Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

Article abstract of DOI:10.1016/j.bmc.2013.02.016

The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C-nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.

Full text of DOI:10.1016/j.bmc.2013.02.016

Accepted Manuscript
Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-gua‐
nine-xanthine phosphoribosyltransferase
Keith Clinch, Douglas R. Crump, Gary B. Evans, Keith Z. Hazleton, Jennifer
M. Mason, Vern L. Schramm, Peter C. Tyler
PII:
S0968-0896(13)00140-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.02.016
BMC 10611
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
6 December 2012
29 January 2013
6 February 2013
Please cite this article as: Clinch, K., Crump, D.R., Evans, G.B., Hazleton, K.Z., Mason, J.M., Schramm, V.L.,
Tyler, P.C., Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine
phosphoribosyltransferase, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2
013.02.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Acyclic phosph(on)ate inhibitors of Plasmodium falciparum
hypoxanthine-guanine-xanthine phosphoribosyltransferase
a
a
a
b
Keith Clinch , Douglas R. Crump , Gary B. Evans , Keith Z. Hazleton , Jennifer M.
a
b
a,
Mason , Vern L. Schramm and Peter C. Tyler *
a
Carbohydrate Chemistry, Industrial Research Ltd, Lower Hutt 5040, New Zealand
b
Department of Biochemistry, Albert Einstein College of Medicine, New York 10461, USA
Keywords: Phosphoribosyltransferase; malaria; HGXPRTase; purine salvage; protozoa
Abstract.
The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis
of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf),
hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts
hypoxanthine to inosine monophosphate and is essential for purine salvage making the
enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic
aza-C- nucleosides and shown that some are potent inhibitors of Pf HGXPRT while
showing excellent selectivity for the Pf versus the human enzyme.
Introduction.
Malaria is endemic in many parts of the world and there were over 200 million cases
1
resulting in >600,000 deaths in 2010. The disease is caused by Plasmodium protozoa of
2
3
which Pf is the most deadly. Drug resistance and the limitations of current vaccines
makes new drug development important for effective malaria control. Several pathogenic
protozoa lack the enzymes for de novo synthesis of purines and are dependent on the
4
-6
purine salvage pathway. In Pf purine salvage requires purine nucleoside phosphorylase
PfPNP) to generate hypoxanthine and then hypoxanthine-guanine-xanthine
phosphoribosyltransferase (Pf HGXPRT) to convert the hypoxanthine to inosine
(
7
8
monophosphate. Inhibition of PfPNP has been shown to kill Pf in cell culture and in an
1

9
Aotus monkey model. We have been interested in extending this work to the discovery
of inhibitors of Pf HGXPRT as they are also expected to be lethal to the parasite. A
number of purine analogues were reported some time ago as potential inhibitors of
1
0-11
Plasmodium berghei, but none showed significant inhibition of the enzyme.
Scheme 1 Possible transition state of the HGXPRTase-catalysed reaction.
Pf HGXPRT is an N-ribosyltransferase and the transition states of such enzymes have
been characterized with strong ribooxacarbenium ion properties and low bond order to
1
2-13
both the purine and the nucleophile.
Kinetic commitment factors have prevented the
transition state analysis of Pf HGXPRT to date but it would be reasonable to predict a
dissociative transition state with ribooxacarbenium ion character similar to that shown
(
Scheme 1). Enzyme inhibitor design based on incorporating features of the transition
1
4-18
state will often lead to exceptionally potent compounds.
Immucillin-H (and G) 5’-
phosphate 1 and 2 are transition state analogue inhibitors of Pf HGXPRT and also of the
1
9-21
human hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
They mimic the
ribooxacarbenium ion positive charge as well as other substrate properties. However,
they are not selective for the parasite enzyme, would be expensive to synthesize and are
unlikely to penetrate the erythrocyte and parasite cell membranes – so ruling them out as
potential malaria therapeutics.
We have been interested in extending inhibitors 1 and 2 to compounds that are easier to
synthesize and with selectivity for the parasite over the human enzyme and were
intrigued by a report of some acyclic nucleoside phosphonates (such as 3) which were
2
2
moderate inhibitors of Pf HGXPRT with some selectivity over the human enzyme. We
2

envisaged that acyclic nucleoside phosphonates such as 4 with a nitrogen to mimic the
ribooxacarbenium ion with a methylene bridge to a 9-deazapurine might capture some
transition state features. After this work was complete, some nitrogen-containing acyclic
2
3
nucleoside phosphonates were described (e.g 5) which offered some improvements but
failed to capture the full benefits of the nitrogen. Here we present the synthesis and
enzyme inhibition properties of a number of acyclic aza-C-nucleoside phosphonates and
phosphates; some of which are potent and selective inhibitors of Pf HGXPRT. A
2
4
preliminary account of the biology of some of these compounds has been reported.
Results and Discussion.
Target Selection and Synthesis.
We decided to prepare a series of linear aminophosphonates and to this end the
commercially available bromoalkyl phthalamides 6 were treated with triethyl phosphite
2
5
26
and then hydrazine hydrate to give the known aminophosphonates 7. Coupling of
2
7
aldehyde 8 with these aminophosphonates and reduction of the resulting imines
afforded 9 – 13 (Scheme 2). Acidic hydrolysis of the O-benzyl group in the deazapurines
provided 14 – 18 and then dealkylation of the phosphonate ester groups gave the target
aza-C-nucleoside mimics 4, 19 – 22. In a similar manner the 8-azapurine analogue 24
was also prepared from 23. While it was likely that the positioning of the nitrogen in
3

compounds 4, 19 – 22 was optimal for mimicking the positive charge of a
ribooxacarbenium ion transition state, we decided to explore the effect of moving the
2
8
nitrogen along the chain. To this end, treatment of 25 with dimethylamine and
formaldehyde in a Mannich reaction afforded 26 (Scheme 3), which after reaction with
methyl iodide and then basic nitromethane gave 27. Reduction of the nitro group
generated amine 28 which underwent reductive alkylation with phosphonate aldehydes
2
9 to give 30. Deprotection as before gave the targets 31 and 32. It was possible that
substitution of the aminophosphonate alkyl chain with hydroxy or hydroxymethyl groups
2
9
would offer improved properties as ribose mimics and so the known azide ()-33 was
reduced to the aminophosphonate ()-34 (Scheme 4). In addition the monobenzyl ether
3
0
3
5 was converted into bromide
o
o
Scheme 2 (a) triethyl phosphite, 120 C; (b) H NNH , EtOH; (c) NaBH , EtOH; (d) 35% aq HCl, 60 C; (e)
4
2
2
4
o
8% HBr 90 C; (f) 2-picoline borane, MeOH.
4

Scheme 3 (a) HCHO, Me N.HCl, NaOAc; (b) MeI; (c) CH NO , NaOMe; (d) CoCl , NaBH ; (e) (EtO) P,
2
3
2
2
4
3
o
o
o
1
60 C; (f) 1M HCl, reflux; (g) NaBH , EtOH; (h) conc HCl, 60 C; (i) 48% HBr, 90 C.
4
5

o
Scheme 4 (a) H , Pd/C; (b) i, NaH, TBDMSCl, ii, Ph P, CBr ; (c) (EtO) P, 175 C; (d) MeOH/35% aq
2
3
4
3
o
o
HCl; (e) MsCl, Et N; (f) NaN , DMF, 80 C; (g) 2-picoline borane, 8, EtOH; (h) i, 35% aq HCl, 70 C, ii,
3
3
o
4
8% HBr, 90 C.
()-36 and then the phosphonate ()-37, from which the aminophosphonate ()-41 was
derived. Reductive alkylation of aminophosphonates ()-34 and ()-41 with 8 gave, after
deprotection, the targets ()-44 and ()-45.
We have observed when designing inhibitors of nucleoside phosphorylases and
1
4, 31-32
hydrolases that serinol can be a good mimic of a ribooxacarbenium ion.
Consequently we wanted to make serinol phosphonate analogues. For these compounds
we chose to derivatise 9-deaza-2,6-dichloropurine (46) via 47 and 48 into aldehyde 49
(
Scheme 5). This was done with the intention that both 9-deazahypoxanthine and 9-
deazaguanine derivatives would be synthesized from 49 or its derivatives. Unfortunately
attempted displacements of the Cl by NH were unsuccessful for those compounds (data
3
3
3
not given). The D-serine-derived vinyl phosphonate 50 was reduced and deprotected to
6

t
Scheme 5 (a) KO Bu, THF; (b) K CO , HCHO, aq dioxane; (c) Dess-Martin periodinane, Et N, THF; (d)
2
3
3
H , Pd/C; (e) aq HCl; (f) 49, 2-picoline borane, MeOH; (g) 8, 2-picoline borane, MeOH; (h) 48% HBr, 80
2
o
.
3
i
i
C; (i) NaH, MeI; (j) BuLi, BF OEt ; (k) i, PrOCON COO Pr, (PhO) P(O)N , Ph P; ii, LiAlH .
2
2
2
3
3
4
7

o
Scheme 6 (a) 2-picoline borane, Et N, MeOH; (b) 48% HBr 80 C.
3
aminophosphonate 51 which was coupled with aldehyde 49 to give 52. This was
hydrogenolyzed and further deprotected to the target 53. The L-serine-derived
phosphonate 54 and the fluorophosphonate 58 were processed in the same way to the
3
4-35
enantiomer 57 and the monofluoro compound 61, while the difluorophosphonate 62
similarly afforded 65. We wanted to be sure that partial racemization had not occurred
during reduction of the double bond in 50 and to this end we prepared 53 by an
alternative method. Treatment of di-tert-butylphosphite with sodium hydride and methyl
iodide gave 66, and after deprotonation of 66 the anion was condensed with epoxide 67 to
give 68. This was converted to the amine 69 under conditions for inversion of
configuration and the amine was converted into 53 as before. Both preparations of 53 had
3
6
the same [] value. Additionally the 9-deazaguanine derivative 70 was coupled to
D
difluorophosphonate 63 (Scheme 6) to afford, after deprotection, the alternative
deazaguanine-containing inhibitor 72.
8

t
Scheme 7 (a) ethanolamine, 2-picoline borane; (b) i, Boc O, ii, (BnO) PN(iPr) , tetrazole, iii, BuOOH; (c)
2
2
2
H , Pd/C; (d) 80% aq TFA; (e) P(OMe) , I , py; (f) i, TmsBr, ii, piperidine; (g) NaBH CN, 8, Et N, MeOH
2
3
2
3
3
o
i
5
0 C; (h) 49, 2-picoline borane, Et N, MeOH; (i) i, (BnO) PN Pr , tetrazole, ii, MCPBA.
3 2 2
9

