The syntheses and properties of tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanine and O6-methylguanine

Article abstract of DOI:10.1039/b516447h

The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of S⁶-methylthioguanine are described. The crystal structures and pK_a values of these and related O⁶-methylguanine analogues are reported. All compounds display higher pK_a values than O ⁶-methylguanine with the sulfur-containing analogues being the more basic and exhibiting higher stability in aqueous solution. In a standard substrate assay with the human repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) only the oxygen-containing analogue displayed activity. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b516447h

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www.rsc.org/obc | Organic & Biomolecular Chemistry
The syntheses and properties of tricyclic pyrrolo[2,3-d]pyrimidine analogues
of S6-methylthioguanine and O6-methylguanine†
Ana R. Hornillo-Araujo,a Adam J. M. Burrell,a Miren K. Aiertza,a Takayuki Shibata,a David M. Hammond,‡a
a
Dolore`s Edmont,§ Harry Adams,a Geoffrey P. Margisonb and David M. Williams*a
Received 21st November 2005, Accepted 28th February 2006
First published as an Advance Article on the web 23rd March 2006
DOI: 10.1039/b516447h
The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of S6-methylthioguanine are
described. The crystal structures and pKa values of these and related O6-methylguanine analogues are
reported. All compounds display higher pKa values than O6-methylguanine with the sulfur-containing
analogues being the more basic and exhibiting higher stability in aqueous solution. In a standard
substrate assay with the human repair protein O6-methylguanine-DNA methyltransferase (MGMT)
only the oxygen-containing analogue displayed activity.
which might undergo mechanism-based covalent cross-linking
to MGMT for subsequent structural investigation. Recently the
crystal structure of such a complex between MGMT and an
oligonucleotide containing N1,O6-ethanoxanthosine (2) (the cross-
Introduction
O6-Methylguanine (1) is one of a number of modified bases
that can arise in DNA following exposure to alkylating agents.
link is shown in Fig. 1) was reported.7,8 This revealed a repair
mechanism involving nucleotide flipping and recognition of the
DNA via the minor groove using a HTH motif. The authors also
report the structure of a C145S mutant of MGMT8 in complex
with O6-methylguanine-containing DNA which reveals a hydrogen
bond between the phenolic OH of Tyr114 and the N-3 position of
the modified base.
The high toxicity of this lesion derives from the ability of the
analogue to mispair with thymine during DNA replication. The
human repair protein O6-methylguanine-DNA methyltransferase1
(MGMT) repairs this lesion by transferring the alkyl group to
an active site cysteine (Cys145) in a stoichiometric, irreversible
reaction in which the protein becomes inactivated.2 MGMT also
protects tumour cells from the action of alkylating agents such as
temozolomide and BCNU that are used in cancer therapy. This
has generated considerable interest in compounds that inactivate
MGMT and thereby sensitise tumour cells to killing by these
agents.3,4 Crystal structures of MGMT have been reported for
the human protein5 and an active, truncated human form,6 and a
mechanism for the repair reaction that is performed by MGMT
has been suggested by Daniels et al.5 In this mechanism, the
nucleophilic thiolate anion of Cys145 reacts with the O6-alkyl
group in an SN2 reaction and Tyr114 is the proposed proton donor
required to regenerate guanine within the damaged DNA.
The initial crystal structures of MGMT were obtained in the
absence of substrate DNA and under such circumstances, the
active site cysteine was observed to be buried in the centre of
the protein, far removed from the damaged guanine base. This has
led to the search for suitable oligonucleotide pseudosubstrates
aCentre for Chemical Biology, Richard Roberts Building, Department of
Chemistry, University of Sheffield, Brook Hill, Sheffield, UK S3 7HF.
E-mail: d.m.williams@sheffield.ac.uk; Fax: +44 (0)114 222 9346; Tel: +44
(0)114 222 9502
bCancer Research UK Carcinogenesis Group, Paterson Institute for Cancer
Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, UK
M20 4BX
† Electronic supplementary information (ESI) available: Absorption spec-
troscopy data for compounds 5–7 and 18; colour crystal structure images
of compounds 4a, 5 and 7; pH titration plots of compounds 5–7 and 18.
See DOI: 10.1039/b516447h
‡ Present address: Fakulta¨t fu¨r Chemie und Pharmazie, Ludwig-
Maximilians-Universita¨t, Butenandtstr. 5-13, 81377 Munich, Germany.
§ Present address: Laboratoire de Chimie Organique Biomole´culaire de
Synthe`se, UMR 5625 CNRS-UM II, Universite´ de Montpellier II, Place
Euge`ne Bataillon, 34095 Montpellier Cedex, France.
Fig. 1 Modes of cross-linking of analogues 2–7 with MGMT Cys145.
