hereinafter
referred to as ‘the standard hydrogenolysis procedure’). The three crude
products described in this section were of sufficient purity to be used
without further purification.
analysis suggested that the starting material had been
consumed, but we could not extract the product into a solvent
to enable the catalyst to be filtered off. This was despite
using catalytic palladium on carbon, alumina, or barium
sulfate supports, and a wide range of hot extraction solvents.
In summary, regiospecific routes have been developed for
the synthesis of MTX-γ-conjugates (2), (3), (4), and (5).
N-[1-Hydroxymethyl-2-[(1-oxohexadecyl)oxy]-1-[(1-
oxohexadecyl)oxymethyl]ethyl] glycinamide (11)
Obtained 12.13 g, 97%, from (7)[10] at 45°C for 19 h. 1H NMR δ
(CDCl3) 7.95, s, TRIS-NH; 4.35 and 4.16, AB, J 11.3 Hz, 2 × CH2O;
3.84, s, HOCH2; 3.57, s, CH2N; 2.36, t, J 7.5 Hz, 2 × CH2CO2;
1.71–1.51, m, 2 × CH2CH2CO2; 1.28, br s, 24 × CH2; 0.88, t, J 6.3 Hz,
2 × CH3. Mass spectrum (ESI+) m/z 656 (M + H, 100%).
N-[1,1-Bis(hydroxymethyl)-2-[(1-oxohexadecyl)oxy]ethyl]glycinamide
(12)
1
Obtained 7.71 g, 98%, from (8)[10] at room temperature for 4.5 h. H
NMR δ (CDCl3) 8.19, br, NH; 4.28, s, CH2O; 3.71, d, and 3.54, AB, J
12.5 Hz, 2 × HOCH2; 3.37, s, CH2N; 2.36, t, J 6.8 Hz, CH2CO2;
1.74–1.53, m, CH2CH2CO2; 1.24, br s, 12 × CH2; 0.88, t, J 6.9 Hz, CH3.
Mass spectrum (CI+) m/z 417 (M + H, 62%), 399 (M – H2O, 100).
N-[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinamide (13)
1
Obtained 4.84 g, 100%, from (9)[10] at room temperature for 19 h. H
NMR δ ((CD3)2SO) 7.95, br s, NH; 4.96, br s, 3 × OH; 4.37, br, NH2;
3.55, s, 3 × CH2O; 3.24, s, NCH2CO.

640
C. L. Francis et al.
Direct Conjugation of MTX (1). Nα-[[4-[[(2,4-Diamino-6-pteridinyl)-
methyl]methylamino]benzoyl]-L-(α- and γ-)glutamyl]-N-[1,1-bis-
(hydroxymethyl)-2-[(1-oxohexadecyl)oxy]ethyl]glycinamide (14) and
(4)
stirred at room temperature for 31 h. The solution was concentrated
under vacuum and the residue dissolved in water (20 mL). The solution
was adjusted to pH 4 with 0.33 N NaOH and the resulting precipitate
was collected, rinsed with water and dried under vacuum at 40°C. This
provided the crude title compound (15) (110 mg. 86%) as a yellow
powder. 1H NMR δ ((CD3)2SO) 12.05, br, CO2H; 8.63, s, H-7; 8.21, d,
J 7.5 Hz, glu-NH; 8.04, br, NH2; 7.73, d, J 8.9 Hz, 2 × ArH; 7.11, br,
NH2; 6.82, d, J 9.0 Hz, 2 × ArH; 4.82, s, CH2N; 4.35–4.18, m, glu-α-H;
3.22, s, NCH3; 2.32, t, J 7.4 Hz, glu-γ-CH2; 2.11–1.75, m, glu-β-CH2;
1.39, s, C(CH3)3. Mass spectrum (ESI+) m/z 511 (M + 1, 44%), 533
(M + Na, 100).
Method A
MTX (1) (200 mg, 0.44 mmol), HOSu (56 mg, 0.48 mmol) and DCC
(110 mg, 0.53 mmol) were dissolved in acetonitrile (6 mL) and DMF
(6 mL). The resulting mixture was stirred at room temperature
overnight. A solution of (12) (200 mg, 0.48 mmol) in DMF (3 mL) was
added and the mixture stirred at room temperature for 6 h. Water (5 mL)
was added, the mixture stirred, and the yellow precipitate was filtered
off. The solid was washed with water and acetonitrile and purified on a
silica flash column eluting with chloroform/methanol/water (70 : 30 : 0
to 65 : 30 : 5) affording a mixture of α- and γ-isomers in a ratio of 56 : 44
(by HPLC).
Method B
The pteroic acid (22)[15] (2.01 g, 6.18 mmol) was suspended in DMF
(40 mL). Triethylamine (1.75 mL, 12.4 mmol) was added with stirring
and the solid dissolved. TPTU (1.84 g, 6.17 mmol) was added and the
reaction mixture was stirred at room temperature under nitrogen for
2.5 h. In a separate vessel, triethylamine (1.05 mL) and (18) (1.40 g,
6.89 mmol) were suspended in DMF (30 mL). The active ester
mixture was added slowly to the triethylamine/(18) suspension (rinsed
in with DMF (3 mL)) and the resulting mixture was stirred at room
temperature under nitrogen for 20 h. The mixture was concentrated
and the syrupy residue triturated with ethyl acetate. The resulting solid
was collected by filtration and stirred in chloroform (ca. 70 mL). The
solid was collected by filtration, washed with fresh chloroform, and
dried at room temperature under vacuum providing the title compound
(15) (2.80 g, 89%) as a dark, orange solid, which was of lower purity
(by NMR and HPLC) than samples of (15) prepared by the earlier
method.
Method B
A similar reaction to above, except omitting HOSu (allowing formation
of the MTX-internal anhydride intermediate), gave a mixture of α- and
γ-isomers in a ratio of 7 : 3 (by HPLC).
Use of the tripalmitoylated amine (10) (dissolved in DCM) in
method B also gave an α-to-γ ratio of 7 : 3.
The above reaction was repeated, with the additional step[11] of
adding DMAP (54 mg, 0.44 mmol) in DCM (0.5 mL) before adding
(10). The mixture was ice-cooled during and after these additions and
worked up by evaporation and by washing a DCM solution of the
residue with 10% aqueous citric acid followed by solvent removal.
HPLC analysis of the crude product revealed a mixture of α- and γ-
isomers in a 7 : 3 ratio.
α-t-Butyl-N-[4-[(benzyloxycarbonyl)methylamino]benzoyl]-L-
glutamate (19)
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
(α-t-butoxy)glutamoyl]-N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-
oxohexadecyl)oxy]methyl]ethyl]glycinamide (23)
A mixture of (16)[17] (2.82 g, 9.9 mmol), thionyl chloride (5 mL) and
toluene (56 mL) was heated under reflux for 4 h. The mixture was
cooled and evaporated to afford the acid chloride (17) as a cream solid,
which was evacuated at room temperature overnight. The crude acid
chloride (17) was dissolved in dioxan (18 mL) and the resulting solution
added dropwise to an ice-cooled solution of (18) (1.928 g, 9.5 mmol)
and diisopropylethylamine (4.4 mL) in a mixed solvent of dioxan
(48 mL) and water (36 mL). The reaction was stirred in an ice bath for
40 min and then at room temperature for 24 h. Dioxan was removed
under vacuum and the remaining solution poured into water (80 mL).
The aqueous solution was acidified with 10% acetic acid to pH 4.5 and
then extracted three times with ethyl acetate. The organic phase was
washed three times with saturated, aqueous, sodium chloride solution
and dried. Removal of the solvent gave the title compound (19) (4.34 g,
99%) as a white solid. A portion was flash chromatographed on silica
gel, eluting with 2–3% methanol in DCM. M.p. 156.5-157.5°C (Found:
C, 63.7; H, 6.5; N, 6.0%. C25H30N2O7 requires C, 63.8; H, 6.4; N,
6.0%). 1H NMR δ (CDCl3) 7.79, d, J 8.5 Hz, 2 × ArH; 7.35, d, J 8.4 Hz,
2 × ArH; 7.31, m, C6H5; 6.99, d, J 7.6 Hz, NH; 5.17, s, OCH2; 4.71, ddd,
J 4.5, 7.9, 8.5 Hz, α-H; 3.34, s, NCH3; 2.61–2.41, m, γ-CH2; 2.41–2.20,
m, and 2.15–1.91, m, β-CH2; 1.48, s, C(CH3)3. Mass spectrum (APCI–)
m/z 469 (M – H, 100%).