Scheme 8 (a) i, (BnO) PN(iPr) , ii, MCPBA, iii, H , Pd/C, iv, aq TFA; (b) i, 2-picoline borane, 8, ii, H ,
2
2
2
2
Pd/C; (c) i, 2-picoline borane, 70, ii, 6M aq HCl.
We decided to make the corresponding phosphate esters of some of the phosphonates
described above to compare the relative inhibitory properties. Reductive amination of
aldehyde 8 with ethanolamine afforded 73 (Scheme 7), which was phosphorylated to 74
3
7
and deprotected to phosphate ester 75. Treatment of the Fmoc-protected serinol 76 with
3
8
a mixture of iodine and trimethylphosphite afforded the monophosphate ()-77 in
moderate yield which was deprotected to the serinol phosphate ()-78. Reductive
amination of aldehyde 8 with amine ()-78 gave ()-79 which was hydrogenolyzed to the
serinol target ()-80. In addition, use of aldehyde 49 in the reductive coupling step
followed by acid hydrolysis gave the 2-chloropurine derivative ()-81. The Tris
3
9
derivative 82 was also phosphorylated and deprotected to the aminoalkyl phosphate 83
which was treated as described for 78 to give 84. The chiral serinol phosphate esters were
4
0-41
also prepared; phosphorylation of serinol derivative 85
and deprotection gave
aminoalcohol phosphate 86 which was coupled with the appropriate 9-carbaldehyde-9-
deazapurine derivatives (8 and 70) under reductive conditions to afford, after
deprotection, targets 87 and 88. The enantiomers 89 and 90 were also prepared from the
4
1
corresponding enantiomer of 86 in the same way.
4
2
The aminoalcohol 91 (Scheme 9) can be viewed as a chain extended serinol derivative.
It was converted using standard procedures into carbamate 92 and then phosphate 93, the
diol 94 and finally the chain-extended target 95.
1
0

Scheme 9 (a) TBDPSCl, imidazole; (b) BnOCOCl, NaHCO ; (c) HOAc/H O/THF; (d) BzCl, py; (e)
3
2
Me CO, Me C(OMe) , TsOH; (f) NaOMe, MeOH; (g) (BnO) PNiPr , tetrazole, then MCPBA; (h) Bu NF,
2
2
2
2
2
4
THF; (i) TFA/THF/H O; (j) H , Pd black; (k) NaBH CN, 8, Et N, MeOH.
2
2
3
3
Biological Results.
The compounds were assayed as inhibitors of PfHGXPRT using spectrophotometric
methods to observe the conversion of either xanthine or guanine to xanthosine-5’-
monophosphate or guanosine-5’-monophosphate in the presence of 5-phospho--D-
ribose-1-pyrophosphate. Analysis of the results from the simple phosphonates with
different chain lengths (4, 19 – 22) showed that 4 is a potent inhibitor and with excellent
selectivity for the parasite over the human enzyme. That 4 is the most potent is consistent
with the fact that there are the same number of bonds between the phosphonate and the
deazapurine in 4 as in immucillin-H phosphate 1. Moving the nitrogen along the chain (in
3
1) was not beneficial and neither was incorporating the 8-azapurine (in 24). Adding the
hydroxyl group to the phosphonate alkyl chain (in 44) may have conferred a small benefit
in that 44 was made as the racemate and so one of the enantiomers may be better.
However, it is a small benefit compared to the extra costs of introducing the chirality.
Interestingly the selectivity margin of 44 was even better than for 4. Adding a
hydroxymethyl group (in 45) was not beneficial for inhibitor potency, however the
serinol phosphonate 53 proved to be an exceptionally potent inhibitor, showing again that
serinol can act as an effective mimic for the ribooxacarbenium ion while the selectivity
1
1

a
Table 1
Activity of synthesized acyclic aza-C-nucleoside phosph(on)ates as inhibitors of Pf and
human (Hs) HGXPRT.
Pf HGXPRT
Hs HGPRT
Pf HGXPRT Hs HGPRT
K (nM)
i
K (nM)
i
K (nM)
i
K (nM)
i
1
2
4
2
2
2
3
3
4
4
5
5
9 > 20,000
0 1,900 ± 100
> 10,000
61 1.2 ± 0.1
65 87 ± 8
420 ± 10
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
4,900 ± 300
> 10,000
> 10,000
> 10,000
> 10,000
24
24
10.6 ± 1.3
72 100 ± 11
75 14,400
80 19 ± 2
1 490 ± 35
2 8,600 ± 900
4 27.6 ± 1.9
1 620 ± 61
2 > 20,000
4 9.1 ± 0.1
5 490 ± 60
81 9,400
84 15,300
87 2.4 ± 0.2
88 14.3 ± 1.7
90 650 ± 150
95 980 ± 70
11,320 ± 1,300
> 10,000
> 10,000
2
4
24
3 0.65 ± 0.04
7 23.4 ± 2.3
380 ± 40
16,000 ± 1,000
a
The K values were determined from analyses assuming competitive inhibition as in ref.
i
2
4.
for Pf over Hs enzymes was a factor of >500 for this compound. The enantiomer 57 was
30 times less active. Use of -fluorophosphonates and --difluorophosphonates has
>
been proposed to change the pKa of the phosphonates to make them better mimics of
phosphates, but in this situation the corresponding compounds 61 and 65 were less potent.
Use of a deazaguanine moiety did not confer an advantage either, as in 72. Making the
phosphate analogue 75 of phosphonate 4 resulted in a surprising loss in activity with the
phosphonate clearly being superior in activity. The phosphate analogues of the serinol
phosphonates showed a similar but less dramatic trend with all being less potent than the
corresponding phosphonates.
Conclusions.
1
2

We have synthesized and tested a range of acyclic aza-C-nucleoside phosph(on)ates and
we discovered some relatively simple compounds that are potent and selective inhibitors
of the Pf HGXPRT enzyme. These compounds offer promise as potential therapeutics for
malaria by blocking purine salvage. The next step will be to devise a membrane
permeable prodrug approach that will deliver drug through the erythrocyte and into the
2
4
parasite. We have described some preliminary experiments to this end and further work
will be published in due course.
Experimental.
General Methods.
Chromatography solvents are distilled prior to use. Anhydrous solvents are those
commercially available. Organic solutions are dried over anhydrous MgSO and
4
evaporated under reduced pressure. Air sensitive reactions are performed under Ar.
Analytical TLC is performed on Merck pre-coated silica gel 60 F254, detection by UV
absorption and/or by heating after dipping in a solution of (NH ) Mo O ·4H O (5 wt%)
4
6
7
24
2
and Ce(SO ) ·4 H O (0.1 wt%) in 5% aq. H SO . Flash column chromatography is
4
2
2
2
4
performed on silica gel (40-63 μm) or on an automated system with continuous gradient
1
3
31
19
1
facility. C-, P- and F-NMR spectra are H decoupled, chemical shifts are in ppm and
1
coupling constants in Hz if not already stated. H NMR in CDCl (internal TMS, δ 0),
3
1
3
CD OD (internal TMS, δ 0), DMSOd (internal TMS, δ 0) or D O (internal HOD) C
3
6
2
3
1
NMR in CDCl (centre line of CDCl ), DMSOd (centre line of DMSOd ) or D O, P
3
3
6
6
2
19
NMR in CDCl or D O (external H PO , δ 0), F NMR in CDCl or D O (external CHF ,
3
2
3
4
3
2
3
1
13
1
1
1
13
δ 0). Assignments of H and C resonances are based on 2D ( H- H DQF-COSY, H- C
HSQC, HMBC) and DEPT experiments. High resolution positive and negative
electrospray mass spectra (ESI-HRMS) are recorded on a Q-TOF Premier tandem mass
spectrometer. Melting points are uncorrected. Microanalyses are performed by the
Campbell Microanalytical Laboratory, University of Otago, New Zealand.
Diethyl {[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}(methyl)amino]methyl}phosphonate (14). Diethyl aminomethylphosphonate (7 n=1,
1
3