We have also been interested in designing pseudosubstrates
of O6-methylguanine for cross-linking to MGMT and have
concentrated our efforts on tricyclic pyrrolo[2,3-d]pyrimidine (7-
deazapurine) analogues. Recently we reported the syntheses9 of
the novel analogues 3a and 3b for which we envisaged covalent
modification via the C5 (C7 of 7-deazapurine) position upon
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reaction with MGMT (Fig. 1). Such compounds would reveal
additional information not present in the existing MGMT–DNA
complex,7,8 in particular interactions between the protein and the
N1 and the amino group of the modified base (see Fig. 1) that
are likely to be important during the repair of O6-methylguanine.
Unfortunately compound 3a is too reactive for incorporation into
DNA since in aq. solution it is hydrolysed to the corresponding O6-
hydroxyethyl-7-deazaguanine derivative. In contrast compound 3b
is relatively stable and displays an IC50 of approximately 1 mM
in a standard MGMT assay.9 Previous studies have shown that
synthetic oligonucleotide substrates containing S6-methylthio-
and Se6-methylselenoguanine analogues are also repaired by
MGMT10 which has encouraged us to prepare the corresponding
thio analogues of 3a and 3b.
Scheme 1 Reagents and conditions: (i) PCC, CH2Cl2, n = 1 (ref. 13),
n = 2 (ref. 9); (ii) TMSBr, DMSO, 0 ◦C to room temp., 4 h; (iii)
2,6-diamino-4(3H)pyrimidinone, aq. NaOAc, n = 1, 77%, n = 2, 31%.
We report here the syntheses of the novel tricyclic sulfur-
containing pyrrolo[2,3-d]pyrimidines 4a and 4b. We also present
the crystal structures of the sulfur-containing pyrrolopyrimidines
4a and 7 together with that of the tricyclic analogue containing the
7-membered oxygen-containing ring (compound 5). In addition
we determine the respective pKa values of the N-methylated
tricyclic pyrrolo[2,3-d]pyrimidine derivatives 5, 6 and 7 and report
on the abilities of these and their unmethylated derivatives (3b, 4a
and 4b) to act as inactivators of MGMT.
To our knowledge these are the first crystal structures of tricyclic
pyrrolo[2,3-d]pyrimidines to be described. There are few reports
of similar compounds in the literature and these include related
nucleoside analogues in which the third ring contains N–N11 or N–
O12 functionality. Analogues of the former compound (triciribine)
display antiviral and antineoplastic properties.
Scheme 2 Reagents and conditions: (i) BCl3, CH2Cl2, −78 ◦C, 6 h, n = 1
(ref. 13), n = 2 (ref. 9); (ii) n = 2, Ph3P, DIAD, DMF, 41%.
conditions using DIAD and Ph3P to afford the tricyclic analogue
3b in 41% yield. This yield was slightly better than that obtained
previously using the combination of DEAD and Ph3P (30% yield).9
Thiation of the compounds 10 and 11 using trifluoroacetic anhy-
dride in pyridine followed by treatment with sodium hydrosulfide15
gave compounds 14 and 15 respectively (Scheme 3). Debenzylation
using boron trichloride afforded the alcohols 16 and 17 respec-
tively. The cyclisation of 16 and 17 using the Mitsunobu reaction
gave the tricyclic sulfur-containing homologues 4a and 4b in 20%
and 30% yield respectively. Compound 4a was also isolated in 22%
during extended treatment of compound 14 with BCl3.
Results and discussion
Previously, compounds 3a and 3b were obtained in two steps
from the appropriate 5-substituted pyrimidine precursors 8 and
9, respectively.9 Compounds 813 and 99 were obtained via Michael
addition of 2,6-diamino-4(3H)pyrimidinone to the appropriate
nitroalkenes which were in turn prepared in 4 steps from 1,3-
propanediol or 1,4-butanediol respectively. Compounds 8 and 9
were subsequently converted (in 4 steps) to the desired tricyclic
pyrrolopyrimidines 3a and 3b.9
Scheme 3 Reagents and conditions: (i) (CF3CO)2O, pyridine, 0 ◦C, 1 h,
then NaSH in DMF, n = 1, 65%, n = 2, 69%; (ii) BCl3, CH2Cl2, −78 ◦C,
9 h, n = 1, 57%, n = 2, 31%; (iii) Ph3P, DIAD, DMF, n = 1, 20%, n = 2,
30%.
In order to develop a more efficient synthesis of 3b that could
also be applied to obtaining the novel sulfur-containing analogues
4a and 4b, we considered an alternative route to 5-substituted
pyrrolo[2,3-d]pyrimidines that has been used in the synthesis of
the queuine base.14 This method, which involves the reaction of an
a-bromoaldehyde with 2,6-diamino-4(3H)pyrimidinone, allowed
the preparation of compounds 10 and 11 in three steps from the
respective diols (Scheme 1).