Method A
The MTX α-ester (15)[13] (11.00 g, 21.5 mmol) was dissolved in DMF
(86 mL). DCM (200 mL) was added. DCC (5.50 g, 26.7 mmol) was
then added and the solution was stirred for 20 min. Amine (10)
(21.32 g, 23.9 mmol) in DCM (70 mL) was then added dropwise over
10 min. DMAP (258 mg, 2.1 mmol) was then added and the resulting
mixture stirred at room temperature under nitrogen for 24 h. The
mixture was filtered and concentrated (rotary/oil pump, 50°C) to
remove DMF. The residue was dissolved in DCM, refiltered and
evaporated. The residue was dissolved in ethyl acetate/DCM (150/20
mL), washed with saturated, aqueous, sodium chloride solution, dried
and evaporated to dryness. The residue was flash chromatographed on
silica gel, eluting with 4–5% methanol in DCM, affording the title
compound (23) (22.01 g, 74%) as a bright yellow solid. M.p. 91-93°C
(Found: C, 67.3; H, 9.7; N, 10.0%. C78H132N10O11 requires C, 67.6; H,
9.6; N, 10.1%). 1H NMR δ ((CD3)2SO/CDCl3, 5 : 2) 8.55, s, H-7; 8.25,
d, J 6.8 Hz, glu-NH; 8.03, t, J 6.2 Hz, gly-NH; 7.87, s, TRIS-NH; 7.72,
d, J 9.4 Hz, 2 × ArH; 7.44, br, NH2; 6.80, d, J 9.4 Hz, 2 × ArH; 6.63, br,
NH2; 4.80, s, CH2N; 4.29, m, 7H, glu-α-H and 3 × CH2O; 3.69, d, J 6.1
Hz, NCH2CO; 3.21, s, NCH3; 2.26, m, 8H, 3 × CH2CO and glu-γ-CH2;
2.08–1.89, m, glu-β-CH2; 1.58–1.40, m, CH2CH2CO; 1.36, s, C(CH3)3;
1.22, br, 36 × CH2; 0.85, t, J 6.6 Hz, CH3; Mass spectrum (APCI+) m/z
1386 (M + H, 100%), 1408 (M + Na, 89).
α-t-Butyl-N-[4-(methylamino)benzoyl]-L-glutamate (20)
Prepared from (19) by the standard hydrogenolysis procedure in 99%
yield as a colourless solid, which was used without further purification.
1H NMR δ (CDCl3): 7.70, d, J 8.8 Hz, 2 × ArH; 6.91, d, J 7.5 Hz, NH;
6.62, d, J 9.0 Hz, 2 × ArH; 4.71, dt, J 4.5, 8.0 Hz, α-H; 2.88, s, NCH3;
2.55–2.42, m, γ-CH2; 2.42–2.20, m, and 2.13–1.92, m, β-CH2, 1.48, s,
C(CH3)3.
Method B
Pteridine bromide (21)[18] (0.220 g, 0.55 mmol) was dissolved in DMF
(8 mL). DCM (12 mL) was added. Amine (28) (0.654 g, 0.54 mmol)
was dissolved in DCM (12 mL) and added dropwise to the pteridine
solution. The resulting solution was stirred at room temperature
overnight. The solution was concentrated under vacuum. The residue
was then dissolved in DCM and the solution was washed twice with
aqueous sodium bicarbonate solution (0.5%), twice with water, dried
and evaporated. The residue was flash chromatographed on silica gel,
eluting with 2–5% methanol in DCM, which provided the title
compound (23) (0.46 g, 61%) as a yellow powder.
α-t-Butyl-N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methyl-
amino]benzoyl]-L-glutamate (15)
Method A
Pteridine bromide.HBr (21)[18] (100 mg, 0.25 mmol) and (20) (87 mg,
0.25 mmol) were mixed in DMF (3 mL) and the resulting solution

Total Synthesis of Methotrexate-γ-TRIS-Fatty Acid Conjugates
641
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
(α-t-butoxy)glutamoyl]-N-[1-hydroxymethyl-2-[(1-oxohexadecyl)-
oxy]-1-[(1-oxohexadecyl)oxymethyl]ethyl]glycinamide (24)
mixture allowed to warm to room temperature at which it was stirred,
under nitrogen, for 5.5 h. The mixture was placed in a refrigerator (4°C)
overnight then diluted with DCM (25 mL) and washed with aqueous,
sodium bicarbonate solution (5% w/v, 35 mL) (pH > 7). Acetic acid
(glacial, 1.8 mL) was added and the mixture shaken (pH ca. 4). The
organic layer was washed with water (2 × 25 mL), dried and evaporated.
The residue was flash chromatographed on silica gel, eluting with
8-30% methanol in DCM to afford the title compound (2) (742 mg,
90%) as a yellow solid. When this reaction was performed on a 15 g
scale, a 78% yield was obtained. M.p. > 220°C (dec.) (Found (sample
dried under vacuum to constant weight): C, 65.1; H, 9.6; N, 10.3%.
C74H124N10O11.2H2O requires C, 65.1; H, 9.4; N, 10.3%) (Found: m/z
The MTX α-ester (15)[13] (3.04 g, 5.95 mmol) was dissolved, with
stirring, in DMF (40 mL). HOSu (0.91 g, 7.80 mmol) was added and
stirred until dissolution occurred. DCC (1.82 g, 8.82 mmol) was then
added and the resulting mixture stirred at room temperature under
nitrogen for 16 h. In a separate vessel, amine (11) (4.29 g, 6.55 mmol)
and triethylamine (1.1 mL, 0.81 g, 8.0 mmol) were dissolved in DCM
(35 mL). This solution was added to that of the MTX active ester and
the resulting mixture stirred for 5 h. The mixture was evaporated and
the residue extracted with DCM. The resultant suspension was filtered
through glass wool (to remove the urea by-product) and the filtrate
was evaporated. The residue was flash chromatographed on silica gel.
Elution with 3–8% methanol in DCM afforded the title compound (24)
(4.17 g, 61%) as a yellow solid. M.p. 97–100°C (Found (sample dried
under vacuum to constant weight): C, 62.9; H, 8.9; N, 12.1%.
1329.959. C74H125N10O11 requires m/z 1329.953). 1H NMR
δ
((CD3)2SO/CDCl3, 5 : 2) 8.51, s, H-7; 7.99–7.88, m, 2H, gly-NH and
glu-NH; 7.91, s, TRIS-NH; 7.67, d, J 8.7 Hz, 2 × ArH; 7.17, br, NH2;
6.75, d, J 8.7 Hz, 2 × ArH; 6.42, br, NH2; 4.75, s, CH2N; 4.26, m, 7H,
glu-α-H and 3 × CH2O; 3.66, d, J 4.7 Hz, NCH2CO; 3.19, s, NCH3;
2.25, m, 8H, 3 × CH2CO and glu-γ-CH2; 2.14–1.84, m, glu-β-CH2;
1.63–1.36, m, CH2CH2CO; 1.20, br, 36 × CH2; 0.85, t, J 6.5 Hz, CH3.
Mass spectrum (ESI+) m/z 1330 (M + H, 100%), 1352 (M + Na, 22);
(ESI–) m/z 1328 (M – 1, 100%).
1
C62H102N10O10.2H2O requires C, 62.9; H, 9.0; N, 11.8%). H NMR δ
((CD3)2SO) 8.55, s, H-7; 8.24, d, J 6.2 Hz, glu-NH; 8.01, J 5.1 Hz,
gly-NH; 7.73, d, J 9.5 Hz, 2 × ArH; 7.52, s, TRIS-NH; 6.80, d, J 9.5
Hz, 2 × ArH; 6.67, br, NH2; 4.92, t, J 5.8 Hz, OH; 4.79, s, ArCH2N;
4.31–4.14, m, 5H, 2 × CH2O and glu-α-H; 3.73–3.58, m, 4H,
NCH2CO and CH2OH; 3.21, s, NCH3; 2.32–2.18, m, 6H, 2 × CH2CO
Method B
The amine (34) (0.287 g, 0.25 mmol) was dissolved with stirring in
DCM (9 mL) and, in a separate vessel, bromomethyl pteridine (21)[18]
(0.10 g, 0.25 mmol) was dissolved with stirring in DMF (3 mL). The
pteridine bromide solution was added to the amine solution and the
resulting mixture stirred at room temperature under nitrogen for 67 h.