2
7
9
8.5 mg, 1.5 eq.) and the aldehyde 8 (100 mg, 1 eq.) are heated to 60 °C in EtOH (6
mL) for 30 min until dissolved. The solution is then cooled to room temperature, NaBH4
29.5 mg, 2eq) is added and the mixture is stirred for 30 min. The mixture is evaporated
(
onto silica gel and flash chromatography (9/1/0.1 v/v/v DCM/MeOH/conc. NH ), gives
3
diethyl [({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-
yl]methyl}amino)methyl]phosphonate (9), (76 mg). This product is heated with conc.
HCl (0.5 mL) at 60 °C for 1 h and the mixture is evaporated in vacuo twice from water
and purified by flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc. NH3)
1
to give 14, (45 mg, 36%). H NMR (500 MHz, MeOD) δ 7.93 (s, 1H), 7.51 (s, 1H),
1
3
4
1
.20-4.14 (m, 4H), 3.26-3.35 (m, 4H), 3.34 (m, 6H). C NMR (125MHz, MeOD) δ155.8,
3
1
45.0,143.3, 129.8, 119.5, 111.8, 64.4, 44.5, 44.4, 43.5, 42.3, 16.7. P NMR (202 MHz,
+
MeOD) δ 22.5. ESI-HRMS for C H N O NaP [M+Na] calcd 337.1042; found
1
2
19
4
4
3
37.1039.
Diethyl {2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]ethyl}phosphonate (15). Using the procedure above for the
1
preparation of compound 14, compound 7, (n=2) is converted to title compound 15. H
NMR (500 MHz, DMSO-d ) δ 7.77 (s,1H), 7.27 (s, 1H), 3.95 (m, 4H), 3.76 (s, 2H), 2.72
6
1
3
(
m, 2H), 2.50 (s, 2H),1.92 (m, 2H), 1.19 (m, 6H). C NMR (125MHz, MeOD) δ153.6,
3
1
1
43.0, 141.3, 125.8, 117.6, 115.3, 60.8, 42.1, 26.0, 24.9, 16.2. P NMR (202 MHz,
+
DMSO-d ) δ30.3. ESI-HRMS for C H N O NaP [M+Na] calcd 351.1198; found
6
13 21
4
4
3
51.1194.
Diethyl {2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]propyl}phosphonate (16). Using the procedure above for the
1
preparation of compound 14, compound 7, (n=3) is converted to title compound 16. H
NMR (500 MHz, D O) δ 8.79 (s,1H), 7.70 (s,1H), 4.29 (s, 2H), 3.98-3.92 (m, 4H), 3.07-
2
1
3
3
1
.04 (m,2H), 1.86-183 (m, 2H), 1.16-1.10 (m,6H). C NMR (125MHz, D O) δ 152.7,
2
3
1
44.8, 133.0, 132.6, 118.2, 103.3, 63.6, 46.7, 40.4, 21.8, 20.7, 18.9, 15.6. P NMR (202
+
MHz, D O) δ 33.4. ESI-HRMS for C H N O P [M+H] calcd 343.1535; found
2
14 24
4
4
3
43.1528.
1
4

Diethyl {2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]butyl}phosphonate (17). Using the procedure above for the
preparation of compound 14, compound 7, (n=4) is converted into first diethyl [4-({[4-
1
(
benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino) butyl]phosphonate (12). H
NMR (500 MHz, CDCl ) δ 8.46 (s, 1H), 7.72 (s, 1H), 7.44-7.27 (bm, 5H), 5.53 (s, 2H),
3
4
6
1
.19 (s, 2H), 4.03-3.98 (m, 4H), 2.80-2.77 (m, 2H), 1.72-1.51 (bm, 6H), 1.30-1.23 (m,
1
3
H). C NMR (125MHz, CDCl ) δ 149.7, 139.4, 130.5, 129.3, 128.5, 128.4, 128.2,
3
3
1
28.1, 127.7, 115.0, 108.5, 67.7, 61.6, 47.1, 41.5, 27.8, 27.7, 25.6, 24.5, 20.9, 16.4. P
+
NMR (202 MHz, CDCl ) δ 31.6. ESI-HRMS for C H N O P [M+H] calcd 447.2161;
3
22 32
4
4
1
found 447.2153. This is then converted to title compound 17. H NMR (500 MHz,
DMSO-d ) δ 8.82 (s, 1H), 7.78 (s, 1H), 4.36 (s, 2H), 4.01-4.07 (m, 4H), 3.10-3.08 (m,
6
2
H), 1.91-1.85 (m, 2H), 1.81-1.75 (m, 2H), 1.65-1.54 (m, 2H), 1.26-1.20 (m, 6H).
Diethyl {2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]pentyl}phosphonate (18). Using the procedure above for the
preparation of compound 14, compound 7, (n=5) is converted first into diethyl [5-({[4-
(
benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino) pentyl]phosphonate (13).
1
H NMR (500 MHz, CDCl ) δ 8.53 (s, 1H), 7.33 (s, 1H), 7.47-7.28 (bm, 5H), 5.57(s, 2H),
3
4
.09-4.00 (m, 4H), 4.01(s, 1H), 1/70-1.51 (bm, 6H), 1.39-1.35(m, 2H), 1.32-1.27 (m, 6H).
1
3
C NMR (125MHz, CDCl ) δ 155.4, 149.5, 149.1, 136.3, 128.5, 128.4, 128.3, 128.0,
3
3
1
1
27.7, 115.3, 113.8, 67.8, 61.4, 48.8, 43.2, 29.0, 28.2, 26.0, 25.0, 22.2, 16.4. P NMR
+
(
202 MHz, CDCl ) δ 32.2. ESI-HRMS for C H N O P [M+H] calcd 461.2318; found
3
23 34
4
4
1
4
61.2311. This is then converted to title compound 18. H NMR (300 MHz, D O) δ 8.53
2
(
s, 1H), 7.68 (s,1H), 4.36 (s, 2H), 4.11-3.97(m, 4H), 3.09-3,01 (m, 2H), 1.90-1.40 (bm,
H), 1.28-1.22 (m, 6H).
8
{[({4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]methyl}phosphonic
acid (19). The diethyl phosphonate (14, 43mg) is heated with 48% HBr for 5 h at 90 °C
and the solution is evaporated in vacuo twice from water. Reverse phase (C )
18
chromatography eluting with water gives title compound (19, 40 mg) as a white water
1
insoluble solid. H NMR (500 MHz, DMSO-d ) δ 8.75 (s, 1H), 7.73 (s, 1H), 3.39 (s, 2H),
6
1
3
3
.36 (s, 2H). C (125 MHz, DMSO-d ) δ 152.3, 144.0, 137.2, 130.7, 117.7, 104.3, 42.2,
6
1
5

3
1
+
4
2
1.0. P (202 MHz, DMSO-d ) δ 11.7. ESI-HRMS for C H N O P [M+H] calcd
8 12 4 4
6
59.0596; found 259.0599.
{[({4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]ethyl}phosphonic
acid (20). Using the procedure for the preparation of compound 19, compound 15 is
1
converted to title compound 20. H NMR (500 MHz, D O) δ 8.69 (s, 1H), 7.69 (s,1H),
2
1
3
4
1
2
.34 (s,2H), 3.23-3.19 (m, 2H), 2.02-1.95 (m, 2H). C NMR (125MHz, D O) δ 153.2,
2
3
1
44.5, 134.8, 132.3, 118.2, 103.6, 42.0, 40.2, 25.0, 23.9. P NMR (202 MHz, D O) δ
2
-
0.6. ESI-HRMS for C H N O P [M-H] calcd 271.0596; found 271.0594.
9
12
4
4
{[({4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}phosphonic
acid (4). Using the procedure for the preparation of compound 19, compound 16 is
1
converted to title compound 4. H NMR (500 MHz, D O) δ 8.68 (s, 1H), 7.69 (s, 1H),
2
1
3
4
.30 (s, 2H), 3.07-3.01 (m, 2H), 1.88-1.80 (m, 2H), 1.74-1.68 (m 2H). C NMR
(
125MHz, D O) δ 152.5, 144.9, 132.8, 131.8, 118.3, 103.0, 47.1, 40.5, 24.1, 23.0, 19.4.
2
3
1
+
P NMR (202 MHz, D O) δ 29.2. ESI-HRMS for C H N O P [M+H] calcd
2
10 16
4
4
2
87.0909; found 287.0904.
{[({4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]butyl}phosphonic
acid (21). Using the procedure for the preparation of compound 19, compound 17 is
1
converted to title compound 21. H NMR (500 MHz, D O) δ 8.93 (s, 1H), 7.75 (s, 1H),
2
1
3
4
.31 (s, 2H), 3.02-3.06 (m, 2H), 1.75-1.67 (m, 4H), 1.55-1.51 (m, 2H). C NMR (125
MHz, D O) δ 152.5, 144.9, 132.8, 131.7, 118.3, 103.0, 46.6, 40.4, 26. 3, 26.2, 25.0, 19.3.
2
3
1
+
P NMR (202 MHz, D O)δ 31.2. ESI-HRMS for C H N O P [M+H] calcd 301.1066;
2
11 18
4
4
found 301.1064.
{[({4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]pentyl}phosphonic
acid (22). Using the procedure for the preparation of compound 19, compound 18 is
1
converted to title compound 22. H NMR (500 MHz, D O) δ 8.93 (s, 1H), 7.76 (s, 1H),
2
4
2
.31 (s, 2H), 3.01-3.05 (m, 2H), 1.71-1.58 (bm, 4H), 1.49-1.42 (m, 2H), 1.38-1.32 (m,
1
3
H). C NMR (125MHz, D O) δ 152.4, 144.9, 132.8, 131.7, 118.3, 103.1, 46.9, 40.3,
2
1
6