In order to obtain information about the physical properties
of the tricyclic analogues, compounds 3b, 4a and 4b were all
converted into their corresponding N-methylated derivatives 5,
6 and 7 which we envisaged as simple model compounds of the
respective nucleosides. Methylation was achieved using sodium
hydride and MeI in DMF. Methylation of the 7-deaza analogue
of O6-methylguanine in the same way furnished the methylated
pyrrolo[2,3-d]pyrimidine analogue 18 of O6-methylguanine.
Debenzylation of 10 and 11 afforded the alcohols 12 and
13 (Scheme 2). Compound 13 was cyclised under Mitsunobu
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Previously we reported that compound 3b is a weak inactivator
of MGMT (IC50 approx. 1 mM).9 To assess the likely potential
of the tricyclic thio compounds in DNA to act as cross-linking
agents to MGMT we used the same standard assay.16 This
assay involves pre-incubation of MGMT with the inactivator,
followed by measurement of the amount of radiolabelling of
the protein which occurs upon the subsequent addition of DNA
containing tritiated O6-methylguanine. However, neither of the
thio-containing analogues 4a nor 4b displayed any activity. The
repair of DNA containing O6-methylguanine by MGMT is
approximately 70 times faster than the analogous reaction with
the same substrate containing S6-methylthioguanine.10 Thus, the
apparent inactivities of 4a and 4b was not completely unexpected
and since the rates of repair of the free base and DNA containing
it vary considerably,2 the compounds 4a and 4b might still be
recognised once incorporated into DNA. All of the N-methylated
compounds 5–7 were inactive. This also was not largely unexpected
since these compounds are bulkier than the free bases and thereby
have decreased access to the MGMT active site which normally is
facilitated following binding of DNA.2 However, 3b does appear
to be a pseudosubstrate of MGMT and clearly further studies
following the incorporation of this compound and the sulfur-
containing analogues into DNA are necessary.
In the context of our ultimate goal of the incorporation of these
analogues into DNA for cross-linking to MGMT we were also
interested in the structures, chemical stabilities and pKa values of
these compounds. In previous studies9 we reported that the ana-
logue 3a is unstable in aqueous solution and undergoes hydrolysis
to the ring-opened 5-hydroxyethyl analogue. Unfortunately we
were unable to obtain crystals of 3a for X-ray analysis. However,
we were able to obtain crystal structures of the N-methylated
derivatives 5 and 7 and the free base 4a (crystals of compound
6, the N-methylated derivative of 4a, were unsuitable for X-ray
analysis). The structures of compounds 4a, 5 and 7 are displayed
as ball and stick structures in Fig. 2 (see ESI† for colour TIFF and
PDB files). The pyrrolopyrimidine C–O and C–S bond lengths in
˚
˚
1.37 A and 1.76 A. This suggests that the ring strain within the
non aromatic ring in compounds 5 and 7 is small. Furthermore, a
gauche arrangement of the methylene protons in these compounds
is observed, which also minimises torsional strain. In compound
4a, the corresponding C–S–C and S–C–C bond angles are 99.3◦
and 117◦ respectively with a C–S bond length of 1.75 A, again
˚
suggesting that this compound is also not particularly strained.
This is reflected in the stabilities of these compounds towards
hydrolysis. Thus, compound 3a is unstable in aqueous solution,9
whereas compounds 3b, 4a and 4b remain unchanged after
overnight treatment with either aqueous or methanolic ammonia
at room temperature. We also note in the crystal structures
of these compounds that the electrophilic carbon, at least in
structures 5 and 7, is displaced somewhat from the plane of
the pyrrolopyrimidine moiety. This is relevant to the preferred
trajectory of nucleophilic addition of the thiolate of Cys145 of
MGMT during the repair reaction. O6-Methylguanine within G:T
mispairs adopts the proximal (to the purine N7) conformation,17
whilst as the nucleoside the distal conformation is preferred.18 In
the crystal structures of MGMT–DNA complexes the guanine
lesion is not base paired and neither the proximal nor distal
conformation would appear to be incompatible with repair.8
The mechanism by which MGMT is proposed to repair O6-
methylguanine lesions in DNA involves protonation of the modi-
fied guanine base by a Tyr114 of the protein.2,5,8 For this reason,
analogues designed to act as substrates for MGMT should ideally
display pKa values similar to or above that of O6-methylguanine
(2.3519). Thus the pKa values of compounds 5–7 and 18 (the N-
methyl derivative of 7-deaza-O6-methylguanine) were determined
by absorption spectroscopy (see experimental for details and Fig. 1
and 2 of the ESI†). The pKa values are displayed in Table 1 and
show that all of the compounds analysed are more basic than O6-
methylguanine and on this basis are not incompatible with repair
by MGMT.