The mixture was evaporated under vacuum and the residue dissolved in
DCM (12 mL). This solution was washed with aqueous sodium
bicarbonate solution (1%, 10 mL) (pH 7–8). Without separating the
layers, aqueous acetic acid (10%) was added dropwise with shaking to
adjust the pH back to 4–5. The organic layer was washed with water
(20 mL), dried and evaporated. The residue was purified by radial
chromatography on a 1 mm silica gel plate. Elution with 8%
methanol/0.8% water in DCM to 15% methanol/1% water in DCM
gave the title compound (2) (0.161 g, 48%) as a yellow solid, identical
to that prepared by the earlier method.
and glu-γ-CH2; 2.07–1.90, m, glu-β-CH2; 1.59–1.41, m,
2 ×
CH2CH2CO; 1.38, s, C(CH3)3; 1.22, br, 24 × CH2; 0.84, t, J 6.6 Hz, 2
× CH3. Mass spectrum (ESI+) m/z 1169 (M + Na, 100%); (ESI–) m/z
1181 (M + Cl–, 100%).
The following compounds were prepared by employing the
appropriate amine in the above procedure. The two products described
in this section were obtained as yellow solids.
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
(α-t-butoxy)glutamoyl]-N-[1,1-bis(hydroxymethyl)-2-[(1-oxohexa-
decyl)oxy]ethyl]glycinamide (25)
Obtained 0.934 g, 61%. M.p. 107–110°C (Found (sample dried under
vacuum to constant weight): C, 59.5; H, 8.0; N, 14.9%.
C46H73N10O9.H2O requires C, 59.6; H, 8.0; N, 15.1%) (Found: m/z
1
909.554. C46H73N10O9 requires m/z 909.556). H NMR δ ((CD3)2SO)
8.58, s, H-7; 8.25, d, J 6.3 Hz, glu-NH; 8.05, t, J 5.3 Hz, gly-NH; 7.75,
d, J 9.5 Hz, 2 × ArH; 7.27, s, TRIS-NH; 6.80, d, J 9.5 Hz, 2 × ArH; 6.78,
br, NH2; 4.86–4.71, m, 4H, ArCH2N and 2 × OH; 4.35–4.18, m, glu-α-
H; 4.20, s, CH2O; 3.65, d, J 6.3 Hz, NCH2CO; 3.57, d, J 5.5 Hz, 2 ×
CH2OH; 3.21, s, NCH3; 2.36–2.18, m, 4H, CH2CO and glu-γ-CH2;
2.10–1.84, m, glu-β-CH2; 1.60–1.39, m, CH2CH2CO; 1.35, s, C(CH3)3;
1.25, br s, 12 × CH2; 0.85, t, J 6.3 Hz, CH3. Mass spectrum (APCI+) m/z
931 (M + Na, 22%), 909 (M + H, 100); (APCI–) m/z 943 (M + Cl–,
100%).
Method C
The pteroic acid (22)[15] (58 mg, 0.18 mmol) was dissolved in DMF
(3 mL). Triethylamine (50 µL, 0.37 mmol) was added with stirring and
the solid dissolved. TPTU (54 mg, 0.18 mmol) was added and the
reaction mixture was stirred at room temperature under nitrogen for
4.5 h. In a separate vessel, triethylamine (30 µL) and (50) (0.20 g,
0.20 mmol) were dissolved in DCM (3 mL). The active ester mixture
was added slowly to the (50)/triethylamine solution and the resulting
mixture (pH 8) stirred at room temperature under nitrogen for 18 h, and
at 40°C for 7.5 h. The mixture was evaporated and the residue radially
chromatographed, eluting with 92 : 8 : 0.4 dichloromethane/methan-
ol/water to give the title compound (2) (0.111 g, 47%) as a yellow
solid.
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
(α-t-butoxy)glutamoyl]-N-[2-hydroxy-1,1-
bis(hydroxymethyl)ethyl]glycinamide (26)
Obtained 0.33 g, 57% (DMF sole reaction solvent), m.p. > 140°C
(gradual dec.) (Found (sample dried under vacuum to constant weight):
C, 50.3; H, 6.2; N, 19.0%. C30H42N10O8.3H2O requires C, 49.7; H, 6.2;
N, 19.3%). 1H NMR δ ((CD3)2SO) 8.56, s, H-7; 8.27, d, J 6.8 Hz, glu-
NH; 8.10, t, J 6.2 Hz, gly-NH; 7.46, br s, NH2; 7.14, s, TRIS-NH;
7.72and 6.82, AA´BB´, J 9.4 Hz, 4 × ArH; 6.62, br s, NH2; 4.79, s,
ArCH2N; 4.69, t, J 6.2 Hz, 3 × OH; 4.34–4.15, m, glu-α-H; 3.70, d, J
6.3 Hz, gly-NCH2CO; 3.52, d, J 6.2 Hz, 3 × CH2OH; 3.21, s, NCH3;
2.37–2.15, m, glu-γ-CH2; 2.09–1.80, m, glu-β-CH2; 1.39, s, C(CH3)3.
Mass spectrum (ESI+) m/z 671 (M + H).
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
γ-glutamyl]-N-[1-hydroxymethyl-2-[(1-oxohexadecyl)oxy]-1-[(1-
oxohexadecyl)oxymethyl]ethyl]glycinamide (3)
Treatment of tert-butyl ester (24) using ‘Method A’ above
(chromatography mobile phase 0–1% water in 1 : 4 methanol/DCM)
afforded the title compound (0.31 g, 56%) as a yellow solid. M.p. >
200°C (dec.) (Found (sample dried under vacuum to constant weight):
C, 60.5; H, 8.4; N, 12.4%. C58H94N10O10.3H2O requires C, 60.8; H, 8.8;
N, 12.2%). 1H NMR δ ((CD3)2SO) 8.52, s, H-7; 8.05, t, J 5.8 Hz, gly-
NH; 7.82–7.59, m, 5H, 2 × ArH and NH2 and TRIS-NH (D2O exchange
revealed 7.67, d, J 8.9 Hz, 2 × ArH); 6.78, d, J 8.9 Hz, 2 × ArH; 4.75, s,
ArCH2N; 4.30–4.41, m, 5H, glu-α-H and 2 × CH2OPm; 3.57–3.73, m,
4H, NCH2CO and CH2OH; 3.17, s, NCH3; 2.10–2.30, m, 6H, 2 ×
CH2CO and glu-γ-CH2; 1.80–2.09, m, glu-β-CH2; 1.36–1.57, m, 2 ×
CH2CH2CO; 1.07–1.33, br, 24 × CH2; 0.82, t, J 6.6 Hz, 2 × CH3. Mass
spectrum (ESI–) m/z 1089 (M – H, 100%).
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
γ-glutamyl]-N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-oxohexadecyl)-
oxy]methyl]ethyl]glycinamide (2)
Method A
TFA (1.7 mL, 22 mmol) was added dropwise to an ice-cooled, stirred
solution of (23) (0.86 g, 0.62 mmol) in DCM (5 mL) and the resulting

642
C. L. Francis et al.
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
γ-glutamyl]-N-[1,1-bis(hydroxymethyl)-2-[(1-oxohexadecyl)oxy]-
ethyl]glycinamide (4)
triethylamine. Further stirring at room temperature for 32 h and
concentration under vacuum gave a dark red, oily residue, which was
triturated with acetonitrile (180 mL) to give a yellow solid. The solid
was dissolved in DMF (20 mL), re-precipitated by dropwise addition of
acetonitrile (100 mL), filtered off, rinsed with acetonitrile and ether,
and then air-dried over night. Purification by reversed-phase
preparative HPLC in 500 mg portions (eluting with 0-8% MeOH/H2O,
see general experimental section), followed by freeze-drying, afforded
the title compound (5) (2.472 g, 52%) as a bright yellow solid.
