Middlesbrough, West Ham and West Brom became the first Premier League academy teams to be eliminated from this year's revamped EFL Trophy competition.
Boro lost 2-1 at Cambridge, West Ham were beaten 3-0 by Wycombe and the Baggies lost 2-0 to Luton - their second defeats of the group stage.
Norwich and Leicester are the first development squads to go through.
League One leaders Scunthorpe and third tier strugglers Coventry City also qualified for the next round.
Tuesday saw 62 teams in action with 13 clubs sealing their place in the second round - an evening once again marked by low attendances at most grounds.
On Monday, English Football League chief executive Shaun Harvey defended the new format of the Checkatrade Trophy which saw 16 development squads introduced into the tournament for 2016-17.
Norwich's academy side followed their 6-1 win at Peterborough a fortnight ago with a 5-0 thumping of Barnet - which eliminated the League Two hosts. That game was watched by 457 fans.
Kairo Mitchell scored the only goal in Leicester's 1-0 win at fourth tier Grimsby which sent the Premier League academy team through and put the Mariners out.
Elsewhere, in the southern section, Wycombe, Crawley, MK Dons, Southend, Luton, Millwall and AFC Wimbledon progressed.
In the northern section, Cambridge and Walsall joined Scunthorpe and Leicester in round two.
Six of the academy clubs' players have to be under 21, with no limits on the other five players in their team.