˚
˚
Table 1 Determined pKa values for compounds 5, 6, 7 and 18
compounds 5 and 7 are 1.35 A and 1.76 A respectively. The C–
O–C bond angle in compound 5 is 118.1◦, whilst the C–S–C bond
angle in 7 is 106.1◦. Both compounds 5 and 7 display a similar C–
C–C bond angle of 112◦ in the non aromatic ring. In comparison,
the mean bond angles in compounds of general structure PhOR
and PhSR found in the Cambridge Crystallographic Data Centre
are 117.6◦ and 103.3◦ with corresponding Ar–X bond lengths of
Compound
Determined pKa value
5
4.05 0.05
4.31 0.03
4.81 0.02
4.27 0.02
6
7
18
Fig. 2 Crystal structures of compounds 4a (left), 5 (centre) and 7 (right). The crystal structure of 4a also contains one molecule of methanol (far left).
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We are currently engaged in the synthesis of DNA containing
analogues 3b, 4a and 4b which will be reported subsequently
together with their biological properties.
2,7,8,9-Tetrahydro-6-oxa-2,3,5-triazabenzo[cd]azulen-4-amine 3b
Diisopropylazodicarboxylate (DIAD) (1.10 g, 1064 lL,
5.40 mmol) was added dropwise to Ph3P (1.42 g, 5.40 mmol) in
dry DMF (30 ml) under Ar at room temp. The mixture was then
stirred at room temp. for 30 min, 13 in dry DMF (20 ml) was added
dropwise over 20 min and the mixture was stirred overnight. After
evaporation the residue was adsorbed onto silica and purified
by column chromatography (10–20% MeOH in CH2Cl2) then
triturated with acetonitrile (10 ml), to give a cream-coloured solid
(337 mg, 41%). Data identical to those described.9
Experimental
All nomenclature was generated using the IUPAC online naming
service (http://www.iupac.org/nomenclature/index.html)
CH3CN, CH2Cl2 and pyridine were dried by heating under reflux
with CaH2 followed by distillation. Dry DMSO and DMF were
purchased from Aldrich. All dry solvents except pyridine were
˚
stored over activated 3 A molecular sieves under Ar. All other
7-Amino-3,4-dihydro-1H-5-thia-1,6,8-triazaacenaphthylene 4a
reagents were purchased from commercial suppliers and used
without purification. Silica gel for column chromatography was
obtained from BDH (particle size 30–60 lm). For TLC pre-coated
Merck Kieselgel 60 F254 aluminium backed plates were used. TLC
systems used were A = 10% MeOH in CH2Cl2; B = CH2Cl2; C =
10% MeOH in EtOAc; D = 20% MeOH in CH2Cl2; E = 15%
MeOH in CH2Cl2.
Melting points were measured on a Gallenkamp Melting Point
Apparatus and are uncorrected. UV–Visible data were obtained
with a VARIAN CARY 50 Probe Spectrometer. Nuclear magnetic
resonance (NMR) spectra were run on a Bruker AC-250 and
AMX-400 spectrometers. 1H spectra were run at 250.13 MHz
or 400.13 MHz respectively and 13C spectra at 62.83 MHz or
100.61 MHz respectively.
Method 1. Compound 16 (65 mg, 0.31 mmol) and Ph3P
(246 mg, 0.93 mmol) were dissolved in dry DMF (30 ml) under
Ar and the solution was cooled to 0 ◦C. DIAD (193 lL,
0.93 mmol) was then added dropwise and the mixture was then
stirred overnight at room temp. The reaction mixture was then
evaporated and the crude product was purified by silica gel
column chromatography (5–10% MeOH in CH2Cl2) and then
recrystallised from MeOH to give dark orange-brown needles
(12 mg, 20%); found: C, 49.96; H, 4.22; N, 28.95; S, 16.76.
C8H8N4S requires C, 49.98; H, 4.19; N, 29.14; S, 16.68%; mp >
350 ◦C (decomp.); Rf (E) 0.65 (fluorescent at 365 nm), 16 = 0.33
(fluorescent at 365 nm); pH = 7.87 (T = 21.4 ◦C); kmax(MeOH)/nm
234.9, 326.2 (log e/dm3 mol−1 cm−1 4.30, 3.53); kmin(MeOH)/nm
219.8, 284.4 (log e/dm3 mol−1 cm−1 4.05, 3.01); luminescence
(MeOH; c = 2.71 × 10−6 M): kExc = 240 nm, kEmm = 405 nm;
kExc = 325 nm, kEmm = 405 nm; dH (d6-DMSO) 2.89 (2H, t, J 6.4,
CH2CH2S), 3.27 (2H, t, J 6.4, CH2S), 6.09 (2H, s, NH2), 6.62
(1H, d, J 1.2, CH-2), 10.77 (1H, s, NH-1) ppm; dC (d6-DMSO)
21.92, 31.02, 107.24, 109.78, 114.73, 150.09, 160.20, 160.34 ppm;
m/z (ES+) 193 ([M + H]+, 100%); acc. mass: 193.0551, C8H9N4S
requires 193.0548.