Method A
Subjecting tert-butyl ester (25) to the above procedure (chloroform as
extraction solvent instead of DCM, chromatography mobile phase
78 : 20 : 2 to 60 : 35 : 5 chloroform/methanol/water) gave the title
compound (0.66 g, 79%) as a yellow solid. M.p. > 170°C (dec.) (Found
(sample dried under vacuum to constant weight): C, 55.8; H, 7.8; N,
15.2%. C42H65N10O9.3H2O requires C, 55.6; H, 7.8; N, 15.4%) (Found:
Nα-[[4-[(Benzyloxycarbonyl)methylamino]benzoyl]-L-(α-t-butoxy)-
glutamoyl]-N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-oxohexadecyl)-
oxy]methyl]ethyl]glycinamide (27)
m/z 853.492. C42H65N10O9 requires m/z 853.494). 1H NMR
δ
((CD3)2SO) 8.57, s, H-7; 8.16, d, J 6.2 Hz, glu-NH; 8.07, t, J 6.3 Hz,
gly-NH; 7.75, d, J 8.8 Hz, 2 × ArH; 7.37, s, TRIS-NH; 6.84, d, J 8.9 Hz,
2 × ArH; 6.62, br, NH2; 4.79, s, ArCH2N; 4.32–4.12, m, glu-α-H; 4.20,
s, CH2O; 3.67, d, J 6.3 Hz, NCH2CO; 3.57, s, 2 × CH2OH; 3.20, s,
NCH3; 2.35-2.15, m, 4H, CH2CO and glu-γ-CH2; 2.13–1.85, m, glu-β-
CH2; 1.60–1.37, m, CH2CH2CO; 1.25, br, 12 × CH2; 0.85, t, J 6.3 Hz,
CH3. Mass spectrum (ESI+) m/z 853 (M + H, 100%).
DCC(0.69g,3.36mmol)andDMAP(22mg)wereaddedtoanice-cooled
solutionof(19)(1.05g,2.24mmol)and(10)(2.00g,2.24 mmol)inDCM
(30 mL). The mixture was stirred at room temperature for 24 h, filtered
and the filtrate concentrated under vacuum. The residue was purified on
a silica flash column (MeOH/DCM, 1 : 100 to 2 : 100) to afford the title
compound (27) (1.748 g, 58%) as a white solid. M.p. 40-41°C (Found:
C,70.2;H,9.9;N,4.4%.C79H132N4O13 requiresC,70.5;H,9.9;N,4.2%).
1H NMR δ (CDCl3) 7.82, d, J 8.8 Hz, 2 × ArH; 7.37, d, J 7.5 Hz, 2 ×
ArH; 7.35, m, C6H5; 7.21, d, J 7.4 Hz, glu-NH; 6.79, t, J 5.3 Hz, gly-
NH; 6.56, s, TRIS-NH; 5.19, s, PhCH2; 4.72–4.56, m, α-CH; 4.41, s, 3
× OCH2; 3.90, dd, J 5.2, 16.7 Hz, and 3.76, dd, J 5.1, 17.3 Hz, NCH2;
3.36, s, NCH3; 2.47–2.31, m, γ-CH2; 2.31, t, J 7.0 Hz, 3 × CH2CO;
2.14–1.95, m, β-CH2; 1.70–1.52, m, 3 × CH2CH2CO; 1.49, s, C(CH3)3;
1.36–1.18, m, 36 × CH2; 0.88, t, J 6.5 Hz, 3 × CH3. Mass spectrum (ESI+)
m/z 1346 (M + H, 2%), 387 (22), 309 (24), 225 (100).
Method B
Prepared from amine (37) and bromomethyl pteridine (21)[18] following
‘Method B’ for compound (2) (but with DMF as sole solvent), which
afforded the title compound (4) (0.264 g, 36%) as a yellow solid.
Method C
TPTU-mediated coupling of pteroic acid (22)[15] with amine (49) as
described in ‘Method C’ for compound (2) (but with DMF as sole
solvent) afforded the title compound (4) (2.67 g, 54%) as a yellow solid.
Nα-[[(4-Methylamino)benzoyl]-L-(α-t-butoxy)glutamoyl]-N-[2-[(1-
oxohexadecyl)oxy]-1,1-bis[[(1-oxohexadecyl)oxy]methyl]ethyl]gly-
cinamide (28)
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
γ-glutamyl]-N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinamide
(5)
Prepared from (27) by the standard hydrogenolysis procedure in 98%
yield as a white solid, the title compound was used without further
purification. 1H NMR δ (CDCl3) 7.69, d, J 8.7 Hz, 2 × ArH; 7.19, t, J
5.7 Hz, gly-NH; 6.95, d, J 6.4 Hz, glu-NH; 6.72, s, TRIS-NH; 6.64, d,
J 8.8 Hz, 2 × ArH; 4.69–4.55, m, α-CH; 4.42, s, 3 × OCH2; 3.91, dd, J
5.6, 16.7 Hz, and 3.80, dd, J 5.4, 16.6 Hz, NCH2; 2.90, s, NCH3;
2.47–2.21, m, 8H, 3 × CH2CO and γ-CH2; 2.06–1.86, m, β-CH2;
1.68–1.51, m, 3 × CH2CH2CO; 1.49, s, C(CH3)3; 1.31–1.25, m, 36 ×
CH2; 0.88, t, J 6.7 Hz, 3 × CH3. Mass spectrum (ESI+) m/z 1233
(M + Na, 30%), 565 (15), 225 (100).
Method A
The tert-butyl ester (26) (0.459 g, 0.68 mmol) was dissolved, with
stirring, in TFA (3 mL). The solution was stirred at room temperature
for 1.5 h and the solvent was evaporated under vacuum. The residue was
dissolved in water and the solution pH (ca. 2) was adjusted to 6 with
aqueous sodium bicarbonate solution (1 g in 75 mL). Aqueous acetic
acid (10%) was added until pH 4 was reached. The solution was freeze-
dried and the residue purified by flash chromatography and then radial
chromatography on silica gel. Elution with 1–3% water in 3 : 5
methanol/chloroform afforded the title compound (5) (0.086 g, 21%) as
a yellow solid. M.p. > 180°C (dec.) (Found (sample dried under vacuum
to constant weight): C, 46.5; H, 6.0; N, 20.8%. C26H35N10O8.3H2O
requires C, 46.7; H, 6.0; N, 21.0%) (Found: m/z 615.263. C26H35N10O8
Nα-[N-(t-Butyloxycarbonyl)-L-(α-benzyloxy)glutamoyl]-N-[2-[(1-
oxohexadecyl)oxy]-1,1-bis[[(1-oxohexadecyl)oxy]methyl]ethyl]-
glycinamide (30)
1
α-Benzyl-N-(t-butyloxycarbonyl)-L-glutamate (29) (2.86 g, 8.4 mmol)
and (10) (7.56 g, 8.4 mmol) were dissolved with stirring in DCM
(60 mL). DCC (2.08 g, 10.08 mmol) was added followed by DMAP
(50 mg) and the resulting mixture stirred at room temperature under
nitrogen for 18.5 h. The mixture was filtered and the filtrate evaporated.
The residue was extracted with hot petroleum spirit (60–80) and filtered
again. The filtrate was evaporated and the residue recrystallized from
petroleum spirit (60–80) to give the title compound (30) (9.31 g, 91%)
as a white solid. M.p. 56–56.5°C (Found: C, 70.6; H, 10.1; N, 3.8%.
C71H125N3O12 requires C, 70.3; H, 10.4; N, 3.5%). 1H NMR δ (CDCl3)
7.35, br s, C6H5; 6.55–6.45, m, 2H, gly-NH and TRIS-NH; 5.32, d, J 6.9
Hz, glu-NH; 5.21, AB J 12.0 Hz and 5.13, AB, J 12.2 Hz, CH2Ph;
4.49–4.22, m, 7H, glu-α-H and 3 × CH2O; 3.84, d, J 5.3 Hz, gly-CH2;
2.31, t, J 7.5 Hz, 8H, 3 × CH2CO and glu-γ-CH2; 2.40–2.10, m and
2.06–1.72, m, glu-β-CH2; 1.72–1.48, m, 3 × CH2CH2CO; 1.43, s,
C(CH3)3; 1.26, br s, 36 × CH2; 0.88, t, J 6.7 Hz, 3 × CH3. Mass spectrum
(ESI+) m/z 1235 (M + Na, 34%), 1113 (21), 122 (100).
requires m/z 615.264). H NMR δ ((CD3)2SO) 8.58, s, H-7; 8.28, d, J
6.3 Hz, NH; 8.11, t, J 6.2 Hz, NH; 7.73, d, J 8.5 Hz, 2 × ArH; 7.78–7.62,
br, and 7.55–7.40, br, NH2; 7.15, s, NH; 6.83, d, J 8.5 Hz, 2 × ArH; 6.63,
s, br, NH; 4.80, s, ArCH2N; 4.94–4.57, br, 3 × OH; 4.35–4.20, m, glu-
α-H; 3.70, d, J 5.8 Hz, NCH2; 3.53, s, 3 × CH2OH; 3.22, s, NCH3;
2.20-2.32, m, glu-γ-CH2; 1.80-2.15, m, glu-β-CH2. Mass spectrum
(ESI+) m/z 615 (M + H, 100%).