3
1
2
6.6, 26.5, 26.3, 25.1, 21.4. P NMR (202 MHz, D O) δ 32.3. ESI-HRMS for
2
+
C H N O P [M+H] calcd 315.1222; found 315.1221.
12
20
4
4
3
-((7-Hydroxy-1H-pyrazolo[4,3-d]pyrimidin-3-yl)methylamino)propylphosphonate
ammonium salt (24). Anhydrous HCl in MeOH (1.0 M, 0.11 mL, 0.11 mmol) is added
dropwise to a solution of the diethyl phosphonate (7, n=3) (100 mg, 0.51 mmol) in
4
3
anhydrous MeOH (2 mL) followed by the addition of the aldehyde 23 (90 mg, 0.34
mmol) and 2-picoline borane complex (50 mg, 0.44 mmol). The resulting suspension is
left to stir for 2 h and the then homogeneous solution absorbed onto silica gel and the
resulting residue subjected to flash chromatography (EtOAc then 5 to 10% v/v MeOH in
CHCl ) to afford diethyl 3-((7-methoxy-2-(tetrahydro-2H-pyran-2-yl)-2H-pyrazolo[4,3-
3
d]pyrimidin-3-yl)methylamino)propylphosphonate (123 mg, 82 % yield) as a white solid,
which is committed to the next step without further characterisation or purification.
Concentrated HCl (1.5 mL, 18 mmol) is added to a solution of this material (90 mg,
0
6
.204 mmol) in MeOH (3 mL, 0.204 mmol) and the resulting mixture heated to reflux for
h. The reaction mixture is concentrated in vacuo and partitioned between chloroform
and water, the water layer is extracted with additional chloroform, the organic layers are
combined, dried and concentrated in vacuo. The residue is dissolved in aqueous
hydrobromic acid (2 mL, 17.68 mmol) and heated for a further 6 h at 80 ºC. The reaction
mixture is concentrated in vacuo and the resulting residue co-distilled with water (2 x 20
mL). Flash chromatography (1:1 v/v dioxane/conc. NH OH) afforded the title compound
4
1
2
4 (35 mg, 60%) as a foam. H NMR (500 MHz, D2O):  = 7.98 (s, 1H), 4.47 (s, 2H),
13
3
.16 (t, J = 6.9 Hz, 2H), 1.91 (septet, J = 7.0 Hz, 2H), 1.56 (m, 2H). C NMR (125 MHz,
D O):  = 155.2, 148.4, 137.5, 135.6, 128.6, 66.6, 48.7, 48.6 (d, J = 13.4 Hz), 40.8, 26.1
2
3
1
(
d, J = 132 Hz), 25.6, 20.7(d, J = 3.8 Hz). P (202 MHz, D O):  = 21.9.
2
{[4-(Benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}dimethylamine (26). The
2
8
deazapurine 25 (178 mg,1 eq.) in dioxane (1 mL) and water (1 mL) with NaOAc (64.6
mg, 1 eq.), dimethylamine hydrochloride (64.6 mg,1 eq.) and formaldehyde (65.5 μL, 1.1
eq., 37% aq) is heated at 95 °C for 4 h and then evaporated under reduced pressure. The
residue is evaporated twice from ammonia in MeOH onto silica gel. Flash
1
chromatography (DCM/MeOH-NH 9:1 v/v) gives title compound 26 (163 mg). H NMR
3
1
7

(500 MHz, MeOD) δ 8.42 (s, 1H), 7.54 (s, 1H), 7.53-7.31 (m, 5H), 5.68 (s, 2H), 3.75 (s,
1
3
2
1
2
H), 2.28 (s, 6H). C NMR (125 MHz, MeOD) δ 157.2, 150.3, 137.8, 131.8, 129.4,
+
16.4, 112.1, 69.1, 52.4, 44.8. ESI-HRMS for C H N O [M+H] calcd 283.1559; found
1
5
19
4
83.1560.
4
2
-(Benzyloxy)-7-(2-nitroethyl)-5H-pyrrolo[3,2-d]pyrimidine (27). The dimethylamine
6 (548 mg,1 eq.) in tetrahydrofuran (25 mL) is treated with methyl iodide (1.2 mL, 10
eq.) at room temperature for 18 h and then the solution is evaporated under high vacuum
to give an orange foam of the quaternary ammonium salt of 26. Nitromethane (3.34 mL,
3
0 eq.) is added to methanol (32 mL) and methanolic sodium methoxide (7 mL, 30%, 20
eq.) and the mixture is stirred under argon for 10 min and then the crude quaternary
ammonium salt is added. The mixture is stirred for 2 h and then the methanol is
evaporated. The residue is diluted with water, the pH adjusted to 3 with 1 N HCl and
extracted 3x with ethyl acetate. The organic extract is washed with brine, dried and
evaporated. Flash chromatography (20-50% v/v EtOAc in hexanes) gives title compound
1
2
7
1
7 (310 mg, 53%). H NMR (500 MHz, DMSO-d ) δ 8.44 (s, 1H), 7.54 (d, 1H), 7.42-
6
1
3
.33 (m, 5H), 5.61 (s, 2H), 4.93 (m, 2H), 3.37 (m, 2H). C NMR (125MHz, DMSO-d ) δ
6
54.9, 148.7, 136.6, 128.8, 128.4, 114.4, 109.7, 75.0, 67.0, 22.1. ESI-HRMS for
+
C H N O Na [M+Na] calcd 321.0964; found 321.0970.
15
14
4
3
2
-[4-(Benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl] ethan-1-amine (28). Sodium
borohydride (393 mg, 10 eq.) is added in small portions to 27 (310 mg 1 eq.) in methanol
20 mL) with CoCl .6H O (495 mg, 2 eq.) at 0 °C and then the mixture is stirred at 0 °C
(
2
2
for 30 min. The product is evaporated onto silica gel and flash chromatography (9/1/0.1
1
and 8/2/0.2 v/v/v DCM/MeOH/conc. NH ) gives title compound 28 (195 mg, 70%). H
3
NMR (500 MHz, MeOD) δ 8.4 (s, 1H), 7.36 (s, 1H), 7.53-7.30 (m, 5H), 5.62 (s, 2H),
1
3
2
1
2
.98-2.92 (m, 4H). C NMR (125MHz, MeOD) δ 157.1, 149.8, 137.9, 129,3,129.6,128.3,
+
16.8, 114.2, 69.1, 43.0, 28.1. ESI-HRMS for C H N O [M+H] calcd 269.1402; found
1
5
17
4
69.1404.
Diethyl (2-oxoethyl)phosphonate (29, n=1) and diethyl (3-oxopropyl)phosphonate
29, n=2). Triethylphosphite (8.4 mL) and 2-(2-bromoethyl)-1,3-dioxolane (10 g) are
(
1
8

heated at 160 °C for 18 h and then evaporated under high vacuum at 100 °C . The residue
is refluxed with 1 N HCl under argon for 15 min and cooled to room temperature. The
product is salted out with solid NaCl and extracted with DCM, washed with saturated
NaHCO , brine, dried and concentrated. Flash chromatography (EtOAc-MeOH 95:5 v/v)
3
gives title compound 29, n=2 (2.5 g). The NMR spectral data was identical with literature
4
4
values. The same method applied to 2-(bromomethyl)-1,3-dioxolane afforded title
4
5
compound 29, n=1 with the same NMR data as reported.
Diethyl [2-({2-[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-
yl]ethyl}amino)ethyl]phosphonate (30, n=2). The amine 28 (67 mg, 1.5 eq) and
phosphonoaldehyde 29, n=1 (40 mg, 1 eq.) in ethanol (2 mL) are heated at 70 °C for 30
min and cooled to room temperature. NaBH (17 mg, 2 eq.) is added and the mixture
4
stirred for 30 min. The mixture is evaporated onto silica gel and subjected to flash
chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc.NH ) to give title
3
1
compound 30, n=2 (30 mg). H NMR (500 MHz, CDCl ) δ 8.48 (s, 1H), 7.48-7.27 (m,
3
5
1
1
2
H), 7.19 (s, 1H), 5.55 (s, 2H), 4.07-4.01 (m, 4H), 3.04-2.95 (m, 6H), 2.08-2.02 (m, 2H),
1
3
.28-1.25 (m, 6H). C NMR (125 MHz, CDCl ) 155.3, 149.1, 148.8, 136.3, 128.4, 127.4,
3
3
1
15.4, 113.7, 67.9, 61.8, 49.1, 42.8, 26.1, 25.0, 24.0, 16.4. P NMR (202 MHz, CDCl )
3
+
9.2. ESI-HRMS for C H N O P [M+H] calcd 433.2005; found 433.1999.
2
1
30
4
4
Diethyl [3-({2-[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-
yl]ethyl}amino)propyl]phosphonate (30, n=3). The amine 28 (60 mg, 1.5 eq.) and
phosphonoaldehyde 29, n=2 (36 mg, 1 eq.) in ethanol (2 mL) are heated at 70 °C for 30
min and cooled to room temperature. NaBH (15 mg, 2 eq.) is added and the mixture
4
stirred for 30 min. Evaporation onto silica gel and flash chromatography (9/1/0.1 and
)
1
8
/2/0.2 v/v/v DCM/MeOH/conc. NH gives title compound 30, n=3 (50 mg). H NMR
3
(
500 MHz, CDCl ) δ 8.53 (s, 1H), 7.26 (s, 1H), 7.49-7.19 (m, 5H), 5.57 (s, 2H), 4.09-
3
1
3
4
.01 (m, 4H), 2.98 (s, 4H), 2.70 (m, 2H), 1.78-1.71 (m, 4H), 1.31-1.26 (m, 6H). C NMR
(
125 MHz, CDCl ) δ 155.3, 149.3, 136.3, 128.5, 126.8, 115.4, 114.8, 67.9, 61.5, 49.7,
3
31
4
9.6, 24.5, 24.0, 22.8, 22.7, 16.4. P NMR (202 MHz, CDCl ) δ 32.1. ESI-HRMS for
3
+
C H N O P [M+H] calcd 447.2161; found 447.2160.
22
32
4
4
1
9