5-Benzyloxy-1-pentanol
Pentane-1,5-diol (157 g, 1.45 mol) was dissolved in dry DMF
◦
(2 L) under Ar and cooled to 0 C. NaH (40.4 g, 1.6 mol, 95%
dispersion in mineral oil) was cautiously added over 60 min and the
reaction was stirred for a further 30 min. Benzyl chloride (1.45 mol,
188.8 g, 215 ml) was then added dropwise and the mixture was
stirred at room temp. for 24 h. The precipitated solid was filtered,
the solvent evaporated and the residue was redissolved in CH2Cl2
(1 L), washed with water (300 ml), dried (MgSO4) and evaporated.
Distillation under ◦reduced pressure gave a colourless oil (218 g,
77%); bp 110–112 C (0.1 mm Hg) (lit20 123 ◦C (0.4 mm Hg)); Rf
(A) 0.68; dH (d6-DMSO) 1.26–1.51 (6H, m, 3 CH2), 3.41 (4H, m,
CH2OH, CH2O), 4.36 (1H, t, OH) 4.42 (2H, s, OCH2Ph), 7.20–
7.40 (5H, m, Ph) ppm; dC (d6-DMSO) 22.78, 29.61, 32.82, 61.13,
70.17, 72.28, 127.76, 127.83 and 128.68, 139.21 ppm; m/z (EI+)
194 (M+); acc. mass: 194.1300, C12H18O2 requires 194.1307.
Method 2. BCl3 (1 M) in heptane, (43.7 mL, 43.7 mmol) was
added dropwise◦to compound 14 (1.50 g, 4.86 mmol) in dry CH2Cl2
◦
(90 ml) at −78 C under Ar. The reaction was stirred at −78 C
for 4 h, then more BCl3 solution (19.5 mL, 19.5 mmol) was added
and stirring was continued for a further 6 h. The mixture was
then warmed to room temp. overnight whilst a solution of EtOH
in CH2Cl2 (220 mL, 1 : 1) was added dropwise. The residue was
purified after evaporation by silica column chromatography (10%
MeOH in CH2Cl2) followed by recrystallisation from MeOH to
give dark orange-brown needles (200 mg, 22%). Compound 16
was also obtained in this reaction (236 mg, 23%).
5-Benzyloxypentanal
A solution of 5-benzyloxy-1-pentanol (100 g, 0.52 mol) in CH2Cl2
(400 ml) was added to a stirred suspension of PCC (226.5 g, 1.13
2,7,8,9-Tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulen-4-amine 4b
R
mol), Hyflo Super Celꢀ (230 g), silica (230 g) and CH2Cl2 (2 L) at
0 ◦C. The reaction was stirred at room temp. for 3 h, then filtered
Preparation analogous to 3b: using 17 (360 mg, 1.61 mmol) in dry
DMF (20 ml) and DIAD (649 mg, 622 lL, 3.21 mmol) and Ph3P
(842 mg, 3.21 mmol) in dry DMF (25 ml). Purification gave a light
brown solid (100 mg, 30%); Rf (A) 0.5; dH (CD3OD) 2.15–2.28
(2H, m, SCH2CH2), 2.92–2.97 (2H, m, SCH2CH2CH2), 3.10–3.13
(2H, m, SCH2), 6.75 (1H, t, J 1.3, CH-6) ppm; dC (d4-CD3OD)
26.43, 30.01, 31.85, 77.35, 97.25, 117.22, 124.98, 152.95, 155.25,
160.51 ppm; m/z (EI+) 206 (M+); acc. mass: 206.0633, C9H10N4S
requires 206.0626 (deviation 3.1 ppm).
and the filtrate was purified on a 3 L column consisting of silica-
R
Hyflo Super Celꢀ and silica and eluted with CH2Cl2 (10 L) to
give a pale yellow oil (67.0 g, 68%); Rf (B) 0.42; dH (d6-DMSO)
1.45–1.65 (4H, m, 2 CH2), 2.46 (2H, m, CH2CHO), 3.43 (2H, m,
CH2O), 4.41 (2H, s, OCH2Ph), 7.20–7.40 (5H, m, Ph), 9.66 (1H,
t, J 1.5, CHO) ppm; dC (d6-DMSO) 18.94, 29.06, 43.19, 69.72,
72.27, 127.86, 128.69, and 129.63, 135.06, 203.86 ppm; m/z (EI+)
192 (M+); acc. mass: 192.1151, C12H16O2 requires 192.1150.
