Synthesis of N-alkyl substituted indolocarbazoles as potent inhibitors of human cytomegalovirus replication

Article abstract of DOI:10.1016/S0960-894X(01)00352-3

The synthesis and antiviral evaluation of unsymmetrical indolocarbazole derivatives of Arcyriaflavin A, substituted with a range of alkyl groups at the indole nitrogen, is described. Structure-activity relationships in this series against human cytomegalovirus (HCMV) replication in cell culture are reported. Compound 4b was identified as potent inhibitor of HCMV (IC₅₀=19 nM), which retained activity against a range of HCMV strains including ganciclovir resistant isolates.

Full text of DOI:10.1016/S0960-894X(01)00352-3

Bioorganic & Medicinal ChemistryLetters 11 (2001) 1993–1995
Synthesis of N-Alkyl Substituted Indolocarbazoles as Potent
Inhibitors of Human Cytomegalovirus Replication
Martin J. Slater,* Robert Baxter, Robert W. Bonser, Stuart Cockerill,
Kam Gohil, Nigel Parry, Ed Robinson, Roger Randall, Clive Yeates,
WendySnowden and Adeline Walters
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
Received 26 March 2001; accepted 16 May2001
Abstract—The synthesis and antiviral evaluation of unsymmetrical indolocarbazole derivatives of Arcyriaflavin A, substituted with
a range of alkyl groups at the indole nitrogen, is described. Structure–activity relationships in this series against human cytomega-
lovirus (HCMV) replication in cell culture are reported. Compound 4b was identified as potent inhibitor of HCMV (IC50=19 nM),
which retained activityagainst a range of HCMV strains including ganciclovir resistant isolates. # 2001 Elsevier Science Ltd. All
rights reserved.
Human cytomegalovirus (HCMV) disease is a common
life-threatening opportunistic viral infection in the
immunocompromised. It is a primarycause of death in
recipients of bone marrow and renal transplants, and is
one of the most prevalent serious infections in AIDS
patients, frequentlygiving rise to pneumonitis and
retinitis.1À6 The introduction of highlyactive anti-
retroviral therapy(HAART) has had a major impact on
the treatment of HCMV disease in HIV-infected indivi-
duals, with the successful suppression of HIV RNA
levels and immune reconstitution permitting dis-
continuation of HCMV maintenance therapywithout
relapse.7À9 How long anti-HCMV therapycan be inter-
rupted and when treatment should be initiated, is likely
to be related to the emergence of HIV resistance and the
potential progression of HIV disease and AIDS. Cur-
rent treatment options for HCMV disease are limited to
ganciclovir (GCV), foscarnet (PFA), cidofovir
(HPMPC) and formivirsen.10À15 While these drugs have
made significant contributions to the treatment of
HCMV disease, all possess significant side effects and
have poor oral bioavailability, requiring intravenous or
intraocular routes of administration. Here, we report on
a group of compounds identified during our search for
an agent with potent anti-HCMV activityand an
improved profile of safetyand bioavailabilit.y We
recentlyreported the discoverythat symmetrical indo-
locarbazoles such as 1 (Arcyriaflavin A) are a new class
of potent, selective inhibitors of HCMV replication
(Fig. 1).16 We explored one aspect of the structure–
activityrelationships (SARs) and discovered that sym-
metrical substitution into the phenyl indole groups with
small lipophilic moieties (e.g., 2,10-difluoro substitu-
tion, 2) was well tolerated.16 We now report the synth-
eses and SARs of unsymmetrical N-alkyl substituted
indolocarbazoles.
Our objective was to investigate the effect on antiviral
activityof substitution at the indole nitrogen, removing
the symmetry in the process. We chose to introduce
simple alkyl groups at the indole nitrogen atoms, since
indolocarbazoles bearing more complex functionalities,
particularlyfuran and pyran derivatives, are known to
be potent inhibitors of protein kinase C (PKC) and/or
topoisomerase I,17,18 properties which must be absent in
*Corresponding author. Tel.: +44-1438-763416; fax: +44-1438-
763616; e-mail: mjs40312@glaxowellcome.co.uk
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00352-3