{
2-[(2-{4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}ethyl)amino]ethyl}phosphonic
acid (31). The diethyl phosphonate 30, n=2 (52 mg) is heated at 60 °C with conc. HCl
0.5 mL) for 1 h, evaporated under reduced pressure twice from water and the heated
(
with 48% HBr (1 mL) at 90 °C for 5 h and evaporated again twice from water. The
product is chromatographed on reverse phase C silica gel eluting with water to give title
1
8
1
compound 31 (39 mg). H NMR (500M Hz, D O) δ 8.78 (s, 1H), 7.50 (s, 1H), 3.29-3.24
2
1
3
(
m, 2H), 3.22-3.18 (m, 2H), 3.05-3.02 (m, 2H), 2.04-1.97 (m, 2H). C NMR (125MHz ,
3
1
D O) δ 152.8, 144.0, 132.2, 130.3, 117.9, 108.0, 47.0, 42.9, 24.9, 23.8, 20.3. P NMR
2
+
(
202 MHz, D O) δ 21.3. ESI-HRMS for C H N O P [M+H] calcd 287.0909; found
2
10 16
4
4
2
87.0912.
{
3-[(2-{4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}ethyl)amino]propyl}phosphonic
acid (32). The diethyl phosphonate 30, n=3 (50 mg) is heated at 60 °C with conc. HCl
0.5 mL) for 1 h and then evaporated under reduced pressure twice from water. The
(
residue is then heated with 48% aq. HBr (1 mL) at 90 °C for 5 h and evaporated again
twice from water. The product is chromatographed on reverse phase C silica gel eluting
1
8
1
with water to give title compound 32 (30mg). H NMR (500 MHz, D O) δ 8.76 (s, 1H),
2
7
.49 (s, 1H), 3.26-3.17 (m, 2H), 3.07-2.98 (m, 4H), 1.88-1.79 (m, 2H), 1.74-1.67 (m, 2H).
1
3
C NMR (125MHz , D O) δ 152.9, 144.0, 132.4, 130.2, 117.9, 108.2, 47.9, 47.2, 24.3,
2
3
1
+
2
3.2, 20.3, 19.5. P NMR (202 MHz, D O) δ 28.3. ESI-HRMS for C H N O P [M+H]
2 11 18 4 4
calcd 301.1066; found 301.1062.
Diethyl (3-amino-2-hydroxypropyl)phosphonate (34). Diethyl (3-azido-2-
2
9
hydroxypropyl)phosphonate (33) (620 mg) is reduced with 10% palladium on charcoal
(
250 mg) in ethanol (10 mL) under hydrogen at 1 atm. for 1 h, filtered and concentrated.
)
Flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc. NH gives title
3
1
compound 34 (388 mg). H NMR (500 MHz, CDCl ) δ 4.18-4.09 (m, 4H), 3,98-3.92 (m,
3
1
3
1
H), 2.88-2.68 (m, 2H), 2.15-1.95 (bm, 3H),1.98-1.89 (m, 2H), 1.35-1.33 (m, 6H). C
3
1
NMR (125 MHz, CDCl ) δ 67.4, 61.9, 48.1, 31.6, 31.0, 16.4. P NMR (202
3
+
MHZ,CDCl )δ 30.0. ESI-HRMS for C H NO P [M+H] calcd 212.1052; found
3
7
19
4
2
12.1049.
2
0

[3-(Benzyloxy)-2-(bromomethyl)propoxy](tert-butyl)dimethylsilane (36). A solution
3
0
of 35 (1.04 g,1 eq.) in dry THF (3 mL) is added to NaH (60%, 127 mg, 5.3 eq.) in dry
THF (12 mL) with ice cooling. The mixture is stirred at room temperature for 30 min and
t
then BuMe SiCl (800 mg, 5.3 eq.) in dry THF (2 mL) is added. After 2 h, water (6 mL)
2
is added and the product extracted with EtOAc, washed with brine, dried and evaporated.
Flash chromatography (hexanes-EtOAc 9:1 v/v) gives 2-[(benzyloxy)methyl]-3-[(tert-
1
butyldimethylsilyl)oxy]propan-1-ol (1.25 g). H NMR (500 MHz, CDCl ) δ 7.37-7.30 (m,
3
5
0
4
3
H), 4.51 (s, 2H), 3.80-3.71 (m, 4H), 3.64-3.53 (m, 2H), 2.07-2.01 (m, 1H), 0.86 (s, 9H),
1
3
.06 (s, 6H). C NMR (125MHz,CDCl ) δ 138.0, 128.4, 127.7, 73.2, 70.0, 64.5, 63.4,
3
+
3.0, 25.9, 18.2, 0.0. ESI-HRMS for C H O NaSi [M+Na] calcd 333.1826; found
1
7
30
3
33.1858. To this material (1.25 g, 1 eq.) in dry DCM (2 mL) and dry pyridine (0.35 mL)
under argon is added CBr (2 g, 1.5 eq.) followed by the dropwise addition of
4
triphenylphosphine (1.07 g, 1.02 eq.) in dry DCM (2 mL). After 2.5 h, ice-cold hexanes
(
25 mL) are added and the precipitate filtered off. Flash chromatography (hexanes and
then hexane with 5% v/v EtOAc) of the concentrated filtrate gives title compound 36
1
(
788 mg). H NMR (500 MHz, CDCl ) δ 7.32-7.21 (m, 5H), 4.45 (s, 2H), 3.67-3.59 (m,
3
1
3
2
H), 3.55-3.41 (m, 2H), 2.16-2.09 (m, 1H), 0.83 (s, 9H), 0.00 (s, 6H). C NMR (125
MHz,CDCl ) δ 138.3, 128.4, 127.6, 73.4, 69.1, 61.7, 43.8, 33.0, 25.9, 18.2, 0. ESI-HRMS
3
+
for C H O NaSi79Br [M+Na] calcd 395.1018; found 395.1020.
1
7
29
2
Diethyl {2-[(benzyloxy)methyl]-3-[(tert-butyldimethylsilyl)oxy] propyl}phosphonate
37). Compound 36 (2.46 g, 1 eq.) and triethylphosphite (3.5 mL, 3 eq.) are heated at
75 °C for 18 h. Concentration of the product under high vacuum at 100 °C and flash
(
1
chromatography (hexanes-EtOAc 20%-100% v/v) of the residue gives title compound 37
1
(
2.07g). H NMR (500 MHz, CDCl ) δ 7.29-7.22 (m, 5H), 4.45 (s, 2H), 4.04-4.03 (4H),
3
3
6
7
.66-3.62 (m,2H), 3.52-3.50 (m,2H), 2.20-2.12 (m,1H), 1.87-1.72 (m, 2H), 1.28-1.24 (m,
1
3
H), 0.84 (s, 9H), 0.0 (s, 6H). C NMR (125 MHz,CDCl ) δ 138.6, 128.3, 127.5, 73.0,
3
3
1
0.2, 63.0, 61.4, 36.6, 25.9, 24.3, 23.1, 18.3, 16.4, 0.0. P NMR (202 MHZ, CDCl )
3
+
δ.32.0. ESI-HRMS for C H O NaSiP [M+Na] calcd 453.2202; found 453.2205.
2
1
39
5
Diethyl [2-(azidomethyl)-3-(benzyloxy)propyl]phosphonate (40). Compound 37 (1.87
g) in MeOH (10 mL) and conc. HCl (10 mL) is stirred at room temperature for 1 h.
2
1