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2-Methyl-7,8,9-tetrahydro-6-oxa-2,3,5-
triazabenzo[cd]azulen-4-amine 5
were then added dropwise via a syringe and the solution was
stirred at room temp. for 4 h. A suspension of 2,6-diamino-
4(3H)pyrimidinone (5.88 g, 44.75 mmol) and sodium acetate
(3.67 g, 44.75 mmol) in water (100 ml) was then added and
the reaction stirred overnight. The mixture was extracted with
EtOAc (4 × 1 L) and the organic layers were washed with brine
(500 ml), dried (Na2SO4) and evaporated. Purification by silica
chromatography (10% MeOH in EtOAc) gave a cream coloured
solid (8.94 g, 77%). Data as described.13
To compound 3b (320 mg, 1.68 mmol) in dry DMF (6.5 ml) under
Ar at 0 ◦C was added NaH (84 mg, 2.1 mmol, 60% dispersion
in oil) and the mixture was stirred for 30 min. MeI (265 mg,
116 lL, 1.86 mmol) was then added dropwise at 0 ◦C and the
mixture was then stirred at room temp. overnight. MeOH (5 ml)
was then added and the mixture was evaporated. The residue was
purified by silica column chromatography (10% MeOH in CH2Cl2)
and recrystallised from CH2Cl2–EtOAc to give cream-coloured
needles (320 mg, 93%); mp 217–219 ◦C; Rf (A) 0.5; found: C,
58.60; H, 5.94; N, 26.28. C10H12ON4 requires C, 58.8; H, 5.9; N,
27.4%; kmax (MeOH)/nm 270.02 (log e/dm3 mol−1 cm−1 3.65); kmin
(MeOH)/nm 280.06 (log e/dm3 mol−1 cm−1 3.51); ksh (MeOH)/nm
261.06 (log e/dm3 mol−1 cm−1 3.62); dH (d6-DMSO) 2.02 (2H, m,
CH2), 2.74 (2H, t, J 5.6, CH2), 3.50 (3H, s, NCH3), 4.32 (2H, m,
OCH2), 6.01 (2H, s, NH2), 6.67 (1H, s, CH-6) ppm; dC (CD3OD)
26.21, 29.19, 31.79, 78.64, 98.43, 117.82, 125.70, 152.98, 155.25,
160.54 ppm; m/z (EI+) 204 (M+); acc. mass: 204.1016, C10H12ON4
requires 204.1011.
2-Amino-5-[3-(benzyloxy)propyl]-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one 11
Preparation analogous to 10: using 5-benzyloxypentanal (6 g,
31.2 mmol), dry CH3CN (100 ml), bromotrimethylsilane
(35.9 mmol, 4.88 ml), dry DMSO (34.3 mmol, 2.44 ml) and
2,6-diamino-4(3H)pyrimidinone (34.3 mmol, 4.51 g) and sodium
acetate (34.3 mmol, 4.51 g) in water (100 ml). Work-up and
purification gave a pink-white solid (2.85 g, 31%). Data as
described.9
2-Amino-5-(3-benzyloxyethyl)-3,7-dihydro-pyrrolo[2,3-
d]pyrimidin-4-thione 14
7-Amino-1-methyl-3,4-dihydro-1H-5-thia-1,6,8-
triazaacenaphthylene 6
Sodium hydrosulfide hydrate (NaSH·xH2O) (14.8 g, 263.8 mmol)
was dried over a naked flame, suspended in dry DMF (200 ml) and
stirred overnight. Compound 10 (2.5 g, 8.8 mmol) was then dried
by co-evaporation of dry pyridine (3 × 20 ml). The resulting syrup
was dissolved in dry pyridine (120 ml) under Ar and cooled to
Preparation analogous to 5: using 4a (200 mg, 1.04 mmol), NaH
(48 mg, 1.20 mol, 60% dispersion in oil) and MeI (75.5 lL,
1.20 mmol) in dry DMF (3 ml). Chromatography gave an orange
foam (188 mg, 88%). Recrystallisation from MeOH afforded
orange needles; mp 48–50 ◦C, Rf (A) 0.48 (product; fluorescent
at 365 nm), 6 = 0.35 (fluorescent at 365 nm); pH = 7.92 (T =
23.8 ◦C); kmin(MeOH)/nm 222.0, 291.7 (log e/dm3 mol−1 cm−1
3.92, 3.31); kmax(MeOH)/nm 238.2, 326.4 (log e/dm3 mol−1 cm−1
0
◦C. Trifluoroacetic anhydride (9.9 mL, 70.4 mmol) was then
added dropwise and the mixture stirred for 1 h at 0 ◦C. The
suspension of NaSH in DMF was then added and stirring
continued at room temp. overnight. The mixture was then poured
into saturated aq. ammonium bicarbonate solution (500 ml) and
stirred vigorously at room temp. overnight. The mixture was then
evaporated and the residue was extracted into MeOH (600 ml).