Method B
Prepared from amine (39) and pteridine bromide (21)[18] using ‘Method
B’ for compound (4), which afforded the title compound (5) (8%) as a
yellow solid.
Method C
The pteroic acid (22)[15] (2.88 g, 8.86 mmol) was dissolved in DMF
(100 mL). TPTU (2.58 g, 8.67 mmol) and triethylamine (2.49 mL,
17.6 mmol) were added portionwise alternately. The mixture was
stirred at room temperature for 2 h, then added dropwise to a stirred
suspension of (48) (3.00 g, 9.79 mmol) and triethylamine (1.00 mL) in
DMF (100 mL). The pH was adjusted to 8 with triethylamine and the
mixture stirred overnight. Most of the DMF (ca. 135 mL) was removed
under vacuum and the concentrated solution adjusted to pH 8 with
Nα-[L-(α-Benzyloxy)glutamoyl]-N-[2-[(1-oxohexadecyl)oxy]-1,1-
bis[[(1-oxohexadecyl)oxy]methyl]ethyl]glycinamide (31)
TFA (0.85 mL, 11 mmol) was added slowly to an ice-cooled, stirred
solution of (30) (0.85 g, 0.70 mmol) in DCM (2 mL) under nitrogen.

Total Synthesis of Methotrexate-γ-TRIS-Fatty Acid Conjugates
643
The resulting mixture was stirred at room temperature under nitrogen
for 2.5 h. The mixture was evaporated under vacuum without heating.
The residue was dissolved in DCM (8 mL) and washed with saturated,
aqueous, sodium bicarbonate solution (5 mL) (pH 8–9) (CAUTION:
gas evolution). The organic layer was dried and evaporated without
heating to afford the title compound (31) (0.77 g, 100%) as an off-white
coloured solid which was used without further purification. 1H NMR δ
(CDCl3) 7.35, br s, C6H5; 6.80–6.72, m, gly-NH; 6.43, s, TRIS-NH;
5.24, s, CH2Ph; 5.15, d, J 5.5 Hz, glu-NH; 4.49–4.31, m, 7H, 3 × CH2O
and glu-α-H; 3.85, d, J 5.2 Hz, gly-CH2; 2.41–2.18, m, 8H, 3 × CH2CO
M.p. 37.5–38°C (Found: C, 71.2; H, 9.5; N, 4.3%. C82H130N4O13
requires C, 71.4; H, 9.5; N, 4.1%). 1H NMR δ (CDCl3) 7.83, d, J 8.4
Hz, 2 × ArH; 7.49, d, J 7.1 Hz, glu-H; 7.43–7.28, m, 2 × C6H5; 7.70, d,
J 8.4 Hz, 2 × ArH; 6.58, t, J 5.1 Hz, gly-NH; 6.53, s, TRIS-NH; 5.25
and 5.18, AB, J 12.1 Hz, CH2Ph (ester); 5.19, s, CH2Ph (Z); 4.86–4.71,
m, glu-α-H; 4.41, s, 3 × CH2O; 3.73, dd, J 4.5, 16.5 Hz and 3.89, dd, J
5.1, 16.5 Hz, gly-CH2; 3.36, s, NCH3; 2.41–2.22, m, 8H, 3 × CH2CO
and glu-γ-CH2; 2.19–1.85, m, glu-β-CH2; 1.70–1.44, m,
3 ×
CH2CH2CO; 1.25, br s, 36 × CH2; 0.88, t, J 6.2 Hz, 3 × CH3. Mass
spectrum (APCI+) m/z 1380 (M + H, 100%), 1402 (M + Na, 37);
(APCI–) m/z 1414 (M + Cl–, 100%).
and glu-γ-CH2; 2.14–1.70, br, glu-β-CH2; 1.70–1.44, m,
3 ×
CH2CH2CO; 1.25, br s, 36 × CH2; 0.88, t, J 6.7 Hz, 3 × CH3. Mass
spectrum (ESI+) m/z 1135 (M + Na, 13%), 1113 (M + H, 31), 1037
(100).
Method B
The carboxylic acid (35)[17] (0.51 g, 0.97 mmol), HOSu (0.13 g, 1.16
mmol), and DCC (0.30 g, 1.45 mmol) were dissolved with stirring in
DCM (13 mL) and the resulting mixture stirred for 3 h. Amine (10)
(0.87 g, 0.97 mmol) was added followed by DMAP (ca. 6 mg). The
resulting mixture was adjusted to 8 with diisopropylethylamine and
stirred at room temperature under nitrogen for 21 h then filtered
through glass wool and the filtrate was evaporated. The residue was
redissolved in petroleum spirit (60–80) and filtered again. The filtrate
was evaporated and the residue purified by radial chromatography on a
silica gel 4 mm plate, eluting with 0–2% methanol in DCM to give the
title compound (1.03 g, 77%) as a white solid.
Nα-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-
(α-benzyloxy)glutamoyl]-N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-
oxohexadecyl)oxy]methyl]ethyl]glycinamide (32)
Method A
The pteroic acid (22)[15] (88 mg, 0.27 mmol) was dissolved with
warming and stirring in DMA (3.5 mL). The solution was cooled to
room temperature and amine (31) (0.30 g, 0.27 mmol) was added. Upon
dissolution, DCC (67 mg, 0.32 mmol) was added followed by DMAP
(4 mg, 0.03 mmol). The resulting mixture was stirred at room
temperature under nitrogen for 47 h. More DCC (30 mg) was added and
the mixture stirred for 5 days. DCM (15 mL) was added and the mixture
was washed with water (3 × 25 mL). The organic layer was dried and
evaporated. The residue was purified by radial chromatography on a
silica gel 1 mm plate, eluting with 1–4% methanol in DCM to give the
The following compounds were prepared by employing the
appropriate amine in analogous procedures, except omitting the DMAP.
Nα-[[4-[(Benzyloxycarbonyl)methylamino]benzoyl]-L-(α-benzyloxy)-
glutamoyl]-N-[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinamide
(38)
title compound (32) (0.154 g, 40%) as
a yellow solid. M.p.
128–128.5°C (Found: C, 68.2; H, 9.3; N, 9.8%. C81H130N10O11 requires
C, 68.5; H, 9.2; N, 9.9%). 1H NMR δ ((CD3)2SO/CDCl3, 5 : 2) 8.55, s,
H-7; 8.41, d, J 7.6 Hz, glu-NH; 7.98, t, J 5.6 Hz, gly-NH; 7.84, s, TRIS-
NH; 7.74, d, J 8.9 Hz, 2 × ArH; 7.35, br s, C6H5; 7.32, br, NH2; 6.79, d,
J 8.9 Hz, 2 × ArH; 6.75, br, NH2; 5.10, s, CH2Ph; 4.79, s, CH2N;
4.51–4.33, m, glu-α-H; 4.23, m, 3 × CH2O; 3.67, d, J 5.7 Hz, NCH2CO;
3.21, s, N-CH3; 2.37–2.15, m, 8H, 3 × CH2CO and glu-γ-CH2;
2.15–1.86, m, glu-β-CH2; 1.61–1.37, m, 3 × CH2CH2CO; 1.21, br, 36
× CH2; 0.84, t, J 6.7 Hz, 3 × CH3. Mass spectrum (ESI+) m/z 1442
(M + Na, 100%), 1420 (M + H, 79).
Obtained 151 mg, 52%, as a colourless gum (using a DCM/DMF mixed
solvent system and ethyl acetate in place of petroleum spirit) (Found:
m/z 687.263. C34H40N4O10Na (M + Na+) requires m/z 687.264). 1H
NMR δ (CDCl3) 7.78, d, J 8.1 Hz, 2H, 2 × ArH; 7.55, d, J 6.8 Hz, NH;
7.4–77.20, m 12H, 2 × C6H5 and 2 × ArH; 7.13, s, NH; 5.22–5.05, m, 2
× OCH2Ph; 4.90–4.65, m, glu-α-H; 4.04–3.68, m, NCH2; 3.67, br s, 3
× OCH2; 3.37, s, NCH3; 2.46–2.20, m, glu-γ-CH2; 2.12–1.86, m, glu-β-
CH2. Mass Spectrum (APCI+) m/z 665 (M + H, 55%), 647 (81), 557
(57), 539 (71), 134 (100).