Evaporation at reduced pressure gives crude diethyl {2-[(benzyloxy)methyl]-3-
hydroxypropyl}phosphonate (38) (1.45 g) which is dissolved in DCM (30 mL) and
triethylamine (1.9 mL, 3 eq.) and cooled to 0 °C. Methanesulfonyl chloride (0.72 mL, 2
eq.) is added and the mixture stirred at room temperature for 1 h, diluted with DCM and
the organic extract washed with satd. NaHCO brine, dried and evaporated to give crude
3
,
mesylate 39. Displacement of the mesylate is effected by heating a solution of crude 39
1.93 g) in DMF (20 mL) with NaN (0.9 g) at 80 °C for 7 h. Addition of water and
(
3
extraction with diethyl ether followed by flash chromatography (50%-100% v/v EtOAc
1
in hexanes) gives title compound 40 (1.06 g). H NMR (500 MHz, CDCl ) δ 7.38-7.27
3
(
m, 5H), 4.53 (s, 2H), 4.14-4.03 (m, 4H), 3.58-3.47 (m, 4H), 2.34-2.25 (m, 1H), 1.88-
1
3
1
.76 (m, 2H), 1.35-1.27 (m, 6H). C NMR (125 MHz, CDCl ) δ 138.0, 129.6, 128.3,
3
+
7
3.2, 70.2, 61.6, 52.6, 34.4, 25.4, 24.3, 16.4. ESI-HRMS for C H N O NaP [M+Na]
15
24
3
4
calcd 364.1402; found 364 1393.
Diethyl [2-(aminomethyl)-3-hydroxypropyl]phosphonate (41). Compound 40 (1.06 g)
in MeOH (10 mL) with 20% Pd(OH) /C (0.5 g) is hydrogenated at 1 atm for 18 h.
2
Filtration and flash chromatography (9/1/0.1 and 8/2/0.2 v/v DCM/MeOH/conc. NH3)
1
gives title compound 41 (300 mg). H NMR (500 MHz, CDCl ) δ 4.22-4.05 (m, 4H),
3
3
.83-3.71 (m, 2H), 3.02-2.81 (m, 2H), 2.25-2.15 (m, 1H), 2.10-1.98 (m, 1H), 1.86-1.71
1
3
(
m, 2H), 1.38-1.31 (m, 6H). C NMR (125 MHz, CDCl ) δ 67.4, 61.7, 46.2, 37.1, 26.2,
3
3
1
+
2
5.1, 16.5. P NMR (202 MHz, CDCl ) δ.31.8. ESI-HRMS for C H NO P [M+H]
3 8 21 4
calcd 226.1208; found 226.1206.
Diethyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)-2-
hydroxypropyl]phosphonate (42). The aminophosphonate 34 (95 mg,1.5 eq.) and
aldehyde 8 (80 mg, 1 eq.) in ethanol (3 mL) are heated at 70 °C for 10 min and then
picoline borane (64 mg, 2 eq.) is added and the mixture stirred for 3.5 h. The product is
evaporated onto silica gel and subjected to flash chromatography (9/1/0.1 and 8/2/0.2
1
v/v/v DCM/MeOH/conc. NH ) to give title compound 42 (20 mg). H NMR (500
3
MHz,CDCl ) δ 8.53 (s, 1H), 7.49-7.35 (m, 5H), 7.26 (s, 1H), 5.58 (s, 2H), 4.16-3.96 (m,
3
1
3
6
H), 2.83-2.64 (m, 2H), 2.02-1.87 (m, 2H), 1.34-1.30(m, 6H). C NMR (125 MHz,
CDCl )δ 155.4, 149.6, 149.0, 136.2, 128.5, 127.3, 115.4, 115.1, 67.9, 65.0, 61.8, 55.1,
3
2
2

3
1
4
3.0, 32.1, 31.0, 16.4. P NMR (202 MHz, CDCl ) δ.29.9. ESI-HRMS for C H N O P
3 21 30 4 5
+
[
M+H] calcd 449.1954; found 449.1951.
Diethyl {2-[({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-
yl]methyl}amino)methyl]-3-hydroxypropyl}phosphonate (43). The aminophosphonate
1 (100 mg, 1.5 eq.) and aldehyde 8 (80 mg, 1 eq.) are heated together in ethanol (3 mL)
4
at 70 °C for 10 min and then picoline borane (64 mg, 2 eq.) is added and heated at 70 °C
for 2 h. Evaporation and flash chromatography (9/1/0.1v/v/v DCM/MeOH/conc. NH3)
1
gives title compound 43 (99 mg). H NMR (500 MHz, CDCl ) δ 8.55 (s, 1H), 7.49-7.33
3
(
m, 5H), 7.26 (s, 1H), 5.58 (s, 2H), 4.13-3.99 (m, 6H), 3.81-3.63 (m, 2H), 3.01-2.81 (m,
1
3
2
H), 2.26-2.19 (m, 1H), 1.75-1.63 (m, 2H), 1.35-1.24 (m, 6H). C NMR (125MHz,
CDCl ) δ 155.3, 149.8, 149.1, 136.2, 128.5, 127.3, 115.4, 114.3, 67.8, 61.7, 53.7, 43.3,
3
3
1
3
4.6, 26.6, 25.5, 16.4. P NMR (202 MHz, CDCl ) δ 31.3. ESI-HRMS for C H N O P
3 22 32 4 5
+
[
M+H] calcd 463.2110; found 463.2103.
{2-Hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}
phosphonic acid (44). Compound 42 (20 mg) is heated with conc. HCl (0.5 mL) at 70 °C
for 30 min and then evaporated under reduced pressure twice from water. The product is
heated with 48% HBr (1 mL) at 90 °C for 5 h and then evaporated under reduced
pressure twice from water. Reverse phase chromatography on C silica gel eluting with
1
8
1
water gives title compound 44 (7 mg). H NMR (500 MHz, D O) δ 8.89 (s, 1H), 7.44 (s,
2
1
3
1
H), 4.38 (s, 2H), 4.24-4.10 (m, 2H), 3.27-2.99 (m, 2H), 2.04-1.97 (m, 2H). C NMR
(
125 MHz, D O ) δ 152.7, 144.9, 132.9, 132.4, 118.2, 103.3, 62.3, 52.0, 40.5, 33.2, 32.1.
2
3
1
+
P NMR (202 MHz, D O) δ 25.0. ESI-HRMS for C H N O P [M+H] calcd 303.0858;
2
10 16
4
5
found 303.0862.
(3-Hydroxy-2-{[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]methyl}propyl)phosphonic acid (45). Compound 43 (45 mg) is
heated at 70 °C in conc. HCl (0.5 mL) for 1 h and evaporated twice from water. The
crude product is the heated with HBr (48%, 1 mL) at 90 °C for 3 h and then evaporated
twice from water. Chromatography on reverse phase C silica gel eluting with water
1
8
1
gives title compound 45 (23 mg). H NMR (500 MHz, D O) δ 8.19 (s, 1H), 7.58 (s, 1H),
2
2
3

4
.30-4.21 (m, 2H), 3.56-3.37 (m, 2H), 3.15-3.04 (m, 2H), 2.19-2.11 (m, 1H), 1.68-1.46
1
3
(
m, 2H). C NMR (125 MHz, D O) δ 152.6, 144.9, 132.8, 118.4, 103.1, 63.8, 49.8, 40.9,
2
3
1
+
3
3.0, 27.3, 26.2. P NMR (202 MHz, D O) δ.27.5. ESI-HRMS for C H N O P [M+H]
2 11 18 4 5
calcd 317.1015; found 317.1007.
4
-(tert-Butoxy)-2-chloro-5H-pyrrolo[3,2-d]pyrimidine (47). 2,4-Dichloro-5H-
4
6
pyrrolo[3,2-d]pyrimidine (46) (0.5 g, 2.66 mmol) and sublimed potassium tert-butoxide
o
(
1.492 g, 13.30 mmol) are stirred in THF (25 mL) at 40 C for 16 h. The solvent is
evaporated, H O (10 mL) added and the mixture extracted with EtOAc (50 mL), washed
2
with brine, then dried and the solvent evaporated to yield a cream coloured solid. Flash
chromatography (EtOAc-Hexanes, 3:7 v/v) gives title compound 47 (0.33 g, 55%) as a
1
colourless solid. H NMR (500 MHz, CDCl ) 8.58 (bs, exchanged to D O, 1H), 7.37 (t, J
3
2
=
3.1, after D O became d, J = 3.1, 1H), 6.56 (dd, J = 3.1, 2.2, after D O became d, J =
2 2
1
3
3
.1, 1H), 1.70 (s, 9H). C NMR (125.7 MHz, CD OD) 157.3 (C), 152.4 (C), 150.4 (C),
3
1
32.0 (CH), 116.3 (C), 102.0 (CH), 85.0 (C), 28.7 (CH ). Referenced to the centre line of
3
(35)
+
CD OD at δ 49.0. ESI-HRMS for C H
ClN O [M+H] calcd 226.0747; found
3
10 13
3
2
26.0752.
[4-(tert-Butoxy)-2-chloro-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methanol (48). Compound
4
7 (0.330 g, 1.462 mmol) is dissolved in a mixture of 1,4-dioxane (6 mL) and H O (3
2
mL) then potassium carbonate (0.525 g, 3.80 mmol) and formaldehyde solution (37% aq.,
o
1
.53 mL, 19.10 mmol) are added and the mixture heated at 80 C for 19 h. The mixture is
cooled, extracted with EtOAc (50 mL), washed with brine, dried then the solvent
evaporated to a gum which is dissolved in 7M NH -MeOH (10 mL) and left at room
3
temperature for 30 min After evaporation of the solvent the residue is flash
chromatographed (EtOAc-hexanes, 7:3 then EtOAc) to give title compound 48 (0.214 g,
1
5
7%) as a colourless solid. H NMR (500 MHz, CD OD) 7.51 (s, 1H), 4.75 (s, 2H), 1.72
3
1
3
(
s, 9H). C NMR (125.7 MHz, CD OD) 157.3 (C), 150.5(C x 2), 131.0 (CH), 116.8 (C),
3
1
4
16.6 (C), 85.0 (C), 55.2 (CH ), 28.8 (CH ). Referenced to the centre line of CD OD at
2
3
3
(35)
+
9.0 ppm. ESI-HRMS for C H
ClN NaO [M+Na] calcd 278.0672; found 278.0671.
3 2
1
1
14
2
4