The MeOH was then evaporated and the residue was triturated
with aq. triethylammonium acetate (0.1 M, 500 mL; pH 5.2), dried
under vacuum and purified by silica column chromatography
(5% MeOH in CH2Cl2) to give an orange foam (1.7 g, 65%);
Rf (D) 0.68 (product) (10 = 0.5211); kmax(MeOH)/nm 235, 264
(log e/dm3 mol−1 cm−1 3.24, 3.17); kmin(MeOH)/nm 228, 251,
299 (log e/dm3 mol−1 cm−1 3.23, 3.13, 2.96); ksh(MeOH)/nm 343
(log e/dm3 mol−1 cm−1 2.98); dH (d6-DMSO) 3.19 (2H, t, J 7.0,
CH2CH2OBn), 3.69 (2H, td, J 4.2 and 2.7, CH2OBn), 4.48 (2H,
d, J 1.8, OCH2Ph), 6.40 (2H, s, NH2), 6.63 (1H, s, CH-6), 7.19–
7.34 (5H, m, CH-Ph), 11.04 (1H, s, NH-3), 11.22 (1H, s, NH-7)
ppm; dC (d6-DMSO) 26.43, 70.84, 71.62, 115.87, 116.19, 127.21,
127.36, 128.15, 128.90, 138.88, 151.84, 152.10, 175.29 ppm; m/z
(ES+) 301 ([M + H]+, 100%); acc. mass: 301.1123, C15H17N4OS,
requires 301.1126.
4.17, 3.48); luminescence (MeOH; c = 2.52 × 10−6 M): kExc
=
240 nm, kEmm = 410 nm; kExc = 325 nm, kEmm = 410 nm; dH (d6-
DMSO) 2.89 (2H, t, J 6.4, CH2CH2S), 3.28 (2H, t, J 6.4, CH2S),
3.35 (3H, s, CH3N), 6.26 (2H, s, NH2), 6.66 (1H, d, J 1.2, CH-2)
ppm; dC (d6-DMSO) 21.58, 30.44, 30.81, 107.19, 109.46, 119.13,
149.22, 160.37, 160.40 ppm; m/z (ES+) 207 ([M + H]+, 100%);
acc. mass: 206.062397, C9H10N4S requires 206.062618.
2-Methyl-7,8,9-tetrahydro-6-thia-2,3,5-triazabenzo[cd]azulen-
4-amine 7
Preparation analogous to 5: using 4b (99 mg, 0.48 mmol), NaH
(24 mg, 0.6 mmol, 60% dispersion in oil) and MeI (36 lL,
0.5 mmol) in dry DMF (2 ml). Chromatography gave a light
brown solid (65 mg, 62%); Rf (A) 0.6; dH (CD3OD) 2.21–2.27
(2H, m, SCH2CH2), 2.91–2.95 (2H, m, SCH2CH2CH2), 3.10–3.12
(2H, m, SCH2), 3.57 (1H, s, NCH3), 6.71 (1H, t, J 1.3, CH-6)
ppm; dC (CD3OD) 29.24, 30.09, 31.30, 111.59, 117.61, 129.49,
152.68, 153.53, 158.32 ppm; m/z (ES+) 221 ([M + H]+); acc. mass:
221.0681, C10H13N4S requires 221.0861.
2-Amino-5-(3-benzyloxypropyl)-3,7-dihydropyrrolo[2,3-
d]pyrimidin-4-thione 15
2-Amino-5-[2-(benzyloxy)ethyl]-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one 10
Preparation analogous to 14: using 11 (16 g, 53.6 mmol),
trifluoroacetic anhydride (90.1 g, 428.8 mmol, 61 ml), dry pyridine
(660 ml) and dried NaSH·xH2O (80 g, 1.42 mol) in dry DMF
(2 L). Work-up and purification gave a light brown solid (11.6 g,
69%); Rf (D) 0.74 0.37 (lit.9 11 = 0.68); dH (d6]-DMSO) 1.85–1.95
A solution of 4-(benzyloxy) butanal13 (7.25 g, 40.7 mmol) in
◦
dry CH3CN (100 ml) was cooled to 0 C. Bromotrimethylsilane
(6.4 mL, 47.0 mmol) and dry DMSO (3.35 mL, 47.0 mmol)
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(2H, m, CH2), 2.88 (2H, t, J 7.6, CH2), 3.44 (2H, t, J 6.4, CH2O),
4.44 (2H, s, OCH2Ph), 6.39 (2H, s, NH2), 6.53 (1H, s, H-6), 7.31
(5H, m, Ph), 10.97 (1H, s, NH), 11.41 (1H, s, NH); dC (d6-DMSO)
23.76, 30.40, 69.40, 71.72, 111.06, 117.15, 119.41, 127.27, 127.45,
127.64, 127.82 and 128.19, 138.78, 148.54, 151.81, 175.33; m/z
(ES+) 315 ([M + H]+); acc. mass 315.1274, C16H19ON4S requires
315.1280.
below 2.20, dilutions of hydrochloric acid were used as described.23
Absorption spectra were measured at 20 ◦C (after 5 min pre-
incubation). The absorbance values were plotted against pH and
the pKa values were determined following non-linear regression
fitting to eqn (1) using Kaleidagraph software (Synergy Software,
Reading, PA, USA).