1994
M.J.Slater et al./ Bioorg.Med.Chem.Lett.11 (2001) 1993–1995
an anti-HCMV agent. We have alreadyestablished that
Arcyriaflavin A is essentially inactive against PKC and
does not intercalate into DNA.16,19
yields and cleaner reaction profiles, particularly in the
case of b-branched substituents. In the case of the iso-
propyl compound 4e for example, no product could be
isolated using DDQ/PTSA, whereas a modest 23%
yield was attainable using light and iodine.
In order to synthesise the mono-alkylated indolocarba-
zole targets, we initiallyprepared the previouslyunre-
ported bis-BOC protected intermediate of 1. Thus,
treatment of 1 with (BOC)2O and sodium hydride in
DMF and subsequent purification bycolumn chroma-
tographyafforded 3 in good yield. This could be readily
derivatised with the more reactive alkylating agents
(methyl iodide, ethyl iodide, benzyl iodide) and depro-
tected in a separate step to afford the targets 4a–c
(Scheme 1). For less reactive alkyl halides, such as n-
propyl iodide and isopropyl iodide, several highly-
fluorescent impurities formed during the alkylation
stage, from which it was verydifficult to isolate the pure
products. It transpires that the BOC groups are not
entirelystable during the reaction conditions and a
mixture of mono- and di-alkylated compounds are
formed over prolonged reaction times. We therefore
sought an alternative approach.
The new indolocarbazoles (4a–g) were assayed for anti-
viral activityin standard plaque reduction assasy
against HSV-1, HSV-2, HCMV, VZV, influenza A and
HIV-1 (Table 1, HCMV data shown only),23 and for
Published approaches to the regiocontrolled introduc-
tion of substituents at the indolocarbazole nitrogens
either take advantage of their differing electronic char-
acter on the staurosporine lactam aglycone, or utilise an
orthogonallyprotected
bis-indole
maleimide
strategy.17,20À22 With a large stock of the bis-indolyl-N-
methyl maleimide 5 available,16 we chose to simply
alkylate this intermediate since the mono- and di-sub-
stituted products were readilyseparated bychromato-
graphyover silica. Subsequent conversion of the mono-
alkyl derivatives (6d–g) to the corresponding maleic
anhydrides, and then to the parent bis-indolyl mal-
Scheme 2. (i) R-X, NaH, DMF; (ii) KOH, MeOH or dioxan; (iii)
NH4OAc, 140 ꢀC; (iv) DDQ, PTSA, toluene, dioxan; (v) I2, hn, iso-
propanol.
eimides (7d–g) was accomplished in good yields as pre-
16
viouslydescribed.
The final cyclisation step was
Table 1. Anti-HCMV activity, PKC inhibition data and cell cyto-
toxicityof indolocarbazoles
achieved either with 2,3-dichloro-5,6-dicyanobenzoqui-
none (DDQ) and catalytic p-toluenesulphonic acid
(PTSA) in toluene and dioxan solvent mixtures under
reflux, or photochemicallyin the presence of iodine in
isopropanol (Scheme 2).16 Generally, we have found the
photochemical method to be preferred, giving higher
Compound
R
HCMV
CCID50
PKC
IC50/mM (Vero cells)/mM % Inhibition
at 100 mM
1
H
Me
Et
Benzyl
nPr
iPr
iBu
CH2CF3
—
0.2
>125
>31
>125
<31
<8
28
<8
<8
<50
47
IC50=10 mM
23
IC50=50 mM
IC50=50 mM
71
IC50=25 mM
Not tested
4a
4b
4c
4d
4e
4f
4g
GCV
0.06
0.019
T10
0.15
0.1
0.03
0.07
3
>500
T10=unable to measure an antiviral effect due to cell toxicityat 10
mM.
Table 2. Activityof 4b against various strains of HCMV in MRC5
cells
Compound
HCMV strain IC50/(mM)
AD169 Towne Davis Xba Fa C8805b 2599R
4b
GCV
0.019
3
0.09
1.5
0.03
1.4
0.02
>10
0.06
ND
0.13
1.6
Scheme 1. (i) 2(BOC)2O, 2NaH, DMF, 70%; (ii) methyl iodide or
ethyl iodide or benzyl bromide, NaH, DMF; (iii) HCl, EtOAc or 90%
aq TFA.
aGCV resistant due to 4 amino acid deletion in UL97 gene.
bGCV resistant due to mutation in DNA polymerase.

M.J.Slater et al./ Bioorg.Med.Chem.Lett.11 (2001) 1993–1995
1995
24
their abilityto inhibit protein kinase C (PKC).
pattern of activitymirrored that observed with the aryl
The
cityassays, Dr. Karen Biron for the provision of virus
strains Xba F and C8805, Dr. Margaret Tisdale for
influenza and HIV assays and Drs. Peter Collins, Eddy
Littler and George Hardyfor advice.
ring substituted series, in that antiviral activitywas spe-
cific for HCMV. All new N-alkyl indolocarbazoles pos-
sessed significant anti-HCMV activity, with the
exception of the N-benzyl compound 4c. The ethyl
derivative 4b was the most potent example, with an IC50
of 19 nM, being two orders of magnitude more active
than ganciclovir. A clear trend is apparent in the cell
cytotoxicity data, with side chains bigger than ethyl
conferring significant cytotoxicity. The ethyl compound
(4b) itself was not toxic, and has an apparent ther-
apeutic index >6500. There is a good separation
between the concentration required to inhibit HCMV
replication in cell culture and that required to inhibit
PKC. No correlation was noted in this series between
HCMV activity, cell cytotoxicity and PKC inhibition.
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dione (4b) as a potent, selective inhibitor of HCMV
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lead for the development of new anti-HCMV agents.
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ment of this particular compound. Our approaches to
applythe SARs described herein in order to design potent
analogues with improved solubilitywill be reported in
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The authors wish to thank Alan Emerson and Chris
Roberts for excellent technical assistance with cytotoxi-