Nα-[[4-[(Benzyloxycarbonyl)methylamino]benzoyl]-L-(α-benzyloxy)-
glutamoyl]-N-[2,2-dimethyl-5-[(1-oxohexadecyl)oxymethyl]-[1,3]di-
oxan-5-yl]glycinamide (42)
Method B
Diisopropylethylamine (0.2 mL, 1.1 mmol) was added to a stirred
solution of (22)[15] (0.20 g, 0.55 mmol) in DMF (8 mL) under nitrogen.
DECP (0.15 mL, 1.1 mmol) was added and the resulting mixture stirred
for 55 min. Diisopropylethylamine (0.2 mL, 1.1 mmol) was added
followed by a solution of (31) (1.22 g, 1.1 mmol) in DMF (3 mL) and
the mixture stirred for 42.5 h. Sodium bicarbonate (100 mg) was added
and the mixture evaporated. The residue was dissolved in DCM
(25 mL) and washed with half-saturated aqueous sodium bicarbonate
solution (25 mL) then water (2 × 25 mL). The organic layer was dried
and evaporated. The residue was purified by radial chromatography on
a silica gel 4 mm plate, eluting with 3–5% methanol in DCM to give the
title compound (32) (0.394 g, 50%) as a yellow solid.
Obtained 10.23 g, 73%, as a white, waxy solid. M.p. 52–53°C (Found:
C, 67.2; H, 8.0; N, 6.0%. C53H74N4O11 requires C, 67.5; H, 7.9; N,
5.9%). 1H NMR δ (CDCl3) 7.82, d, J 8.5 Hz, 2 × ArH; 7.52, d, J 7.3 Hz,
glu-NH; 7.45–7.27, m, 12H, 2 × C6H5 and 2 × ArH; 6.59–6.42, m, 2H,
gly-NH and TRIS-NH; 5.24 and 5.17, AB, J 12.1 Hz, CH2Ph; 5.18, s,
CH2Ph; 4.88–4.78, m, glu-α-H; 4.43, s, CH2O-palmitoyl; 4.24 and
4.22, AB, J 12.1 Hz, CH2O; and 3.92, dd, J 5.5, 12.1 Hz, and 3.82–3.49,
m, 4H, CH2O and gly-CH2; 3.35, s, NCH3; 2.44–2.22, m, 4H, CH2CO
and glu-γ-CH2; 2.21–2.02, m, glu-β-CH2; 1.71–1.51, m, CH2CH2CO;
1.47, s, CH3; 1.40, s, CH3; 1.24, br, 12 × CH2; 0.87, t, J 6.7 Hz, CH3.
Mass spectrum (APCI+) m/z 943 (M + H, 28%), 885 (99), 777 (100);
(APCI–) m/z 977 (M + Cl–, 100).
Nα-[[4-[(Benzyloxycarbonyl)methylamino]benzoyl]-L-(α-
benzyloxy)glutamoyl]-N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-
oxohexadecyl)oxy]methyl]ethyl]glycinamide (33)
Nα-[[4-[(Benzyloxycarbonyl)methylamino]benzoyl]-L-(α-
benzyloxy)glutamoyl]-N-[1,1-bis(hydroxymethyl)-2-[(1-
oxohexadecyl)oxy]ethyl]glycinamide (36)
Method A
Obtained 0.19 g, 42%, as a clear, colourless, viscous oil (Found: C, 66.3;
H,7.7;N,6.2%.C50H70N4O11 requiresC,66.5;H,7.8;N,6.2%).1HNMR
δ ((CD3)2SO) 8.84, d, J 7.4 Hz, glu-NH; 8.07, t, J 5.5 Hz, gly-NH; 7.87,
d, J 8.7 Hz, 2 × ArH; 7.44, d, J 8.7 Hz, 2 × ArH; 7.40–7.30, m, 10H, 2
× C6H5; 7.28, s, TRIS-NH; 5.13, s, 2 × CH2Ph; 4.79, t, J 5.5 Hz, 2 × OH;
4.53–4.37, m, glu-α-H; 4.18, s, CH2O; 3.68, d, J 5.5 Hz, gly-CH2; 3.56,
d, J 5.8 Hz, 2 × CH2OH; 3.29, s, NCH3; 2.39–2.20, m, 4H, CH2CO and
glu-γ-CH2; 2.20–1.85, m, glu-β-CH2; 1.62–1.36, m, CH2CH2CO; 1.22,
br, 12 × CH2; 0.84, t, J 6.6 Hz, CH3. Mass spectrum (APCI+) m/z 903
(M + H, 69%), 885 (M – H2O, 100); (APCI–) m/z 937 (M + Cl–, 100).
The amine (31) (0.74 g, 0.67 mmol) was dissolved in DCM (8 mL).
Diisopropylethylamine (0.2 mL) was added and the stirred solution was
cooled in an ice-bath. A solution of chloride (17) in dry DCM (3 mL)
was added slowly. The resulting mixture was allowed to warm to room
temperature and stirred for 5 h. The mixture was transferred to a
separating funnel and washed with water (2 × 20 mL). The organic layer
was dried and evaporated. The residue was purified by radial
chromatography on a silica gel 2 mm plate, eluting with 0–2% methanol
in DCM to give the title compound (33) (0.62 g, 68%) as a white solid.

644
C. L. Francis et al.
Compound (36) was also prepared by the hydrolysis of acetonide
was stirred at room temperature for 3.5 h. The suspension was filtered
and the collected solid was rinsed with acetonitrile. The filtrate was
mixed with triethylamine (1.00 mL) and added dropwise to a solution
of freshly prepared (13) (17.06 mmol) in DMF (50 mL). The reaction
pH was adjusted to 8 with triethylamine. The mixture was stirred at
room temperature overnight and filtered through a glass filter paper,
rinsing the collected solid with acetonitrile. The filtrate was
concentrated under vacuum to a volume of about 15 mL. More
acetonitrile (100 mL) was added and a white precipitate formed. The
suspension was stirred at room temperature until all the gum-like
material broke up. The mixture was filtered and the filtrate was
concentrated to give a colourless gum, which was purified on a silica
flash column (5 cm × 20 cm, preconditioned with DCM, eluted with
MeOH/DCM 5 : 100) to afford the title compound (44) (3.77 g, 65%) as
a white wax (Found: m/z 532.232. C26H34N3O9 requires m/z 532.229).
1H NMR δ ((CD3)2SO) 8.07, t, J 6.3 Hz, NH; 7.82, d, J 6.3 Hz, NH;
7.45–7.22, m, 10H, 2 × C6H5; 7.16, s, NH; 5.13, s, PhCH2; 5.04, m,
PhCH2; 4.68, t, J 6.3 Hz, 3 × OH; 4.18–4.04, m, glu-α-H; 3.70, d, J 6.3
Hz, NCH2; 3.52, d, J 6.3 Hz, 3 × CH2OH; 2.25, t, J 6.2 Hz, glu-γ-CH2;
2.12–1.68, m, glu-β-CH2. Mass Spectrum (APCI+): m/z 532 (M + H,
15%), 514 (100), 406 (50), 91 (27).
(42) by the following method. The substrate (42) (1.24 g, 1.31 mmol)
was dissolved in dioxan (30 mL) with stirring. Water (6 mL) was added
to the stirred solution, followed by p-toluenesulfonic acid monohydrate
(60 mg). The mixture was heated at 45°C for 5 h, stored in a freezer
overnight then stirred at 45–50°C for 2 h. The mixture was concentrated
under vacuum and the residue partitioned between DCM and half-
concentrated, saturated, sodium chloride solution. The aqueous layer
was extracted with DCM (× 2), and the combined organic layers washed
with saturated, aqueous, sodium chloride solution, dried, and then
evaporated. The residue was radially chromatographed on a 4 mm silica
gel plate, eluting with 2% methanol in DCM to return the starting
material (42) (50 mg, 4%) and give the title compound (36) (0.91 g,
77%) as a clear, colourless, viscous oil.