4
-(tert-Butoxy)-2-chloro-5H-pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (49). Dess-
Martin periodinane (0.390 g, 0.921 mmol) is added to a mixture of triethylamine (0.47
mL, 3.35 mmol) and alcohol 48 (0.214 g, 0.837 mmol) in THF (6 mL) and the mixture is
stirred at room temperature for 40 min. EtOAc (50 mL), sat.aq. NaHCO (3 mL) and
3
water (3 mL) are added successively and the mixture filtered through Celite. The organic
layer is separated and washed with brine then dried and the solvent evaporated to leave a
solid which is dissolved in a 1:1 v/v mixture of MeOH/CHCl , silica gel added and the
3
solvent evaporated. The residue is flash chromatographed (EtOAc-hexanes,4:6 then 1:1
v/v) to give title compound 49 as a colourless solid which is triturated with a little EtOAc
1
and dried (0.179 g, 84%). A sample is recrystallized from EtOAc. H NMR (500 MHz,
1
3
DMSO d ) 13.08 (bs, 1H), 10.06 (s, 1H), 8.41 (s, 1H), 1.70 (s, 9H). C NMR (125.7
6
MHz, DMSO d ) 183.8 (CH), 156.2 (C), 151.0 (C), 149.6 (C), 137.7 (CH), 116.8 (C),
6
1
15.9 (C), 84.5 (C), 28.2 (CH ). Referenced to the centre line of DMSOd at δ 39.7. ESI-
3 6
(35)
+
HRMS for C H
ClN NaO [M+Na] calcd 276.0516; found 276.0511.
3 2
1
1
12
Diethyl [(3S)-3-amino-4-hydroxybutyl]phosphonate hydrochloride (51). Compound
3
3
5
0 (0.460 g, 1.27 mmol) and 10% Pd-C (50 mg) are stirred in EtOH (20 mL) under a
hydrogen atmosphere at ambient pressure and temperature for 6 h. The mixture is filtered
through Celite and the solvent evaporated. The residue is dissolved in a mixture of EtOH
(
3 mL) and 37% aq. HCl (2 mL) and left at room temperature for 1.5 h then the solvent is
2
2
evaporated to give title compound 51 as a colourless gum (330 mg, 100%). [α] +5.3 (c
D
1
0
3
.57 MeOH). H NMR (500 MHz, D O) 4.28-4.17 (m, 4H), 3.91 (dd, J = 12.5, 3.7, 1H),
2
.74 (dd, J = 12.5, 6.4, 1H), 3.48 (m, 1H), 2.14-1.93 (m, 4H), 1.40 (t, J = 7.1, 6H).
13
Referenced to HOD at 4.79 ppm. C NMR (125.7 MHz, D O), 64.3 (d, J = 6.6, CH ),
2
2
6
0.7 (CH ), 53.5 (d, J = 18.2, CH), 22.3 (d, J = 4.1, CH ), 21.0 (d, J = 141.2, CH ), 16.2
2 2 2
3
1
(
d, J = 5.7, CH ). Referenced to internal CH CN at δ 1.47. P NMR (202.4 MHz, D O),
3 3 2
+
3
3.3 (s). ESI-HRMS for C H NO P [M-HCl+H] calcd 226.1208; found 226.1213.
8 21 4
Diethyl [(3R)-3-amino-4-hydroxybutyl]phosphonate hydrochloride (55). Compound
3
3
5
4 (0.35 g, 0.96 mmol) is treated as described above for the preparation of 51 to give
2
2
title compound 55 (0.25 g, 99%). [α] D -5.1 (c 0.63, MeOH). ESI-HRMS for C H NO P
8
21
4
2
5

+
1
13
31
[
M-HCl+H] calcd 226.1208; found 226.1205. The H, C and P NMR spectra are
identical to those of the enantiomer 51.
Diethyl [(3S)-3-[({2-chloro-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]-4-hydroxybutyl]phosphonate (52). A mixture of 51 (0.055 g, 0.591
mmol), triethylamine (0.030 mL, 0.211 mmol), aldehyde 49 (0.1 g, 0.394 mmol) and 2-
picoline borane complex (0.055 g, 0.512 mmol) are stirred together in MeOH (4 mL) at
room temperature for 16 h. The solvent is evaporated and the residue chromatographed
on silica gel (CHCl -MeOH-7M NH in MeOH, 9.6:0.2:0.2 v/v/v) to give intermediate
3
3
diethyl [(3S)-3-({[4-(tert-butoxy)-2-chloro-5H-pyrrolo[3,2-d]pyrimidin-7-
yl]methyl}amino)-4-hydroxybutyl]phosphonate as a colourless gum (108 mg) which is
dissolved in 1:1 mixture of 37% aq. HCl-THF (3 mL) and left at room temperature for 10
min. The solvent is evaporated and the residue dissolved in EtOH and neutralized with
Amberlyst A21 resin. After filtering off the resin, the filtrate is evaporated and the
residue is chromatographed on silica gel (CHCl -MeOH-7M NH in MeOH,
3
3
7
.0:2.75:0.25 v/v/v) to give title compound 52 (0.070 g, 44%) as a colourless amorphous
2
2
1
solid. [α] D +10.3 (c 0.555, MeOH) H NMR (500 MHz, CD OD) 7.37 (s, 1H), 4.28 (d. J
3
=
13.7, 1H), 4.23 (d, J = 13.7, 1H), 4.13-4.05 (m, 4H), 3.93 (dd, J = 12.3, 3.6, 1H), 3.72
1
3
(
dd, J = 12.3, 4.6, 1H), 3.22 (m, 1H), 2.04-1.90 (m, 4H), 1.31 (dt, J = 7.0, 0.5, 6H). C
NMR (125.7 MHz, CD OD) 162.5 (C), 149.7 (C), 146.4 (C), 129.3 (CH), 119.4 (C),
3
1
07.7 (C), 63.5 (d, J = 6.4, CH ), 60.1 (CH ), 59.7 (d, J = 17.5, CH), 40.4 (CH ), 22.8 (d,
2 2 2
J = 3.7, CH ), 22.2 (d, J = 142.3, CH ), 16.7 (d, J = 5.8, CH ). Referenced to the centre
2
2
3
3
1
line of CD OD at δ 49.0. P NMR (202.3 MHz, CD OD), 31.7 (s). ESI-HRMS for
3
3
(35)
+
C H
ClN O P [M+H] calcd 407.1251; found 407.1250.
4 5
1
5
25
Diethyl [(3R)-3-[({2-chloro-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]-4-hydroxybutyl]phosphonate (56). In the same way as that
described for the synthesis of enantiomer (52 (Scheme 18), compound 55 (0.083 g, 0.317
mmol) is treated with triethylamine (0.030 mL, 0.211 mmol), aldehyde 49 (0.054 g, 0.21
mmol) and 2-picoline borane complex (0.029 g, 0.275 mmol) to give after treatment with
2
2
a 1:1 mixture of 37% aq. HCl-MeOH, title compound 56 (0.039 g, 45%). [α] D -10.6 (c
2
6

(
35)
+
0
4
.455, MeOH). ESI-HRMS for C H
ClN O P [M+H] calcd 407.1251; found
4 5
1
5
25
1
13
31
07.1250. The H, C and P NMR spectra are identical to those of the enantiomer 52.
Diethyl [(1R/S, 3S)-3-amino-1-fluoro-4-hydroxybutyl]phosphonate hydrochloride
3
3
(
59). The -fluoro-vinylphosphonate 58 (0.222 g, 0.582 mmol) and 10% Pd/C (50 mg)
are stirred in EtOH (10 mL) under a hydrogen atmosphere at ambient temperature and
pressure for 5.5 h. The mixture is filtered through Celite and the solvent evaporated to a
colourless gum (225 mg) which is dissolved in a mixture of EtOH (3 mL) and 37% aq.
HCl (1 mL) and left at room temperature for 1.5 h. The solvent is evaporated to give title
1
compound 59 as an ~1:1 mixture of diastereomers as a dark yellow gum. H NMR (500
MHz, D O), 5.38 (m, 0.5H), 5.28 (m, 0.5H), 4.38-4.30 (m, 4H), 3.96 (t, J = 3.4, 0.5H),
2
3
.94 (t, J = 3.4, 0.5H), 3.81-3.70 (m, 2H), 2.46-2.24 (m, 2H), 1.43 (t, J = 7.1, 6H).
1
3
Referenced to HOD at δ 4.79. C NMR (125.7 MHz, D O) 86.9 (dd, J = 175.6, 173.7,
2
CHF), 85.9 (dd, J = 177.2, 173.3, CHF), 66.0 (d, J = 7.0, CH ), 65.8 (d, J = 6.7, CH ),
2
2
6
1.6 (CH ), 61.0 (CH ), 51.3 (d, J = 16.0, CH), 50.6 (d, J = 14.7 CH), 29.9 (d, J = 19.8,
2 2
CH ), 29.8 (d, J = 19.2, CH ), 16.3 (d, J = 4.9, CH ). Referenced to internal CH CN at δ
2
2
3
3
3
1
19
1
.47. P NMR (202. MHz, D O), 18.0 (d, J = 76.0), 17.8 (d, J = 76.0). F NMR (470.5
2
+
MHz, D O) -209.7 (d, J = 76.0). ESI-HRMS for C H FNO P [M-HCl+H] calcd
2
8
20
4
2
44.1114; found 244.1111. The NMR spectra indicated 59 is a ~1:1 mixture of
diastereomers.
Diethyl [(1R/S, 3S)-3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-
yl]methyl}amino)-1-fluoro-4-hydroxybutyl]phosphonate (60). Compound 59 (0.157 g,
0
.561 mmol), aldehyde 8 (0.113 g, 0.401 mmol), Et N (0.034 mL, 0.241 mmol) and 2-
3
picoline borane complex (0.056 g, 0.521 mmol) are stirred together in MeOH (15 mL) at
o
5
7
0 C for 24 h. The solvent is evaporated and the residue flash chromatographed (CHCl -
3
M NH in MeOH, 99:1 then 99:2 v/v) to give title compound 60 as an ~1:1 mixture of
3
1
diastereomers as a colourless gum. H NMR (500 MHz, CD OD) 8.42 (s, 1H), 7.52-7.49
3
(
m, 3H), 7.39-7.29 (m, 3H), 5.61 (s, 2H), 5.13 (ddt, J = 46.4, 4.8, 2.4, 0.5H), 5.07 (ddt, J
46.7, 6.9, 3.4, 0.5H), 4.23-4.12 (m, 4H), 4.08 (d, J = 13.5, 0.5H), 4.01 (s, 1H), 3.98 (d,
J = 13.5, 0.5H), 3.72 (dt, J = 11.5, 4.7, 1H), 3.56 (dt, J = 10.7, 5.5, 1H), 2.97 (pent, J =
=
1
3
5
.6, 0.5H), 2.91 (sext, J = 4.6, 0.5H), 2.14-1.81 (m, 2H), 1.36-1.30 (m, 6H). C NMR
2
7