ꢀ
ꢁ
Ka
A = AI + (AM − AI) ·
(1)
Ka + [H+]
2-Amino-5-(2-hydroxyethyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidine-4-thione 16
Where A is the absorbance at the measured wavelength, AI is the
absorbance of the cation and AM is the absorbance of the neutral
molecule.
BCl3 (1 M) in heptane, (33.6 mL, 33.6 mmol) was added dropwise
to a solution of compound 14 (1.12 g, 3.73 mmol) in dry
CH2Cl2 (80 ml) at −78 ◦C under Ar and the reaction was
stirred at that temp. for 9 h. The mixture was then warmed
to room temp. overnight whilst a mixture of EtOH–CH2Cl2
(200 mL, 1 : 1) was added dropwise. The mixture was then
evaporated, redissolved in ethanol (50 ml) and neutralized with
aq. sodium hydroxide solution (4 M). The residue was purified
after evaporation by silica column chromatography (10% MeOH
in CH2Cl2) to give a pale brown solid (447 mg, 57%); Rf
(E) 0.30, 14 = 0.62; kmax(MeOH)/nm 234.6, 270.6, 347.1 (log
e/dm3 mol−1 cm−1 4.11, 3.84, 3.86); kmin(MeOH)/nm 225.0, 251.6,
302.2 (log e/dm3 mol−1 cm−1 4.00, 3.56, 3.24); ksh(MeOH)/nm
285.2 (log e/dm3 mol−1 cm−1 3.73); dH (d6-DMSO) 2.98 (2H, t, J
7.0, CH2CH2OH), 3.60 (2H, t, J 7.0, CH2OH), 4.43 (1H, t, J 5.2,
OH), 6.41 (2H, bs, NH2), 6.59 (1H, bs, CH-6), 11.01 (1H, bs, NH-
3), 11.43 (1H, bs, NH-7) ppm; dC (d6-DMSO) 29.80, 62.06, 111.14,
116.56, 117.97, 148.40, 151.77, 175.27 ppm; m/z (ES+) 211 ([M +
H]+, 100%); acc. mass: 211.0651, C8H11N4OS requires 211.0654.
Crystal structure determination of compound 4a
(C8H8N4S·CH3OH)¶
Crystal data. C9H12N4OS, M = 224.29, monoclinic, a =
3
˚
˚
8.6545(12), b = 15.734(2), c = 7.7979(11) A, U = 1055.1(3) A , T =
150(2) K, space group P21/c, Z = 4, l(Mo–Ka) = 0.286 mm−1,
11534 reflections measured, 2391 unique (Rint = 0.0276) which
were used in all calculations. The final wR(F2) was 0.0948 (all
data).
Crystal structure determination of compound 5
Crystal data. C10H12N4O, M = 204.24, orthorhombic, a =
3
˚
˚
7.9420(17), b = 13.862(3), c = 8.4623(19) A, U = 931.6(4) A , T =
150(2) K, space group Pna21, Z = 4, l(Mo–Ka) = 0.100 mm−1,
9204 reflections measured, 1141 unique (Rint = 0.0369) which
were used in all calculations. The final wR(F2) was 0.1186 (all
data).
Crystal structure determination of compound 7
2-Amino-5-(3-hydroxypropyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-thione 17
Crystal data. C10H12N4S, M = 220.30, monoclinic, a =
3
˚
˚
7.8532(12), b = 12.4259(19), c = 11.0895(17) A, U = 1024.4(3) A ,
T = 150(2) K, space group P21/c, Z = 4, l(Mo–Ka) = 0.286 mm−1,
8569 reflections measured, 2325 unique (Rint = 0.0305) which were
used in all calculations. The final wR(F2) was 0.1328 (all data).
Preparation analogous to 16: using 15 (0.5 g, 1.59 mmol), CH2Cl2
(25 ml), BCl3 in heptane (14.2 ml, 14.18 mmol). Work-up and
purification gave a pale brown solid (110 mg, 31%); Rf (D) 0.53;
dH (d6-DMSO) 1.70–1.80 (2H, m, CH2), 2.79 (2H, t, J 6.4, CH2),
3.35 (2H, t, J 6.4, CH2OH), 4.32 (1H, t, OH), 6.37 (2H, bs, NH2),
6.55 (1H, s, H-6), 10.92 (1H, bs, NH), 11.36 (1H, bs, NH); dC
(d6-DMSO) 22.72, 34.01, 60.72, 99.30, 113.65, 118.64, 151.68,
152.74, 175.91; m/z (ES+) 247 ([M + Na]+); acc. mass: 247.0635,
C9H12ON4SNa requires 247.0630.
Acknowledgements
We are grateful to Dr Brian Taylor and Ms Sue Bradshaw for NMR
data, Mr Simon Thorpe for mass spectra. We are also grateful to
the EPSRC for financial support.
2-Amino-4-methoxy-7-methyl-7H-pyrrolo[2,3-d]pyrimidine 18
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Org. Biomol. Chem., 2006, 4, 1723–1729 | 1729
©