N-(2,2-Dimethyl-5-[(1-oxohexadecyl)oxymethyl]-[1,3]dioxan-5-yl)-
glycinamide (41)
Prepared from (40)[10] by the standard hydrogenolysis procedure in
quantitative yield as a white, waxy solid, which was used without
further purification. 1H NMR δ (CDCl3) 7.60, s, TRIS-NH; 4.48, s,
CH2O; 4.32 and 3.79, AB, J 12.4 Hz, 2 × CH2O; 3.31, s, CH2N; 2.54,
br, NH2; 2.34, t, J 7.3 Hz, OCOCH2; 1.72–1.54, m, CH2CH2CO; 1.50,
s, CH3; 1.41, s, CH3; 1.25, br, 12 × CH2; 0.87, t, J 6.5 Hz, CH3. Mass
spectrum (APCI+) m/z 457 (M + H, 32%), 399 (100); (APCI–) m/z 491
(M + Cl–, 100).
Nα-[L-γ-Glutamyl]-N-[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]-
glycinamide (48)
Prepared from (44) by the standard hydrogenolysis procedure in
quantitative yield as a white solid, which was used without further
purification. 1H NMR δ ((CD3)2SO) 8.30, t, J 5.6 Hz, gly-NH; 7.47, s,
TRIS-NH; 5.60–4.90, br, NH2; 3.71, and 3.62 AB, J 5.9, 16.9 Hz,
NHCH2; 3.60–3.12, m, 7H, 3 × OCH2 and glu-α-CH; 2.31, t, J 6.5 Hz,
glu-γ-CH2; 1.98–1.80, m, glu-β-CH2.
Nα-[(4-methylamino)benzoyl-L-γ-glutamyl]-N-[2-[(1-oxohexadecyl)-
oxy]-1,1-bis[[(1-oxohexadecyl)oxy]methyl]ethyl]glycinamide (34)
Prepared from (33) by the standard hydrogenolysis procedure in 99%
yield as a white solid, which was used without further purification. 1H
NMR δ ((CD3)2SO/CDCl3, 5 : 2) 8.11, d, J 7.5 Hz, glu-NH; 7.99, t, J 5.5
Hz, gly-NH; 7.82, s, TRIS-NH; 7.66, d, J 8.5 Hz, 2 × ArH; 6.48, d, J 8.5
Hz, 2 × ArH; 6.05, m, NH; 4.26, m, 7H, glu-α-H and 3 × CH2O; 3.67,
d, J 5.5 Hz, gly-CH2; 2.71, d, J 4.0 Hz, NCH3; 2.24, t, J 7.1 Hz, 8H, glu-
γ-CH2 and 3 × CH2CO; 2.30–1.83, m, glu-β-CH2; 1.62–1.37, m, 3 ×
CH2CH2CO; 1.25, br s, 36 × CH2; 0.83, t, J 6.8 Hz, 3 × CH3. Mass
spectrum (APCI+) m/z 1156 (M + H, 100%), 1178 (M + Na, 79);
(APCI–) m/z 1154 (M + Cl–, 100%).
Nα-[N-(Benzyloxycarbonyl)-L-(α-benzyloxy)glutamoyl]-N-[1,1-bis-
(hydroxymethyl)-2-[(1-oxohexadecyl)oxy]ethyl]glycinamide (45)
The carboxylic acid (43) (2.51 g, 6.76 mmol) and HOSu (1.18 g,
10.25 mmol) were dissolved in DCM (63 mL). DCC (2.01 g, 9.74 mmol)
was added and the resulting mixture was stirred for 2 h. The reaction
mixture was filtered and the filtrate was mixed with triethylamine
(0.5 mL) and added dropwise into a stirred solution of (12) (3.14 g,
7.54 mmol) in DCM (63 mL). Triethylamine (0.5 mL) was added and
the reaction mixture was stirred at room temperature for 3 h. More
triethylamine (2 drops) was added and the reaction mixture was stirred
at 4°C overnight. The reaction mixture was evaporated under vacuum
togiveasemisolidwhichwaspurifiedbyradialchromatographyonsilica
gel, eluting with 0–2.5% methanol in DCM to give the title compound
(45) (4.07 g, 66%) as a white wax (Found: C, 65.2; H, 8.2; N, 5.4%.
C42H63N3O10 requires C, 65.5; H, 8.3; N, 5.5%) (Found: m/z 770.462.
C42H64N3O10 requires m/z 770.459). 1H NMR δ (CDCl3) 7.37, br s, 2 ×
C6H5; 6.83, s, TRIS-NH; 6.44–6.31, m, gly-NH; 5.72, d, J 8.5 Hz, glu-
NH; 5.18, s, CH2Ph; 5.10, s, CH2Ph; 4.52–4.36, m, glu-α-H; 4.29, s,
CH2O; 3.99–3.49, m, 6H, CH2N and 2 × CH2OH; 2.39–2.18, m, 4H,
CH2CO and glu-γ-CH2; 2.02–1.82, m, glu-β-CH2; 1.75–1.42, m,
CH2CH2CO2; 1.28, br s, 12 × CH2; 0.88, t, J 6.8 Hz, CH3. Mass spectrum
(APCI+) m/z 770 (M + H, 53%), 752 (M – H2O, 100); (APCI–) m/z 804
(M + Cl–,100%). An82%yieldwasobtainedonasmaller(800mg)scale.
The following compounds were prepared by employing the
appropriate amine in the above procedure.
The following compounds were prepared by employing the
appropriate N-benzyloxycarbonyl and benzyl ester protected substrate
in the above procedure. The two crude products described in this section
were of sufficient purity to be used without further purification.
Nα-[(4-Methylamino)benzoyl-L-γ-glutamyl]-N-[1,1-bis(hydroxy-
methyl)-2-[(1-oxohexadecyl)oxy]ethyl]glycinamide (37)
Obtained 4.26 g, 97%, as a white solid. 1H NMR ((CD3)2SO) δ 8.16, d,
J 7.5 Hz, glu-NH; 8.07, t, J 5.8 Hz, gly-NH; 7.67, d, J 8.8 Hz, 2 × ArH;
7.29, s, TRIS-NH; 6.52, d, J 8.8 Hz, 2 × ArH; 6.28–6.14, m, NHCH3;
4.37–4.20, m, glu-α-H; 4.18, s, CH2O; 3.67, d, J 5.5 Hz, gly-CH2; 3.56,
s, 4H, 2 × CH2OH; 2.70, d, J 4.9 Hz, NCH3; 2.26, t, J 7.5 Hz, 4H, glu-
γ-CH2 and CH2CO; 2.15–1.80, m, glu-β-CH2; 1.60–1.38, m,
CH2CH2CO; 1.22, br s, 12 × CH2; 0.84, t, J 6.5 Hz, CH3. Mass
spectrum (APCI+) m/z 679 (M + H, 79%), 661 (100); (APCI–) m/z 677
(M + Cl–, 100).
Nα-[(4-Methylamino)benzoyl-L-γ-glutamyl]-N-[2-hydroxy-1,1-
bis(hydroxymethyl)ethyl]glycinamide (39)
Nα-[N-(Benzyloxycarbonyl)-L-(α-benzyloxy)glutamoyl]-N-[1-
hydroxymethyl-2-[(1-oxohexadecyl)oxy]-1-[(1-oxohexadecyl)-
oxymethyl]ethyl]glycinamide (46)
Obtained 286 mg, 97%, as a white foamy solid (1H n.m.r. displayed the
desired product mixed with ethanol in ca. 1 : 1 mole ratio). 1H NMR δ
((CD3)2SO) 8.18, d, J 6.9 Hz, glu-NH; 8.12, t, J 6.3 Hz, NH; 7.68, d, J
8.8 Hz, 2 × ArH; 7.17, s, NH; 6.53, d, J 8.7 Hz, 2 × ArH; 6.30–6.20, m,
NH; 4.40–4.23, m, α-CH: 3.72, d, J 6.3 Hz, glu-CH2; 3.53, s, 3 × OCH2;
2.72, d, J 6.2 Hz, NCH3; 2.28, t, J 6.3 Hz, γ-CH2; 2.17–1.85, m, β-CH2.