(
(
125.7 MHz, CD OD) 157.0 (C), 150.1 (CH), 150.0 (CH), 149.6 (C), 137.9 (C), 130.3
3
CH), 129.5 (CH), 129.4 (CH), 129.2 (CH), 116.7 (C), 115.2 (C), 114.8 (C), 87.8 (dd, J =
1
6
77.5, 171.9 CHF), 87.5 (dd, J = 177.5, 171.9 CHF), 69.0 (CH ), 64.8 (t, J = 8.4, CH ),
2 2
4.5 (t, J = 7.5, CH ), 64.1 (CH ), 63.7 (CH ), 56.4 (d, J = 13.6, CH), 55.7 (d, J = 13.9,
2
2
2
CH), 41.3 (CH ), 41.2 (CH ), 33.8 (d, J = 20.1, CH ), 32.7 (d, J = 19.7, CH ), 16.7 (CH ).
2
2
2
2
3
3
1
Referenced to the centre line of CD OD at δ 49.0. P NMR (202.3 MHz, CD OD) 19.0
3
3
1
9
(
d, J = 77.6), 18.5 (d, J = 77.6). F NMR (470.5 MHz, CD OD) -209.9 (d, J = 77.7), -
3
+
2
4
10.7 (d, J = 77.4). ESI-HRMS for C H FN O P [M+H] calcd 481.2016; found
22
31
4
5
81.2014. The NMR spectra indicated 60 is a ~1:1 mixture of diastereomers.
Diethyl [(3S)-3-amino-1,1-difluoro-4-hydroxybutyl]phosphonate hydrochloride (63).
34-35
Compound 62
(0.345 g, 0.860 mmol) is dissolved in a mixture of EtOH (3 mL) and
3
7% aq. HCl (1 mL) and left to stand at room temperature for 1.5 h. The solvent is
1
evaporated to give title compound 63 as a yellow gum (0.256 g, 100%). H NMR (500
MHz, CDCl ) 8.24 (bs, 3H), 4.60 (bs, 1H), 4.35-4.28 (m, 4H), 4.02 (d, J = 9.1, 1H), 3.92-
3
1
3
3
.81 (m, 2H), 2.91-2.74 (m, 1H), 2.63-2.47 (m, 1H), 1.39 (dt, J = 7.0, 1.0, 6H). C NMR
(
125.7 MHz, CDCl ) 119.3 (dt, J = 261.3, 216.5, CF ), 65.5 (d, J = 4.5, CH ), 61.5 (CH ),
3
2
2
2
4
8.2 (CH), 33.5 (m, CH ), 16.3 (d, J = 5.0, CH ). Referenced to the centre line of CDCl
2 3
3
3
1
19
at δ 77.0. P NMR (202.3 MHz, CDCl ), 5.4 (t, J = 104.9). F NMR (470.5 MHz,
3
CDCl ) -107.7 (dd, J = 301.1, 102.5), -111.2 (dd, J = 301.3, 105.9). ESI-HRMS for
3
+
C H F NO P [M-HCl+H] calcd 262.1020; found 262.1017.
8
19
2
4
Diethyl [(3S)-3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)-
,1-difluoro-4-hydroxybutyl]phosphonate (64). Compound 63 (0.140 g, 0.470 mmol),
1
aldehyde 8 (0.095 g, 0.336 mmol), Et N (0.042 mL, 0.302 mmol) and 2-picoline borane
3
o
complex (0.047 g, 0.437 mmol) are stirred in MeOH (15 mL) at 50 C for 24 h. The
solvent is evaporated and the residue flash chromatographed (CHCl -7M NH in MeOH,
3
3
2
2
9
9:1 then 98:2 v/v) to give title compound 64 as a colourless gum (0.088 g, 53%). [α] D
1
+
7.5 (c 0.475, MeOH). H NMR (500 MHz, CD OD) 8.42 (s, 1H), 7.53 (s, 1H), 7.51 (d,
3
J = 7.3, 2H), 7.38-7.30 (m, 3H), 5.60 (s, 2H), 4.29-4.22 (m, 4H), 4.04 (d, J = 13.6, 1H),
3
1
.99 (d, J = 13.6, 1H), 3.73 (dd, J = 11.3, 4.3 1H), 3.53 (dd, J = 11.3, 6.2, 1H), 3.18 (m,
1
3
H), 2.34-2.24 (m, 2H), 1.34 (t, J = 7.0, 6H). C NMR (125.7 MHz, CD OD) 157.0 (C),
3
2
8

1
1
6
50.1 (CH), 149.5 (C), 137.8 (C), 130.4 (CH), 129.5 (CH), 129.4 (CH), 129.2 (CH),
22.0 (dt, J = 259.1, 218.5, CF ), 116.7 (C), 114.5 (C), 69.0 (CH ), 66.2 (d, J = 6.7, CH ),
2
2
2
4.2 (CH ), 53.6 (CH), 41.1 (CH ), 36.3 (dt, J = 19.8, 14.3, CH ), 16.7 (CH ). Referenced
2
2
2
3
3
1
to the centre line of CD OD at δ 49.0. P NMR (202.4 MHz, CD OD) 6.7 (t, J = 109.9).
3
3
1
9
F NMR (470.5 MHz, CD OD) -110.3 (dd, J = 299.3, 109.9, -111.6 (dd, J = 299.3,
3
+
1
09.9). ESI-HRMS C H F N O P [M+H] calcd 499.1922; found 499.1927.
22
30
2
4
5
[(3S)-4-Hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-
d]pyrimidin-7-yl}methyl)amino]butyl]phosphonic acid hydrobromide (53).
Compound 52 (0.060 g, 0.147 mmol) is dissolved in EtOH (5 mL), 10% Pd-C (40 mg)
added and the mixture stirred under a hydrogen atmosphere at ambient pressure and
temperature for 3 h. The mixture is filtered through Celite and the solvent evaporated to
give diethyl [(3S)-4-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-
yl}methyl)amino]butyl]phosphonate hydrochloride (0.055 g, 91%) as a colourless solid.
2
2
1
[
α] +12.2 (c 0.54, MeOH). H NMR (500 MHz, CD OD) 7.97 (s, 1H), 7.67 (bs, 1H),
D
3
4
1
2
1
.47 (d, J = 13.9, 1H), 4.43 (d, J = 13.9, 1H), 4.15-4.06 (m, 4H), 4.00 (dd, J = 12.4, 2.9,
H), 3.76 (dd, J = 12.4, 4.1, 1H), 3.35 (m, partially hidden by residual CD OD, 1H),
3
1
3
.11-1.88 (m, 4H), 1.32 (t, J = 7.1, 6H). C NMR (125.7 MHz, CD OD) 155.7 (C),
3
45.2 (C), 143.8 (CH), 130.8 (CH), 119.6 (C), 108.0 (C), 63.6 (d, J = 4.3, CH ), 59.6 (d,
2
J = 17.7, CH), 59.0 (CH ), 39.7 (CH ), 22.3 (d, J = 3.7, CH ), 22.2 (d, J = 142.7, CH ),
2
2
2
2
3
1
1
6.7 (d, J = 5.8, CH ). Referenced to the centre line of CD OD at δ 49.0. P NMR (202.3
3 3
+
MHz, CD OD) 31.2 (s). ESI-HRMS for C H N O P [M-HCl+H] calcd 373.1641;
3
15 26
4
5
o
found 373.1642. This material (0.052 g, 0.127 mmol) is heated at 80 C in hydrobromic
acid (48% aq., 1.002 mL, 12.72 mmol) for 4 h. The solvent is evaporated and the residue
eluted from a column of RP 18 silica gel (H O) to afford title compound 53 as a
2
2
3
1
colourless glass which slowly crystallized. [α] D +9.3 (c 0.355, H O). H NMR (500
2
MHz, D O + DCl): δ 8.93 (s, 1H), 7.84 (s, 1H), 4.51 (d, J = 14.7 Hz, 1H), 4.48 (d, J =
2
1
1
4.7 Hz, 1H), 3.97 (dd, J = 13.1, 2.7 Hz, 1H), 3.81 (dd, J = 13.1, 4.7 Hz, 1H), 3.46 (m,
13
H), 2.08-1.77 (m, 4H). Referenced to HOD at 4.79 ppm. C NMR (125.7 MHz, D O
2
+DCl) 153.4 (C), 145.2 (CH), 133.8 (C) 133.7 (CH), 118.8 (C), 104.0 (C), 59.8 (d, J =
1
7.1 Hz, CH), 58.5 (CH ), 38.9 (CH ), 23.3 (d, J = 137.2 Hz, CH ), 21.5 (d, J = 3.2 Hz,
2
2
2
2
9