Obtained 1.00 g, 83%, as a white solid. M.p. 43-44°C (Found: C, 69.0;
1
H, 9.6; N, 4.3%. C58H93N3O11 requires C, 69.1; H, 9.3; N, 4.2%). H
NMR δ (CDCl3) 7.35, br s, 10H, 2 × C6H5; 6.69, s, TRIS-NH; 6.40, m,
gly-NH; 5.70, d, J 8.0 Hz, glu-NH; 5.17, s, CH2Ph; 5.10, s, CH2Ph;
4.50–4.34, m, glu-α-H; 4.38 and 4.23, AB, J 11.5 Hz; 2 × CH2O;
3.98–3.69, m, gly-CH2; 3.77, s, CH2OH; 2.39–2.17, 4H, m, CH2CO and
glu-γ-CH2; 2.07–1.82, m, glu-β-CH2; 1.69–1.49, m, CH2CH2CO; 1.25,
br s, 24 × CH2; 0.88, t, J 6.4 Hz, 2 × CH3. Mass spectrum (APCI+) m/z
1008 (M + H, 55%), 990 (100); (APCI–) m/z 1042 (M + Cl–, 100%).
Nα-[N-(Benzyloxycarbonyl)-L-(α-benzyloxy)glutamoyl]-N-[2-
hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinamide (44)
The acid (43) (4.04 g, 11 mmol) and HOSu (1.87 g, 16 mmol) were
dissolved in acetonitrile (60 mL) at room temperature. A solution of
DCC (3.35 g, 16 mmol) in acetonitrile (10 mL) was added. The reaction

Total Synthesis of Methotrexate-γ-TRIS-Fatty Acid Conjugates
645
Nα-[N-(Benzyloxycarbonyl)-L-(α-benzyloxy)glutamoyl]-N-[2-[(1-
oxohexadecyl)oxy]-1,1-bis[[(1-oxohexadecyl)oxy]methyl]ethyl]-
glycinamide (47)
[2] G. D. Weinstein, J. L. McCullough, E. Olsen, Arch. Dermatol.
1989, 125, 227.
[3] X. E. Wells, V. J. Bender, C. L. Francis, H. M. He-Williams, M.
K. Manthey, M. J. Moghaddam, W. G. Reilly, R. G. Whittaker,
Drug Dev. Res. 1999, 46, 302.
Obtained 1.30 g, 87%, as a white wax (Found: C, 71.4; H, 10.2; N,
3.4%. C74H123N3O12 requires C, 71.3; H, 9.9; N, 3.4%). 1H NMR δ
(CDCl3) 7.35, br s, 2 × C6H5; 6.50, s, TRIS-NH; 6.29, br, gly-NH; 5.68,
d, J 7.9 Hz, glu-NH; 5.17, s, CH2Ph; 5.11, s, CH2Ph; 4.41, m, 7H, glu-
α-H and 3 × CH2O; 3.87–3.76, m, gly-CH2; 2.38–2.12, m, 8H, 3 ×
CH2CO and glu-γ-CH2; 2.12–1.84, br, glu-β-CH2; 1.69–1.46, m,
CH2CH2CO; 1.25, br s, 36 × CH2; 0.9, t, J 6.5 Hz, 3 × CH3. Mass
spectrum (APCI+) m/z 1246 (M + H, 78%), 1171 (27), 1138 (100);
(APCI–) m/z 1282 (M + Cl–, 35%), 1172 (68), 1154 (100).
[4] R. G. Whittaker, P. J. Hayes, V. J. Bender, Pept. Res. 1993, 6,
125.
[5] R. G. Whittaker, V. J. Bender, W. G. Reilly, PCT Int. Appl. WO
95 04,030, 1995 (Chem. Abstr. 1995, 123, 199403).
[6] R. G. Whittaker, V. J. Bender, W. G. Reilly, M. J. Moghaddam,
PCT Int. Appl. WO 96 22,303, 1996 (Chem. Abstr. 1996, 125,
221435).
[7] F. H. Cameron, M. J. Moghaddam, V. J. Bender, R. G.
Whittaker, T. J. Lockett, Biochim. Biophys. Acta 1999, 1417,
37.
Nα-[L-γ-Glutamyl]-N-[1,1-bis(hydroxymethyl)-2-[(1-oxohexadecyl)-
oxy]ethyl]glycinamide (49)
[8] T. Lockett, W. Reilly, M. Manthey, X. Wells, F. Cameron,
M. Moghaddam, J. Johnston, K. Smith, C. Francis, Q.
Yang, R. Whittaker, Clin. Exp. Pharmacol. Physiol. 2000,
27, 563.
[9] R. G. Whittaker, X. E. Wells, W. G. Reilly, PCT Int. Appl. WO 00
34,281, 2000 (Chem. Abstr. 2000, 133, 30964).
[10] R. Adhikari, C. L. Francis, G. W. Simpson, Q. Yang, Aust. J.
Chem. 2002, 55, 629.
[11] D. M. G. Martin, P. S. Siedlecki, Org. Proc. Res. Dev. 2000, 4,
259.
[12] X. Huang, X. Luo, Y. Roupioz, J. W. Keillor, J. Org. Chem. 1997,
62, 8821.
Prepared from (45) by the standard hydrogenolysis procedure in 93%
yield as a light grey solid, which was used without further purification.
1H NMR δ ((CD3)2SO) 8.26, t, J 5.8 Hz, gly-NH; 7.56, s, TRIS-NH;
5.35, b, NH2; 4.31 and 4.18, AB, J 10.7 Hz, CH2O; 3.75–3.25, m, 7H,
glu-α-H and CH2N and 2 × CH2OH; 2.40–2.13, m, 4H, CH2CO2 and
glu-γ-CH2; 2.04–1.74, m, glu-β-CH2; 1.62–1.37, m, CH2CH2CO2;
1.27, br s, 12 × CH2; 0.88, t, J 6.7 Hz, CH3. Mass spectrum (ESI+) m/z
546 (M + H, 100%).
Nα-[L-γ-Glutamyl]-N-[2-[(1-oxohexadecyl)oxy]-1,1-bis[[(1-oxohexa-
decyl)oxy]methyl]ethyl]glycinamide (50)
Prepared from (47) by the standard hydrogenolysis procedure as a white
solid (0.32 g, 97%). 1H NMR δ ((CD3)2SO/CDCl3, 2 : 1) 8.12, m, gly-
NH; 7.93, s, TRIS-NH; 4.27, s, 3 × CH2O; 3.90–3.51, m, glu-α-H and
CH2N; 2.40–2.18, m, 8H, 3 × CH2CO2 and glu-γ-CH2; 2.10–1.91, m,
glu-β-CH2; 1.60–1.35, m, 3 × CH2CH2CO2; 1.22, br s, 36 × CH2; 0.84,
t, J 6.7 Hz, 3 × CH3. Mass spectrum (ESI+) m/z 1023 (M + H, 100%).
[13] A. Rosowsky, R. Forsch, J. Uren, M. Wick, J. Med. Chem. 1981,
24, 1450.
[14] A. Rosowsky, J. H. Freisheim, H. Bader, R. Forsch, S. S.
Susten, C. A. Cucchi, E. Frei III, J. Med. Chem. 1985, 28,
660.
[15] J. Kralovec, G. Spencer, A. H. Blair, M. Mammen, M. Singh, T.
Ghose, J. Med. Chem. 1989, 32, 2426.
Acknowledgments
[16] S. C. Fu, M. Reiner, T. L. Loo, J. Org. Chem. 1975, 30, 1277.
[17] J. R. Piper, J. A. Montgomery, F. M. Sirotnak, P. L. Chello, J.
Med. Chem. 1982, 25, 182.
[18] J. R. Piper, J. A. Montgomery, J. Org. Chem. 1977, 42, 208.
[19] J. R. Piper, C. A. Johnson, G. M. Otter, F. M. Sirotnak, J. Med.
Chem. 1992, 35, 3002.
The authors acknowledge the financial support of F.H.
Faulding and Co. Limited, 1538 Main North Road, Salisbury
South, S.A. 5106, and the excellent technical assistance of
Marianne Bliese and Grace Broadhead. We thank Dr Vera
Bender for some preliminary experiments.
[20] A. Rosowsky, J. E. Wright, C. M. Vaidya, R. A. Forsch, H. Bader,
J. Med. Chem. 2000, 43, 1620.
[21] E. C. Taylor, P. J. Harrington, S. R. Fletcher, G. P. Beardsley, R.
G. Moran, J. Med. Chem. 1985, 28, 914.
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